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PubMed1984

Structural effects in alkyl nitrite oxidation of human hemoglobin.

Oxidations of hemoglobin in oxygen-saturated and deoxygenated media by 10 structurally variant alkyl nitrites have been examined in kinetic detail. Pronounced structural influences on rate constants, whose values span a range of 80 in oxidations of both oxy- and deoxyhemoglobin, have been observed. tert-Butyl nitrite provides the slowest oxidation rate that for deoxyhemoglobin terminates after only half of the available iron(II) heme units have been oxidized. Activation parameters have been determined for oxidations of oxyhemoglobin and deoxyhemoglobin by ethyl, isopropyl, and neopentyl nitrites from kinetic evaluation of these reactions as a function of temperature. The differences in free energies of activation (delta G) between hemoglobin R and T states range from 1.8 to 2.9 kcal/mol for the three alkyl nitrites examined. The composite data portray alkyl nitrite oxidations as inner sphere electron transfer processes whose kinetic characteristics reflect the ligand binding properties of hemoglobin. A sulfhydryl-induced alkyl nitrite oxidation of oxyhemoglobin that is most pronounced in oxygen-saturated media has been observed, and its cause has been traced to nitrosyl exchange of alkyl nitrites with the beta-93 cysteine sulfhydryl group of hemoglobin.

Hemoglobin A↗
PubMed1982

Ether phospholipids in guinea pig polymorphonuclear leukocytes and macrophages. Occurrence of high levels of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine.

Significant proportions of the choline phosphoglycerides (CPG) were found to contain alkyl either-type moieties (e.g., 1-O-alkyl-2-acyl-glycero-3-phosphocholine) in both guinea pig peritoneal exudate polymorphonuclear leukocytes (16.4%) and macrophages (13.5%). High proportions of the ethanolamine phosphoglycerides (EPG) contained alkenyl either moieties in both cells (37.2 and 41.2%), while the proportions of the CPG containing alkenyl moieties and of the EPG containing alkyl moieties were shown to be small. The either phospholipid composition as well as the fatty chain profiles of these two types of cells had relatively similar patterns. However, the fatty chains at the 1- and 2-positions for alkenyl either, alkyl ether and diacyl phosphoglycerides showed considerable differences. The amount of 16:0 at the 1-position was higher in alkyl compounds than that in diacyl compounds of the CPG. This was also the case in either-containing and diacyl EPG. The most predominant fatty acids at the 2-position was 18:2, in each lipid class, except for the alkenyl CPG. The amounts of 20:4 and other polyunsaturated fatty acids were low in every lipid class, though ether compounds contained higher amounts of 20:4 than diacyl compounds, particularly for EPG.

Animals↗
PubMed1980

Enzymatic synthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, a hypotensive and platelet-aggregating lipid.

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine, derived chemically from choline plasmalogens of beef heart, has been shown to possess powerful antihypertensive activity (Blank, M. L., Synder, F., Byers, L. W., Brooks, B., and Muirhead, E. E. (1979) Biochem. Biophys. Res. Commun. 90, 1194-1200) and to be an extremely potent platelet-activating factor (Demopoulos, C. C., Pinckard, R. N., and Hanahan, D. J. (1979) J. Biol. Chem. 254, 9355-9358). In the present study, microsomal preparations of rat spleen were shown to synthesize 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine by an acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase reaction; the acetyltransferase appears to be different from the acyltransferase responsible for the transfer of palmitate to glycerolipids. The apparent Km for acetyl-CoA was 67 microM; the optimal concentration of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine was 30 microM. Higher concentrations of the lyso substrate were inhibitory. When acetyl-CoA (100 microM) and 1-hexadecyl-sn-glycero-3-phosphocholine (30 microM) were incubated wih spleen microsomes under optimal conditions, the specific activity was approximately 10 nmol of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine/min/mg of protein. Although the reaction was inhibited by the addition of EDTA or EGTA to the incubation mixture, the acetyltransferase did not appear to require a divalent cation. An acyl analog, 1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine, also served as a substrate for the acetyltransferase; however, the unnatural isomer, 3-alkyl-2-lyso-sn-glycero-1-phosphocholine, did not. The acetyltransferase activity was found in a variety of tissues, but the spleen had the highest activity of the tissues examined; lung, lymph nodes, and thymus also had high activities. Identification of the product and the possible physiological importance of the pathway are discussed.

Acetyltransferases↗
PubMed1994

Retrovirus-mediated gene transfer of rat glutathione S-transferase Yc confers alkylating drug resistance in NIH 3T3 mouse fibroblasts.

A major limitation to successful cancer treatment is the existence of drug resistance. While several mechanisms of drug resistance have now been well characterized, mechanisms of resistance to alkylating drugs have remained less well defined. Several experimental models of alkylator resistance have implicated isoforms of glutathione S-transferase (GST) but transfection experiments using cloned isoforms of GST have yielded conflicting results. While there are several plausible explanations for these apparently contradictory findings, the issue that clonal variability might potentially confound the results of conventional transfection experiments has been raised. To address this issue properly, we have studied rat GST-Yc expression and drug sensitivity to alkylating drugs in populations of mouse NIH 3T3 fibroblasts following either transfection or transduction with an N2-based retrovirus vector. In comparison with cells treated with an antisense vector, Yc-transfected and Yc-transduced populations of NIH 3T3 cells expressed increased levels of GST-Yc mRNA (Northern blot), increased levels of immunodetectable GST-Yc (Western blot), and, respectively, 1.4- and 1.9-fold increases in total GST activity and 6.1- and 8.3-fold increases in glutathione peroxidase activity (associated with the Yc subunit). Yc-transfected and Yc-transduced cell populations were, respectively, 5.8- (P < 0.001) and 2.4-fold (P < 0.05) resistant to chlorambucil and 10.8- (P < 0.01) and 5.4-fold (P < 0.001) resistant to mechlorethamine. The range of resistance of clonal isolates from either population was 1.8-6.0-fold for chlorambucil and 4.6-6.1-fold for mechlorethamine (P < 0.05). In contrast, these cells showed unaltered sensitivity to the antimetabolite methotrexate, a nonalkylating drug. These results clearly demonstrate that the rat GTS-Yc is able to confer alkylating drug resistance in mouse fibroblasts. The ability to confer alkylating drug resistance following retrovirus-mediated gene transfer also raises the possibility of using GST-Yc somatic gene transfer to confer protection to the hematopoietic system in a gene therapy strategy applicable to cancer.

3T3 Cells↗
PubMed1975

Naturally-ocurring low molecular weight IgM in patients with rheumatoid arthritis, systemic lupus erythematosus, and macroglobulinemia. II. Structural studies and comparison of some physicochemical properties of reduced and alkylated IgM, and low molecular weight IgM.

Some physicochemical properties of naturally occurring low molecular weight IgM (LMW IgM), IgM and their subunits obtained by reduction-alkylation were compared. On the basis of analytical ultracentrifugation, SDS polyacrylamide electrophoresis, analytical Sephadex chromatography, and heavy (H) and light (L) chain composition purified LMW IgM had an S20,W constant of approximately 8S, a molecular weight of 195,000 to 205,000 and a structural formula H2L2. Reduction and alkylation of IgM (IgM RA) by two techniques produces two components which could be distinguished by analytical ultracentrifugation, sucrose density ultracentrifugation, and gel filtration studies. IgM RA-cys had an estimated molecular weight similar to LMW IgM and IgM-2ME had physical properties similar to IgG. The molecular weights of H and L chains of IgM and LMW IgM were similar: 69,000 to 75,500 and 26,000 to 27,000, respectively. Unlike LMW IgM and IgM RA-cys, the less dense IgM RA reduced with 2ME dissociated into half subunits, and free H and L chains in SDS gel electrophoresis, indicating that it lacked intra-subunit disulfide bonds. J chain was not detected in LMW IgM or in the chromatographically purified reduced-alkylated subunits from a patient whose IgM contained J chain. Carbohydrate analyses of IgM, LMW IgM, and reduced-alkylated IgM products did not demonstrate differences in individual sugars or in the percentage of total carbohydrate. The different sedimentation and gel filtration properties of the two components obtained by reduction-alkylation of IgM could not be attributed to variations in peptide chain or carbohydrate content, rather they are probably dependent on conformational changes resulting from variable, reduction of intra-subunits disulfide bonds.

Antigen-Antibody Reactions↗
PubMedDate not supplied

[Bis-chloromethyl ether and carcinogenesis of alkylating agents].

Bis-Chloro-Methyl-Ether is an alkylating agent and a recognised carcinogen. It can form spontaneously from the reaction of chloridric acid with formaldehyde. In the past it was extensively used as a chemical intermediate in organic synthesis, particularly as a crosslinking agent in the manufacture of ion-exchange resins. Recently, since its carcinogenicity has been proved in animal studies and confirmed in epidemiological studies of occupationally exposed cohorts, its use has been consistently reduced. A characteristic association has been observed between BCME exposure and a peculiar lung cancer histotype (oat cell carcinoma). In spite of these data, little information is available on the molecular alterations related to BCME exposure and possibly to its carcinogenic activity. Some suggestions can reasonably be obtained considering how the class of alkylating agents acts. They form adducts, binding different positions of DNA bases. The reaction that seems more relevant for mutagenesis and carcinogenesis is the alkylation at the atom O6 of guanine in DNA, which is followed by mis-coding and GC-->AT transition mutation. This kind of alteration determines the activation of a group of enzyme like DNA repair, mismatch repair, excision repair and a specific one, methyl guanine methyl transferase (MGMT). This last repair protein transfers alkyl groups from the O6 position of guanine to an internal cysteine residue, inactivating itself. Thus, the possibility for the cell to eliminate alkylated DNA bases depends strictly upon the cellular content of MGMT. In this view reduced or absent levels of the enzyme are associated with an increased number of adducts and hence increased risk of DNA mutations and cancer. At the moment no molecular studies in vivo have been performed to verify this hypothesis. The peculiar association BCME-oat cell carcinoma, the most chemosensitive tumor, need further investigation.

Air Pollutants, Occupational↗
PubMed1992

Mutagenesis and DNA repair for alkylation damages in Escherichia coli K-12.

In this work we report on the isolation of an Escherichia coli K-12 mutation, which confers a high sensitivity to bacteria cells to mutagenesis by simple monofunctional alkylating agents. The mutation emerged spontaneously from a bacterial strain that already proved useful in various mutagenicity studies. By monitoring the influence of such a mutation on the frequency of induced mutation by ethylating (EMS, DES, ENU, ENNG) vs. methylating (MMS, DMS, MNU, MNNG) compounds, and on the in vivo repair capacity for different alkyl-DNA lesions (O6-alkG, N7-alkG, N3-meA), we conclude that the mutation should affect the gene (ogt) that encodes constitutive DNA repair alkyltransferase (ATase). Thus in the presence of ada, differences in mutagenicity were observed only with ethylating agents; the sensitization of cells to both the ethylating and methylating partners requiring, by contrast, the absence of the ada protein. These results support the reported in vitro substrate specificities for both ogt and ada ATases. The parental cells exhibited biphasic dose-response curves in accordance with the idea of low basal level saturation attributed to the uninducible ogt ATase. Deficient bacterial derivatives showed, by contrast, linear mutation induction responses. The in vivo removal of alkylated bases from DNA was measured in bacterial strains deficient in the excision repair pathway (delta uvrB) and unable to induce the adaptive response (ada::Tn10). The very low initial levels for O6-meG and O6-etG (1.1 and 0.2 molecules per cell, respectively) were readily repaired by the parental cells but remained unchanged in the hypermutable derivatives. This result suggests that in the absence of nucleotide excision repair and of the adaptive response, no alternative pathway, other than ogt, is available for the repair of the major mutagenic lesion, O6-alkG, at least during the first 4 hours after alkylation. Comparatively, no differences were found in the capacity to repair the major lethal adduct, N3-meA, in agreement with the fact that no effect on cell survival was detected. In conclusion, we propose that the biological significance of the ogt protein relies mainly on its ability to prevent mutagenesis by low levels of bulkier ethylation products (especially in the absence of uvr excision repair.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkylating Agents↗
PubMed2000

Ras-related GTPase RhoB forces alkylation-induced apoptotic cell death.

rhoB encoding a Ras-related GTPase is immediate-early inducible by genotoxic treatments. To address the question of the physiological role of RhoB in cellular defense, cells stably overexpressing wild-type RhoB protein were generated. Overexpression of RhoB renders cells hypersensitive to the killing effect of alkylating agents including antineoplastic drugs but not to UV-light and doxorubicin. As compared to control cells, RhoB overexpressing cells revealed an increase in the frequency of alkylation-induced apoptotic cell death. This indicates that RhoB is involved in modulating apoptotic signaling. Furthermore, overexpression of RhoB resulted in a prolonged transient block to DNA replication upon MMS treatment. UV-induced replication blockage was not affected by RhoB. Based on the data we suggest RhoB to be a novel regulatory factor which takes influence on the level of cytotoxicity of DNA damaging drugs and forces cells to alkylation-induced apoptosis. The data indicate that this might be due to RhoB mediated delay in cell cycle progression upon alkylation treatment.

3T3 Cells↗
PubMed1996

N-alkylvaline levels in globin as a new type of biomarker in risk assessment of alkylating agents.

Adducts with the N-terminal valine of erythrocyte globin can serve as individual biomarkers of systemic and cellular exposure to endogenous and exogenous alkylating agents. In contrast to "detoxification markers" of this kind of mecapturic acids derived from alkylation of glutathione, individual N-alkylations of valine in globin reflect the formally "toxifying" part of the stress due to alkylating agents transformed into the ultimate toxicant. Thus, in contrast to the traditional methods of biological monitoring this approach enables a better evaluation of systemic exposure to reactive agents, adapted more sensibly to the exposure situation over the whole life span of erythrocytes, and it can serve as a specific biomarker of exposure for the purpose of health surveillance in occupational medicine. An individual evaluation of exposures in comparison with the range of corresponding background levels is discussed from the point of view of supplementary risk assessment in medical surveillance of occupationally exposed persons.

Acrylonitrile↗
PubMed2003

Identification of alkylation-sensitive target chaperone proteins and their reactivity with natural products containing Michael acceptor.

Molecular chaperones have a crucial role in the folding of nascent polypeptides in endoplasmic reticulum. Some of them are known to be sensitive to the modification by electrophilic metabolites of organic pro-toxicants. In order to identify chaperone proteins sensitive to alkyators, ER extract was subjected to alkylation by 4-acetamido-4'-maleimidyl-stilbene-2,2'-disulfonate (AMS), and subsequent SDS-PAGE analyses. Protein spots, with molecular mass of 160, 100, 57 and 36 kDa, were found to be sensitive to AMS alkylation, and one abundant chaperon protein was identified to be protein disulfide isomerase (PDI) in comparison with the purified PDI. To see the reactivity of PDI with cysteine alkylators, the reduced form (PDIred) of PDI was incubated with various alkylators containing Michael acceptor structure for 30 min at 38 degrees C at pH 6.3, and the remaining activity was determined by the insulin reduction assay. Iodoacetamide or N-ethylmaleimide at 0.1 mM remarkably inactivated PDIred with N-ethylmaleimide being more potent than iodoacetamide. A partial inactivation of PDIoxid was expressed by iodoacetamide, but not N-ethylmaleimide (NEM) at pH 6.3. Of Michael acceptor compounds tested, 1,4-benzoquinone (IC50, 15 microM) was the most potent, followed by 4-hydroxy-2-nonenal and 1,4-naphthoquinone. In contrast, 1,2-naphthoquinone, devoid of a remarkable inactivation action, was effective to cause the oxidative conversion of PDIred to PDIoxid. Thus, the action of Michael acceptor compounds differed greatly depending on their structure. Based on these, it is proposed that PDI, one of chaperone proteins in ER, could be susceptible to endogenous or xenobiotic Michael acceptor compounds in vivo system.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed2001

In vivo repair of ENU-induced oxygen alkylation damage by the nucleotide excision repair mechanism in Drosophila melanogaster.

DNA damage caused by oxygen alkylation of bases (mainly at O6-G, O4-T and O2-T positions in DNA) has been correlated with the mutagenic and carcinogenic potency of monofunctional alkylating agents. In all kinds of organisms, repair of O6-alkylG is carried out mainly by the enzyme O6-methyl guanine-DNA methyltransferase (MGMT). However, little is known about the repair of the O-alkylT adducts or about the contribution of nucleotide excision repair (NER) to this process, especially in higher eukaryotes. To study the influence of the NER system on the repair of O-alkylation damage, the molecular mutation spectrum induced by N-ethyl-N-nitrosourea (ENU) in an NER-deficient Drosophila strain, carrying a mutation at the mus201 locus, was obtained and compared with a previously published spectrum for NER-proficient conditions. This comparison reveals a clear increase in the frequency of base pair changes, including GC --> AT and AT --> GC transitions and AT --> TA transversions. In addition, one deletion and two frameshift mutations, not found under NER-proficient conditions, were isolated in the NER-deficient mutant. The results demonstrate that: (1) N-alkylation damage contributes considerably (more than 20%) to the mutagenic activity of ENU under NER-deficient conditions, confirming that the NER system repairs this kind of damage; and (2) that in germ cells of Drosophila in vivo, NER seems to repair O6-ethylguanine and/or O2-ethylcytosine, O4-ethylthymine, and possibly also O2-ethylthymine.

Alkylating Agents↗
PubMed1976

Sites of alkylation of poly(U) by agents of varying carcinogenicity and stability of products.

Several alkylating agents of widely varying reported carcinogenicity (dimethylsulfate, diethylsulfate, ethylmethanesulfonate, methylnitrosourea, ethylnitrosourea and ethylnitrosoguanidine) were reacted with poly(U) at pH values ranging from 4.5 to 7.5. All nucleophilic centers (internal phosphate groups, ribose hydroxyls, and O2, N-3 and O4 sites of the uracil base) were found reactive, though to different extents, at neutrality and in slightly acid solution. The distribution of products is a function of the alkylating agent and pH. The nitroso compounds are more reactive toward oxygens than are dialkylsulfates and alkylalkanesulfonates. The ratio of N : O alkyl products is strongly pH dependent, primarily due to the N-3 being most reactive at the higher pH values, while the diester is most reactive at the lower pH values. The extent of reaction of the O2, O4 or 2'-O or ribose is not greatly affected over the pH range tested. At pH 5.0 alkyl ribophosphotriesters mainly lose alchol to re-form a stable phosphodiester. With increasing OH- concentration, the favored reaction is chain scission at the 3'-O-P bond.

Alkylating Agents↗
PubMed1981

Physical association modeling of DNA alkylation. Some DNA conformational aspects.

The widespread occurrence of physical binding between biological macromolecules and small molecules has prompted us to hypothesize that physical binding contributes to DNA alkylation specificity. The preferred physical binding sites for a CH+3-like test probe were predicted for several sequences of DNA using molecular mechanics free space calculation methods. Sequences containing A = T basepairs direct physical binding to the minor groove, whereas sequences containing G identical to C basepairs direct physical binding to the major groove. Physical binding calculations were also performed for model 'unwound' DNA conformations. The results of the test probe studies were subsequently employed as starting points to predict the preferred physical binding sites for the more complicated case of an actual alkylating agent, the dimethylaziridinium ion. These studies demonstrate that physical binding specificity is highly dependent upon DNA sequence and conformation, and correlates well with the DNA alkylation site specificity observed for alkylating agents in th dimethylaziridine class.

Adenine↗
PubMed1994

Chromosome aberrations: persistence of alkylation damage and modulation by O6-alkylguanine-DNA alkyltransferase.

Alkylating agents produce a spectrum of DNA lesions alkylated at different sites on the molecule. These lesions differ in their propensities to cause effects such as cytotoxicity, mutations and sister-chromatid exchanges. We have used our observations that some methylating agents produce increasing levels of chromosome aberrations (abs) through successive cell cycles in Chinese hamster ovary cells, but not in normal human cells, to begin a study of which alkylated products are most likely to lead to chromosome abs, and in particular which adducts persist in DNA and cause abs after the first cell cycle. We previously observed increasing yields of abs with successive cell cycles in CHO-WBL cells treated with dimethyl nitrosamine (DMN), e.g., at 10 mM DMN, 8.8% cells with abs at first metaphase (M1) and 26.0% at third metaphase (M3) after treatment. Here we tested 4 methylating agents and their ethyl analogs in CHO cells, normal human fibroblasts (L136), and human lymphocytes. We sampled cells at several times after treating for 3 h (CHO and lymphocytes) or 4.5 h (L136). S9 metabolic activation was used for DMN and diethyl nitrosamine. BrdUrd labeling was used to identify cells in M1, M2 and M3. The methylating agents were more potent aberration (ab) inducers than ethylating agents, on a molar basis. In CHO cells, yields of abs were maintained or increased through up to 3 cell cycles after treatment with DMN, methyl methanesulfonate, methyl nitrosourea and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG). With ethylating agents the ab yields in CHO cells were similar or lower in second and third cycles. In contrast, there was no evidence for persistence of lesions leading to abs in either human cell type; ab yields were markedly decreased with subsequent cell cycles for all agents. Normal human cells are proficient in repair of alkylation at the O6 site of guanine by O6-alkylguanine-DNA alkyltransferase (AGT), whereas CHO cells lack AGT activity. To explore the role of repair by AGT on the lesions involved in production of abs, we studied L136 cells, with and without O6-benzylguanine (BZG), a specific inhibitor of AGT. With MNNG, inhibition of AGT resulted in higher ab yield and production of abs through later cell cycles, so that human fibroblasts now behaved similarly to CHO cells. Preliminary data from the reciprocal experiment in CHO cells engineered to express high levels of AGT revealed a greatly decreased ab response to MNNG. In addition, the low ab yields observed were similar through later cycles or increased only slightly.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkylating Agents↗
PubMed1985

Nuclear proteins damage by alkylating agents with different degrees of carcinogenicity.

We have tried to establish a correlation between the carcinogenic potency of two alkylating compounds and specific target sites in chromatin. We have therefore compared the nuclear metabolism of radioactively-labelled methylmethanesulfonate (MMS), a relatively weak carcinogen and N-methylnitrosourea (MNU), a highly potent carcinogen in cultured primary hepatocytes which have, high microsomal drug-metabolizing activity and in V79 Chinese hamster cells which have low microsomal enzymatic activity. The modification of specific amino acid residues in acid-soluble nuclear proteins (H) and non-histone nuclear proteins (NH) was studied after exposing the cells to various doses of alkylating agents overnight. We found that at all doses, mainly the cysteine (Cys), but also to a lower extent the histidine (His) residues are methylated in both H and NH protein fractions by MMS. At high doses of MMS, traces of methylarginine and methylated lysines could be detected. MNU predominantly methylates lysine and arginine residues, the former being found mostly in H, the latter in NH. Although both hepatocytes and V79 cells metabolized radioactively-labelled carcinogen, a higher percentage of counts were incorporated by the hepatocytes; 'unusually' methylated amino acids were detectable in the hepatocyte proteins with relatively low doses of the alkylating agents but not in V79 cells. In the presence of exogenous microsomes, during exposure of V79 cells to the alkylating agents, the amount of amino acid methylation is qualitatively and quantitatively similar to that found in hepatocytes. Our data suggest a specific mechanism of protein methylation, at the level of target amino acids, for carcinogens with different potencies similar to what has been found for DNA bases. A component of the microsomal fraction (S9) may be able to enhance this effect.

Alkylating Agents↗
PubMed2000

DNA binding and alkylation by the "left half" of azinomycin B.

Azinomycin B (also known as carzinophilin A) contains two electrophilic functional groups-an epoxide and an aziridine residue-that react with nucleophilic sites in duplex DNA to form cross-links at 5'-dGNT and 5'-dGNC sequences. Although the aziridine residue of azinomycin is undoubtedly required for cross-link formation, analogues containing an intact epoxide group but no aziridine residue retain significant biological activity. Azinomycin epoxide analogues (e.g., 5 and 6) are of interest due to their potent biological activity and because there is evidence that azinomycin may decompose in vivo to yield such compounds. To investigate the chemical events underlying the toxicity of azinomycin epoxides, DNA binding and alkylation by synthetic analogues of azinomycin B (6, 8, and 9) that comprise the naphthalene-containing "left half" of the antibiotic have been investigated. The epoxide-containing analogue of azinomycin (6) efficiently alkylates guanosine residues in duplex DNA. DNA alkylation by 6 is facilitated by noncovalent binding of the compound to the double helix. The results of UV-vis absorbance, fluorescence spectroscopy, DNA winding, viscometry, and equilibrium dialysis experiments indicate that the naphthalene group of azinomycin binds to DNA via intercalation. Equilibrium dialysis experiments provide an estimated binding constant of (1.3 +/- 0.3) x 10(3) M(-)(1) for the association of a nonalkylating azinomycin analogue (9) with duplex DNA. The DNA-binding and alkylating properties of the azinomycin epoxide 6 provide a basis for understanding the cytotoxicity of azinomycin analogues which contain an epoxide residue but no aziridine group and may provide insight into the mechanisms by which azinomycin forms interstrand DNA cross-links.

Alkylation↗
PubMed1991

Synthesis and evaluation of DNA-targeted spatially separated bis(aniline mustards) as potential alkylating agents with enhanced DNA cross-linking capability.

DNA-targeted separated bis-mustards were synthesized by attaching aniline mono-mustards at the 4- and 9-positions of the DNA-intercalating ligand 9-aminoacridine-4-carboxamide, with the intention of improving the low cross-link to monoadduct ratio found with most alkylating agents. The geometry of these compounds requires that, when the acridine binds to DNA by intercalation, one alkylating moiety is delivered to each DNA groove. Gel electrophoretic studies show that only one arm of these compounds (probably that attached to the 9-position) alkylates DNA, such alkylation occurring specifically in the major groove at the N7 of guanines. Cell-line studies confirm that the mode of cytotoxicity of these compounds (unlike that of untargeted aniline bis-mustards of comparable reactivity) is due to bulky DNA monoadduct formation. It is concluded that more information is required about the exact orientation of the initial monoadducts before ligands with specific DNA cross-linking ability can be designed.

Alkylating Agents↗
PubMed1977

Sites in nucleic acids reacting with alkylating agents of differing carcinogenicity of mutagenicity.

The site of alkylation of a nucleic acid, in vivo, is greatly dependent on the type of alkylating agent. Most alkylating agents of low mutagenicity or carcinogenicity (such as dimethylsulfate) react primarily with the ring nitrogens. The carcinogenic N-nitroso compounds have a great affinity for alkylating oxygens and react with all ring oxygens as well as the phosphodiesters and, in the case of RNA, with the 2'-O of ribose. Ethylating agents, though in absolute terms less reactive than the corresponding methylating agents, show even greater affinity toward oxygens. It appears that the ethyl nitroso compounds that are carcinogenic are also the most reactive with oxygens.

Alkylating Agents↗