Repair of sulfur mustard-induced DNA damage in mammalian cells measured by a host cell reactivation assay.

DNA damage is thought to be the initial event that causes sulfur mustard (SM) toxicity, while the ability of cells to repair this damage is thought to provide a degree of natural protection. To investigate the repair process, we have damaged plasmids containing the firefly luciferase gene with either SM or its monofunctional analog, 2-chloroethyl ethyl sulfide (CEES). Damaged plasmids were transfected into wild-type and nucleotide excision repair (NER) deficient Chinese hamster ovary cells; these cells were also transfected with a second reporter plasmid containing RENILLA: luciferase as an internal control on the efficiency of transfection. Transfected cells were incubated at 37 degrees C for 27 h and then both firefly and RENILLA: luciferase intensities were measured on the same samples with the dual luciferase reporter assay. Bioluminescence in lysates from cells transfected with damaged plasmid, expressed as a percentage of the bioluminescence from cells transfected with undamaged plasmid, is increased by host cell repair activity. The results show that NER-competent cells have a higher reactivation capacity than NER-deficient cells for plasmids damaged by either SM or CEES. Significantly, NER-competent cells are also more resistant to the toxic effects of SM and CEES, indicating that NER is not only proficient in repairing DNA damage caused by either agent but also in decreasing their toxicity. This host cell repair assay can now be used to determine what other cellular mechanisms protect cells from mustard toxicity and under what conditions these mechanisms are most effective.

Role of base excision repair in protecting cells from the toxicity of chloroethylnitrosoureas.

The chloroethylnitrosoureas react extensively with cellular DNA to produce a variety of DNA adducts, including a deoxycytidine-deoxyguanosine (dC-dG) cross-link that is clearly cytotoxic. It is now well established that O6-alkylguanine-DNA-alkyltransferase can prevent formation of this dC-dG cross-link and thereby diminish the toxicity of the chloroethylnitrosoureas. Besides alkyltransferase, DNA glycosylases from various species can also contribute to cellular resistance to the chloroethylnitrosoureas, but the mechanism for this increased resistance has not been established. It is known, however, that several chloroethylnitrosoureas-modified DNA bases, including the exocyclic adduct, N2,3-ethanoguanine, are released by Escherichia coli 3-methyladenine DNA glycosylase II. In the study described here, we examined the possibility that this enzyme might act on the exocyclic intermediate in dC-dG formation, 1,O6-ethanodeoxyguanosine, and prevent-dC-dG cross-linking in this way. However, the presence of E. coli 3-methyladenine DNA glycosylase II does not decrease the amount of dC-dG cross-link formed when chloroethylnitrosourea reacts with DNA, and we conclude that this enzyme does not recognize 1,O6-ethanodeoxyguanosine. Therefore, its contribution to resistance probably resides in its action on other nitrosourea-induced DNA modifications.

Hypothermia causes a reversible, p53-mediated cell cycle arrest in cultured fibroblasts.

Normal human fibroblasts grown in cell culture undergo a reversible growth arrest when incubated at 28 degrees C. During incubation at 28 degrees C, levels of p53 and p21 rise in these cells and cell cycle analysis shows that they have undergone a cell cycle arrest. To examine the importance of p53 in mediating this arrest, mouse embryo fibroblasts that are either wild-type or that are defective in p53 were also subjected to hypothermia. Only those cells with wild-type p53 undergo a cell cycle arrest, indicating that p53 has a role in mediating this response. Because many tumor cells have defective p53, this suggests that hypothermia may increase the selective toxicity of chemotherapeutic agents for tumor cells.

Fpg protein releases a ring-opened N-7 guanine adduct from DNA that has been modified by sulfur mustard.

Transfection of the Escherichia coli fpg gene into Chinese hamster ovary cells has been reported to enhance survival after exposure to aziridine (C. Cussac and F.Laval, 1996, Nucleic Acids Res., 24, 1742-1746). This result suggests that Fpg protein protects cells from toxicity by removing ring-opened N-7 guanine adducts from DNA, and raises the possibility that Fpg protein would offer protection from other agents that alkylate the N-7 position of guanine. Since the major adduct formed by sulfur mustard in DNA is 7-hydroxyethyl-thioethylguanine (HETEG), we have investigated the action of Fpg protein on the ring-opened form of this adduct (ro-HETEG). A substrate containing ro-HETEG was prepared by alkaline treatment of DNA modified by [14C]sulfur mustard. Fpg protein purified from an over-producing strain of E. coli released ro-HETEG from this substrate in an enzyme- and time-dependent manner, and at a rate that is similar to that at which it releases ring-opened 7-methylguanine. Thus, Fpg protein acts efficiently on ro-HETEG, and may offer some protection against the toxic action of sulfur mustard.

The chloroethylnitrosoureas: sensitivity and resistance to cancer chemotherapy at the molecular level.

The chloroethylnitrosoureas were developed in a synthetic program that began with the observation that N-methyl-N'-nitro-N-nitrosoguanidine was an effective agent against L1210 cells. The antitumor activity of the chloroethylnitrosoureas is based on their reactions with DNA, especially the formation of a cytosine-guanine crosslink in DNA. Resistance occurs when the enzyme, O6-alkylguanine-DNA alkyltransferase, repairs an intermediate in crosslink formation. Inhibition of O6-alkylguanine-DNA alkyltransferase often restores sensitivity to the chloroethlylnitrosoureas although evidence is accumulating that other repair mechanisms may also contribute to the resistance phenomenon. Continuing investigations in this field center on finding agents whose reactions with DNA are more specific, on elucidating other resistance mechanisms, and on overcoming resistance by developing new inhibitors of repair enzymes.

Release of sulfur mustard-modified DNA bases by Escherichia coli 3-methyladenine DNA glycosylase II.

The toxic effects of sulfur mustard have been attributed to DNA modification with the formation of 7-hydroxyethylthioethyl guanine, 3-hydroxyethylthioethyl adenine and the cross-link, di-(2-guanin-7-yl-ethyl)sulfide. To investigate the action of bacterial 3-methyladenine DNA glycosylase II (Gly II) on these adducts, calf thymus DNA was modified with [14C]sulfur mustard and used as a substrate for Gly II. Gly II releases both 3-hydroxyethylthioethyl adenine and 7-hydroxyethylthioethyl guanine from this substrate. In comparison with the activity of Gly II towards methylated DNA, 3-hydroxyethylthioethyl adenine is released somewhat more slowly than 3-methyladenine, while 7-hydroxyethylthioethyl guanine is released much more readily than 7-methylguanine. Glycosylase action may play a role in protecting cells from the toxic effects of sulfur mustard.

A 32P-postlabeling method for the detection of adducts in the DNA of human fibroblasts exposed to sulfur mustard.

Since the toxicities of sulfur mustard are attributed to DNA alkylation, levels of DNA modification in exposed cells should correlate with the intensity of exposure. We have found that 32P-postlabeling can be used successfully to detect the major adduct, 7-hydroxyethylthio- ethyldeoxyguanosine 5'-phosphate (HETEpdG), that is formed in DNA by sulfur mustard. This method has been used to establish a correlation between exposure and adduct formation in human fibroblasts grown in cell culture and exposed to sulfur mustard concentrations between 2.5 and 15 microM. DNA was recovered from these cells using a salt precipitation method to remove proteins and was found to have an HETEpdG content which increased linearly with SM concentration. This relationship shows that one HETEpdG per 10(6) nucleotides is produced at a SM concentration of 2.3 microM. Growth of fibroblast cells, assayed by trypan-blue exclusion, is somewhat inhibited by 2 microM SM, indicating that 32P-postlabeling has the requisite sensitivity to detect adducts at levels of SM that are minimally toxic.

A 32P-postlabeling method for detecting unstable N-7-substituted deoxyguanosine adducts in DNA.

Many antitumor agents, including the mustards, form N-7 deoxyguanosine adducts in DNA that are difficult to quantitate by the 32P-postlabeling procedure because of their instability. We have developed a method that is successful for the analysis of such adducts using, as a prototype mustard, 14C-labeled bis(2-chloroethyl)sulfide. This agent forms the unstable product 7-hydroxyethylthioethyldeoxyguanosine in DNA. By performing enzymatic digestions to 3'-deoxynucleotides at 10 degrees C, including a second N-7-substituted guanine deoxynucleotide as an internal standard, removing most of the unmodified nucleotides and [32P]ATP on disposable anion columns, and measuring the labeled products after separation on a C18 column, we are able to detect 1 unstable N-7 deoxyguanosine adduct in 10(7) normal nucleotides with good precision.

Detection of sulfur mustard-induced DNA modifications.

Sulfur mustard is acutely toxic to the skin, eyes, and respiratory tract, and is considered carcinogenic to humans by the IARC. Since all of these toxicities are thought to be initiated by DNA alkylation, the level of DNA damage should serve as a biomarker for exposure. To develop methods of detecting this damage, DNA was modified by [14C]-labeled sulfur mustard and DNA adducts were released by mild acid hydrolysis. Radioactivity co-eluted on HPLC analysis with marker 7-(2-hydroxyethylthioethyl) guanine and 3-(2-hydroxyethylthio-ethyl) adenine synthesized from 2-chloroethyl 2-hydroxy-ethyl sulfide. Unambiguous identification of the major adduct, 7-(2-hydroxy-ethylthioethyl) guanine, was provided by gas chromatography combined with mass spectrometric detection. The most abundant adduct, 7-(2-hydroxyethyl-thioethyl) guanine, accounted for 61% of the total alkylation and could be detected as a fluorescent HPLC peak with a detection limit of 10 pmol. To demonstrate the applicability of this method to biological samples, DNA was extracted from the white blood cells of human blood exposed to 131 microM sulfur mustard in vitro and shown to contain 470 pmol of 7-(2-hydroxyethylthio-ethyl) guanine per mg of DNA.

Protection against chloroethylnitrosourea cytotoxicity by eukaryotic 3-methyladenine DNA glycosylase.

A eukaryotic 3-methyladenine DNA glycosylase gene, the Saccharomyces cerevisiae MAG gene, was shown to prevent N-(2-chloroethyl)-N-nitrosourea toxicity. Disruption of the MAG gene by insertion of the URA3 gene increased the sensitivity of S. cerevisiae cells to N-(2-chloroethyl)-N-nitrosourea, and the expression of MAG in glycosylase-deficient Escherichia coli cells protected against the cytotoxic effects of N-(2-chloroethyl)-N-nitrosourea. Extracts of E. coli cells that contain and express the MAG gene released 7-hydroxyethylguanine and 7-chloroethylguanine from N-(2-chloroethyl)-N-nitrosourea-modified DNA in a protein- and time-dependent manner. The ability of a eukaryotic glycosylase to protect cells from the cytotoxic effects of a haloethylnitrosourea and to release N-(2-chloroethyl)-N-nitrosourea-induced DNA modifications suggests that mammalian glycosylases may play a role in the resistance of tumor cells to the antitumor effects of the haloethylnitrosoureas.

Synthesis of the prototype DNA-protein cross-link, 1-(guan-1-yl)-2-(cysteine-S-yl)ethane, and its role in the reactions of the haloethylnitrosoureas.

The haloethylnitrosoureas form a cytotoxic DNA cross-link in a series of reactions which involves initial alkylation of the O6 position of guanine and rearrangement to the intermediate, 1,O6-ethanoguanine; 1,O6-ethanoguanine then reacts with a neighboring cytosine base. O6-Alkylguanine-DNA alkyltransferase can interrupt this process after the initial alkylation step by removing the alkyl group from the O6 position of guanine. Recent evidence suggests that the O6-alkylguanine-DNA alkyltransferase also recognizes 1,O6-ethanoguanine as a substrate, becoming bound to DNA when it interacts with that intermediate. It has also been shown that glutathione becomes bound to haloethylnitrosourea-treated DNA, apparently through chemical interaction with 1,O6-ethanoguanine. Since both of these reactions involve the thiol group of cysteine, we have examined the reaction of cysteine with 1,O6-ethanoguanine, characterizing the prototype DNA-protein cross-link, 1-(3-cytosinyl),2-(1-guanyl)ethane, which is formed in this reaction. These results establish a competitive reaction with 1,O6-ethanoguanine as a likely route to protein-DNA cross-linking.

Identification of the cross-link between human O6-methylguanine-DNA methyltransferase and chloroethylnitrosourea-treated DNA.

Chloroethylnitrosoureas induce reactive O6-guanine adducts in DNA that can form either interstrand cross-links or a covalent complex with the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). To test our hypothesis that these end-products are formed from the common precursor, 1-O6-ethanoguanine, we compared the kinetics of interstrand cross-link formation with those of decay of MGMT complex forming capacity. The half-lives of these processes were identical. Our hypothesis also predicts that the linkage between DNA and MGMT is 1-(guan-1-yl)-2-(cystein-S-yl)ethane. This notion was tested by forming the complex with 35S-labeled recombinant human MGMT and a chloroethylnitrosourea-treated oligodeoxynucleotide. After degradation by depurination and proteolytic digestion, the identity of the [35S]cysteine-guanine linkage was confirmed by comparison with the synthetic marker compound using high performance liquid chromatography and UV spectrometry. These results strengthen the hypothesis that DNA interstrand cross-links and DNA-MGMT complex both arise from the same precursor. The data also suggest that 1-O6-ethanoguanine is a good substrate for MGMT such that, under certain conditions in vivo, DNA-MGMT complex formation may constitute a significant secondary lesion.

Release of N2,3-ethenoguanine from chloroacetaldehyde-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II.

The human carcinogen vinyl chloride is metabolized in the liver to reactive intermediates which form N2,3-ethenoguanine in DNA. N2,3-Ethenoguanine is known to cause G----A transitions during DNA replication in Escherichia coli, and its formation may be a carcinogenic event in higher organisms. To investigate the repair of N2,3-ethenoguanine, we have prepared an N2,3-etheno[14C]guanine-containing DNA substrate by nick-translating DNA with [14C]dGTP and modifying the product with chloroacetaldehyde. E. coli 3-methyladenine DNA glycosylase II, purified from cells which carry the plasmid pYN1000, releases N2,3-ethenoguanine from chloroacetaldehyde-modified DNA in a protein- and time-dependent manner. This finding widens the known substrate specificity of glycosylase II to include a modified base which may be associated with the carcinogenic process. Similar enzymatic activity in eukaryotic cell might protect them from exposure to metabolites of vinyl chloride.

The formation and enzymatic repair of DNA modifications caused by the haloethylnitrosoureas and related compounds.

The haloethylnitrosoureas are both useful antitumor agents and known carcinogens. These biological activities are believed to be associated with DNA modification, and some biologically significant lesions have been identified in DNA exposed to these agents. At the same time, DNA repair is a cause of resistance to treatment by these agents, and may also serve as protection against their carcinogenic effects.

Release of N2,3-ethanoguanine from haloethylnitrosourea-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II.

3-Methyladenine DNA glycosylase II (Gly II), purified from Escherichia coli cells which carry the plasmid PYN1000, has been tested for its ability to release N2,3-ethanoguanine from DNA modified by the antitumor agent N-[2-chloroethyl-1,2-14C]-N'-cyclohexyl-N-nitrosourea ([14C]CCNU). Gly II has been shown to release N2,3-ethanoguanine in a protein- and time-dependent manner at a rate that exceeds the rate at which this enzyme releases other alkylated bases from [14C]CCNU-modified DNA. This finding widens the known substrate specificity for Gly II to include a modified base which bears an exocyclic ring structure, a class of modifications caused by a variety of chemical carcinogens.

3-Methyladenine DNA glycosylase activity in a glial cell line sensitive to the haloethylnitrosoureas in comparison with a resistant cell line.

Extracts of a glial cell line (SF-126) which is sensitive to the cytotoxic effect of the haloethylnitrosoureas and of a cell line (SF-188) which is resistant to these agents have been tested for their ability to release methylated bases from a DNA substrate which has been modified with [3H]dimethyl sulfate. In comparison with the sensitive cell line, extracts from the resistant cell line have 2-3-fold higher enzymatic activity. High performance liquid chromatography profiles of the bases which are released by these extracts show that the activity is specific for 3-methyladenine, suggesting that the resistant cells contain elevated levels of 3-methyladenine DNA glycosylase. Previous studies have shown that these cells also contain elevated levels of O6-alkylguanine-DNA alkyl-transferase, suggesting that both enzyme activities may be involved in the resistance of this cell line to the haloethylnitrosoureas.

Release of 7-alkylguanines from N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA by 3-methyladenine DNA glycosylase II.

Purified bacterial 3-methyladenine DNA glycosylase II releases four 7-alkylguanines from [3H]N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA: 7-(2-hydroxyethyl)guanine,1,2-bis(7-guanyl)ethane, 7-(2-chloroethyl)guanine, and 7-(2-ethoxyethyl)guanine. 7-(2-Ethoxyethyl)guanine, a new compound, is formed as a result of an interaction with ethanol, a common solvent for the 2-haloethylnitrosoureas. Of the four 7-alkylguanines which are released from [3H]N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA, 7-(2-hydroxyethyl)guanine is released at a rate very much slower than the other three. As shown by a study of the spontaneous decomposition of the corresponding 7-alkyl-deoxyguanines, differences in chemical stability do not appear to explain the slow release of 7-(2-hydroxyethyl)guanine. In view of previous results showing a difference in the distribution of alkylation products between sensitive and resistant glial cell lines, the broad specificity of this enzyme suggests that glycosylase activity could play a role in cellular resistance to 2-haloethylnitrosoureas.

Development of monoclonal antibodies recognizing 7-(2-hydroxyethyl)guanine and imidazole ring-opened 7-(2-hydroxyethyl)guanine.

7-(2-Hydroxyethyl)guanine (7HEG) is of biological interest because it is formed in vivo by reaction of DNA with ethylene oxide (EO). Furthermore, the major DNA adduct of vinyl chloride, 7-(2-oxyethyl)guanine, can be converted to this adduct by reduction. Two monoclonal antibodies (9E2, 4A5) recognizing 7HEG have been developed from BALB/c mice immunized with the adduct coupled to keyhole limpet hemocyanin. In addition, another antibody (8E10) was developed against the imidazole ring-opened form of the adduct (ro-7HEG). ELISAs were used to determine the sensitivity and specificity of these antibodies. With antibody 9E2, 50% inhibition of antibody binding in the competitive ELISA was at 54 pmol of the modified base 7HEG/well and 67 pmol 7HEGR/well, while with antibody 4A5, the values were 3.6 pmol 7HEG/well and 6.7 pmol 7HEGR/well. Antibody 8E10 gave 50% inhibition at 48 pmol ro-7HEGR/well. Neither antibody 9E2 nor 8E10 cross-reacted with unmodified DNA or with the normal nucleosides at the highest concentration tested. However, antibody 4A5 had a low affinity for deoxyguanosine (50% inhibition at 31,000 pmol). Sensitivity of adduct measurement can be increased 3- to 10-fold using an ELISA with fluorescence endpoint detection. These antibodies have been used to determine the level of adducts in DNA modified in vitro with [3H]- or [14C]EO. Because of the cross-reactivity of the most sensitive antibody, 4A5, with deoxyguanosine, a combined HPLC/immunoassay method was developed to quantitate 7HEG in DNA. The limit of sensitivity of this method is dependent upon the amount of DNA available for analysis. Using 30 fmol as the lowest detectable amount (20% inhibition) in the fluorescent ELISA with antibody 4A5 and 100 micrograms of DNA assayed per well, adduct levels of 1/10(7) nucleotide can be determined. This method was applied to DNA adduct detection in EO-treated myeloma cells and whole blood. Antibody 8E10 was also used in immunohistochemical studies to visualize ring-opened adducts in cells treated with EO followed by high pH. These antibodies will be used for the detection and quantitation of adducts in human samples.

Formation of N2,3-ethanoguanine in DNA after in vitro treatment with the therapeutic agent, N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea.

HPLC analyses of the bases released by acid from N-(2-chloroethyl)-N-nitrosourea-treated DNA and N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-treated DNA show the presence of a new guanine adduct, N2,3-ethanoguanine. This derivative can be synthesized at the monomer level by treating 2-hydroxyethylguanine with thionyl chloride. The product of this reaction, purified by HPLC, has been shown to have a mol. wt corresponding to ethanoguanine by mass spectrometry; NMR spectrometry also supports this structural assignment. The UV and fluorescence spectra are very similar to those of N2,3-ethenoguanine, providing evidence that the ethano bridge is attached between N2 and 3 positions. Proof that the derivative is N2,3-ethanoguanine comes from the fact that it can be converted to N2,3-ethenoguanine by dehydrogenation on a palladium catalyst. The discovery of this new derivative raises to four the number of tricylic derivatives that have been isolated from DNA treated with 2-haloethylnitrosoureas. The new adduct, N2,3-ethanoguanine, is closely related to an etheno adduct formed by chloroacetaldehyde, a metabolite of the human carcinogen vinyl chloride, and may have relevance to either the therapeutic or carcinogenic actions of the 2-haloethylnitrosoureas.