Nucleotide Adducts Research Articles

Cytochrome P4501A induction, benzo[a]pyrene metabolism, and nucleotide adduct formation in fish hepatoma cells: Effect of preexposure to 3,3′,4,4′,5‐pentachlorobiphenyl

AbstractIn PLHC‐1 hepatoma cells, benzo[a]pyrene (B[a]P) caused a maximum induction of cytochrome P4501A (CYP1A) activity, measured as ethoxyresorufin O‐deethylation (EROD), after 4 to 8 h of exposure, depending on the B[a]P concentration. The decline of EROD activity at longer exposure times was probably caused by the rapid metabolism of B[a]P in this system (57% metabolism within 4 h incubation). In subsequent experiments, PLHC‐1 cells were preinduced with PCB 126 for 24 h and then received a dose of 10, 100, or 1,000 nM 3H‐B[a]P. A 1‐nM concentration of PCB 126 caused an 80‐fold induction of CYP1A activity, resulting in an increase in B[a]P metabolism of less than 10%, except at the highest concentration of B[a]P (1,000 nM), where a 50% increase was observed. In another experiment, an 80‐fold induction of CYP1A activity caused a 20% increase in the metabolism of B[a]P (100 nM), and RNA adduct formation was increased approximately twofold. These results indicate that, at exposure concentrations up to 100 nM B[a]P, CYP1A activity is not rate limiting for B[a]P metabolism. Furthermore, CYP1A seems to also be specifically involved in B[a]P activation in PLHC‐1 cells. However, CYP1A induction causes only a relatively small increase in activation, probably because of the action of other enzymes involved in B[a]P activation and deactivation.

A new modification of the 32P-post-labeling method to recover IQ-DNA adducts as mononucleotides.

To obtain accurate estimates of DNA adduct levels yielded by genotoxic compounds, it is essential to completely digest adducted nucleotides to mononucleotides. We previously developed a suitable method, called modified method I, to obtain DNA adducts of heterocyclic amines as 32P-labeled-mononucleoside adduct 5'-phosphate forms, by use of nuclease P1 (NP1) and phosphodiesterase I (PDEI) to digest adducted oligonucleotides. In this study, we applied method I to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-DNA adduct analysis and found that one of the IQ-DNA adducts, 5-(deoxyguanosin-N2-yI)-2-amino-3-methylimidazo[4,5-f]quinoline 3',5'-diphosphate (pdGp-N2-IQ), was resistant to the 3'-phosphatase activity of NP1, but sensitive to that of T4 polynucleotide kinase (PNK). DNA obtained from the liver of rats fed IQ was 32P-labeled by the standard method and the 32P-labeled nucleotides obtained were incubated with PNK and NP1 to remove 3'-phosphate groups and then digested with PDEI. Three spots were obtained. One major spot was identified as N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline 5'-phosphate (pdG-C8-IQ) and a second abundant adduct as pdG-N2-IQ. The third spot, of which the structure is unknown, was minor. The new method is called modified method II. Modified method II could be applicable to a wide variety of chemicals.

Mass spectrometric analysis of 2-deoxyribonucleoside and 2'-deoxyribonucleotide adducts with aldehydes derived from lipid peroxidation.

An important emerging issue in chemical carcinogenesis is the role that products of endogenous metabolism play in formation of covalently modified DNA. One example is the formation of alpha, beta-unsaturated aldehydes as a result of endogenous and drug-stimulated lipid peroxidation. Malondialdehyde (MDA), crotonaldehyde (CR), 2-hexenal (HX), and 4-hydroxy-2-nonenal (HNE) react covalently with 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) residues on DNA to form promutagenic cyclic adducts that may be important in the etiology of cancer in humans and animals. The accurate quantification of such adducts provides a powerful tool in molecular epidemiology for assessing carcinogenic risks from various lifestyle choices (e.g. diet, drug use) in humans. 32P-Postlabeling is recognized as one of the most sensitive methods available for detection of DNA adducts in human tissues, but without adequate validation such methodology can yield inaccurate quantitative measurements. We have used LC separations in conjunction with electrospray ionization MS and tandem MS (triple quadrupole and hybrid quadrupole-orthogonal acceleration time of flight analyzers) to characterize MDA-, CR-, HX- and HNE-modified dG and nucleotide (3'- and 5'-monophosphate; 3',5'-bisphosphate) adducts. These data have been used to validate 32P-postlabeling methods for quantification of low level MDA-dG adducts formed in DNA of human and animal tissues. Availability of reliable methods for quantification of endogenous DNA damage in humans and animals is essential for determining unknown etiologies of cancer and for the assessment of cancer risks in humans.

Absence of DNA Adduct Formation by Phenobarbital, Polychlorinated Biphenyls, and Chlordane in Mouse Liver Using the32P-Postlabeling Assay

Phenobarbital (PB), polychlorinated biphenyls (PCBs), and chlordane (CLD) increase liver tumor incidences in rodents, and all are tumor promoters. Most indirect tests for DNA reactivity, including mutagenicity and chromosomal damage, have been negative with these agents. Consequently, the modes of action for tumorigenesis by these compounds are not believed to involve direct DNA reactivity; however, only limited information from direct tests is available for the lack of DNA adduct formation. PB, PCBs, and CLD were tested for DNA adduct formation in the liver of male and female B6C3F1mice after either single or 2-week dietary exposures. Single gavage dose levels were as follows: PB, 200 mg/kg; PCBs, 50 mg/kg; and CLD, 50 mg/kg. Dietary dose levels were as follows: PB, 1000 ppm; PCBs, 200 ppm and CLD, 200 ppm. Animals were killed 24 h following the end of test-substance administration. DNA was extracted from the liver, and DNA adduct concentrations were enriched using either 1-butanol extraction of adducted nucleotides or nuclease P1digestion of unadducted nucleotides. Using this protocol, none of the three test compounds produced DNA adducts detected by32P-postlabeling. Similar negative results were obtained for DNA from the livers of both male and female mice receiving either single or 2-week exposures. The two positive controls, benzidine for the 1-butanol extraction procedure and 2-acetylaminofluorene for the nuclease P1procedure, showed the expected patterns of DNA adducts. These results support the conclusion that the carcinogenicity of PB, PCBs, and CLD in experimental animals is not the result of direct DNA reactivity, but involves epigenetic mechanisms.

Identification of adenine nucleotide-containing metabolites of bisphosphonate drugs using ion-pair liquid chromatography–electrospray mass spectrometry

Bisphosphonates are synthetic pyrophosphate analogues, which are used as therapeutic drugs for the treatment of metabolic bone disorders. Some of these bisphosphonates can be metabolised in cells into non-hydrolysable nucleotide analogues. In this paper, we describe an ion-pairing high-performance liquid chromatography method that is compatible with negative ion electrospray mass spectrometry for the separation of these metabolites. Tandem mass spectrometry and collision-induced dissociation (CID) were used for identification of the metabolites. The CID mass spectra of bisphosphonate–adenine nucleotide adducts are very informative, because major fragment ions are formed by cleavage of the bisphosphonate moiety from the conjugate. The method was used for detection of the nucleotide metabolites of clodronate, tiludronate and etidronate in extracts from mammalian cells after treatment with bisphosphonates.

Combination of high-performance liquid chromatography and thin-layer chromatography separation of five adducted nucleotides isolated from liver resulting from intraperitoneal administration with 7H-dibenzo[ c, g]carbazole to mice

7H-Dibenzo[ c, g]carbazole, DBC, is a potent environmental liver carcinogen. Liver DNA from mice treated with DBC exhibited seven distinct DBC-DNA adducts as detected by 32P-postlabeling using multidimensional TLC. To improve quantitation and chemically characterize the adducts, DNA samples were hydrlyzed, 32P-postlabeled and the adducts were separated from the unadducted normal nucleotides on TLC using a D1 solvent, 0.65 M sodium phosphate (pH 6.8). Adducts were eluted from the TLC plates with 4.0 M pyridinium formate, concentrated, resuspended in 50% aqueous methanol and injected onto the HPLC; five individual adduct peaks were resolved and collected by this method. This approach will prove useful to decrease analysis time and improve chemical characterization of tightly clustered DNA adducts generated in vivo.

Comparisons of the binding of [ 14C]radiolabelled tamoxifen or toremifene to rat DNA using accelerator mass spectrometry

Tamoxifen, widely used as adjuvant therapy in the treatment of breast cancer, is now undergoing trials as a cancer chemopreventative agent. Previous work has shown an association between 32P-postlabelled adducts in rat liver DNA and the development of liver tumours. With the use of accelerator mass spectrometry, [ 14C]tamoxifen was shown to bind to liver DNA of female rats in a dose-dependent manner and was linear over 0.1–1 mg/kg, compatible with the therapeutic dose used in women (20 mg/person per day). Radiolabel could also be detected in extrahepatic organs, including reproductive and GI-tract, where levels were about 18 and 46%, respectively those seen in liver. Following enzymatic hydrolysis of liver DNA, normal nucleotides by HPLC showed <2% incorporation of the [ 14C]radioactivity while >80% appeared as non-polar products. In contrast, when animals were given an equivalent dose of [ 14C]toremifene, binding to DNA was an order of magnitude lower than that seen with tamoxifen and no evidence of non-polar adducted nucleotides following HPLC. However, in vitro, using human, rat or mouse liver microsomal preparations, NADPH-dependent binding of both toremifene and tamoxifen to calf thymus DNA could be demonstrated, suggesting that under favourable circumstances toremifene is capable of undergoing conversion to reactive intermediates.

Sequence specific mutagenesis of the major (+)-anti-benzo[a]pyrene diol epoxide-DNA adduct at a mutational hot spot in vitro and in Escherichia coli cells.

In the supF gene, most (+)-anti-benzo[a]pyrene diol epoxide ((+)-anti-B[a]PDE) mutagenesis hot spots in Escherichia coli are in 5'-GG sequences [Rodriguez and Loechler (1993) Carcinogenesis 14, 373-383]. A major hot spot was detected at G1 in the sequence 5'-GCG1G2-CCAAAG, whereas G2 yielded very few mutants. In order to investigate the details of such sequence context effects of (+)-anti-B[a]PDE mutagenesis, we have constructed 25-mer oligonucleotides and single-stranded M13 genomes containing the above decamer sequence, in which the trans-N2-dG adduct induced by (+)-anti-B[a]PDE [(+)-trans-anti-B[a]P-N2-dG] at G1 or G2 was introduced. In vitro DNA synthesis on the adducted 25-mers was strongly blocked at each site, although the 3'-->5' exonuclease-deficient Klenow fragment could incorporate a nucleotide opposite the adduct in the presence of Mn2+. For both sites purine nucleotides were preferred. The ratio Vmax/K(m) indicated that the efficiency of incorporation of dGTP opposite these sites was very similar, but dATP incorporation opposite the adduct at G1 was five-fold more efficient than that at G2. For each site, further extension beyond the adducted nucleotide was investigated by annealing four different primers, in which only the nucleotide opposite the adducted deoxyguanosine was altered. Significant extension was only observed when deoxyadenosine was located opposite adducted G1. When the M13 genomes containing the (+)-trans-anti-B[a]P-N2-dG were replicated in E. coli, survival of each adducted genome was less than 1% as compared to the unadducted genome. Upon induction of SOS, viability increased 2-6-fold. DNA sequencing showed no base substitutions in the progeny from SOS-uninduced cells, although small deletions in a quasipalindromic sequence occurred with the adduct being located at either site. However, following SOS induction, up to 40% targeted base substitutions were detected when the adduct was located at G1, while approximately 12% of the progeny were mutants with the adduct at G2. Most base substitutions were targeted G-->T transversions. We conclude that (+)-trans-anti-B[a]P-N2-dG is a highly mutagenic and replication blocking lesion. In addition, the biological consequence of this adduct depends on whether it is located at G1 or G2, suggesting that sequence context plays a major role in the mutagenic processing of this adduct.

Postlabeling detection of DNA adducts of antitumor alkylating agents.

The sensitivity for DNA adduct formation by antitumor alkylating agents (mechlorethamine, cisplatin and adozelesin) of the postlabeling technique and thin-layer chromatography was studied. Three DNAs were used: a double-stranded 20-bp oligonucleotide of defined sequence, calf thymus DNA and murine leukemia L1210 cellular DNA. With high concentrations of mechlorethamine, there was a marked decrease in normal dGp, a lesser decrease in dAp and dCp and no change in dTp. Using 2D mapping PEI-cellulose thin-layer chromatography analyses, it was found that six mechlorethamine: DNA adducts were produced after a short exposure to mechlorethamine. After an extended time at relatively high drug concentrations there was an alteration in the mechlorethamine: DNA adduct pattern that may reflect the conversion of monoadducts to crosslinked adducts. Similar observations were made with cisplatin and adozelesin. When murine leukemia L1210 cells were treated with 50 microM mechlorethamine or 50 microM cisplatin for 1 h, six or more mechlorethamine: DNA adducts and five cisplatin: DNA adducts were detected. After allowing 6 h. for repair of potentially lethal damage, several adducts were no longer detectable and others appeared with diminished intensity. Nuclease P(1) dephosphorylates normal nucleotides at relatively low enzyme concentrations with variation depending upon the nucleotide. In general, considerably lower concentrations of nuclease P1 were required to dephosphorylate the normal nucleotides than to dephosphorylate the antitumor alkylating agent: nucleotide adducts, thus allowing increased sensitivity of the postlabeling assay. The sensitivity of detection of antitumor alkylating agent: DNA adducts in DNA from treated L1210 cells approached one adduct per 10(7)-10(8) nucleotides. These results suggest that the postlabeling technique may be sufficiently sensitive and specific for the study of the clinically effective levels of antitumor alkylating agents.

Detection and identification of benzo[a]pyrene diol epoxide adducts to DNA utilizing capillary electrophoresis-electrospray mass spectrometry.

Capillary zone electrophoresis (CZE) coupled with negative ion electrospray mass spectrometry (ES-MS) is used for the detection and identification of adducts formed from the reaction of DNA with (+/-)-anti-7,8,9,10-tetrahydrobenzo[a]pyrene-7,8-diol 9,10-epoxide (BPDE),an active metabolite of benzo[a]pyrene (BaP). Results presented in this paper demonstrate low nanogram detection limits ( < 10 ng or < 15 pmol) for normal scan spectra and collision-induced dissociation spectra of the main nucleotide adduct formed from this reaction. (BPDE reacts predominantly with the exocyclic amino group of guanine.) Exploitation of selective reaction monitoring (SRM) produces detection limits in the low picogram range ( < 85 pg or < 130 fmol). The application of sample stacking significantly increases the concentration detection limit (to approximately 10(-8) M). Nucleotide adducts are negatively charged at most pHs and are therefore ideally suited to the stacking process used in this research. These techniques have been applied to the analysis of the adducts formed from the in vitro reaction of BPDE with DNA. In addition it is shown that CZE-ES-MS, combined with solid-phase sample cleanup, can detect adducts at levels of four adducts in 10(7) unmodified bases or less.

Stabilization of monofunctional platinum–nucleotide adducts: reactions of N-acetyl-L-methionine complexes with guanosine 5′-monophosphate and guanylyl(3′–5′)guanosine

The complexes [Pt(en)MeCO-Met-S)Cl]NO31, [Pt(en)(MeCO-Met-S)2][NO3]22(en = ethane-1,2-diamine, MeCO-Met =N-acetyl-L-methionine) and their 15N analogues (1n and 2n) have been prepared and characterized by 1H, 13C and two-dimensional [1H, 15N] spectroscopy. Complex 1(half-life 3.9 h at 310 K) hydrolysed more slowly than [Pt(en)Cl2], whereas 2 was stable in water. The reaction of 1n with guanosine 5′-monophosphate (5′-GMP) gave a stable mixed-ligand complex [Pt([15N]en){MeCO-Met(1–)-S}(5′-GMP-N7)]+, and the reaction with guanylyl(3′-5′)guanosine (GpG) gave two different monofunctional adducts [Pt([15N]en){MeCO-Met(1–)-S}(GpG-N7)]+, due to platination of either 3′- or 5′-G, with a preferential formation of one over the other (ratio 60:40). During the initial stages of the reaction the chelated complex [Pt([15N]en){MeCO-Met(2–)-S,N}]3 was also observed, which subsequently reacted with 5′-GMP or GpG via ring opening to give monofunctional adducts. Reactions of complex 2 with 5′-GMP and GpG also lead to such adducts, with release of MeCO-Met. Little conversion of monofunctional adducts into bifunctional adducts was observed. Methionine and its derivatives could play a role in the trapping of monofunctional adducts of platinum anticancer drugs with DNA in vivo.

Acridine Adduct of [60]Fullerene with Enhanced DNA-Cleaving Activity

Photochemical cleavage of DNA in the presence of C{sub 60} and a C{sub 60} acridine derivative is reported. It is found that the intercalator-linked C{sub 60} is more effective in photochemical DNA cleavage. Structural simulations of the adducted nucleotides are reported.

(E)-2-hexenal-induced DNA damage and formation of cyclic 1,N2-(1,3-propano)-2'-deoxyguanosine adducts in mammalian cells.

(E)-2-Hexenal (hexenal), a natural flavor compound, acts as directly genotoxic agent and forms cyclic 1,N2-propano adducts with deoxyguanosine. Formation of this adduct in isolated DNA and in cells was studied with a modified 32P-postlabeling procedure including HPLC separation, nuclease P1 enrichment, two-dimensional TLC of adducted nucleotide bisphosphates on PEI-cellulose, and quantification of adduct spots by liquid scintillation counting. Adduct formation with the more reactive crotonaldehyde was included for comparison. Synthesized adducted dG-3'-phosphates served as external standards for identification and quantification. In calf thymus DNA, hexenal (0.2 mM) shows a time dependent formation of adducts, yielding 1.55 pmol/mumol of DNA at 5 h incubation. With crotonaldehyde (0.2 mM) the adduct rate was about 10-fold higher. Hexenal also generated 1,N2-propano-dG adducts in the human lymphoblastoid Namalva cell line (0.2 mM, 1 h, 86 fmol/mumol of DNA) and in primary rat colon mucosa cells (0.4 mM, 30 min, 50 fmol/mumol of DNA). In primary colon mucosa cells from rats and humans, hexenal and crotonaldehyde (0.4 mM, 30 min) induced DNA damage, detected by single cell microgel electrophoresis (comet assay). In primary rat gastric mucosa cells, hexenal was only weakly active, inducing detectable DNA damage in 20% of cells at 0.8 mM concentration. In contrast, primary mucosa cells from rat esophagus were as sensitive as colon cells. After single oral application of hexenal to rats (up to 320 mg/kg body wt) DNA damage was not detectable in gastrointestinal mucosa. Analysis of hexenal in selected flavored foods revealed concentrations up to 14 ppm (0.14 mM) that are comparable to its natural occurrence in some fruits and vegetables (up to 30 ppm). Thus, the concentration range selected for the toxicological studies described here clearly is relevant: Hexenal, at concentrations found in food, exerts genotoxic effects in cells from rat and human gastrointestinal tract.

Effect of pH of the T4 Polynucleotide Kinase Reaction on the 32P-Postlabeling Assay of DNA Adducts

Among the currently available methods for detecting bulky hydrophobic compounds bound to DNA, the 32P-postlabeling is the most sensitive and most widely practiced. One of the key reaction steps of this assay is the polynucleotide kinase (PNK)-catalyzed incorporation of 32 P i into nucleotides. To ensure high sensitivity and reproducibility of the assay, it is imperative that the PNK reaction be conducted under optimal conditions. The PNK reaction is generally conducted at pH 9.6 in the 32P assay. We have investigated pH profiles of the PNK reactions using both normal (2′-deoxyadenosine 3′-monophosphate) and adducted (benzo[ a]pyrene- and cigarette smoke condensate-modified) nucleotides. The optimum pH range for the PNK reaction was between 7.4 and 8.4 with both types of nucleotides. Higher pH levels such as 9.4 and 9.6 were detrimental to the PNK reaction, yielding only about one-third or less of the labeling obtained at the optimum pH. Based on these results, the PNK reaction in the 32P-postlabeling assay should be conducted in the pH range between 7.8 and 8.0 to achieve quantitative labeling of adducted nucleotides.

Synthesis and characterization of adducts of alachlor and 2-chloro-N-(2,6-diethylphenyl)acetamide with 2'-deoxyguanosine, thymidine, and their 3'-monophosphates.

Adducts of the preemergence herbicide 2-chloro-N-(methoxymethyl)-N-(2,6-diethylphenyl)-acetamide (alachlor) and 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA) with 2'-deoxyguanosine, thymidine, 2'-deoxyguanosine 3'-monophosphate, and thymidine 3'-monophosphate have been synthesized and characterized. Under mildly basic conditions alachlor and CDEPA form N-1 adducts with 2'-deoxyguanosine and N-3 adducts with thymidine as a result of chlorine displacement. In addition, alachlor formed an N-7 adduct with 2'-deoxyguanosine, 7-[[(N-(methoxymethyl)-N-(2,6-diethylphenyl)carbamoyl]methyl]guani ne. N-1 adducts of alachlor and CDEPA with 2'-deoxyguanosine 3'-monophosphate and N-3 adducts with thymidine 3'-monophosphate are also described. In addition to spectroscopic data, structural proof included the dephosphorylation of each nucleotide adduct to its corresponding nucleoside adduct by nuclease P1. Alachlor and alachlor adducts but not CDEPA and CDEPA adducts exhibited rotational isomerism as evidenced by proton and 13C NMR studies. These rotamers were attributed to hindered rotation about the shortened N-carbonyl bond. Computational methods employing molecular mechanics and quantum mechanics were used to characterize the structures and energies of these rotamers to account for the patterns of duplicate NMR resonances observed.

32P-postlabeling of bile components: bulky adduct-like behavior in polyethyleneimine-cellulose thin layer chromatography.

The 32P-postlabeling assay has been used widely in carcinogen-DNA adduct analysis because of its sensitivity and reproducibility. Cloned T4 polynucleotide kinase (PNK), routinely used in this assay, phosphorylates the 5'-OH groups of adducted nucleotides in the presence of [gamma-32P]ATP. However, as an exception to this property, PNK has been reported to phosphorylate non-adducted carcinogen metabolites, such as tetrol derivatives of benzo[a]pyrene and chrysene. Also, PNK phosphorylates both 5'-OH and 3'-OH groups of safrole-adducted deoxydinucleoside monophosphates having an unmodified purine in the 3'-position. In the present study we show that T4 PNK catalyzed the transfer of [32P]phosphate from [gamma-32P]ATP to rat bile components or purified bile acids (derivatives of 3 alpha-hydroxy-5 beta-cholanic acid) in the absence of nucleic acids or nucleases. However, labeling of the bile acids appeared over 100,000-fold less efficient than labeling of 2'-deoxyadenosine-5'-monophosphate. There was no reaction in the absence of bile components or PNK. Dehydrocholic acid, which lacks hydroxyl groups, was resistant to phosphorylation. On polyethyleneimine-cellulose TLC maps, 32P-labeled rat bile extract gave an array of non-polar radioactive spots which resembled carcinogen-DNA adducts, while 32P-labeled purified bile acids each gave a single spot. These 32P-labeled products liberated 32Pi upon incubation with prostatic acid phosphatases. Two of the radioactive spots obtained from rat bile were identified as phosphorylated taurocholic and taurodeoxycholic acids by co-chromatography with 32P-labeled standards. These findings demonstrate for the first time that PNK is able to phosphorylate natural products other than nucleotides and further emphasize the need to rule out contamination with bile acids and possibly other bulky/hydrophobic alcohols when analyzing DNA samples by 32P-postlabeling.

Class-specific immunoadsorption purification for polycyclic aromatic hydrocarbon-DNA adducts.

An immunoenrichment procedure has been developed for applications in the detection and identification of a broad range of polycyclic aromatic hydrocarbon (PAH)-DNA adducts at very low abundance. The procedures are based on a monoclonal antibody raised to r-7,trans-8,trans-9-trihydroxy-cis-10-(N2-deoxyguanosyl-5'-phospha te)- 7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE-N2-dG) which has been tested for cross-reactivity towards DNA and proteins (bovine serum albumin, chicken gamma globulin and human globin) covalently modified with a range of PAH diol-epoxides. The antibody recognised DNA adducted with the diol-epoxides of benzo[a]pyrene, benz[a]anthracene, chrysene, dibenz[a,h]anthracene or picene. The antibody also cross-reacted with the 7,8,9,10-tetraol derived from benzo[a]pyrene and the 1,2,3,4-tetraols of benz[a]anthracene and chrysene. The degree of cross-reactivity was greatest for PAH adducts with structural features similar to anti-BPDE-N2-dG proximate to the base attachment. The antibody also recognised a range of PAHs adducted to human globin; these included adducts of benzo[a]pyrene, benz[a]anthracene and chrysene diol-epoxides. This wide range of recognition provides good evidence for the class-specific recognition of PAH adducts by the antibody. When immobilized on Sepharose 4B and used in the immunoadsorption purification of adducted nucleotides, the antibody selectively enriched adducts of benzo[a]pyrene, benz[a]anthracene and chrysene from normal nucleotides. Quantitative measurements with [14C]benzo[a]pyrene-DNA adducts showed that the immobilized antibody was able to enrich benzo[a]pyrene adducts from a DNA hydrolysate containing adducts at a level of 1 adduct/10(10) normal nucleotides. In addition, this immunoadsorption technique was effective in enriching a mixture of DNA adducts formed in the skin of CF1 mice treated cutaneously with a mixture of [3H]benzo[a]pyrene and [3H]chrysene. Class-specific immunoenrichment procedures for DNA adducts are important in assisting the identification of genotoxic components in complex mixtures. The performance characteristics of this immobilized antibody suggest that it may be suitable for application in the detection, identification and monitoring of human exposures to low levels of PAHs.

Improved thin-layer chromatographic separation of 32P-postlabeled DNA adducts

DNA adducts represent the putative initiating event in the chemical carcinogenesis process. 32P-Postlabeling is one of several assays which have been developed for the sensitive detection of DNA adducts. An integral part of the 32P-postlabeling assay is the separation of adducted nucleotides by multidirectional, multisolvent, anion-exchange polyethyleneimine—cellulose thin-layer chromatography. Standard since the introduction of this assay has been the use of high-salt, high-urea solvents for the resolution of adducts during the D3 and D4 phases of the chromatography. Urea solvents are able to separate adducts resulting from a number of chemicals, however, they are time-consuming, retain a lot of background noise, may push adducts into inadequately resolved diagonal radioactive zones, and may not separate adducts of similar structure. In this study we introduce the use of a dilute ammonium hydroxide solvent for D4 chromatography and compare it to other standard solvents such as lithium chloride—Tris · HCl—urea, sodium phosphate—Tris · HCl—urea, and isopropanol—4 M ammonium hydroxide for adduct separation, resolution, recovery, retention of background noise, and chromatography development time. We found that 0.2 M ammonium hydroxide worked well for the recovery, separation, and resolution of a wide array of adducts derived from highly lipophilic polycyclic aromatic hydrocarbons and aromatic amines. In addition, this solvent required much less time (< 1 4 ) as compared to the other solvents and more importantly allowed the separation of adducts which otherwise comigrated and were not visible when using the other three D4 solvents. Background radioactivity per cm 2 of the thin layers was also reduced two- to three-fold with the ammonia solvent versus the urea-based solvents. Thus, the use of this dilute ammonia system provides powerful resolution in a time-efficient manner.

32P-postlabelling analysis of DNA adducts from Fischer-344 rats administered 2,4-diaminotoluene

Using 32P-postlabelling and thin layer chromatography, DNA adduct formation by the potent animal carcinogen 2,4-diaminotoluene in Fischer-344 rats was investigated. DNA from four different organs, liver, mammary gland, kidney and lung, were examined for adducts following single administration of this compound. DNA binding was detected in all four organs, with each producing one major and two minor adduct spots on autoradiograms. The adducts induced were qualitatively identical among the different organs, but quantitative differences were observed. The two target organs of 2,4-diaminotoluene induced carcinogenesis, the liver and mammary gland produced higher adduct yields, with levels up to 30-times higher than those for the two non-target organs. Since the liver is the principal target for 2,4-diaminotoluene induced carcinogenesis, we further examined DNA adducts from this site for the effects of different doses and time points. DNA binding in liver was detected following doses as low as 4.1 μmol/kg. At the highest concentration examined (2046 μmol/kg), the level of the major adduct was 29.2 adducted nucleotides per 10 7 total nucleotides. The yields for the two minor adducts were approximately one-tenth that for the major adduct. Following a 410 μmol/kg dose, DNA adduct removal over time was examined. DNA adduct removal exhibited biphasic kinetics, with a rapid initial phase followed by a slower rate of elimination. Up to 60% of maximum adduct levels persisted after 2 weeks. DNA binding by 2,4-diaminotoluene was also compared to that by its weakly carcinogenic analog, 2,4-dinitrotoluene. The two compounds produced identical adduct patterns, suggesting that they share common metabolites and adducts. Adduct yields from 2,4-dinitrotoluene, however, were lower. The results of our studies suggest that the differences in carcinogenic potency between 2,4-diaminotoluene and 2,4-dinitrotoluene, as well as the organotropic effects of 2,4-diaminotoluene may be explained, in part, by quantitative differences in the extent of DNA adduct formation.

Molecular orbital studies of the hydrolysis reactions of benzo[a]pyrene diol epoxides

Kinetic studies of the hydrolysis of benzo[a]pyrene diol epoxides have shown that hydrolysis proceeds through an acid-catalyzed SN1 mechanism and that the rate-determining step is the formation of a benzylic carbocation. The formation of this carbocation indicates that the diol epoxide can react with nucleophilic sites on DNA as an alkylating agent. Previous work has indicated that the kinetic data could be accurately fit to kobsd = kH [H+] + k0, in which kH [H+] is the pseudo first-order rate constant for acid-catalyzed hydrolysis and k0 is the rate constant for spontaneous hydrolysis. The observation made in several laboratories, that DNA catalyzes the hydrolysis of carcinogenic diol epoxides, is of direct interest in light of our recent calculation indicating that the local environment of DNA has a high concentration of hydrogen ions. The significance of acidic domains around nucleic acids is primarily in the ability of the protons to catalyze reactions. We have hypothesized that the high concentration of hydrogen ions at the DNA surface is responsible for the catalysis and that the generation of benzylic carbocations at the DNA surface can result in either nontoxic tetraols or in mutagenic nucleotide adducts, depending on the nucleophile with which the carbocation reacts. The calculations presented here are concerned with qualitatively understanding the rate constants in terms of the mechanisms of the acid-catalyzed and the spontaneous (H2O-catalyzed) hydrolyses. © 1992 John Wiley & Sons, Inc.