Induction of oral mucosal tumors in hamsters and rats treated with methyl(acetoxymethyl)nitrosamine.

An oral tumor model has been developed in inbred Syrian golden hamsters by continuous applications every 2 weeks of methyl(acetoxymethyl)nitrosamine [(DMN-OAC) CAS: 56856-83-8; methylnitrosaminomethyl ester acetic acid] at 2 mg/kg body weight alone or by a single application of DMN-OAC followed by continuous twice weekly applications of 12-O-tetradecanoylphorbol 13-acetate (TPA) (1 microgram/animal). Similar studies were done in the W rat buccal mucosa. In the hamsters treated continuously with DMN-OAC, 100% of the tumors were observed in the cheek pouch; none were observed at other sites. In contrast, in the rats treated similarly, only a 67% tumor incidence was observed, of which only 42% were oral tumors. A promoter effect of TPA was observed in hamster cheek pouch tumors induced by DMN-OAC, whereas rat oral mucosa did not respond to TPA treatment.

Mechanism of esophageal tumor induction in rats by N-nitrosomethylbenzylamine and its ring-methylated analog N-nitrosomethyl(4-methylbenzyl)amine.

The metabolism of the esophageal carcinogen N-nitrosomethylbenzylamine (MBN) and its ring-methylated analog N-nitrosomethyl(4-methylbenzyl)amine (4-MeMBN) was investigated in male Wistar rats. When given in the drinking water, both compounds have been shown to induce a high incidence of esophageal carcinomas but, after systemic administration of equimolar doses, 4-MeMBN is considerably less toxic and carcinogenic than is MBN. Following a single i.v. injection, 4-MeMBN disappeared from serum faster than did MBN. After 5 hr, neither compound was detectable in serum. Within 12 hr after a single i.v. injection (0.017 mmol/kg) of [methyl-14C]-MBN, 49% of the radioactivity was exhaled as 14CO2, and less than 5% was in the urine, compared with only 13% as 14CO2 and 65% in the urine after an equimolar dose of 4-Me[methyl-14C]MBN. The urinary metabolite of 4-MeMBN was identified as its benzoic acid derivative. Methylation of DNA purines 4 hr after a single i.v. injection (0.017 mmol/kg) of [methyl-14C]MBN was highest in the esophagus (344 mumol 7-methylguanine per mol guanine), followed by liver, lung, and forestomach. Considerably less DNA methylation was produced by an equimolar dose of 4-MeMBN, with highest values in liver, followed by esophagus (22 mumol 7-methylguanine per mol guanine) and lung. However, s.c. injections of equitoxic doses of MBN (18 mg/kg) and 4-MeMBN (394 mg/kg) produced similar amounts of 7-methylguanine in esophageal nucleic acids. These data indicate that the lower toxicity and carcinogenicity of 4-MeMBN after systemic administration are due to the rapid formation (mainly in the liver) and excretion via the urine of its benzoic acid derivative. The strong carcinogenic effect of orally administered 4-MeMBN can be explained by direct uptake from the drinking water into the esophageal mucosa. Following a single i.v. injection (0.017 mmol/kg) of [methylene-14C]MBN and 4-Me[methylene-14C]MBN, no benzylated bases were detectable in rat tissues. This indicates that the bioactivation of these compounds is initiated predominantly by hydroxylation at the methylene bridge leading to a methylating rather than a benzylating intermediate as the ultimate carcinogen.

Organ and cell specificity of DNA methylation by N-nitrosomethylamylamine in rats.

N-Nitrosomethylamylamine (NMAA) is a potent carcinogen in rodents with the esophagus as the principal target organ. The present study aims at an assessment of DNA methylation by NMAA in various rat tissues and an identification of cell populations actively involved in its bioactivation. Adult male F344 rats received a single i.p. dose of N-nitroso[methyl-14C]amylamine (0.1 mmol/kg). After 6 h organs were removed and the DNA was extracted, hydrolyzed in 0.1 M HCl, and subjected to radiochromatography on Sephasorb-HP. Highest levels of DNA alkylation were found in esophagus (798 mumol 7-methylguanine/mol mol guanine), followed by nasal epithelium (672 mumol) and liver (624 mumol). Trachea, lung, forestomach, and kidney had considerably lower levels of alkylation and in glandular stomach, spleen, and duodenum, values were close to the limit of detection. Specific target cell populations were identified autoradiographically and by immunohistochemistry using a rabbit antiserum to O6-methyldeoxyguanosine. In the esophagus, NMAA was selectively metabolized by the basal cells of the mucosa. In the respiratory tract, O6-methyldeoxyguanosine was almost exclusively present in the tracheal and bronchiolar epithelia. In the nasal cavity, labeled nuclei were found in both the olfactory and the respiratory epithelium and in the serous glands. Our studies indicate that NMAA and related asymmetrical nitrosamines are, in addition to liver, preferentially metabolized in tissues derived from the ventral entoderm, including the upper respiratory and gastrointestinal tract.

Detection of DNA adducts formed by aristolochic acid in renal tissue from patients with Chinese herbs nephropathy.

A unique type of rapidly progressive renal fibrosis, designated Chinese herbs nephropathy (CHN), has been described in young Belgian women who had followed a slimming regimen including recently introduced Chinese herbs (Stephania tetrandra and Magnolia officinalis). Aristolochic acid (AA), a known nephrotoxin and carcinogen, was suspected as its causal factor. To substantiate this hypothesis, renal tissue from five patients with CHN and six patients with other renal diseases was analyzed for the presence of AA-derived DNA adducts, a described biomarker of AA exposure associated with its carcinogenic and mutagenic activity. Using the 32P-postlabeling method, a major distinct DNA adduct spot was found in all five cases of CHN and identified by cochromatographic analyses with authentic markers as the deoxyadenosine adduct of AA-I [7-(deoxyadenosin-N6-yl)-aristolactam I], the major component of the plant extract AA. This DNA adduct was absent in the six control cases. The 7-(deoxyadenosin-N6-yl)-aristolactam I adduct levels in CHN ranged from 0.7 to 5.3/10(7) nucleotides. Our data demonstrate that AA is implicated in CHN. They suggest a mechanism for the urothelial atypia and cancers observed in this disease and raise the possibility that a DNA mutation is responsible for the kidney-destructive fibrotic process.

Aristolochic acid activates ras genes in rat tumors at deoxyadenosine residues.

Aristolochic acid I (AAI), a nitrophenanthrene derivative, is the major component of the carcinogenic plant extract aristolochic acid, which has been used as a medicine since antiquity. Long term oral administration of AAI to male Wistar rats induces multiple tumors, mainly in the forestomach, ear duct, and small intestine. The presence of activated transforming genes was investigated in various tumors of 18 AAI treated rats, namely in 14 squamous cell carcinomas of the forestomach, 7 squamous cell carcinomas of the ear duct, 8 tumors of the small intestine, 3 tumors of the pancreas, 1 adenocarcinoma of the kidney, 1 lymphoma, and 2 metastases in the lung and the pancreas. By utilizing the tumorigenicity assay and Southern blot analysis, we have detected an activated c-Ha-ras gene in the DNAs of 5 of 5 squamous cell carcinomas of the forestomach. Direct sequencing of amplified material revealed an AT----TA transversion mutation at the second position of codon 61 of the c-Ha-ras gene (CAA to CTA) in all transfectants as well as in the 5 original rat tumors. Enzymatic amplification of ras sequences followed by selective oligonucleotide hybridization detected identical mutations in 93% (13 of 14) of forestomach tumors, in 100% (7 of 7) of ear duct tumors, and in the lung metastasis. Among those tumors tested, we had 4 cases in which the forestomach tumors and the ear duct tumors originated from the same rat, showing the same mutation in both tissues. Moreover, similar mutations were demonstrated at c-Ki-ras codon 61 in 1 of 7 ear duct tumors (CAA to CAT) and in 1 of 8 tumors of the small intestine (CAA to CTA) as well as at c-N-ras 61 (CAA to CTA) in a pancreatic metastasis. Additional transfection experiments of some tumors scoring negative for ras gene mutations in dot blot analyses revealed a CAA to CTA transversion at codon 61 of the c-Ha-ras gene in 1 forestomach tumor as well as at codon 61 of the c-N-ras in 1 hyperplasia of the pancreas and in 1 lymphoma. The apparent selectivity for mutations at adenine residues in AAI induced tumors is consistent with the identification of an N6-deoxyadenosine-AAI adduct formed by reaction of AAI with DNA in vitro, suggesting that carcinogen-deoxyadenosine adducts are the critical lesions in the tumor initiation by aristolochic acid.

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.

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.

Sugar-linked dithiocarbamates as modulators of metabolic and genotoxic properties of N-nitroso compounds.

A series of putative anticarcinogenic and antimutagenic compounds was synthesized on the basis of tetraethylthiuram disulfide (disulfiram) and its metabolite, diethyldithiocarbamate (DDTC). Diallyldithiocarbamate was synthesized in order to combine the anticarcinogenic properties of diallyl sulfide, a known inhibitor of chemical carcinogenesis from Allium species, and those of DDTC. Several sugar-linked dithiocarbamates (SDTCs) were prepared using glucose, cellobiose, and lactose as glycosyl donors and DDTC and diallyldithiocarbamate as acceptors. All the S--glycoside bonds of SDTCs were very stable under physiological conditions in vitro. At low nitrosamine concentrations, glucose-DDTC inhibited microsomal nitrosamine dealkylases in vitro. In vivo these enzymes were also inhibited 4 h after i.p. administration of glucose-DDTC or lactose-DDTC to rats (1.7 mmol/kg); after 24 h, the values had returned to control levels. Glucose-DDTC induced the activity of glutathione-related enzymes. Concomitant treatment of rats with glucose-DDTC and N-nitrosodiethylamine (NDEA) led to a depression of the oxidative metabolism of [14C]NDEA to 14CO2 but increased the elimination of unchanged [14C]NDEA in the urine. Furthermore, glucose-DDTC totally inhibited the formation of DNA single-strand breaks induced by NDEA. All these effects may contribute to possible antimutagenic and anticarcinogenic actions of the dithiocarbamates investigated.

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.

Activating mutations at codon 61 of the c-Ha-ras gene in thin-tissue sections of tumors induced by aristolochic acid in rats and mice.

The plant extract aristolochic acid, which consists mainly of aristolochic acid I (AAI) and aristolochic acid II (AAII), induces tumors in rats and mice. Thin-tissue sections of rat tumors induced by AAI and of mouse tumors induced by aristolochic acid, were analyzed for c-Ha-ras mutations in codon 61. Areas of neoplastic and histologically normal tissue were manually scraped out and separated. Using the polymerase chain reaction (PCR) and mutation detection by selective oligonucleotide hybridization, we observed AT----TA transversion mutations in DNA of neoplastic portions, but not in DNA of adjacent normal tissue in both rat and mouse tumors.

Effect of site-specifically located aristolochic acid DNA adducts on in vitro DNA synthesis by human DNA polymerase alpha.

In order to examine the effect of purine adducts of the plant carcinogen aristolochic acid (AA) on DNA replication four 30-mer templates were prepared which contained single site-specific AA lesions. The oligonucleotides were isolated by HPLC and shown to contain the two known aristolochic acid I-DNA adducts (dA-AAI, dG-AAI) or the two known aristolochic acid II-DNA adducts (dA-AAII, dG-AAII) at position 27 from the 3' end by 32P-postlabeling. These adducts templates were replicated in primer (23-mer) extension reactions catalysed by human DNA polymerase alpha. Both AAI-DNA adducts (dA-AAI, dG-AAI) blocked DNA synthesis predominantly (80-95%) at the nucleotide 3' to the adduct, although primer extension to the full length of the template was found with unmodified control templates. Increasing dNTP concentrations had only a small effect on the DNA synthesis and translesional synthesis was negligible. In contrast, both AAII-DNA adducts showed marked differences in primer extension reactions. Blocking of DNA synthesis by the dA-AAII adduct was strongly dNTP dependent. With increasing dNTP concentrations 27 and 28 nucleotide products, indicating termination of DNA synthesis after incorporation of a nucleotide opposite this adduct and incorporation of an additional nucleotide accumulated. Only the dG-AAII adducted template allowed substantial translesional synthesis to the full length of the template (up to 25%). When a 26-mer primer was used to examine nucleotide incorporation directly across from the four purine adducts, we found no detectable incorporation of nucleotides for the dA-AAI adduct, whereas the dG-AAI adduct and both AAII-adducts (dA-AAII and dG-AAII) allowed preferential incorporation of the correct nucleotide. These results indicate that for human polymerase alpha three AA purine adducts (dA-AAI, dG-AAI and dA-AAII) provide severe blocks to DNA replication and that dG-AAII, which allows translesional synthesis, may not be a very efficient mutagenic lesion.

Mutagenicity of the two main components of commercially available carcinogenic aristolochic acid in Salmonella typhimurium.

One of the 2 main components of the commercially available carcinogenic aristolochic acid (AA) was isolated, the other was enriched. Three different aristolochic acid samples (AAI 99% pure; AAI 65% + AAII 35%; AAI 32% + AAII 68%) were assayed for mutagenic activity in Salmonella typhimurium TA1537, TA100 and TA100 NR with and without the addition of a metabolizing mixture. The two main components (AAI and AAII) were direct mutagens in Salmonella strains TA1537 and TA100 with almost equal mutagenic potency. In TA100 NR the aristolochic acid samples showed no or only a very low level of biological activity, indicating the necessity of nitroreduction for the bioactivation of the samples. These findings suggest that both AAI as well as AAII can be used in further studies to elucidate the metabolism of aristolochic acid.

Carcinogenicity of di(N-nitroso)-perhydropyrimidine (DNPP) after intraperitoneal application to rats.

Male Sprague-Dawley rats received 30 mg/kg body wt. (group I) or 15 mg/kg (group II) di(N-nitroso)-perhydropyrimidine (DNPP) by the intraperitoneal route once a week for life. The DNPP treatment significantly reduced the life expectancy in both groups. DNPP induced tumors of the esophagus (27% incidence in group I, 33% in group II) and possibly also of the liver (4% incidence in group I, 8% in group II).

Peroxidase oxidizes N-nitrosomethylaniline to ultimate carcinogens(s) binding to DNA and transfer RNA in vitro.

Carcinogenic N-nitrosomethylaniline is oxidized in vitro by horseradish peroxidase in the presence of H2O2 to ultimate carcinogens, which bind to DNA and transfer RNA (tRNA). tRNA is more accessible for modification by the activated carcinogen studied. The modification of nucleic acid by N-nitrosomethylaniline metabolite(s) formed by peroxidase is inhibited by some compounds of physiological importance (ascorbate, glutathine) and by radical trapping agents (nitrosobenzene, methyl viologen). 32P-postlabeling assay of DNA and tRNA modified by N-nitrosomethylaniline activated by peroxidase shows covalent adduct formation with nucleic acids. The role of peroxidases in the activation of N-nitrosamines leading to organ and/or cell specificity of these carcinogens is discussed.

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.

Detoxication products of the carcinogenic azodye Sudan I (solvent yellow 14) bind to nucleic acids after activation by peroxidase.

The C-hydroxyderivatives of the carcinogenic dye Sudan I, 1-phenylazo-2,6-dihydroxynaphthalene and 1-(4-hydroxyphenylazo)-2-hydroxynaphthalene, which are considered to be detoxication products of this dye bind to DNA or tRNA after oxidation into active metabolites by peroxidase and H2O2 in vitro. The 32P-postlabeling analysis of DNA modified by active metabolites of both Sudan I derivatives provides evidence that the covalent binding to DNA is the principal type of DNA modification. Since the urinary bladder is rich in peroxidases, the participation of these enzymes in activation of detoxicating products of Sudan I may be involved in the initiation of Sudan I-carcinogenesis in this organ.

The structural basis for the mutagenicity of aristolochic acid.

Molecular orbital calculations with aristolochic acid I (AAI) and the model compounds 8-nitro-1-naphthoic acid (1,8NNA) and 3-nitro-2-naphthoic acid (2,3NNA) confirm a similar conformation of the nitro and carboxyl groups in these molecules. The ortho isomer 2,3NNA is not mutagenic in the Salmonella strains TA 100 or TA 1537, but the peri-substituted 1,8NNA shows mutagenic activity similar to AAI in TA 100, although it is only weakly active in TA 1537. We propose a mechanism of activation via a cyclic nitrenium ion with an aristolactam structure which is possible only in peri-substituted nitro carboxylic acids.

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.

Effects of disulfiram on mixed function oxidase system and trace element concentration in the liver of rats.

Disulfiram (DSF), an inhibitor of chemically induced carcinogenesis, and its metabolite diethyldithiocarbamate (DDTC) have been investigated for their influence on trace element distribution and on certain enzymes of the drug metabolizing system in the livers of phenobarbital (PB) treated rats. Both substances diminished the PB induced enzyme response in liver microsomes, DDTC being more effective (-85%) than DSF (-60%). The copper, cobalt and zinc content of the livers of DSF treated animals were increased by factors of 6, 3 and 1.5 respectively as compared to controls, while DDTC treatment had no influence on liver trace element content. A correlation between enzyme inhibition and enhanced trace element uptake of the liver after DSF administration could not be observed. The change of trace element transport into the liver during DSF treatment is discussed.

Antitumor-activity and distribution of Beta-d-lactosylisophosphoramide mustard - a potential cytostatic agent exploiting lectin mediated transport.

Affinity chromatography of the Walker carcinosarcoma 256 B tumor revealed a lactose-specific lectin of 24 kDa. In a chemotherapeutical experiment, B-D-lactosylisophosphoramide mustard (corresponding lectin present in tumor tissue) proved to be significantly more active than B-D-maltosylisophosphoramide mustard (no corresponding lectin present), although both compounds differ only marginally in their sugar parts. Whole body autoradiography after application of C-14-labeled B-D-lactosylisophosphoramide mustard in tumor bearing rats shows an accumulation of radioactivity in kidneys, thymus, central nervous system and in the tumor. Binding between the isolated lectin and the lactose-conjugated drug is shown in vitro; its activation outside tumor cells is experimentally excluded.