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Aldehydes are abundantly present in tobacco smoke and in urban air pollution and are endogenously generated as products of the lipid peroxidation process. These molecules can react with DNA bases forming mutagenic exocyclic adducts, which have been used as biomarkers of aldehyde exposure and as potential tools for the study of inflammation, metal storage diseases, neurodegenerative disorders, and cancer. High-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) provides a highly precise, specific and ultrasensitive method for the detection of exocyclic DNA adducts. Here we present and describe a validated micro-HPLC-Electro Spray Ionization (ESI)-MS/MS method for the quantification of 1,N2-propanodGuo, an adduct produced following the reaction between 2'-deoxyguanosine and acetaldehyde or crotonaldehyde.

Stable-isotope-dilution tandem mass spectrometry is the most advanced technique used for quantitative determination of a wide spectrum of endogenously generated DNA nucleobase modifications. It is regarded as a gold standard for such analyses. Here, we consider the requirements for reliable identification and quantification of DNA adducts/modifications, whether endogenously derived or not, and discuss how their quantification can provide information on the mechanism of action and the biological relevance of individual nucleobase modifications. A clinical application of such measurements will only be possible after a full validation of the assay and once we have gained a better understanding of the exact role that these DNA modifications play in disease pathogenesis. Once these prerequisites are satisfied, DNA modification measurements may be helpful as clinical parameters for treatment monitoring, for risk group identification and for the development of prevention strategies.

Bile acids (BAs) play two vital roles in living organisms, as they are involved in (1) the secretion of cholesterol from liver, and (2) the lipid digestion/absorption in the intestine. Abnormal bile acid synthesis or secretion can lead to severe liver disorders. Even though there is extensive literature on the mass spectrometric determination of BAs in biofluids and tissue homogenates, there are no reports on the spatial distribution in the biliary network of the liver. Here, we demonstrate the application of high mass resolution/mass accuracy matrix-assisted laser desorption/ionization (MALDI)-Fourier-transform ion cyclotron resonance (FTICR) to MS imaging (MSI) of BAs at high spatial resolutions (pixel size, 25 μm). The results show chemical heterogeneity of the mouse liver sections with a number of branching biliary and blood ducts. In addition to ion signals from deprotonation of the BA molecules, MALDI-MSI generated several further intense signals at larger m/z for the BAs. These signals were spatially co-localized with the deprotonated molecules and easily misinterpreted as additional products of BA biotransformations. In-depth analysis of accurate mass shifts and additional electrospray ionization and MALDI-FTICR experiments, however, confirmed them as proton-bound dimers. Interestingly, dimers of bile acids, but also unusual mixed dimers of different taurine-conjugated bile acids and free taurine, were identified. Since formation of these complexes will negatively influence signal intensities of the desired [M – H]- ions and significantly complicate mass spectral interpretations, two simple broadband techniques were proposed for non-selective dissociation of dimers that lead to increased signals for the deprotonated BAs. Graphical Abstractᅟ Electronic supplementary material The online version of this article (10.1007/s13361-017-1886-6) contains supplementary material, which is available to authorized users.

INTRODUCTION: Acetaminophen protein adducts in the circulation are a specific biomarker of acetaminophen oxidation, and may be a more sensitive measure of impending hepatic injury following overdose than alanine transaminase (ALT). We performed an exploratory analytical substudy of adducts during a clinical trial (NACSTOP) of abbreviated (12-hour) versus control (20-hour) acetylcysteine to identify any signal of diminished antidotal effectiveness with shortened therapy. METHODS: We measured adducts at 0, 12, and 20 hours from a convenience sample of subjects enrolled in the cluster-controlled NACSTOP trial evaluating a 12-hour (“abbreviated”; 200 mg/kg over 4 hours, 50 mg/kg over 8 hours) vs 20-hour acetylcysteine regimen (“control”; 200 mg/kg over 4 hours, 100 mg/kg over 16 hours). Adducts were assayed using high-performance liquid chromatography/mass spectrometry. RESULTS: Median ALT 20 hours after the initiation of acetylcysteine was 12 U/L (IQR 8,14) in the abbreviated 12-hour regimen group (N = 8), compared with the control group 16 U/L (IQR 11,21; N = 21) (p = 0.46). Adduct concentrations were similarly low in both groups: abbreviated [(0.005 μmol/L, IQR (0,0.14)] and control [(0.005 μmol/L, IQR (0,0.05)] (p = 0.61). CONCLUSIONS: There were minimal to no acetaminophen protein adducts detected. These findings further support discontinuing acetylcysteine when acetaminophen concentrations are low and liver function tests normal after 12 hours of treatment.

In DNA adduct analysis, the 32P-postlabeling technique is a powerful tool due to its high detection sensitivity. It is performed by enzymatic digestion of DNA samples, enrichment of the adduct nucleotides, and then 5'-labeling with 32P. This method is particularly useful for detection of bulky adducts. An additional advantage is that only a small amount of DNA is required for detecting DNA adducts. This chapter describes the experimental procedure for separation and detection of DNA adducts by polyacrylamide gel electrophoresis, which is an attractive method for visually assessing differences in adduct formation between samples.

Binary and ternary complexes of copper(II) with l-glutamic acid (Glu) and diamines 1,3-diaminopropane and 1,4-diaminobutane, putrescine (tn, Put), as well as adducts formed in the metal-free systems, have been investigated in aqueous solutions. The types of complexes formed and their overall stability constants were established on the basis of computer analysis of potentiometric results. The reaction centers and the modes of interaction were identified on the basis of spectroscopic studies (NMR, Vis and EPR). In the ligands studied the interaction centers are the oxygen atoms from carboxyl groups, nitrogen atom from the amine group of glutamic acid and the nitrogen atoms from amine groups of the diamines. The centers of noncovalent interaction in the adducts that formed in the metal-free systems are also potential sites of metal ion coordination, which is important in biological systems. In the Glu–diamine systems, molecular complexes of the (Glu)Hx(diamine) type are formed. In the (Glu)H2(tn) adduct, in contrast to the corresponding complex with Put, an inversion effect was observed in which the first deprotonated amine group of tn became a negative reaction center and interacted with the protonated amine groups from Glu. Depending on the pH, the amine groups from the diamine can be either a positive or a negative center of interaction. In the Cu(Glu)2 species the first molecule of Glu takes part in metallation through all functional groups, whereas the second molecule makes a “glycine-like” coordination with the Cu(II) ions that is only through two functional groups. According to the results, introduction of Cu(II) ions into metal-free systems (Glu–diamine) changes the character of interactions between the bioligands in the complexes that form in Cu(II)–Glu–diamine systems and no ML…L′ type complexes are formed. However, in the ternary systems only the heteroligand complexes Cu(Glu)(diamine) and Cu(Glu)(diamine)(OH) are observed.

For many years it was puzzling that metal atoms, which were not considered good Lewis acids, could form adducts with CO, considered to be a poor donor toward most Lewis acids. An example is the hexacarbonylchromium, Cr(CO)6. On the other hand, it was stressed that chromium, having 24 electrons, by accepting 6x2 electrons from 6 CO molecules, will fill its orbitals to a total of 36 electrons, which is the electron configuration of krypton. The LCAO approximation method (linear combination of atomic orbitals) helped to explain the stability of these adducts as being due to a two-way acid-base interaction between the metal and CO: the metal synergically interacts with CO, acting as an acid and a base, while CO simultaneously behaves as a donor and acceptor. In 1955 Ernst Otto Fischer, University of Munich, showed that an increase of the number of chromium electrons to 36 can be achieved by three π-electron pairs of benzene, like, e.g., in benzenetricarbonylchromium, (C6H6)Cr(CO)3. Cyclopentadienyl, C5H5 -, has also been known to form compounds with metal atoms.

Since the discovery of C60 by Kroto et al.1 and its subsequent isolation by Kratschmer et al.2 a number of applications of this molecule and its derivatives have been found. The major areas include electronic applications, which still is a topic of much research effort.3 Another area that has received considerable attention is to make fullerenes water-soluble to facilitate their use in biological and biomedical applications. These applications emerge from the fullerenes absorbing strongly in the ultraviolet and moderately in the visible range of the spectrum, leading to the formation of singlet oxygen with almost 100% efficiency.4 The high reactivity of fullerenes towards free radicals has led to these being recognized as a ‘free radical sponge’.5 These two properties (illustrated schematically in Figure 1a and Figure 1b) have been the base of a number of biomedical applications.

Chlorination of water for human consumption is important for disease prevention. However, halogenated by-products, such as trihalomethanes (THM), can be formed during this process (Bunn et al 1975). THM production raises public health concerns related to potential human cancer risk (Fawell, 2000). Chloroform (trichloromethane: TCM) is a byproduct that is frequently detected at relatively high concentrations (IARC, 1991), i.e. 0.1 to 300 μg/litre in finished drinking water (Uden and Miller, 1983) and up to 1 mg/litre in chlorinated swimming pool water (Lahl et al 1981).

Epichlorohydrin (ECH, 1-chloro-2,3-epoxypropane) is a chemical used industrially in the manufacture of e.g. epoxy resins, glycidyl ethers, ion exchangers, pharmaceuticals, paper, textiles and coatings. ECH has been shown to induce mutagenic and clastogenic effects in various test systems. It has been shown to be carcinogenic in animals and classified as probably carcinogenic to humans. Recently, ECH was shown to induce formation of DNA adducts in humans exposed to ECH (Plna et al., 2000). A review article concerning the toxicology of ECH has been written by Giri (1997)

Cyclooxygenase-2 (COX-2) catalyzes the oxygenation of arachidonic acid into the prostaglandin endoperoxide, PGH2. PGH2 is then processed by different isomerases into prostaglandins and thromboxane A2. PGH2 also undergoes rearrangement in aqueous solution into PGE2, PGD2 and into the levuglandins (LG) E2 and D2 1. LG’s are highly reactive γ-ketoaldehydes that have been shown to form adducts and cross-links with proteins 2, 3, primarily through their reaction with the e-amine of lysine. The potential that these levuglandin adducts of proteins could be biologically important may be inferred from the knowledge that lipid-modification of proteins is known to influence their function and cellular localization 4–7.

Ecteinascidin 743 (Et 743), a natural product derived from the Caribbean tunicate Eteinascidia turbinata, is a potent antitumor agent currently in phase II clinical trials. Et 743 binds in the minor groove of DNA, forming covalent adducts by reacting with N2 of guanine. Although DNA is considered to be the macromolecular receptor for Et 743, the precise mechanism by which Et 743 exerts its remarkable antitumor activity has not yet been elucidated. The aim of this study is to provide a rationale for the antitumor activity of Et 743 by studying its fundamental interactions with DNA at the molecular level.

Ethenoadducts have attracted considerable interest when they were detected in the reactions between DNA and occupational carcinogens such as vinyl chloride, urethane and acrylonitrile1. The deleterious effects of ethenobases were further attested by studies showing their high mutagenic potential2,3. Interestingly, background levels of 3,N 4-etheno2’-deoxycytidine (3,N 4-ɛdC) and 1,N 6-etheno-2’-deoxyadenosine (1,N 6-ɛdA) were detected in tissue DNA from untreated rodents and humans4. However, the origin of the adduct background is still open to debate. It is strongly suggested that increased oxidative stress and lipid peroxidation (LP) are implicated in this process1. In fact, most of LP final products are very efficient alkylating agents. Among these products, malonaldehyde and HNE, are the most extensively studied. However, only HNE was shown to form DNA ethenoadducts5. Due to the lack of information about other LP products, the bulk of the lesions have been attributed to HNE. Even though, other products are reported to be very cytotoxic and can also play an important role in the genotoxic effects associated with LP.

It has been proposed that the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) may be involved in the causation of human lung cancer (Hecht and Hoffmann, 1988), but direct evidence for the involvement of NNK in human lung cancer is lacking (Hecht and Tricker, 1999). In the A/J mouse lung tumor model, a single intraperitoneal (i.p.) injection of 10 μmole [2.07 mg] NNK/mouse results in 7–12 lung tumors per mouse after 16 weeks (Hecht et al., 1989). The initial events in NNK-induced A/J mouse lung tumorigenesis are believed to be metabolism of NNK to biological reactive intermediates (BRI) with the potential to react (via methylation of DNA) to produce O6-methylguanine (O6MeG), GC→AT transitional mispairing, and subsequent activation of the K-ras proto-oncogene (Ronai et al., 1993). α-Hydroxylation of the methylene carbon atoms adjacent to the N-nitroso group in NNK and NNAL yields unstable BM which spontaneously decompose to methanediazohydroxide with the potential to react with DNA to produce 7-methylguanine (7-MeG), O4-methylthymidine (O4MeT) and O6MeG adducts. α-Hydroxylation of the NNK methyl group yields a BM with the potential to pyridyloxobutylate DNA, while α-methyl hydroxylation of NNAL is not known to result in DNA adduct formation. Other metabolic transformations represent detoxification pathways for NNK and NNAL.

Malondialdehyde (MDA) is a toxic and mutagenic metabolite produced by lipid peroxidation, and prostaglandin biosynthesis. MDA induces frameshift mutations in tester strains of Salmonella typhimurium. It reacts with DNA, and at physiological pH the major adduct is a pyrimidopurinone formed by reaction with guanine: M1G [3-(2’-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-α]-purin-10(3H)-one]. When site-specifically incorporated into a duplex oligodeoxynucleotide containing a frameshift-prone (CG)3, repeat derived from the Salmonella typhimurium hisd3052 gene, spontaneous opening of M1G to the N2-(3-oxo-1propenyl)-dG species occurred. In this work d(ATCGCMCGGCATG), (M=M1G) was annealed to d(CATGCCGCGAT) to model the putative strand slippage intermediate which would precede a two base deletion in the (CG), iterated repeat. 1H NMR studies indicate that in contrast to the duplex DNA structure, M1G remains intact. A single bulge conformation exists. M1G and its 3’-neighbor cytosine are unpaired. The M1G is intrahelical and stacked, whereas the unpaired cytosine is poorly stacked and appears to be extrahelical.

BBR3464 is a novel Phase I clinical agent based on a triplatinum structure (Figure 1), trans-[bis trans-diaminechloroplatinum(μ-1,6-hexanediamine) ]diamineplatinum tetranitrate salt. Its DNA binding is characterized by a high percentage of interstrand cross-links and the ability to induce the B -- Z conformation in poly(dG.dC).poly(dG.dC). To help characterize these novel DNA adducts further, we have begun a program to model the types of interactions and the ensuing conformational changes induced by covalent binding of BBR3464 to DNA.

The Diels-Alder reaction of α-pyrones with alkenes and alkynes is well known:1 some 40 years ago, Wittig and co-workers reported that the reaction of α-pyrone (1a) with benzyne (2) led to formation of naphthalene (4a).2 In fact, the initial adduct is the bicyclic intermediate 3a, which undergoes a retro-Diels-Alder reaction, losing CO2 to give the aromatic compound 4a. The intermediate 3a was not detected because its conversion to 4a is favoured on both enthalpic and entropic grounds. In the intervening years the synthetic potential of this transformation for construction of aromatic molecules from α-pyrones has hardly been exploited.3 Here we describe its application in the synthesis of some interesting compounds.

The mulberry tree, a typical plant of the genus Morus, has been widely cultivated in China and Japan. Its leaves are indispensable as food for silkworms. On the other hand, the root bark of the mulberry tree, Morus alba L. and other plants of the genus Morus, has been used as an antiphlogistic, diuretic, expectorant, and laxative in the Chinese herbal medicine called “Sang-Bai-Pi” (“Sohakuhi” in Japanese).1 In the pharmacological field, several researchers have reported on the hypotensive effect of the extract. Considering these reports, it was suggested that the hypotensive constituents were made up of a mixture of many phenolic constituents of the mulberry tree and related plants. About 70 kinds of new phenolic compounds could be isolated from the Japanese cultivated mulberry tree and Chinese crude drug “Sang-Bai-Pi”. Among them, a hypotensive compound, kuwanon G (1), was isolated in 0.2 percent yield from the root bark.2 Furthermore, kuwanon G is considered to be formed through an enzymatic Diels-Alder type reaction of a chalcone (2) and dehydrokuwanon C (3) or its equivalent (fig. 1). Subsequently, about 40 kinds of Diels-Alder type adducts have been isolated from moraceous plants.3–5

Ingest of disulfiram blocks the metabolism of acetaldehyde, the product of ethanol metabolism, by inhibiting hepatic mitochondrial aldehyde dehydrogenase (ALDH2), a key ene by virtue of its low Km for acetaldehyde (Mascher & Kikuta, 1992; Greenfield & Pietruszko, 1977). Disulfiram is rapidly reduced in vivo to N,N-diethyldithiocarbamate (DDC) (Cobby et al., 1977) which is further metabolized as shown in Scheme 1. The general consensus is that disulfiram is too short-lived in vivo to account for the inhibition of ALDH2 that one its metabolites is the ultimate inhibitor (Yourick & Faiman, 1991; Hart & man, 1992).

Free radicals produce a number of lesions in DNA such as base lesions, sugar lesions, single-strand breaks, double-strand breaks, abasic sites and DNA-protein cross-links by a variety of mechanisms. Hydroxyl radical, hydrated electron and H atom react with the heterocyclic bases in DNA by addition. In oxygenated systems, adduct radicals of pyrimidines are converted into corresponding peroxyl radicals with oxygen. Hydroxyl radical reacts with purines at diffusion-controlled rates by addition to C4-, C5- and C8-positions. A fraction of hydroxyl radicals reacts with the sugar moiety in DNA by abstraction of H atoms from all five carbon atoms, producing sugar radicals. Further reactions of DNA radicals result in formation of numerous products. The types and yields of DNA modifications profoundly depend on the free radical-generating system, experimental conditions, and the presence or absence of oxygen. In chromatin, DNA-protein cross-links are formed by combination of two radicals, or by radical addition reactions. A number of analytical techniques have been used to identify and quantify a variety of products of DNA. Many pyrimidine and purine lesions have been identified and quantified in cells and tissues. Evidence indicates that most products arise as a result of reactions of hydroxyl radical with DNA constituents in cells. A number of DNA lesions possess premutagenic properties. The biological consequences of DNA modifications in cells are largely unknown and await further exploration.

Use of anthocyanins as coloring agents in food is limited, since it is well known that they lose their original color in the presence of nucleophiles. This phenomenon occurs in aqueous alkaline media (OH− addition), but most of all in the presence of bisulfite, commonly used as a preservative. Even though this has been observed for many years, the mechanism is not thoroughly understood. The chalcone form (3), in equilibrium1,2 with the carbinol (2) resulting from hydroxyl addition on the flavylium (1), was postulated. Addition of bisulfite readily occurs, as it does with carbonyls leading to a chalcone-bisulfite adduct.3,4 More recent works, by means of UV-visible,5 kinetic,6,7 and thermodynamic measurements,8 have shown that adducts could result from nucleophilic addition of SO2 either on the C-2 or C-4 carbon of the flavylium, thus refuting the necessity of opening into a chalcone (fig. 1).

The pH-dependence and solvent isotope effects of dealkylation in diastereomeric adducts of Electric eel (Ee) acetylcholinesterase (AChE), fetal bovine serum (FBS) AChE (1), mouse (Mo) AChE and human (Hu) butyrylcholinesterase (BChE) inactivated with P(S)C(S) and P(S)C(R) 2-(3, 3-dimethylbutyl) methylphosphonofluoridate (soman) were studied at 25.0 ± 0.1 EC with Mo AChE and at 4.0 ± 0.1 EC with the other cholinesterases.(2) Analogous studies were carried out with the E202Q mutant of Mo AChE and the E197Q, E197D, E197G and W82A mutants of Hu BChE at 25.0 ± 0.1 EC (2). Best fit parameters for the asymmetric bell-shaped curves observed for the adducts of Ee, FBS and Mo AChE are pK1 = pK2 = 4–4.9 and pK3 = 5.2–6.6. These pKs are consistent with the participation of two carboxylate ions, possibly from E327 and E199, and His440H+ in catalysis of the dealkylation reaction by AChEs. The E199Q mutant of Mo AChE inactivated with the soman diastereomers yielded a single pK with values of 5.5–5.8. Nearly symmetric pH curves for soman-inhibited Hu BChE and its E197D mutant gave pK2 of 3.7–4.6 and pK2 of 7.3–8, but for the corresponding adducts of the E199Q mutant pK2 is a low 5. The dealkylation in soman-inhibited Hu BChE is then consistent with participation of one carboxylate ion side chain and His447H+. Maximal rate constants (kmax) are 1–6 min−1 for AChEs and 2 min−1 for Hu BChE at 25.0 EC. The solvent isotope effects at the pH maxima are 1.1–1.3 indicating unlikely proton transfer or preprotonation at the enzymic transition states for the dealkylation reaction.

The molecular masses of the bacterially expressed recombinant HuAChE and its conjugates with series of alkyl methylphosphonofluoridates and with diisopropyl fluorophosphate (DFP), were measured by electrospray - ionization mass spectrometry (ESMS). The mass of HuAChE (measured as 64700 Da, calc. 64695 Da) increased, following reactions with sarin, isobutyl methyl phosphorofluoridate (IBMPF), 1, 2-dimethylpropyl methyl phosphorofluoridate (DMPF), soman and DFP, by 120, 140, 150, 160 and 160 Da respectively. These values were in excellent agreement with the calculated masses of the adducts and reflected both the addition of the phosphonyl moiety and the gradual mass increase due to branching of the alkoxy substituent. The composition of the phosphyl adducts change with time to yield a common product with molecular mass of 64780 Da, which is consistent with dealkylation of the phosphonyl moieties. By sequential ESMS measurements we were able to estimate the kinetics of evolution of the aged product of the HuAChE-soman adduct (t1/2 ≈ 50 sec, at pH 6.0). This rate is in good agreement with that determined by kinetic measurement of the development of non-reactivability under similar conditions. It is important to note that in agreement with the accepted mechanism of dealkylation, only two molecular species are evident in the sequential ESMS measurements during the aging process.

Prostaglandin endoperoxide synthase (PGHS, EC 1.14.99.1) catalyzes the first two steps of prostaglandin biosynthesis.1 Its cyclooxygenase activity oxygenates arachidonic acid to form PGG2; the peroxidase activity of the enzyme then reduces PGG2 in the presence of a reducing substrate to the corresponding alcohol PGH2.2,3 The cyclooxygenase activity is inhibited by a structurally diverse class of compounds known as non-steroidal antiinflammatory drugs (NSAID’s).4 Inhibition of PGHS by NSAID’s is believed to be the biochemical basis for their antiinflammatory properties.5 Covalent modification of PGHS is responsible for enzyme inactivation by aspirin4, acylimidazoles6, acyl-N-hydroxysuccinimides6,7, and N-alkylmaleimides.8 However, a large excess of inhibitor (~300 fold) and prolonged incubation times (~120 min) are required to achieve significant inhibition in all of these cases. In the present study, we have tethered to the maleimido moiety, a series of substrate and inhibitor mimics that are capable of binding at the fatty acid substrate site. Maleimide derivatives linked to a series of medium length fatty acids were found to exhibit much more potent cyclooxygenase inactivation. The most potent of these inhibitors, N-(carboxyheptyl)maleimide, inhibits enyzme activity within seconds after mixing with a stoichiometric amount of PGHS protein. Varying the length of the alkyl chain in N-(carboxyalkyl)maleimides or removal of the carboxylate dramatically reduces their potency as rapid PGHS inhibitors. In addition to the structure-activity relationships, the mechanism of inhibition of PGHS by N-(carboxyheptyl)maleimide was explored by subsequent inhibition studies with radiolabeled N-(carboxyheptyl)maleimide. Although incubation of apoPGHS with radiolabeled inhibitor led to the incorporation of radioactivity in the protein, subsequent attempts to identify the amino acid residue(s) by peptide mapping were unsuccessful, presumably due to the instability of the enzyme/inhibitor adduct.

The photosensitized oxidation of foods is initiated by natural pigments or sensitizers producing an excited state which may follow Type I or Type II reaction pathways1. The interaction of sensitizer with food components such as protein or fat, generating free radicals, is called Type I; The reaction of excited sensitizer producing singlet oxygen is called Type II2. Dairy products are excellent sources of riboflavin3. Photochemical reactions of riboflavin and various flavin compounds have been studied in several systems4, 5, 6. Riboflavin has been shown to favor a Type I mechanism because it is easily oxidized or reduced and its high water solubility minimizes its interaction with oxygen which is readily more soluble in lipids7. The triplet state of riboflavin has been suggested to generate singlet oxygen in milk during exposure to light8. ESR can detect the formation of singlet oxygen by reaction with 2,2,6,6-tetramethyl-4-piperidone (TMPD), forming a stable nitroxide radical adduct, 2,2,6,6-tetramethyl-4-piperidone-l-oxyl (TAN or TEMPONE)9, 10.

In rats and humans, inhalation kinetics of n-hexane (HEX) and n-heptane (HEP) were compared with urinary excretion of 2,5-hexanedione (HDO) and 2,5-heptanedione (HPDO), respectively. Furthermore, the reactivities of HDO and HPDO with Nα-acetyl-L-lysine towards the formation of pyrrolyc adducts was studied. By means of the data gained, the potency of HEP for inducing peripheral neuropathy is compared with the well known one of HEX. In rats, kinetic analysis revealed two different metabolic processes for HEX and HEP, one process characterized by high affinity and low capacity (maximal rate of metabolism Vmaxl: HEX 84, HEP 112 μmol/h/kg) and one by low affinity and high capacity (Vmax2: HEX 456 μmol/h/kg). For HEP, Vmax2 cannot be given, since the deviation from linearity of the curve representing the rate of metabolism versus the exposure concentration was too small within the concentration range studied of up to 10000 ppm. Urinary excretion of HDO resulting from exposure to HEX correlated with the first process, whereas the corresponding excretion of HPDO as a metabolite of HEP correlated with the second process. In humans, rates of metabolism of HEX and HEP increased linearly with the exposure concentrations up to the tested values of 300 ppm (HEX) and 500 ppm (HEP), the pulmonary retention at steady state being 23% (HEX) and 35% (HEP) at rest. Of totally metabolized HEX during and after HEX exposure to 300 ppm, about 0.5% was excreted as HDO in urine. Of totally metabolized HEP during and after HEP exposure up to 500 ppm, only about 0.01% was excreted as HPDO in urine. Background excretion of HPDO was found in urine of rats and of both γ-diketones in urine of humans; the sources are still unknown. In rats, urinary excretion of HPDO resulting from exposure to 500 ppm HEP was about 7 times less and in humans about 4 times less than that of HDO resulting from exposure to 50 ppm HEX over the same time span. In vitro, the rate of pyrrole formation from the reaction of HPDO with Na-acetyl-L-lysine was about half that obtained with HDO. This indicates a lower neurotoxic potency of HPDO. From our findings it becomes intelligible that HEP was not neurotoxic in rats in contrast to HEX. Furthermore, for humans we also conclude the neurotoxic potency of HEP to be significantly lower than that of HEX.

Vinyl chloride is recognized as a human carcinogen. 2-Chlorooxirane, its biological reactive intermediate formed by the P450 enzyme system in the liver, and the rearrangement product chloroacetaldehyde give rise to the etheno (e) DNA adducts. This type of modified DNA bases appears to be the molecular basis for the genotoxic effects of the parent compound, and site specific mutagenesis studies (Basu et al., 1993) show its mutagenicity. There is evidence that e adducts might not only be formed by vinyl chloride metabolites but also be present at a certain level due to endogeneous sources (Misra et al., 1994).

Antibodies to a variety of oxidized DNA bases have been generated in a number of laboratories including our own (see Table I). Most of these antibodies have been elicited using protein-conjugated haptens of interest. In general, the antibodies have reasonable affinity such that appropriate sensitivity in the various assays can be achieved. The difficulty with antibodies that recognize oxidized DNA bases is that the oxidized bases do not differ largely from their unoxidized derivatives. Thus, the specificity for detecting lesions in DNA must be high especially when one considers the low level of damaged compared to undamaged bases. Even if the sensitivity of the assay can be amplified, cross-reactivity of the antibody with the unoxidized base in DNA remains an obstacle to successful detection of low levels of the oxidized base. This particular problem is not seen as often when antibodies are elicited to the chemical adducts that have features that vary quite dramatically from the unadducted base. An additional consideration is that the lesion must be stable to the procedures used during preparation of the immunogen and during DNA denaturation. The latter is usually necessary since the antibodies often do not recognize the lesion as well in duplex DNA. Despite these shortcomings, antibodies to oxidized bases have been effectively utilized to detect these lesions in oxidized or ionizing radiation-treated DNA in vitro.

The 32P-postlabeling assay was originally devised by Randerath et al. (1981) to measure carcinogen-DNA adducts. In the procedure, DNA is first digested by micrococcal nuclease and calf spleen phosphodiesterase to give nucleoside 3′-monophosphates (normal and modified) that are subsequently labeled by incubation with [γ-32P]-ATP and T4 polynucleotide kinase. The radiolabeled compounds are then separated by two-dimensional TLC. The assay has two important advantages. First, there is no requirement for prelabeling the DNA, which makes the assay useful for the study of DNA lesions in tissues. Second, because of the availability of [γ-32P]-ATP of high specific activity, the assay permits detection at the femtomole level. There are, however, two major drawbacks:(1) the polynucleotide kinase must be able to act on the modified nucleoside 3′-monophosphate and (2) the resulting labeled modified nucleoside diphosphate must be separable from the high background of normal nucleoside diphosphates. These problems are well illustrated in the reports of efforts to detect thymine glycols, a well-known oxidative base lesion, in irradiated DNA (Reddy et al., 1991; Hegi et al., 1989). Furthermore, lesions that involve base loss, such as abasic sites and deoxyribose fragments, cannot be detected by this approach.

Quantitative imunoassays such as radioimmunoassay (RIA) and enzyme-linked immunosorbant assay (ELISA) are sensitive and reliable procedures used to measure UV photoproducts in purified sample DNA (Eggset et al., 1983; Strickland, 1985; Mitchell and Nairn, 1989; Matsunaga et al., 1990; Wani and Arezina, 1991; Vink et al., 1993). Competitive immunoassays have distinct advantages over other procedures in the analysis of DNA damage in human and environmental samples. For instance, unlike spectroscopic analyses, immunoassays are not limited to certain classes of adducts, and unlike HPLC and 32P-postlabeling, immunoanalysis of DNA damage does not require hydrolysis of sample DNA which can decrease the signal-to-noise ratio and alter chemical structure. ELISAs and RIAs require minimal sample manipulation and have been performed on crude cell lysates. In addition, sample DNA does not require prelabeling with a radioactive tracer and antibody binding is quasi-independent of molecular weight (i.e., DNA degradation). Immunoassays are thus readily applied to a variety of biological materials and have typically been used to measure DNA damage in cell and organ cultures, normal tissues and tumor biopsies, and various other samples including buccal cells, bone marrow aspirates, and peripheral blood lymphocytes. RIA is a sensitive and facile technique for measuring genotoxic damage in DNA.

Reactive intermediates can be monitored in vivo through their reaction products (adducts) with macromolecules. Sensitive methods, based on gas chromatography/mass spectrometry which permit structural identification and quantification, have been developed for the determination of adducts to hemoglobin (Hb). In studies of exposed animals, occupationally exposed workers and smokers a number of Hb adducts have been observed in unexposed control individuals. Methods for the determination of Hb adducts have, particularly through the use of tandem mass spectrometry, reached a sensitivity permitting studies of adducts from reactive intermediates due to “background exposure”. N-(2-Hydroxethyl)valine (HOEtVal) is one of the background Hb adducts observed. This adduct has been used as a model in studies of sources of background adducts and determinants of their levels.

Cyproterone acetate (CPA) [Fig. 1] is a sex steroid with strong antiandrogenic and gestagenic action, which is widely used in human therapy. Upon long term feeding CPA causes liver tumors in rats [1]; this activity has been attributed to tumor promotion. The safety of the therapeutic use of the steroid has been questioned recently, since findings from our laboratories indicate that CPA exhibits genotoxic activity. CPA induces DNA repair synthesis in cultured hepatocytes from female rats [2] and the formation of CPA-derived DNA adducts in rat liver and in hepatocytes [3].

Whereas the molecular basis for the antitumor activity of cisplatin is not yet established, there are numerous experimental results implicating DNA binding in the antitumor mechanism. Whatever happens between platinum binding to DNA and the cell death, the hypothesis that the structural distortion plays a role in the processing of the Pt-DNA adducts does not seem unlikely, and provides the pharmacological motivation for structural studies on these adducts.

It is generally believed that the formation of DNA adducts by covalent interaction of electrophilic species of carcinogens (ultimate carcinogens) with macromolecules, particularly DNA, is an essential first step in the multistage process of carcinogenesis (Miller and Miller, 1981). Until the early 1980s, use of radiolabeled carcinogens was the main method to determine the binding of chemical carcinogens to DNA. Since then several methods have been utilized for nonradio-labeled carcinogens. The assays that are practiced currently are based on specific antibodies, fluorescence properties of adducts, gas chromatography/mass spectrometry, and 32P-postlabeling. These assays require a few micrograms to several hundred micrograms of DNA, with a detection limit of 1 adduct per 106 to 1010 nucleotides, depending on the method (Beach and Gupta, 1992). The 32P-postlabeling assay has emerged as the major tool for measuring DNA adducts because of its ultrasensitivity and applicability to theoretically any DNA-damaging agent, irrespective of its chemical nature, including unknowns. Over 100 individual agents or complex mixtures have been tested in rodents and aquatic systems in vivo and rodent and human cells in vitro (Beach and Gupta, 1992).

Since the discovery of the antitumor activity of cis-diamminedichloroplatinum(II) (cisplatin), it is generally accepted that the cytotoxic action of platinum drugs is the consequence of their interaction with cellular DNA. Therefore, knowledge about the formation and persistence of DNA damage may help to understand the working mechanism of these compounds and may eventually lead to the development of better Pt-containing antitumor drugs.

Formaldehyde is oxidized in mammalian tissues and in several other sources to formate in two consecutive reactions catalyzed by separate enzymes (Uotila and Koivusalo, 1974a; 1974b) (Scheme 1). Formaldehyde and glutathione react first non-enzymically to form a hemithioacetal adduct (S-hydroxymethylglutathione) (Reaction 1). The adduct is the substrate of formaldehyde dehydrogenase (EC 1.2.1.1) and is oxidized in the NAD-depend-ent reaction to S-formylglutathione (Reaction 2). Formaldehyde dehydrogenase has been shown to be identical with the class III alcohol dehydrogenase (Koivusalo et al., 1989). For a comprehensive review on formaldehyde dehydrogenase, see Uotila and Koivusalo (1989). The hydrolysis of S-formylglutathione to reduced glutathione and formate (Reaction 3) is catalyzed by a specific enzyme, S-formylglutathione hydrolase (EC 3.1.2.12). Formate can be oxidized by the catalase reaction or is transferred to tetrahydrofolate to form 10-formyl-tetrahydrofolate. Formate is thus incorporated into the reactions of the C-1 metabolism by which it can be oxidized to carbon dioxide in the 10-tetrahydrofolate dehydrogenase reaction (Kutzbach and Stokstad, 1968).

Psoralen plus UVA (long wavelength ultraviolet radiation) (PUVA) produces three types of adducts in DNA: (i) intercalation of the psoralen molecule between flat, stacked base-pairs, (ii) formation of a monoadduct by UVA radiation-dependent covalent bonding of the psoralen molecule to a base, primarily via a cyclobutane ring to a thymine, on one strand of the DNA, and (iii) formation of a DNA interstrand cross-link by a second UVA radiation-dependent covalent bonding of the psoralen molecule to a base on the opposite strand of the DNA molecule. The latter adduct (iii) is thought to be the most important biologically.

Some biological properties of bovine and recombinant human Cu, Zn superoxide dismutase (bSOD and rhSOD)-poly-ethylene glycol (PEG) adducts prepared by coupling 1–9 strands of high molecular weight PEG (35,000–120,000 daltons) are compared to SOD adducts coupled with 7 or 15 strands of low molecular weight PEG (5,000 daltons).

We use a novel assay to detect strand specific repair in genes after cellular exposure to cisplatin at IC50 levels. Single stranded DNA capable of hybridizing to gene specific probes is generated enzymatically by the 3′–5′ exonuclease activity of 74 DNA polymerise. in the presence of cisplatin adducts, the exonuclease activity of this enzyme is blocked, preventing the formation of single stranded DNA, and thus lowering the amount of complementary sequence available for probe hybridization.1,2

Exposure to genotoxic carcinogens results in the formation of covalently bound adducts between the genotoxin and DNA, which may cause mutation and cytogenetic alterations. The dose of the genotoxic carcinogenic compounds involved in the exposure may be monitored by quantitative analysis of DNA adducts, and these data may also be used as an indicator of genotoxic risk. The analytical techniques required for adduct measurements need to be of exceptional sensitivity and include 32P-postlabelling, mass spectrometry and immunoassay. Exposure to alkylating carcinogens may also be monitored by measurement of adducts formed with amino acids in haemoglobin (Hb). Since, for a variety of compounds, the extent of protein-adduct formation relates quantitatively with that of DNA adducts, measurement of the former may indicate the biologically-effective dose of the compound received.

Most known chemical carcinogens are electrophilically reactive compounds (RX) or are formed in vivo from non-reactive precursors (A) (Miller and Miller, 1966). The electrophiles react with nucleophilic atoms (Y) of biomacromolecules giving rise to adducts (RY): $$A \to RX\xrightarrow[{({k_y})}]{{ + {Y^ - }}}RY + {X^ - }$$ (1) The measurement of adducts to sufficiently stable macromolecules may be used for identification of electrophiles in vivo and for dose monitoring as a basis for risk estimation. For reasons to be discussed below, the Stockholm group has used hemoglobin (Hb) for in vivo dose monitoring of chemical carcinogens in animals and humans.

The combination of FeII(DPAH)2 (DPAH2 = 2,6-dicarboxyl pyridine) and O2 in a 2:1 pyridine/acetic acid solution results in a rapid autoxidation to produce HOOH and FeIII(DPA)(DPAH) (k1, 1.8±0.5 M-1s-1). The resultant HOOH reacts with excess FeII(DPAH)2 via a nucleophilic addition to give a Fenton reagent [(DPAH)2-FeIIOOH + pyH+] (1) that reacts with (a) excess FeII(DPAH)2 to give FeIII(DPA)(DPAH) [k2, (2±1) x 103 M-1s-1], (b) excess c-C6H12 and PhSeSePh (a carbon radical trap) to give c-C6H11SePh [kinetic-isotope-effect (\(\matrix{ {\{ {\rm{F}}{{\rm{e}}^{{\rm{II}}}}{{({\rm{PA}})}_2} + {\rm{HOOH}}} & {\mathrel{\mathop{\kern0pt\longrightarrow} \limits_{{{({\rm{py}})}_2}{\rm{HOAc}}}^{{\rm{k}},2 \times {{10}^3}{{\rm{M}}^{ - 1}}{{\rm{s}}^{ - 1}}}} } & {[{{({\rm{PA}})}_2} - {\rm{F}}{{\rm{e}}^{{\rm{II}}}}} \cr } {\rm{OOH + py}}{{\rm{H}}^{\rm{ + }}}](1)\} \)), 1.7], and (c) excess O2 to form an adduct [(DPAH)2-FeIIIOOH(O2) + pyH+](5), which reacts with excess c-C6H12 via an intermediate [(DPAH)2FeIV(OH)(OOC6H11)] (6) and FeII(DPAH)2 to give c-C6H10(O) [KIE, 2.6] and FeIII(DPA)(DPAH) [32 mM FeII(DPAH)2,O2 (1 atm, 3.4 mM), and 1 M c-C6H12 yield 6 mM c-C6H10(O)]. The related FeII(PA)2 complex in combination with t-BuOOH (or HOOH) catalytically activates O2 to oxygenate hydrocarbons {e.g., c-C6H12 → c-C6H10(O) [9 O2 turnovers per FeII(PA)2]; PhCH2CH3 → PhC(O)CH3 (25 O2 turnovers); c-C6H10 → c-C6H8(O) (5 O2 turnovers); and PhCH(Me)2 → PhC(O)Me, Ph(Me)2COH, and Ph(Me)C=CH2 (5 O2 turnovers). With large t-BuOOH (or HOOH)/FeII(PA)2 ratios spontaneous decomposition occurs to give free O2 that is incorporated into the substrates. The first-formed intermediate is a one-to-one t-BuOOH/FeLx adduct {[(PA)2-FeIIOOBu-t + pyH+] (1)}, which reacts with (a) excess t-BuOOH to give O2 and t-BuOH, (b) excess FeII(PA)2 to give (PA)2FeIIIOH(Bu-t), (c) excess c-C6H12 to give (c-C6H11)py [KIE, 4.6 with t-BuOOH and 1.7 with HOOH], and (d) O2 to form an adduct, [(PA)2-FeIII(O2)(OOBu-t) + pyH+](5), that reacts with c-C6H12 to form c-C6H10(O) [KIE, 8.7 (2.5 with HOOH)]. When PhCH2Me or c-C6H10 are the substrates (RH), 5 reacts to form [(PA)2FeIV(OH)(OOR)] (6), which in turn reacts with RH and O2 in a catalytic cycle to give PhC(O)Me or c-C6H8(O) [2-3 O2 turnovers per FeII(PA)2]. The FeII(bpy) 2 2+ , FeII(OPPh3) 4 2+ , (Cl8TPP)FeII(py)2, FeIIICl3, and CuI(bpy) 2 + complexes in MeCN are similar effective catalysts.