Evaluation of genetic risks of alkylating agents IV. Quantitative determination of alkylated amino acids in haemoglobin as a measure of the dose after-treatment of mice with methyl methanesulfonate.

The present study explores the possibilities of using specific amino acids in haemoglobin for tissue dosimetry of alkylating agents. The well-known directly alkylating compound methyl methanesulfonate has been used as a model compound.In one experiment ³H-labelled methyl methanesulfonate was given to mice intraperitoneally at three dose levels. The degree of alkylation of haemoglobin exhibited a linear dependence on the quantity of methyl methanesulfonate injected. The degree of alkylation of guanine-N-7 in DNA indicated a slight positive deviation from linearity at high doses.After a single injection the degree of alkylation of cysteine-S and histidine-N-3 in haemoglobin decreased linearly with time reaching the value zero after about 40 days (the life-time of the erythrocytes in the mouse). This demonstrates a stability of these alkylated products, which is fundamental to their use as integral dose monitors.In a second experiment mice were treated with methyl methanesulfonate once a week over a period of 8 weeks. The experiment demonstrated an accumulation of alkylated groups in haemoglobin in agreement with expectation.A method for the quantitative determination of S-methylcysteine in a protein hydrolysate by gas chromatography was developed.     

Identification of consistent alkylation of cysteine-less peptides in a proteomics experiment

In a proteomics experiment, reduction and alkylation of proteins prior to enzymatic digestion ensures high sequence coverage of that protein during a database search. However, the alkylation procedure uses an excess of an alkylating agent such as iodoacetamide (IAA). Therefore, although other amino acids are alkylated, these modified peptides are not identified in a database search. Here we show that a large proportion of peptides are mono- and di-alkylated by IAA and therefore not identified via a database search. The first alkylation consistently takes place at the N-terminal amino acid. Therefore, we propose that during the database search conducted during a proteomics experiment, one should have the option of searching for any alkylated peptide at the N-terminal amino acid.     

The action of mono- and di-functional sulphur mustards on the ribonucleic acid-containing bacteriophage μ2

Bacteriophage μ2 is inactivated by both mono- and di-functional sulphur mustards at relatively low extents of alkylation. No degradation of alkylated RNA was detected. Cross-linking of RNA to protein was observed with the difunctional agent, but this reaction was only a minor contribution to the inactivation. Analyses of the reaction products in bacteriophage RNA showed that, at the mean lethal doses, more than one mono-alkylation of guanine had occurred but the sum total of other types of RNA alkylation was close to a single event. The results therefore suggest that inactivation results from the mono-alkylation of adenine or cytosine. In experiments with the difunctional agent cross-linking of RNA bases or of RNA to protein also prevented replication, the existence of these reactions accounting for the greater sensitivity of the bacteriophage to this agent.     

Fluorescence study of DNA alkylation by epoxides

A simple fluorescence assay was devised to measure alkylation of guanine. The assay was tested with simple epoxides: propylene oxide, glycidol, epichlorohydrin, trichloropropylene oxide and styrene oxide, which are known to vary considerably in their mutagenic potency. The order of reactivity parallelled the mutagenic potency, trichloropropylene oxide being the most reactive alkylating agent. Each epoxide alkylated deoxyguanosine faster than single-stranded DNA, at equal concentrations of guanine. Single-stranded DNA was alkylated substantially faster than was double-stranded DNA. The reaction products with each substrate were analysed by thin-layer chromatography and exhibited similar Rf-values. It was concluded that polymers, particularly double-stranded DNA, reacted slower than deoxyguanosine due to the properties of polymers in solution rather than the unavailability of reactive sites for alkylation.     

Methylation of nuclear proteins by dimethylnitrosamine and by methionine in the rat in vivo

1. The incorporation of methyl groups into histones from dimethylnitrosamine and from methionine was studied by injection of the labelled compounds, isolation of rat liver and kidney histones, and analysis of hydrolysates by column chromatography. 2. Labelled methionine gave rise to labelled in-N-methyl-lysine, di-in-N-methyl-lysine and an amino acid presumed to be omega-N-methyl-arginine. 3. Administration of labelled dimethylnitrosamine gave rise to labelled S-methylcysteine, 1-methylhistidine, 3-methylhistidine and in-N-methyl-lysine derived from the alkylating metabolite of dimethylnitrosamine. In addition, labelled formaldehyde released by metabolism of dimethylnitrosamine leads to the formation of labelled S-adenosylmethionine, and hence to labelling of in-N-methyl-lysine, di-in-N-methyl-lysine and omega-N-methylarginine by enzymic methylation. 4. The formation of in-N-methyl-lysine by alkylation of liver histones was confirmed by using doubly labelled dimethylnitrosamine to discriminate between direct chemical alkylation and enzymic methylation via S-adenosylmethionine. These experiments also suggested the possibility that methionine residues in the histones were alkylated to give methylmethionine sulphonium residues. 5. The extent of alkylation of liver histones was maximal at about 5h after dosing and declined between 5 and 24h. The methylated amino acids resulting from direct chemical alkylation were preferentially lost: this is ascribed to necrosis of the more highly alkylated cells. 6. Liver histones were about four times as alkylated as kidney histones; the extent of alkylation of liver histones was similar to that of liver total nuclear proteins. 7. Methyl methanesulphonate (120mg/kg) alkylated liver histones to a greater extent than did dimethylnitrosamine. Diethylnitrosamine also alkylated liver histones. 8. The results are discussed with regard to the possible effects of alkylation on histone function, and the possible role of histone alkylation in carcinogenesis by the three compounds.     

The sequence specificity of alkylation for a series of benzoic acid mustard and imidazole-containing distamycin analogues: the importance of local sequence conformation.

The covalent sequence specificity of a series of nitrogen mustard and imidazole-containing analogues of distamycin was determined using modified sequencing techniques. The analogues tether benzoic acid mustard (BAM) and possess either one, two or three imidazole units. Examination of the alkylation specificity revealed that BAM produced guanine-N7 lesions in a pattern similar to conventional nitrogen mustards. The monoimidazole-BAM conjugate also produced guanine-N7 alkylation in a similar pattern to BAM, but at a 100-fold lower dose. The diimidazole and triimidazole conjugates did not produce detectable guanine-N7 alkylation but only alkylated at selected sites in the minor groove. Unexpectedly, the alkylation specificity at equivalent doses was nearly identical to that found for the previously reported pyrrole-BAM conjugates. The consensus sequence, 5'-TTTTGPuwas strongly alkylated by the triimidazole conjugate in preference to other similar sites including three occurrences of 5'-TTTTAA. Footprinting studies were carried out to examine the non-covalent DNA binding interactions. These studies revealed that the tripyrrole- BAM conjugate bound non-covalently to the same AT-rich sites as distamycin. In contrast, whereas the Im3lexitropsin bound non-covalently to GC-rich sequences, the triimidazole-BAM conjugate did not detectably footprint to either GC- or AT-rich regions at equivalent doses. The results indicate that the alkylation event is not solely dictated by the non-covalent binding and might be influenced by a unique sequence dependent conformational feature of the consensus sequence 5'-TTTTGPu.     

Host-cell reactivation of alkylated T7 bacteriophage.

Purified T7 phage, treated with methyl methanesulfonate, was assayed on Escherichia coli K-12 host cells deficient in base excision repair. Phage survival, measured immediately after alkylation or following incubation to induce depurination, was lowest on a mutant defective in the polymerase activity of DNA polymerase I (p3478). Strains defective in endonuclease for apurinic sites (AB3027, BW2001) gave a significantly higher level of phage survival, as did the strain defective in the 5'--3' exonuclease activity of DNA polymerase I (RS5065). Highest survival of alkylated T7 phage was observed on the two wild-type strains (AB1157, W3110). These results show that alkylated T7 phage is subject to repair via the base excision repair pathway.     

Effect of alkylation on the secondary structure of DNA

S1 nuclease hydrolysis and bezoylated naphthoylated DEAE-cellulose (BND-cellulose) chromatography have been used to demonstrate that alkylation of DNA by dimethyl sulfate at neutral pH leads to the production of partially denatured molecules under conditions where no significant depurination occurs. DNA was alkylated with increasing concentrations of the alkylating agent, and subjected to enzymatic degradation and binding to BND cellulose. An increasing degree of DNA hydrolysis and adherence to BND cellulose was seen. On hydroxyapatite chromatography the alkylated DNA still eluted at the position of double-stranded molecules suggesting the presence of partially denatured regions. The presence of salt had a preventive effect on such denaturation.     

Properties of 3-methyladenine-DNA glycosylase from Escherichia coli.

An Escherichia coli enzyme that releases 3-methyladenine and 3-ethyladenine in free form from alkylated DNA has been purified 2800-fold in 7% yield. The enzyme does not liberate several other alkylation products from DNA, including 7-methylguanine,O6-methylguanine, 7-methyladenine, N6-methyladenine, 7-ethylguanine, O6-ethylguanine, and the arylalkylated purine derivatives obtained by treatment of DNA with 7-bromomethyl-12-methylbenz[a]anthracene. The reaction of the enzyme with alkylated DNA leads to the introduction of apurinic sites but no chain breaks (less than one incision per ten apurinic sites), and there is no detectable nuclease activity with native DNA, depurinated DNA, ultraviolet-irradiated DNA, or X-irradiated DNA as potential substrates. The enzyme is termed 3-methyladenine-DNA glycosylase. It is a small protein, Mr = 19 000, that does not require divalent metal ions, phosphate, or other cofactors in order to cleave base-sugar bonds in alkylated DNA.     

Induction of resistance to alkylating agents in E. coli: the ada+ gene product serves both as a regulatory protein and as an enzyme for repair of mutagenic damage.

The expression of several inducible enzymes for repair of alkylated DNA in Escherichia coli is controlled by the ada+ gene. This regulatory gene has been cloned into a multicopy plasmid and shown to code for a 37-kd protein. Antibodies raised against homogeneous O6-methylguanine-DNA methyltransferase (the main repair activity for mutagenic damage in alkylated DNA) were found to cross-react with this 37-kd protein. Cell extracts from several independently derived ada mutants contain variable amounts of an altered 37-kd protein after an inducing alkylation treatment. In addition, an 18-kd protein identical with the previously isolated O6-methyl-guanine-DNA methyltransferase has been identified as a product of the ada+ gene. The smaller polypeptide is derived from the 37-kd protein by proteolytic processing.     

Regulatory and essential light-chain interactions in scallop myosin. I. Protection of essential light-chain thiol groups by regulatory light-chains

The reactivity of the thiol groups of the essential light-chains of scallop myosin is greatly reduced by the presence of regulatory light-chains on myosin. The thiol groups of the essential light-chains react with iodoacetate only if the regulatory light-chains have been removed by treatment with EDTA. No alkylation of the essential light-chains could be detected in myosins containing regulatory light-chains (untreated or reconstituted myosins) after an overnight incubation with excess iodoacetate at 4 °C. In contrast, similar treatment alkylated two to three thiol groups of essential light-chains in desensitized myosins from which the regulatory light-chains had been removed. In addition, up to seven of the 20 heavy-chain thiols were also alkylated; however, the reactivity of the heavy-chain thiols did not depend on the presence of the regulatory light-chains. ATPase activities were not inhibited by alkylation with iodoacetate. Regulatory light-chains also protected essential light-chain thiols against reaction with N-iodoacetyl-N^′-(l-sulfo-5-naphthyl) ethylenediamine and against dansylation at pH 6.7, although treatment with these reagents caused a considerable loss of ATPase activities. Rebinding of the regulatory light-chains was impaired by alkylation. The results indicate an extensive interaction between the regulatory and the essential light-chains in scallop myosin.     

Alkylation of DNA by melphalan: investigation of capillary liquid chromatography-electrospray ionization tandem mass spectrometry in the study of the adducts at the nucleoside level

Nitrogen mustards are among the oldest cancer chemotherapeutic agents and remain the drugs of choice for treatment of many human cancers. A serious complication of treatment with nitrogen mustards is the increased risk of a secondary leukaemia in long-term survivors because not all alkylating agent interactions with DNA result in cell death. In an earlier study 2'-deoxy-5'-mononucleotide/melphalan adducts have been analysed by us by LC-ES MSMS. In this work we want to present the first results of the analysis of the corresponding 2'-deoxynucleoside/melphalan adducts from DNA hydrolysates by column switching/capillary LC-ES tandem mass spectrometry. Nucleosides, compared to nucleotides, give better chromatographic results and show a good sensitivity under electrospray (+) [ES(+)] ionisation. Several adducts were identified under ES(+) conditions. Mono-alkylated nucleoside adducts alkylated at the base moiety were identified for dGuo, dCyd and dAdo. Structures were identified by recording the low-energy CAD product ion scans. Also a mono-alkylated nucleotide pdA with alkylation position at the phosphate moiety could be detected. This proves that in the case of phosphate alkylation the enzymatic dephosphorylation reaction was inhibited. A Jurkat cell suspension was treated with melphalan (1 mM) and incubated at 37 degrees C (5% CO(2)). After 6 and 48 h, the DNA was isolated and enzymatically hydrolysed. The corresponding nucleoside pool was evaluated with the developed LC-MS method. In the 48-h experiment, one adduct could be identified as a N-7 alkylated dGuo. In the 6-h experiment, no adducts could be found. Additional experiments were done wherein Jurkat-DNA, isolated from a non-treated cell culture, was treated with melphalan. These results were analogous with the data found in melphalan-treated calf thymus DNA. Additionally, we tried to determine the exact alkylation position by interpreting high-resolution fragmentation spectra.     

Alkylation of deoxyribonucleic acid in vivo in various organs of C57BL mice by the carcinogens N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and ethyl methanesulphonate in relation to induction of thymic lymphoma. Some applications of high-pressure liquid chromatography.

1. Methods were developed for analysis of alkylpurines, O2-alkylcytosines, and representative phosphotriesters [alkyl derivatives of thymidylyl(3'-5')thymidine], in DNA alkylated in vivo, using high-pressure liquid chromatography. 2. The patterns of alkylation products in DNA in vivo at short times were closely similar to those found for reactions in vitro. Alkylation by the nitrosoureas was complete in vivo within 1 h, but with ethyl methanesulphonate was maximal at 2--4h. 3. The time course of persistence of alkylation products in vivo was determined for several tissues. In addition to the rapid loss of 3- and 7-alkyladenines reported previously for all tissues, a relatively rapid loss of O6-alkylguanines from DNA of liver was found which was more rapid at lower doses. In brain, lung and kidney, excision of O6-alkylguanine was much less marked, but was not entirely excluded by the data. In thymus, bone marrow and small bowel, all alkylated bases were lost with half-lives of 12--24h, at non-cytotoxic doses of alkylation. 4. No evidence for any marked excision of other minor products from alkylated DNA in vivo was found; thus 1-methyladenine, O2-ethylcytosine (found in appreciable amount only with N-ethyl-N-nitrosourea), 3-methylguanine, and dTp(Alk)dT persisted in alkylated DNA, including DNA of liver. 5. The induction of thymic lymphoma was determined over the range of single doses by intraperitoneal injection up to about 60% of the LD50 values, and related to the extent of alkylation of target tissues thymus and bone marrow. With N-methyl-N-nitrosourea over 90% tumour yield was attained at 60 mg/kg, and with N-ethyl-N-nitrosourea up to 52% at 240 mg/kg, but with ethyl methanesulphonate at up to 400 mg/kg only a few per cent of tumours were obtained. 6. The carcinogenic effectiveness of the agents was positively correlated with the extents of alkylation of guanine in DNA of target tissues at the O-6 atom. On the basis that at doses giving equal carcinogenic response these extents of alkylation would be equal, the chemical analyses showed that the ratio of equipotent doses to that for N-methyl-N-nitrosourea would be, for N-ethyl-N-nitrosourea, 5.3 for ethyl methanesulphonate about 21, and for methyl methanesulphonate [Frei & Lawley (1976) Chem.-Biol. Interact. 13, 215--222] about 144. These predictions were in reasonably good agreement with the observed dose-response data for these agents.     

Vinyldiaminotriazine-acridine conjugate as G-quadruplex alkylating agent

Higher-order structures of nucleic acids have become widely noted for their biological consequences and the discovery of an alkylating small molecule for these structures has been of interest due to its therapeutic potential. We previously developed the vinyldiaminotriazine (VDAT)-acridine conjugate as a T-T mismatch alkylating agent. In this report, we focused on the finding of the alkylation to the G-quadruplex (G4) DNA with VDAT-acridine conjugates. The VDAT-acridine conjugates exhibited a considerable alkylation ability to G4 under mild conditions. Moreover, the investigation of properties with the alkylated G4 revealed that alkylation by this conjugate significantly increased the stability of the G4 structure. This study provides a starting point in the further development of selective G4 alkylating small molecules.     

Mass spectrometric measurement of differential reactivity of cysteine to localize protein-ligand binding sites. Application to tubulin-binding drugs

A new method for localizing binding sites of noncovalent drugs on proteins is presented. We have developed an accurate and high-throughput method based on the mass spectrometric measurement of differential reaction yield of cysteine alkylation (MS-DRC). This method, essentially a semiquantitative footprinting approach, is applicable to any type of ligand targeting cysteine-rich proteins because the method measures the reactivity change of each cysteine toward an alkylating agent instead of monitoring the drug itself. Thus, no modification of the drug is needed. In this study, the method is evaluated using tubulin as a model system. Tubulin and drug-treated tubulin were alkylated separately with several alkylating reagents, followed by proteolysis and high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) and HPLC-MS. Relative alkylation yields of each cysteine toward the reagents were measured by mass spectrometric quantitation. The reaction yields of each cysteine of two samples were compared to detect a particular cysteine (or cysteines) for which reaction yield was markedly decreased following drug binding. Monobromobimane (mBrB) showed the highest differential.Thus, the MS-DRC method with mBrB was evaluated with various tubulin agents, including the covalent agent T138067 and the noncovalent agents colchicine, podophyllotoxin, and 2-methoxyestradiol. Conformational changes induced by drug binding, as well as sites of direct binding, may be identified.     

Identification of the globin chain and the amino acid residue responsible for polymerization of bullfrog (Rana catesbeiana) hemoglobin with iodo[14C]acetamide.

The bullfrog (Rana catesbeiana) major hemoglobin dissociates into its constituent globin chains (alpha and beta) which are separated by Sulfopropyl-Sephadex C-25 column chromatography after alkylation with iodo[14C]acetamide. Each globin chain has two cysteine residues and those of the beta-globin chain in the tetramer are preferentially alkylated with iodoacetamide.     

CARBOXYMETHYLATION OF SULPHYDRYL GROUPS IN PROTEOLIPIDS1

(1) The sulphydryl groups of brain white matter proteolipids were studied by alkylation with iodoacetic acid and iodoacetamide in an organic solvent medium. To make sterically hindered sulphydryl groups available, the reaction was also carried out in the presence of sodium dodecyl sulphate. (2) In all cases, iodoacetamide was a better alkylating agent than was iodoacetic acid. (3) Only minimal alkylation of crude white matter proteolipids was obtained in the absence of detergent; addition of sodium dodecyl sulphate increased the availablity of SH groups. (4) Purified proteolipids prepared by column chromatography were alkylated to a lesser degree than were crude proteolipids. (5) Prior reduction with mercaptoethanol resulted in the quantitative conversion of cysteine to S-carboxymethylcysteine with either alkylating agent and in both preparations. (6) The possibility of a conformational difference between the protein in the crude and purified preparations is discussed.     

Isolation and identification of products from alkylation of nucleic acids: ethyl- and isopropyl-purines.

Ethylation and isopropylation of guanine in alkaline solution, or of adenine in formic acid, by alkyl methanesulphonates gave the following products: 1-, N2-, 3-, O6-, 7- and 9-alkylguanines; 1-, 3-, 7- and 9-alkyladenines. The products were identified from their characteristic u.v-absorption spectra, by comparison with either known ethyladenines or with the corresponding known methyladenines, and were also characterized by mass spectrometry. Their chromatographic properties on paper, t.l.c. and various columns were determined. DNA was alkylated in neutral solution with 14C-labelled alkyl methanesulphonates and the ratios of the alkylpurines formed were obtained, and compared for alkylation by methyl, ethyl and isopropyl methanesulphonates and by N-methyl-N-nitrosourea. The extents of alkylation at O-6 of guanine relative to those at N-7 of guanine varied with the reactivity of the methylating agents according to the predictions of Swain & Scott (1953) relating nucleophilicity of the groups alkylated with the substrate constants of the alkylating agents. The relative extents of alkylation at N-3 of adenine did not follow this correlation.