Correction: Combination of inverse electron-demand Diels-Alder reaction with highly efficient oxime ligation expands the toolbox of site-selective peptide conjugations.

Correction for 'Combination of inverse electron-demand Diels-Alder reaction with highly efficient oxime ligation expands the toolbox of site-selective peptide conjugations' by S. Hörner, et al., Chem. Commun., 2015, DOI: 10.1039/c5cc03434e.

Combination of inverse electron-demand Diels-Alder reaction with highly efficient oxime ligation expands the toolbox of site-selective peptide conjugations.

A modular approach combining inverse electron-demand Diels-Alder coupling (DARinv) and oxime ligation expands the toolbox of bioorthogonal peptide chemistry. Applicability of versatile site-specific bifunctional building blocks is demonstrated by generation of defined conjugates comprising linear, cystine-bridged and multi-disulfide functional peptides as well as their conjugation with hybrid silsesquioxane nanoparticles.

Induction of DNA breaks and apoptosis in crosslink-hypersensitive V79 cells by the cytostatic drug beta-D-glucosyl-ifosfamide mustard.

To study molecular aspects of cytotoxicity of the anticancer drug beta-D-glucose-ifosfamide mustard we investigated the potential of the agent to induce apoptosis and DNA breakage. Since beta-D-glucose-ifosfamide mustard generates DNA interstrand crosslinks, we used as an in vitro model system a pair of isogenic Chinese hamster V79 cells differing in their sensitivity to crosslinking agents. CL-V5B cells are dramatically more sensitive (30-fold based on D(10) values) to the cytotoxic effects of beta-D-glucose-ifosfamide mustard as compared to parental V79B cells. After 48 h of pulse-treatment with the agent, sensitive cells but not the resistant parental line undergo apoptosis and necrosis, with apoptosis being the predominant form of cell death (70 and 20% of apoptosis and necrosis, respectively). Apoptosis increased as a function of dose and was accompanied by induction of DNA double-strand breaks in the hypersensitive cells. Furthermore, a strong decline in the level of Bcl-2 protein and activation of caspases-3, -8 and -9 were observed. The resistant parental cells were refractory to all these parameters. Bcl-2 decline in the sensitive cells preceded apoptosis, and transfection-mediated overexpression of Bcl-2 protected at least in part from apoptosis. From the data we hypothesize that non-repaired crosslinks induced by beta-D-glucose-ifosfamide mustard are transformed into double-strand breaks which trigger apoptosis via a Bcl-2 dependent pathway.

Monosaccharide-linked inhibitors of O(6)-methylguanine-DNA methyltransferase (MGMT): synthesis, molecular modeling, and structure-activity relationships.

A series of potential inhibitors of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) were synthesized, characterized in detail by NMR, and tested for their ability to deplete MGMT activity in vitro. The new compounds, omega-[O(6)-R-guan-9-yl]-(CH(2))(n)-beta-d-glucosides with R = benzyl or 4-bromothenyl and omega = n = 2, 4,. 12, were compared with the established inhibitors O(6)-benzylguanine (O(6)-BG), 8-aza-O(6)-benzylguanine (8-aza-BG), and O(6)-(4-bromothenyl)guanine (4-BTG), which exhibit in an in vitro assay IC(50) values of 0.62, 0.038, and 0.009 microM, respectively. Potential advantages of the glucosides are improved water solubility and selective uptake in tumor cells. The 4-BTG glucosides with n = 2, 4, 6 show moderate inhibition with an IC(50) of ca. 0.5 microM, while glucosides derived from BG and 8-aza-BG showed significantly poorer inhibition compared to the parent compounds. The 4-BTG glucosides with n = 8, 10, 12 were effective inhibitors with IC(50) values of ca. 0.03 microM. To understand this behavior, extensive molecular modeling studies were performed using the published crystal structure of MGMT (PDB entry: ). The inhibitor molecules were docked into the BG binding pocket, and molecular dynamics simulations with explicit water molecules were carried out. Stabilization energies for the interactions of specific regions of the inhibitor and individual amino acid residues were calculated. The alkyl spacer is located in a cleft along helix 6 of MGMT. With increasing spacer length there is increasing interaction with several amino acid residues which play an important role in the proposed nucleotide flipping mechanism required for DNA repair.

Evidence for reductive activation of carcinogenic aristolochic acids by prostaglandin H synthase -- (32)P-postlabeling analysis of DNA adduct formation.

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, is implicated in an unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN), which can develop to urothelial cancer. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual susceptibility to this natural carcinogen. We examined the ability of prostaglandin H synthase (PHS) to activate AA to metabolites forming DNA adducts with the nuclease P1 and 1-butanol extraction enrichment procedure of the (32)P-postlabeling assay. PHS is a prominent enzyme in the kidney and urothelial tissues. Ram seminal vesicle (RSV) microsomes, which contain high levels of PHS, generated AA-DNA adduct patterns reproducing those found in renal tissues in CHN patients. 7-(Deoxyadenosin-N(6)-yl)aristolactam I, 7-(deoxyguanosin-N(2)-yl)aristolactam I and 7-(deoxyadenosin-N(6)-yl)aristolactam II were identified as AA-DNA adducts formed by AAI. Two adducts, 7-(deoxyguanosin-N(2)-yl)aristolactam II and 7-(deoxyadenosin-N(6)-yl)aristolactam II, were generated from AAII. According to the structures of the DNA adducts identified, nitroreduction is the crucial pathway in the metabolic activation of AA. The identity of PHS as the activating enzyme in RSV microsomes was proven with different cofactors and inhibitors. Only indomethacin, a selective inhibitor of PHS, significantly decreased the amount of adducts formed by RSV microsomes. The inhibitor of NADPH:CYP reductase (alpha-lipoic acid) and some selective inhibitors of cytochromes P450 (CYP) were not effective. Likewise, only cofactors of PHS, arachidonic acid and hydrogen peroxide, supported the DNA adduct formation of AAI and AAII, while NADPH and NADH were ineffective. These results demonstrate a key role of PHS in the activation pathway of AAI and AAII in the RSV microsomal system and were corroborated with the purified enzyme, namely ovine PHS-1. The results presented here are the first report demonstrating a reductive activation of nitroaromatic compounds by PHS-1.

Human enzymes involved in the metabolic activation of carcinogenic aristolochic acids: evidence for reductive activation by cytochromes P450 1A1 and 1A2.

Aristolochic acid (AA), a naturally occurring nephrotoxin and rodent carcinogen, has recently been associated with the development of urothelial cancer in humans. Determining the capability of humans to metabolize AA and understanding, which human enzymes are involved in AA activation is important in the assessment of individual susceptibility. Using the nuclease P1-enhanced version of the (32)P-postlabeling assay, we compared the ability of human, minipig and rat hepatic microsomal samples to activate AA to metabolites forming DNA adducts. Human microsomes generated AA-DNA adduct profiles reproducing those found in renal tissues from humans exposed to AA. Identical patterns of AA-DNA adducts were also observed when AA was activated by minipig and rat microsomes. Therefore, microsomes of both animals are suitable in vitro systems mimicking the enzymatic activation of AA in humans. To define the role of specific P450 enzymes and NADPH:P450 reductase in the activation of AA by human microsomes we investigated the modulation of AA-DNA adduct formation by specific inducers or selective inhibitors of P450s and cofactors or inhibitors of NADPH:P450 reductase. The inducer of P450 1A1/2, beta-naphthoflavone, significantly stimulated the levels of AA-DNA adducts formed by rat microsomes, but inducers of P450 2B1/2 and 2E1 had no such effect. Furthermore, only inhibitors of the P450 1A subfamily (alpha-naphthoflavone, furafylline) significantly decreased the amount of adducts formed by microsomes from humans, minipigs and rats. alpha-Lipoic acid, an inhibitor of NADPH:P450 reductase, inhibited adduct formation too, but to a lower extent. On the basis of these results, we attribute most of the microsomal activation of AA to P450 1A1 and 1A2, although a role of NADPH:P450 reductase cannot be ruled out. With purified enzymes (recombinant P450 1A1/2 and NADPH:P450 reductase) and microsomes from baculovirus transfected insect cells expressing recombinant human P450 1A1/2 and NADPH:P450 reductase, the participation of these enzymes in the formation of AA-DNA adducts was confirmed. These results are the first report on the activation of AA by human enzymes and clearly demonstrate the role of P450 1A1, 1A2, and NADPH:P450 reductase in catalyzing the reductive activation of AA.

Inactivation of O(6)-methylguanine-DNA methyltransferase by glucose-conjugated inhibitors.

The DNA-repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is a decisive determinant of resistance of tumor cells to methylating and chloroethylating anti-cancer drugs. Therefore, selective inhibition of MGMT in tumors is expected to cause tumor sensitization. Several inhibitors of MGMT have been developed which function in both tumors and normal tissue. To deplete MGMT preferentially in tumors, strategies to target the inhibitor to the tumor tissue need to be developed. Here, we report on the properties of glucose-conjugated MGMT inhibitors that might be useful for tumor targeting since tumor cells frequently over-express glucose transporter. O(6)-Benzylguanine (O6BG), 8-aza-O(6)-benzylguanine, O(6)-(4-bromothenyl)-guanine (O6BTG) and the corresponding spacer-linked beta-D-glucose conjugates were analyzed comparatively for MGMT-inhibitory activity. Substitution at the N9 position of the purine moiety resulted generally in a reduction in the efficiency with which the inhibitors blocked MGMT. However, the inhibitory activity of the O6BTG conjugates increased with increasing spacer length, and O6BTG conjugated with a C8 spacer with beta-D-glucose was nearly as effective as O6BTG on its own. MGMT was inhibited by the conjugates both in crude cell extracts and upon treatment of intact HeLa cells, indicating efficient uptake of the glucose conjugates into cells. Since the O6BTG-C8-D-glucose conjugate 8-[O(6)-(4-bromothenyl)-guan-9-yl]-octyl-beta-D-glucoside was highly efficient at MGMT inhibition in a non-toxic concentration range, the drug might be a useful tool for specific tumor sensitization.

DNA adduct formation by the ubiquitous environmental contaminant 3-nitrobenzanthrone in rats determined by (32)P-postlabeling.

Diesel exhaust is known to induce tumors in animals and is suspected of being carcinogenic in humans. Of the compounds found in diesel exhaust and in airborne particulate matter, 3-nitrobenzanthrone (3-NBA), is a particularly powerful mutagen. We investigated the capacity of 3-NBA to form DNA adducts in vivo that could be used as agent-specific biomarkers of exposure. Female Sprague-Dawley rats were treated orally with 2 mg/kg body weight of 3-NBA, and DNA from various organs was analyzed by (32)P-postlabeling. High levels of 3-NBA-specific adducts were detectable in all organs. Both enrichment versions nuclease P1 digestion and n-butanol extraction resulted in patterns consisting of either 3 or 4 adducts remarkably similar in all tissues examined. The highest level of DNA adducts was found in the small intestine (38 adducts per 10(8) nucleotides) followed by forestomach, glandular stomach, kidney, liver, lung and bladder. To provide information on the nature of the adducts formed in vivo in rats, DNA adducts were cochromatographed in 2 independent systems with standardized deoxyguanosine adducts and deoxyadenosine adducts produced by reaction of 3-NBA in the presence of xanthine oxidase with deoxyribonucleoside 3'-monophosphates in vitro. In both systems, each of the rat adducts comigrated either with a deoxyguanosine or a deoxyadenosine-derived 3-NBA adduct. Our results demonstrate that 3-NBA binds covalently to DNA after metabolic activation, forming multiple DNA adducts in vivo, all of which are products derived from reductive metabolites bound to the purine bases (deoxyguanosine 60% and deoxyadenosine 40%).

Mode of action of methotrexate-albumin in a human T-cell leukemia line and activity against an MTX-resistant clone.

Limitations of low mol. wt anticancer drugs are short tumor exposure times and toxicity to normal tissue. Methotrexate (MTX) covalently linked to human serum albumin (HSA) as a macromolecular carrier caused tumor regressions concomitant with a favorable toxicity profile in a clinical phase I trial (Hartung et aL, Clin. Cancer Res., 1999, 5, 753). We examined the uptake, intracellular degradation, metabolism and thymidylate synthase (TS) inhibition of MTX-HSA in the T-cell leukemia line CCRF-CEM and the MTX transport resistant clone CCRF-CEM/MTX. The number of MTX molecules per albumin molecule was determined by electrospray mass spectrometry. A loading ratio (LR) of approximately 1.4 mol MTX/albumin revealed intact complexes with one and two MTX molecules/albumin. In the complex with an LR of 5.7, albumin with up to 16 MTX molecules was seen. MTX-HSA was taken up by CCRF-CEM cells via endocytosis and cleaved by lysosomal enzymes. Liberated MTX was a poor substrate of folylpolyglutamate synthetase and was exported into the medium. TS was inhibited and cell survival was impaired by MTX-HSA in a time- and concentration-dependent manner. CCRF-CEM/MTX cells exhibited a growth inhibition of 30-40% after MTX-HSA treatment, but no TS inhibition. The alternative uptake route via endocytosis enables MTX-HSA to overcome transport resistance to MTX.

The anticancer agent ellipticine on activation by cytochrome P450 forms covalent DNA adducts.

Ellipticine is a potent antitumor agent whose mechanism of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Using [3H]-labeled ellipticine, we observed substantial microsome (cytochrome P450)-dependent binding of ellipticine to DNA. In rat, rabbit, minipig, and human microsomes, in reconstituted systems with isolated cytochromes P450 and in Supersomes containing recombinantly expressed human cytochromes P450, we could show that ellipticine forms a covalent DNA adduct detected by [32P]-postlabeling. The most potent human enzyme is CYP3A4, followed by CYP1A1, CYP1A2, CYP1B1, and CYP2C9. Another minor adduct is formed independent of enzymatic activation. The [32P]-postlabeling analysis of DNA modified by activated ellipticine confirms the covalent binding to DNA as an important type of DNA modification. The DNA adduct formation we describe is a novel mechanism for the ellipticine action and might in part explain its tumor specificity.

Comparative analysis of methotrexate polyglutamates in lymphoblast preparations from bone marrow and blood, and the contribution of residual red blood cells.

PURPOSE: Blasts isolated from bone marrow aspirates or blood samples of patients with acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML) were compared for uptake of methotrexate (MTX) and formation of MTX polyglutamates (MTX-Glu(n)). Red blood cells (RBC) from the same patient samples were also analyzed. METHODS: Blasts were isolated by standard density centrifugation. RBC were prepared from the pellet of the same centrifugation. MTX-Glu(n) were analyzed by means of HPLC and radiochemical quantification. RESULTS: In lymphoblasts isolated from blood, the distribution patterns of MTX-Glu(n) were the same as in bone marrow lymphoblasts, but the total amount of MTX-Glu(n) accumulated in blood lymphoblasts was reduced by 41% 51% when compared to the same number of bone marrow lymphoblasts of the same patient. RBC accumulated MTX but no formation of MTX-Glu(n) occurred. CONCLUSIONS: The determination of MTX and MTX-Glu(n) in lymphoblasts isolated from blood samples of patients with common ALL provides qualitative information on the capacity of the blasts to form MTX-Glu(n) since distribution patterns of MTX and MTX-Glu(n) parallel that of bone marrow lymphoblasts. The amounts of MTX-Glu(n) accumulated, however, were much lower in blood lymphoblasts. Blood lymphoblasts are therefore not useful for a quantitative analysis of MTX-Glu(n). The contribution of RBC to MTX and MTX-Glu(n) in vitro is only marginal and residual RBC in lymphoblast preparations from bone marrow can therefore be ignored.

Subacute NO generation induced by Alzheimer's beta-amyloid in the living brain: reversal by inhibition of the inducible NO synthase.

Glial activation contiguous to deposits of amyloid peptide (Abeta) is a characteristic feature in Alzheimer's disease. We performed complementary in vitro and in vivo experiments to study the extent, kinetics, and mechanisms of microglial generation of nitric oxide (NO) induced by challenge with Abeta. We showed that Abeta fibrils dose-dependently induced a marked release of stable metabolites of NO in vivo that was strikingly similar regarding extent and temporal profile to the one in the parallel designed microglial cell culture experiments. However, costimulation with interferon gamma, which was a prerequisite for Abeta-induced NO generation in vitro, was not required in vivo, demonstrating that factors are present in the living brain that activate glial cells synergistically with Abeta. Therefore, in Alzheimer's disease, deposits of Abeta fibrils alone may be sufficient to induce a chronic release of neurotoxic microglial products, explaining the progressive neurodegeneration associated with this disease. Our observation that systemic administration of selective iNOS inhibitors abolishes Abeta-induced NO generation in vivo may have implications for therapy of Alzheimer's disease.

Synthesis of fluorescently labeled alkylated DNA adduct standards and separation by capillary electrophoresis.

DNA adducts are regarded as individual internal dosimeters for the exposure to chemical carcinogens. To date, the most sensitive method for DNA adduct analysis is the radioactive 32P-postlabeling method, which allows the detection of one adduct in 10(10) unmodified nucleotides in microg amounts of DNA. However, this technique suffers from disadvantages such as working with radioactive phosphorus and time-consuming chromatographic separation procedures. In addition, the simultaneous detection of adducts from different classes of carcinogens in a DNA sample is difficult. In order to overcome these drawbacks, we are developing a new detection method, comprising fluorescence labeling of DNA adducts, capillary electrophoretic (CE) separation, and on-line detection by monitoring laser-induced fluorescence (LIF). So far, we have evaluated the separation power and the detection limit of CE with fluorescently labeled standard compounds such as unmodified nucleotides or alkylated thymidines. For this purpose, we developed a universal method for labeling 5'-OH-mononucleosid-3'-dicyanoethyl-phosphates with fluorescent dyes based on the phosphoramidite technology for DNA synthesis. The separation of N3-methylated, N3-, O2- and O4-butylated thymidines from the unmodified nucleotide within a few minutes recommends CE-LIF as a powerful method for DNA adduct analysis.

Biodegradability of antineoplastic compounds in screening tests: influence of glucosidation and of stereochemistry.

Some pharmaceuticals such as antineoplastics are carcinogenic, mutagenic, teratogenic and fetotoxic. Antineoplastics and their metabolites are excreted by patients into waste water. In laboratory testing the frequently used isomeric anti-tumour agents cyclophosphamide (CP) and ifosfamide (IF) were shown to be not biodegradable. They are not eliminated in municipal sewage treatment plants and therefore detected in their effluents. Structural related compounds are beta-D-glucosylisophosphoramidmustard (beta-D-Glc-IPM; INN = glufosfamide) and beta-L-glucosylisophosphoramidmustard (beta-L-Glc-IPM). beta-L-Glc-IPM has no antineoplastic effects whereas beta-D-Glc-IPM is active against tumours. In contrast to IF and CP and almost all other investigated antineoplastics beta-D-Glc-IPM is inherently biodegradable. Improved biodegradability of beta-D-Glc-IPM compared to IF shows that reducing the impact of pharmaceuticals on the aquatic environment is feasible by changing the chemical structure of a given compound exerting a similar mode of action and therapeutic activity. Stereochemistry may be crucial for pharmaceutical activity of the compounds as well as for its biodegradability in the environment.

Synthesis of antioxidative and anti-inflammatory drugs glucoconjugates.

Glucoconjugates of (+/-)-ibuprofen, (+/-)-alpha-tocopherol (vitamin E), gentisic acid, gallic acid, 2,6-bis(tert-butyl)-4-thiophenol, and N-acetyl-L-cysteine were prepared with the objective of increasing the bioavailability of such antioxidant and anti-inflammatory drugs. The O-glucosides were synthesized using benzylated alpha-D-glucopyranosyl trichloracetimidate as glycosyl donor. For the synthesis of the S-glucosides, the glycosyl donor 1,2,3,4,6-penta-O-acetyl-beta-D-glucopyranose provided higher yields than the corresponding O-acetylated imidate.

Using polymerase arrest to detect DNA binding specificity of aristolochic acid in the mouse H-ras gene.

The distribution of DNA adducts formed by the two main components, aristolochic acid I (AAI) and aristolochic acid II (AAII), of the carcinogenic plant extract aristolochic acid (AA) was examined in a plasmid containing exon 2 of the mouse c-H-ras gene by a polymerase arrest assay. AAI and AAII were reacted with plasmid DNA by reductive activation and the resulting DNA adducts were identified as the previously characterized adenine adducts (dA-AAI and dA-AAII) and guanine adducts (dG-AAI and dG-AAII) by the (32)P-post-labeling method. In addition, a structurally unknown adduct was detected in AAII-modified DNA and shown to be derived from reaction with cytosine (dC-AAII). Sites at which DNA polymerase progress along the template was blocked were assumed to be at the nucleotide 3' to the adduct. Polymerase arrest spectra showed a preference for reaction with purine bases in the mouse H-ras gene for both activated compounds, consistent with previous results that purine adducts are the principal reaction products of AAI and AAII with DNA. Despite the structural similarities among AAI-DNA and AAII-DNA adducts, however, the polymerase arrest spectra produced by the AAs were different. According to the (32)P-post-labeling analyses reductively activated AAI showed a strong preference for reacting with guanine residues in plasmid DNA, however, the polymerase arrest assay revealed arrest sites preferentially at adenine residues. In contrast, activated AAII reacted preferentially with adenine rather than guanine residues and to a lesser extent with cytosine but DNA polymerase was arrested at guanine as well as adenine and cytosine residues with nearly the same average relative intensity. Thus, the polymerase arrest spectra obtained with the AA-adducted ras sequence do not reflect the DNA adduct distribution in plasmid DNA as determined by (32)P-post-labeling. Arrest sites of DNA polymerase associated with cytosine residues confirmed the presence of a cytosine adduct in DNA modified by AAII. For both compounds adduct distribution was not random; instead, regions with adduct hot spots and cold spots were observed. Results from nearest neighbor binding analysis indicated that flanking pyrimidines displayed the greatest effect on polymerase arrest and therefore on DNA binding by AA.

Analysis of catechins and caffeine in tea extracts by micellar electrokinetic chromatography.

Cancer chemotherapy is a new and important medical science and much interest has been focused on catechins, not only for their antioxidant activity, but also because of their known antimutagenic and antitumorigenic properties. Green tea and black tea, which are among the most popular beverages consumed worldwide, contain many different catechins. We developed an analytical method capable of separating six different catechins and caffeine in tea by micellar electrokinetic chromatography in only 20 min without extensive sample preparation. Furthermore, we compared the amount of catechins and caffeine in several teas and different preparation modes.

Mechanisms of resistance to methotrexate in childhood acute lymphoblastic leukemia: circumvention of thymidylate synthase inhibition.

PURPOSE: In about 25% of patients suffering from acute lymphoblastic leukemia (ALL) treatment failures occur that are most likely due to development of resistance to methotrexate (MTX). Blasts from patients with ALL were evaluated for MTX uptake, formation of long-chain MTX polyglutamates (MTX-Glu5+6), cytotoxicity and thymidylate synthase inhibition by MTX and compared to blasts from patients with acute myelogenous leukemia (AML). METHODS: Radioactively labeled MTX-Glu(n) were analyzed by means of HPLC. Thymidylate synthase activity was measured by a tritium-release assay. Cytotoxicity was determined by trypan blue exclusion. RESULTS: In most ALL blasts (n = 9) large amounts of MTX-Glu5+6 (1.06-7.03 pmol/10(7) cells) and high cytotoxicity (43.5% 92.7%) were found, while in others small amounts of MTX-Glu5+6 (0.0-0.39 pmol/10(7) cells) caused only weak cytotoxicity (6.0% 27.9%) (n = 5, 2 relapsed patients). Resistance to MTX in blasts from AML patients (n = 5) was also caused by reduced synthesis of MTX-Glu5s+6 (0.0-0.42 pmol/10(7) cells). In contrast, some ALL blasts (n = 7, 4 relapsed patients) were able to survive MTX treatment despite large amounts of MTX-Glu5+6 (1.5-5.05 pmol/10(7) cells) and extensive thymidylate synthase inhibition. CONCLUSIONS: Since the majority of ALL patients were examined at first diagnosis, an inherent mechanism of resistance seems most likely. We propose a mechanism based on the switch of thymidylate synthesis to the salvage pathway.

Direct evidence for the formation of deoxyribonucleotide adducts from carcinogenic N-nitroso-N-methylaniline revealed by the 32P-postlabeling technique.

N-Nitroso-N-methylaniline (NMA) is an esophageal carcinogen in the rat. NMA forms a benzenediazonium ion (BDI) during microsomal cytochrome P-450 2B1 (CYP2B1) catalyzed metabolism. Using the nuclease P1-enhanced version of the 32P-postlabeling assay we investigated the formation of adducts by NMA with deoxyadenosine 3'-monophosphate (dAp) and deoxyguanosine 3'-monophosphate (dGp). 32P-postlabeling analysis of dAp and dGp, which were modified by NMA activated with microsomes of rats pretreated with phenobarbital (PB), and directly labeled resulted in each case in the appearance of one single adduct spot. Quantitative analysis of adducts revealed that the extent of dGp modification by activated NMA was more than 23 times greater than the extent of modification of dAp. The results suggest strongly that BDI, derived from NMA by CYP2B1 present in PB microsomes, participates in the formation of dAp and dGp adducts.

The profile of urinary metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in rats is determined by its pulmonary metabolism.

Metabolism of the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rats was compared to metabolism in primary lung and liver cells. Untreated rats and rats pretreated with phenobarbital, acetone or phenethyl isothiocyanate (PEITC) were used for all experiments. Also the influence of [-]-1-methyl-2-[3-pyridyl]-pyrrolidine (nicotine) administered concomitantly with NNK, or incubated with isolated cells, upon NNK metabolism was investigated and found to be only marginal upon alpha-hydroxylation and pyridine N-oxidation in vivo. In hepatocytes nicotine inhibited NNK pyridine N-oxidation, alpha-hydroxylation and glucuronidation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), whereas in lung cells the influence of nicotine was not as pronounced. In vivo phenobarbital induced alpha-hydroxylation and pyridine N-oxidation. In vitro the effects of the modulators were most pronounced upon hepatocytes, where phenobarbital greatly induced pyridine N-oxidation and PEITC inhibited alpha-hydroxylation. NNAL was conjugated to its beta-glucuronide in lung cells at four times higher rates than in hepatocytes. The ratios of the sum of N-oxides to the sum of alpha-hydroxylation products in vivo were similar to those in lung cells, especially at low NNK concentrations (1 microM), while in hepatocytes alpha-hydroxylation was more pronounced. The same correlation of metabolism in isolated lung cells with whole rats was observed if oxidative NNAL metabolism was related to oxidative NNK metabolism. Here hepatocytes showed a much higher formation of NNAL oxidation products than either lung cells formed, or rats excreted in urine. This was true despite a lower rate of metabolism in the lung than in liver if based on cell number, the rate based on mg protein was four times higher in lung than liver. Only after phenobarbital treatment was the contribution of hepatic metabolism to excreted metabolites important. In conclusion the lung which is also the target of NNK carcinogenesis, and not the liver, is the organ with the most important contribution to NNK and NNAL metabolism at concentrations relevant to human exposure.