Extent Of Adduct Formation Research Articles

Toxicokinetic relationship between the adducts in globin and their cleavage products in the urine: Implications for human biomonitoring

Globin adducts of various chemicals, persisting in organism over the whole lifetime of erythrocytes, have been used as biomarkers of cumulative exposures to parent compounds. After removal of aged erythrocytes from the bloodstream, cleavage products of these adducts are excreted with urine as alternative, non-invasively accessible biomarkers. In our biomonitoring studies on workers exposed to ethylene oxide, its adduct with globin, N-(2-hydroxyethyl)valine, and the related urinary cleavage product N-(2-hydroxyethyl)-L-valyl-L-leucine have been determined. To describe a toxicokinetic relationship between the above types of biomarkers, a general compartmental model for simulation of formation and removal of globin adducts has been constructed in the form of code in R statistical computing environment. The essential input variables include lifetime of erythrocytes, extent of adduct formation following a single defined exposure, and parameters of exposure scenario, while other possible variables are optional. It was shown that both biomarkers reflect the past exposures differently as the adduct level in globin is a mean value of adduct levels across all compartments (subpopulations of erythrocytes of the same age) while excretion of cleavage products reflects the adduct level in the oldest compartment. Application of the model to various scenarios of continuous exposure demonstrated its usefulness for human biomonitoring data interpretation.

Behavior of Malondialdehyde and Its Whey Protein Adducts during In Vitro Simulated Gastrointestinal Digestion.

The behavior of malondialdehyde and its whey protein adducts in aqueous buffer and fully hydrogenated coconut oil-in-water emulsions stabilized by Tween 20 or by whey protein was studied during in vitro gastrointestinal digestion. The malondialdehyde levels during in vitro digestion depended upon the kind of sample, the location of the whey protein, and the extent of adduct formation before digestion. During gastric digestion, degradation of acid-labile malondialdehyde-whey protein adducts as well as formation of new malondialdehyde adducts with hydrolyzed whey protein was suggested to occur, in addition to the earlier described equilibria with respect to the aldol self-condensation of malondialdehyde and its hydrolytic cleavage. After in vitro digestion, both malondialdehyde and its adducts were present in the digest with malondialdehyde recoveries varying between 55 and 86% depending upon the model system studied. To conclude, the reactivity of malondialdehyde toward (hydrolyzed) proteins does not stop at the point of ingestion.

Effects of Oxygen Partial Pressure on 4-Hydroxy-2-Nonenal Induced Oxymyoglobin Oxidation

4-hydroxyl-2-nonenal (HNE) is a lipid oxidation product that can increase oxymyoglobin oxidation. However, limited research has evaluated the role of oxygen partial pressure in HNE-induced metmyoglobin formation. Therefore, the objective of was to compare the effects of atmospheric and high-oxygen partial pressure on HNE-induced oxymyoglobin oxidation in vitro. Oxymyoglobin was incubated with or without HNE at atmospheric (20% O2) or high-oxygen (80% O2) partial pressure. Metmyoglobin formation was measured after 0, 48, and 96 h of incubation at 4°C, and mass spectrometry was utilized to characterize the covalent binding of HNE to myoglobin. High-oxygen condition (80% O2) increased (P < 0.05) HNE-induced oxymyoglobin oxidation compared with the atmospheric partial pressure condition (20% O2). However, HNE was bound to myoglobin at both high-oxygen and atmospheric partial pressure conditions, with no differences (P > 0.05) in the extent of adduct formation. These results suggest that high-oxygen conditions had no effect on extent of HNE-binding, but can increase oxymyoglobin oxidation.

Abstract 5242: Further evidence that the DNA-interactive Pyrrolobenzodiazepine (PBD) Dimer SJG-136 works through a transcription factor inhibition mechanism

Abstract The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are sequence-selective DNA minor-groove interacting agents. The PBD dimer SJG-136 has currently been investigated in Phase II clinical trials in ovarian cancer and leukaemia in the UK and the USA. More recently, PBD dimer analogues are being attached to tumour-targeting antibodies to create Antibody-Drug Conjugates (ADCs), some which are now in Phase 3 clinical trials with many others in pre-clinical and clinical development. Transcription factors (TFs) are sequence-specific DNA-interacting proteins that bind to consensus DNA sequences, thereby controlling transcription. TFs regulate processes such as cell differentiation, proliferation and apoptosis. The interaction of a small-molecule with the consensus DNA recognition sequences of TFs can prevent a TF from interacting with its cognate sequence, thereby inhibiting the expression of genes critical for the survival and proliferation of cancer cells. There is growing evidence that PBDs may exert, at least in part, their pharmacological effect through TF inhibition in addition to the arrest of the replication fork, DNA strand breakage, and inhibition of enzymes including endonucleases and RNA polymerases. For this reason, there is now interest in using PBDs as the basis for a small-molecule strategy to target specific DNA sequences for TF inhibition as a novel anticancer therapy. We have developed a reversed-phase HPLC/MS method as a tool to evaluate the interaction of DNA-binding PBD molecules with oligonucleotides of varying lengths and sequences. Using this methodology, we have demonstrated that the PBD dimer SJG-136 binds to the cognate sequences of the oncogenic transcription factors NF-κB, EGR-1, AP-1 and STAT3. Surprisingly, significant differences in the rate and extent of adduct formation between the different cognate sequences were observed which may explain, at least in part, the differences in potency of SJG-136 in various tumour cell lines. Furthermore, an RT PCR study has been carried out using the human tumour cell lines MDA-MB-231 (breast) and HT-29 (colon) to see whether these transcription factors are affected in vitro. The results were consistent with the HPLC-MS studies in that SJG-136 was shown to significantly down-regulate a number of AP-1- and STAT3-dependent genes such as Bcl-2, VEGF, p53 and survivin. These findings add significantly to knowledge of the mechanism of action of SJG-136 and related PBD compounds, and could be relevant for the correct interpretation of clinical activity of molecules of this type both as standalone agents and as ADC payloads. Citation Format: Julia Mantaj, Paul J. Jackson, David E. Thurston, Khondaker Miraz Rahman. Further evidence that the DNA-interactive Pyrrolobenzodiazepine (PBD) Dimer SJG-136 works through a transcription factor inhibition mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5242. doi:10.1158/1538-7445.AM2017-5242

Covalent modification of cytochrome c by reactive metabolites of furan.

Metabolism of the hepatotoxicant furan leads to protein adduct formation in the target organ. The initial bioactivation step involves cytochrome P450-catalyzed oxidation of furan, generating cis-2-butene-1,4-dial (BDA). BDA reacts with lysine to form pyrrolin-2-one adducts. Metabolic studies indicate that BDA also reacts with glutathione (GSH) to generate 2-(S-glutathionyl)butanedial (GSH-BDA), which then reacts with lysine to form GSH-BDA-lysine cross-links. To explore the relative reactivity of these two reactive intermediates, cytochrome c was reacted with BDA in the presence and absence of GSH. As judged by MALDI-TOF mass spectrometry, BDA reacts extensively with cytochrome c to form adducts that add 66 Da to the protein, consistent with the formation of pyrrolinone adducts. Addition of GSH to the reaction mixture reduced the overall extent of adduct formation. The mass of the adducted protein was shifted by 355 Da as expected for GSH-BDA-protein cross-link formation. LC-MS/MS analysis of the tryptic digests of the alkylated protein indicated that the majority of adducts occurred on lysine residues, with BDA reacting less selectively than GSH-BDA. Both types of adducts may contribute to the toxic effects of furan.

Abstract 2230: Interaction of SJG-136 with cognate sequences of oncogenic transcription factors.

Abstract The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are sequence-selective DNA minor-groove interacting agents. They have a chiral centre at their C11a(S)-position which provides them with the appropriate 3-dimensional shape to fit securely within the DNA minor-groove. They also possess a “soft” electrophilic imine moiety at their N10-C11 position which can form an aminal linkage with the C2-NH2 group of a guanine base. The PBD dimer SJG-136 is presently in Phase II clinical trials in ovarian cancer and leukaemia. Transcription factors act through direct or indirect binding to specific DNA consensus sequences within gene regulatory regions, or by forming a complex with other proteins which then act as transcription factors. Covalent or non-covalent interaction of small molecules with key consensus DNA bases can prevent a transcription factor from recognizing its cognate sequence, thus preventing expression of genes critical for the survival and proliferation of cancer cells. The cognate sequences of a number of oncogenic transcription factors are unusually rich in GC sequences and provide ideal alkylation substrates (for both inter- and intrastrand cross-links and mono-alkylation) for both PBD dimers (e.g. SJG-136) and PBD monomers, and there is growing evidence that PBDs exert their pharmacological effects through this mechanism. We have developed a reversed-phase ion pair HPLC/MS method as a tool to evaluate the interaction of DNA-binding molecules with oligonucleotides of varying length and sequences. Using this methodology along with custom-designed oligonucleotides containing the cognate sequences of the transcription factors NFκB, AP-1, EGR-1 and Stat3, all of which contain ideal GC-rich binding sequences for SJG-136, we have observed both kinetic and thermodynamic adduct formation with SJG-136. Crucially, we observed a significant difference in rate and extent of adduct formation between the different cognate sequences which could partly explain the differences in activity of SJG-136 toward different tumour cell lines. For example, SJG-136 formed an adduct rapidly with the NFκB sequence (>50% adduct within 3 hours), moderately with the STAT3 (>35% adduct within 3 hours) and EGR-1 cognate sequences (>30% adduct within 3 hours) while showing insignificant adduct formation with the AP-1 cognate sequence (< 5% adduct within 3 hours) even though their GC contents are comparable. These findings add to knowledge of the possible mechanism of SJG-136, and could help with interpretation of the activity of SJG-136 in various biochemical and pharmacological assays. Citation Format: Julia Mantaj, David E. Thurston, Khondaker M. Rahman. Interaction of SJG-136 with cognate sequences of oncogenic transcription factors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2230. doi:10.1158/1538-7445.AM2013-2230

Transplatin-Conjugated Triplex-Forming Oligonucleotides Form Adducts with Both Strands of DNA

Triplex-forming oligonucleotides (TFOs) can bind to polypurine x polypyrimidine tracts in DNA and, as a consequence, perturb the normal functioning of a targeted gene. The effectiveness of such antigene TFOs can potentially be enhanced by covalent attachment of the TFO to its DNA target. Here, we report that attachment of N-7-platinated guanine nucleosides to the 3'- and/or 5'-ends of oligopyrimidine TFOs enables these TFOs to form highly stable adducts with target DNA deoxyguanosines or deoxyadenosines that are adjacent to the TFO binding site. Such adduct formation stably anchors the TFO to its target. Depending on the sequences adjacent to the TFO binding site, adduct formation can occur on either strand of the DNA. Adduct formation by 3',5'-bis-platinated TFOs can result in the formation of an interstrand cross-link between both strands of the DNA duplex. Formation of the adducts, which could be reversed by treatment with sodium cyanide, was dependent upon the ability of the TFO to bind to DNA and appeared to occur at a rate slower than that at which the TFO bound to the DNA duplex. The extent of adduct formation at 37 degrees C by platinated deoxyribo-TFOs diminished as the pH was increased from 6.5 to 7.4. In contrast, high levels (approximately 86%) of adduct formation by platinated 2'-O-methylribo-TFOs were observed at both pH 6.5 and pH 7.4. Platinated 2'-O-methylribo-TFOs were also shown to bind to plasmid DNA and inhibit transcription in vitro, and to inhibit plasmid replication in E. coli cells. These results suggest that platinum-conjugated TFOs may be good candidates for use as antigene agents.

In Vitro Screening of 50 Highly Prescribed Drugs for Thiol Adduct Formation—Comparison of Potential for Drug-Induced Toxicity and Extent of Adduct Formation

Reactive metabolite formation has been associated with drug-induced liver, skin, and hematopoietic toxicity of many drugs that has resulted in serious clinical toxicity, leading to clinical development failure, black box warnings, or, in some cases, withdrawal from the market. In vitro and in vivo screening for reactive metabolite formation has been proposed and widely adopted in the pharmaceutical industry with the aim of minimizing the property and thus the risk of drug-induced toxicity (DIT). One of the most common screening methods is in vitro thiol trapping of reactive metabolites. Although it is well-documented that many hepatotoxins form thiol adducts, there is no literature describing the adduct formation potential of safer drugs that are widely used. The objective of this study was to quantitatively assess the thiol adduct formation potential of 50 drugs (10 associated with DIT and 40 not associated) and document apparent differences in adduct formation between toxic and safer drugs. Dansyl glutathione was used as a trapping agent to aid the quantitation of adducts following in vitro incubation of drugs with human liver microsomes in the presence and absence of NADPH. Metabolic turnover of these drugs was also monitored by LC/UV. Overall, 15 out of the 50 drugs screened formed detectable levels of thiol adducts. There were general trends toward more positive findings in the DIT group vs the non-DIT group. These trends became more marked when the relative amount of thiol adducts was taken into account and improved further when dose and total daily reactive metabolite burdens were considered. In conclusion, there appears to be a general trend between the extent of thiol adduct formation and the potential for DIT, which would support the preclinical measurement and minimization of the property through screening of thiol adduct formation as part of an overall discovery optimization paradigm.

Mass spectral analyses of hemoglobin adducts formed after in vitro exposure of erythrocytes to hydroxymethylvinyl ketone

Hydroxymethylvinyl ketone (HMVK) is a reactive oxidation product of 3-butene-1,2-diol, a metabolite of 1,3-butadiene. The potential for HMVK (0.1 and 1mM) to form hemoglobin (Hb) adducts in erythrocytes from Sprague-Dawley rats was investigated at physiological conditions (pH 7.4, 37 degrees C) using electrospray ionization mass spectrometry (ESI/MS). With the 0.1mM HMVK globin samples, the results indicate HMVK adduction on the alpha2, beta2 and beta3 chains. With the 1.0mM HMVK globin samples, adducts were detected on the beta2 and beta3 chains. However, no correlation was observed between incubation time and the extent of adduct formation, and additional adducts were detected when globin samples were fractionated by HPLC before the ESI/MS analyses. For specific localizations of adducts on the globin chains, trypsin digested peptides from the 1mM HMVK globin samples were subjected to liquid chromatography/mass spectrometry analyses. The results, which are consistent with formation of HMVK adducts on several specific peptides within the alpha- and beta-chains, suggest selectivity in the interaction of HMVK with the different cysteine residues in Hb. Because adducts were also detected in peptides containing no cysteine residues and multiple HMVK moieties were detected on some of the cysteine-containing peptides, the results suggest other amino acids may be also reactive with HMVK. Adduct profiles and their relative intensities were consistent between the 1 and 2h samples providing evidence for the HMVK reactions being fast and selective. The finding that fewer peptides were adducted in the 0.1mM HMVK globin samples provides further evidence for selectivity of the HMVK reaction. Collectively, the results show HMVK readily and selectively forms adducts on Hb. Characterization of these adducts will facilitate development of useful biomarkers of exposure to HMVK and its precursor 1,3-butadiene.

Formation of benzo[a]pyrene diol epoxide-DNA adducts at specific guanines within K-ras and p53 gene sequences: stable isotope-labeling mass spectrometry approach.

The mutagenicity of a prominent tobacco carcinogen, benzo[a]pyrene (B[a]P), is believed to result from chemical reactions between its diol epoxide metabolite, (+)-anti-7r,8t-dihydroxy-c9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), and DNA, producing promutagenic lesions, e.g., (+)-trans-anti-7R,8S,9S-trihydroxy-10S-(N(2)-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (N(2)-BPDE-dG). Previous studies used the DNA repair enzyme UvrABC endonuclease in combination with ligation-mediated PCR (LMPCR) to demonstrate an increased reactivity of BPDE toward guanine nucleobases within codons 157, 248, and 273 of the p53 tumor suppressor gene (Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. Science 274, 430-432). These sites are also "hot spots" for mutations observed in lung tumors of smokers, suggesting an involvement of B[a]P in the initiation of lung cancer. However, the LMPCR approach relies on the ability of the repair enzyme to excise BPDE-induced lesions, and thus the slowly repaired lesions may escape detection. Furthermore, BPDE-DNA adduct structure and stereochemistry cannot be determined. In the present work, we performed a direct quantitative analysis of N(2)-BPDE-dG originating from specific guanine nucleobases within p53- and K-ras-derived DNA sequences by using a stable isotope labeling-mass spectrometry approach recently developed in our laboratory. (15)N-labeled dG was placed at defined positions within DNA sequences derived from the K-ras proto-oncogene and p53 tumor suppressor gene, the two genes most frequently mutated in smoking-induced lung cancer. (15)N-labeled DNA was annealed to the complementary strands, followed by BPDE treatment and liquid chromatography-electrospray ionization tandem mass spectrometry analysis (HPLC-ESI-MS/MS) of N(2)-BPDE-dG lesions. The extent of adduct formation at (15)N-labeled guanine was determined directly from the HPLC-ESI-MS/MS peak area ratios of (15)N-N(2)-BPDE-dG and N(2)-BPDE-dG. BPDE-induced guanine adducts were produced nonrandomly along K-ras and p53 gene-derived DNA sequences, with over 5-fold differences in adduct formation depending on sequence context. N(2)-BPDE-dG yield was enhanced by the presence of 5-Me substituent at the cytosine base-paired with the target guanine nucleobase, an endogenous DNA modification characteristic for CpG dinucleotides within the p53 gene. In the K-ras-derived DNA sequence, the majority of N(2)-BPDE-dG adducts originated from the first position of the codon 12 (GGT), consistent with the large number of G --> T transversions observed at this nucleotide in smoking-induced lung cancer. On the contrary, the pattern of N(2)-BPDE-dG formation within the p53 exon 5 sequences did not correlate with the mutational spectrum in lung cancer, suggesting that factors other than N(2)-BPDE-dG formation are responsible for these mutations. The stable isotope labeling HPLC-ESI-MS/MS approach described in this work is universally applicable to studies of modifications to isolated DNA by other carcinogens and alkylating drugs.

Implications of cytosine methylation on (+)-anti-Benzo[a]pyrene 7, 8-dihydrodiol 9,10-epoxide N(2)-dG adduct formation in 5'-d(CGT), 5'-d(CGA), and 5'-d(CGC) sequence contexts of single- and double-stranded oligonucleotides.

Covalent binding of (+)-anti-benzo[a]pyrene 7,8-dihydrodiol 9, 10-epoxide (anti-BPDE) to the N(2)-amino group of deoxyguanine in the oligonucleotides 5'-d(CCTATCGXTATCC) and 5'-d(CCTATm(5)CGXTATCC) (X being T, A, or C) has been studied. The extent of formation of the (+)-trans-anti-BPDE-N(2)-dG adduct in single-stranded 13-mer oligonucleotides with 5'-d(m(5)CGT) and 5'-d(m(5)CGA) sequence contexts was significantly higher (1.5- and 2.4-fold, respectively) relative to that of the nonmethylated sequences. With the 5'-d(CGC) sequence context, m(5)dC had no significant effect on adduct formation. When the reaction was allowed to proceed in the presence of oligonucleotide duplexes (composed of a 13-mer parent strand and a 9-mer complement), a significant increase in the extent of adduct formation was observed with 5'-d(m(5)CGT)/d(CGA) and 5'-d(m(5)CGA)/d(CGT), but not with 5'-d(CGC)/d(GCG), relative to those of the nonmethylated duplexes. Independent of sequence context, no clear effect of m(5)dC on diol epoxide binding to the opposite dG in the complementary strand was observed. The level of diol epoxide binding to the dG target in the 13-mer oligonucleotides is in general higher in single-stranded sequences than in the duplexes. With 5'-d(CGA) and 5'-d(m(5)CGA), for instance, adduct yields were 3- and 4-fold higher, respectively. The thermodynamic stability of the (+)-trans-anti-BPDE-N(2)-dG adduct in the 5'-d(m(5)CGT)-containing duplex (composed of a 13-mer parent strand and a full complement) was substantially higher than in the 5'-d(CGT)/d(GCA) sequence context. The stimulating effect of cytosine methylation on the formation of DNA adducts of anti-BPDE has previously been demonstrated in other experimental systems. The increase in yield could possibly be rationalized in terms of prestacking of the pyrenyl ring system with the nucleobases prior to the nucleophilic addition reaction of the exocyclic amino group. The results from induced circular dichroism studies with the (+)-trans-anti-BPDE-N(2)-dG adduct in the 5'-d(m(5)CGT)-containing duplex are consistent with substantial heterogeneity of adduct conformations, including both external minor groove-localized and intercalated structures.

Chromatographic Characterization of Hemoglobin Benzo[a]pyrene-7,8-diol-9,10-epoxide Adducts

The formation of hemoglobin–carcinogen adducts has been detected in carcinogen-treated animals and in human populations. Although polynuclear aromatic hydrocarbons are ubiquitous in the human environment and DNA–aromatic hydrocarbon adducts have been detected in human tissue, the occurrence of hemoglobin–polynuclear aromatic hydrocarbon adducts in humans has not been thoroughly described. In this study we examined the effects of reaction conditions on the extent ofin vitroreaction of human hemoglobin and (+) [3H]benzo[a]pyrene-7,8-diol-9,10-epoxide (anti)(BPDE), a metabolite thought to be largely responsible for the carcinogenic effect of benzo[a]pyrene. The chromatographic properties of the resulting hemoglobin–BPDE adducts were examined by conventional DEAE-cellulose ion exchange liquid chromatography and by reversed phase high performance liquid chromatography. Several adducts were formed which were chromatographically resolved from hemoglobin and from the individual globins. Some adducts were basic and some acidic relative to unaltered hemoglobin, suggesting adduct formation by reaction at carboxyl and basic nitrogen groups, respectively. Alteration of the ion - chromatographic properties of the adducts by an ionic sulfhydryl reagent, together with only a moderate effect of pH on the extent of adduct formation, indicated that the adducts were not formed via reaction with the β93cysteine sulfhydryl group. The chromatographic techniques employed may be applicable for the characterization and analysis of other hemoglobin–carcinogen adducts.

Effect of DNA base sequence on the configuration of deoxyadenosine adducts formed by the Fjord region diol epoxide, (+)-(1R,2S,3R,4S)-3,4-dihydroxy-1,2-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene.

The carcinogen (+)-(1R,2S,3R,4S)-3,4-dihydroxy-1,2-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene (in which the 4-OH group and epoxide oxygen are cis) was reacted with duplexes formed from self-complementary oligodeoxyribonucleotides, producing 1S or 1R configured adducts through trans or cis epoxide ring opening, respectively, by the exocyclic amino group of a central target A. Sequences containing 5'-AT-3' generated much higher S vs R ratios than the average of 3.38 observed with calf thymus DNA samples, while sequences containing 5'-TA-3' generated much lower ratios. Sequences with G in the position immediately 5' to the central AT or TA, and C in the position immediately 3', generated moderately higher ratios than did sequences with adjacent 5'C and 3'G. When thymidine was replaced by deoxyuridine in several sequences, the ratios of S vs R configured dA adducts and dA adducts vs dG adducts were substantially and uniformly reduced, but otherwise varied with the choice of nearest neighbors in patterns similar to those observed with the T containing sequences. Two hypothetical mechanisms are proposed to explain the effect of nearest neighbors on the S vs R dA adduct ratio; in both, diol epoxide intercalation precedes covalent bonding. In one mechanism, intercalation to the 5' side of the target A yields an S adduct while intercalation to the 3' side yields an R adduct, and the extent of adduct formation follows the nearest neighbor series G > C > T.(ABSTRACT TRUNCATED AT 250 WORDS)

Predictability of the covalent binding of acidic drugs in man

Although metabolism via glucuronide conjugation has generally been considered a detoxification route for carboxylic acids, the newly discovered chemical reactivity of these conjugates, leading to covalent binding with proteins, is consistent with the toxicity observed for drugs containing the carboxylic acid moiety. Here we report that degradation rates (intramolecular rearrangement and hydrolysis) for 9 drug glucuronide metabolites show an excellent correlation (r 2=0.995) with the extents of drug covalent binding to albumin in vitro. Furthermore, this binding capacity is predictable based on chemical structure of the acid and depends on the degree of substitution at the carbon alpha to the carboxylic acid. The in vivo covalent binding in humans for these drugs is also predictable (r 2=0.873) when the extent of adduct formation is corrected for the measured plasma glucuronide concentrations. These results suggest that the structure of a carboxylic acid drug may predict the degree to which the corresponding acyl glucuronides will form covalent adducts that probably/possibly lead to toxicity. This information could be a useful adjunct in drug design.

Formation of DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) in male Fischer-344 rats

2-Amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) is known to induce colon tumors in male Fischer-344 rats. Using 32P-postlabeling assays, we have examined PhiP-DNA adduct formation in various organs and white blood cells (WBCs) of the male Fischer-344 rat 24 h after a single oral dose of 0, 0.5, 5 or 50 mg PhIP/kg. Three PhIP-DNA adducts were detected in WBCs and in all organs, except in the liver and stomach which had only two adducts. The extent of adduct formation was dose-related, but at 0.5 mg/kg no adducts could be detected in any of the organs. At 50 mg/kg, adduct levels, expressed as relative adduct labeling values (RAL × 10 7, or adducts per 10 7 nucleotides assuming complete labeling) were highest in the large intestine (5.66), followed by WBCs (5.04), stomach (1.44), small intestine (1.32), kidney (1.16), liver (0.67) and lungs (0.52). It is concluded that orally administered PhIP forms high levels of specific DNA adducts in the large intestine, the target organ in PhIP carcinogenesis in the male Fischer-344 rat, and that the high level of adducts in WBCs indicates that significant amounts of the ultimate carcinogenic form of PhIP are present in the circulation.

Mutation spectrum and sequence alkylation selectivity resulting from modification of bacteriophage M13mp18 DNA with S-(2-chloroethyl)glutathione. Evidence for a role of S-(2-N7-guanyl)ethyl)glutathione as a mutagenic lesion formed from ethylene dibromide.

The major DNA adduct (greater than 95% total) resulting from the bioactivation of ethylene dibromide by conjugation with GSH is S-(2-(N7-guanyl)ethyl)GSH. The mutagenic potential of this adduct has been uncertain, however, because the observed mutagenicity might be caused by other adducts present at much lower levels, e.g. S-(2-N1-adenyl)ethyl)GSH. To assess the formation of other potential adducts, S-(2-(N3-deoxycytidyl)ethyl)GSH, S-(2-(O6-deoxyguanosyl)ethyl)GSH, and S-(2-(N2-deoxyguanosyl)ethyl)GSH were prepared and used as standards in the analysis of calf thymus DNA modified by treatment with [1,2-14C]ethylene dibromide and GSH in the presence of rat liver cytosol; only minor amounts (less than 0.2%) were found. A forward mutation assay in (repair-deficient) Salmonella typhimurium TA100 and sequence analysis were utilized to determine the type, site, and frequency of mutations in a portion of the lacZ gene resulting from in vitro modification of bacteriophage M13mp18 DNA with S-(2-chloroethyl)GSH, an analog of the ethylene dibromide-GSH conjugate. An adduct level of approximately 8 nmol (mg DNA)-1 resulted in a 10-fold increase in mutation frequency relative to the spontaneous level. The spectrum of spontaneous mutations was quite varied, but the spectrum of S-(2-chloroethyl)GSH-induced mutations consisted primarily of base substitutions of which G:C to A:T transitions accounted for 75% (70% of the total mutations). All available evidence implicates S-(2-(N7-guanyl)ethyl)GSH as the cause of these mutations inasmuch as the levels of the minor adducts are not consistent with the mutation frequency observed in this system. The sequence selectivity of alkylation was determined by treatment of end-labeled lac DNA fragments with S-(2-chloroethyl)GSH, cleavage of the DNA at adduct sites, and electrophoretic analysis. Comparison of the sequence selectivity with the mutation spectrum revealed no obligate relationship between the extent of adduct formation and the number of mutations which resulted at different sites. We suggest that the mechanism of mutagenesis involves DNA sequence-dependent alterations in the interaction of the polymerase with the (modified) template and incoming nucleotide.

Reactivation of psoralen-reacted plasmid DNA in Fanconi anemia, xeroderma pigmentosum, and normal human fibroblast cells

We have used a host cell reactivation system to study the effect of 8-methoxypsoralen (8-MOP) reaction on CAT (chloramphenicol acetyltransferase) and NEO (aminoglycoside phosphotransferase) expression in normal human cells, as well as two cell lines with possible DNA repair-processing defects. Plasmid DNA was treated with psoralen plus near-ultraviolet (NUV) irradiation. The reacted plasmids, pSV2cat and pSV2neo, were transfected into Fanconi anemia (FA), xeroderma pigmentosum (XP), and normal human fibroblast cells for transient or stable assay. The cells were assayed for CAT activity at various times after transfection or selected for G418 resistance. The extent of adduct formation required to inhibit expression was much less (difference of D37 greater than 2.5) in FA or XP cells compared to normal. We conclude that in FA and XP cells, the reactivation of CAT was much less than in normal cells. The possibility of differential DNA uptake and/or degradation in transient assay was ruled out by analysis of plasmid DNA recovered from transfected cells. The data of the two independent assays indicate that FA and XP cells are deficient in cross-linked DNA repair.

In vivo formation and repair of O6 in human leukocyte DNA after intravenous exposure to dacarbazine

Blood leukocyte DNA obtained from 11 Hodgkin's disease patients undergoing ABVD chemotherapy was analysed for the presence of the precarcinogenic adduct O6-methylguanine (O6-meG) at various times (1-2 h up to 49 h) after i.v. treatment with the methylating drug dacarbazine. Adduct formation was detected in all but one of the patients examined at levels ranging up to 0.45 fmol/micrograms DNA (7.2 x 10(-7) mol/mol guanine). The levels of the adduct decreased by approximately 30% over the 24 h following exposure and were usually not detectable 49 h after exposure. In five out of seven individuals examined after more than one treatment, consistent methylation responses were noted, while in the remaining two cases the responses were mixed. No correlation between the extent of adduct formation and lymphocyte levels of the repair enzyme O6-alkylguanine-DNA alkyltransferase was observed. The average extent of O6-meG formation 1 h after dacarbazine treatment was (4.3 +/- 3.1) x 10(-2) fmol/micrograms DNA per mg/kg dose [( 1.2 +/- 0.8) x 10(-3) fmol/micrograms DNA per mg/m2 dose)]. Following exposure of rats to similar doses of dacarbazine, the corresponding levels of adduct in blood leukocyte DNA were 1.1 x 10(-2) fmol/micrograms DNA per mg/kg dose (2.6 x 10(-3) fmol/micrograms DNA per mg/m2 dose).

Base and conformational specificity of an amine modification of DNA.

We have investigated the site and conformational preference of the reaction of a formaldehyde/amine reagent with DNA. Previous investigations of this laboratory have established that this reagent will react with native DNA, placing a positively charged amine moiety on the duplex that will survive exhaustive dialysis. The resulting adduct is duplex and base stacked in character, possessing B backbone geometry with a higher average winding angle and exhibiting remarkable stability with respect to the A-form, Z-form, or the single-strand denaturated species. In this current investigation, we have found that the stability of the adduct is dramatically reduced if the DNA is converted to mononucleotides, thus obviating the usual approach of nuclease digestion and chromatography for the identification of the modified nucleotides. Using indirect approaches, we have established that the reactive site that survives removal of the equilibrium concentrations of CH2O and amine is the exocyclic amino group of the guanine bases. This conclusion is based on (1) the positive correlation between GC content and the extent of adduct formation under standard reaction conditions (27 degrees C, 0.63M CH2O, 0.007M n-butylamine, pH 7); (2) decreases in the level of substitution of amine in DNA, which has this site blocked by trinitrobenzene modification; and (3) failure of poly(dI-dC) to retain amine upon dialysis. Raman spectra of the derivatized poly(dG-dC) show enhanced 2'-endo B character, with no marked shifts in the position of any of the lines, indicating the absence of any ring structures involving the N7 and the 06 of G. In standard reaction mixtures, other sites may react but this phenomenon appears to be minimal under conditions that do not favor fluctuational opening of base pairs. In the latter case, excess loading of amine on high GC content polymers produces a CD spectrum that is similar to one produced by poly(dA-dT) in the "X"-form [M. Vorlickova, E. Minyat, and J. Kypr (1984) Biopolymers 23, 1-4]. This conformation is lost, however, upon removal of excess reagents by dialysis and cannot be reestablished, in the absence of unbound amine and formaldehyde. The reaction is specific for the B-form of polynucleotides as demonstrated by the failure of poly(dG-m5dC) in the stable Z-form to exhibit substantial reaction. The B-form of this polymer will react readily with the retention of 0.23 moles amine/mole nucleotide under our standard reaction conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction between poly(N‐vinylpyrrolidone) and poly(acrylic acid)s: Influence of hydrogen and cupric ions on the adduct formation

AbstractInterpolymer adduct formation between poly(N‐vinylpyrrolidone) (PVP) and poly(methacrylic acid) (PMAA) is mainly due to hydrogen bonding. It is found that the interpolymer adduct formation is enhanced in the presence of Cu(II). A simple turbidity measurement making use of a spectrophotofluorometer is described for the study of the interpolymer adduct formation. Enhanced adduct formation in the presence of Cu(II) is described by the empirical relation d[PAd]/D[PVP] = k × 104α[Cu(II)]α, where PAd represents the interpolymer adduct and α and k are constants. Similar results have been obtained in the case of interpolymer adduct formation between poly(acrylic acid) (PAA) and PVP. In the above expression α signifies the influence of chelation on Cu(II)–PAA/PMAA–PVP‐type complex formation and k is the extent of PVP–PAA/PMAA interaction. The enhancement of adduct formation in the presence of Cu(II) is more in PAA than in PMAA. Turbidity and viscosity measurements further indicate that the influence of Cu(II) on interpolymer adduct formation between PVP and PMAA or PAA is more in the case of PAA than PMAA, as PAA is a better chelating ligand. But the extent of adduct formation is more in the case of PMAA in the absence of Cu(II) ions due to hydrophobic interactions exerted by methyl groups.