Inactivation and repair of double-stranded DNA damaged by the aziridinyl nitroimidazole RSU 1069.

Incubation of RSU 1069 in the presence of biologically active double-stranded phi X174 DNA resulted in, depending on pH, ionic strength and concentration of drug, inactivation of the DNA. A variety of lesions are induced including a high number of single-strand breaks and alkali-labile lesions, which are at most partly lethal. The main inactivating damage consists probably of base damage, induced by alkylation. A considerable part of the damage induced by RSU 1069 can be repaired by the various repair enzymes of the bacterial host of the phi X174 DNA. Finally the damage (pattern) depends considerably on the ionic composition of the reaction solution, which can be explained by an equilibrium model presented in this paper.

Mutational specificity of gamma-rays differs for the same target in plasmid DNA and double-stranded (RF) M13 bacteriophage DNA.

In the lacZ alpha gene of a pUC plasmid a 144 bp insert was cloned as target for mutagenesis. Irradiation of the plasmid in a diluted aqueous solution by 60Co gamma-rays under oxic conditions leads to a very specific mutation spectrum. The predominant type of mutation was a C/G to A/T transversion (29 out of 47 mutants) whereas C/G to G/C transversions were found 7 times and C/G to T/A transitions 10 times. Only one frameshift could be observed which was a deletion of an A/T base pair. The mutations were not randomly distributed along the mutation target but show a strong preference for a certain DNA sequence in which two thirds of the mutations were scored. In this DNA area a hotspot (24 of the 47 mutants) for mutagenesis was located and within 6 bp next to this hotspot another seven mutations were scored. The mutation spectrum in the same mutation target as part of double-stranded (RF) M13 phage was published before. In both systems the mutational hotspot is located at the same site, but the predominant type of mutation is different. In the M13 system the C/G to G/C transversion was the most important event.

Interaction of RSU 1069 and 1137 with DNA in vitro. Biological implications and mechanistic aspects.

We have examined the capacity of the nitroimidazole aziridine antitumour drug RSU 1069 to react with DNA in vitro in order to get a better understanding of its mechanism of action. Moreover, we have utilized biologically active phi X174 DNA to investigate the biological relevance of the chemical DNA modification induced by the drug. Incubation of RSU 1069 in the presence of single-stranded phi X174 DNA resulted in extensive inactivation of the DNA, which is dependent on the concentration of drug and temperature. Only about 2% of the inactivating damage can be attributed to strand breakage. The main damage most probably consists of base damage, of which a part is non-lethal and alkali-labile which in turn can be converted into lethal lesion and subsequently into a break applying a post-incubation alkali treatment. Furthermore, from the dependence of the inactivation and also the formation of breaks on pH and ionic strength, it is concluded that the reaction most probably takes place between a protonated RSU 1069 and a negative DNA coil and that the damage pattern reflects the difference in reactivity of RSU 1069 with the phosphate groups and the bases in DNA. Comparison between RSU 1069 and its ring-open hydrolysis product RSU 1137 revealed that (lethal) damage induced in the DNA must be ascribed to the alkylating properties of the aziridine moiety.

Mutations induced by 60Co gamma-irradiation in double-stranded M13 bacteriophage DNA in nitrous-oxide saturated solutions are characterized by a high specificity.

Irradiation of double-stranded M13 mp10 DNA in a diluted aqueous solution under N2O leads to a very specific mutation spectrum. Fifteen of 28 mutations induced in a 144 base pair (bp) target are C/G to G/C transversions, the other five bp substitutions are C/G to A/T transversions. Six mutations were single bp deletions, one is a large deletion of 180 bp and one is a 10 bp duplication which is probably from spontaneous origin. The mutations are not randomly distributed throughout the 144 bp mutation target but concentrated around two sites. The differences and similarities with the radiation-induced mutation spectrum previously obtained under oxygen are discussed.

Kinetics and mechanism of electron transfer between purines and pyrimidines, their dinucleotides and polynucleotides after reaction with hydrated electrons; a pulse radiolysis study.

The radical spectra of mixtures of thymidine 5'-monophosphate (TMP) or uridine 5'-monophosphate (UMP) with adenine 5'-monophosphate (AMP) after hydrated electron attack, measured from 5 to 3000 microsecond after pulse radiolysis, can only be described in terms of the radical spectra of the nucleotides if an electron transfer is taken into account from the purine radical anion to the pyrimidine, resulting in the formation of a pyrimidine radical anion. From analysis of the spectra of the dinucleoside phosphates ApU, dApT and dCpdA after eaq- attack it follows that the electron-donating species is the purine radical anion (A-.) rather than the protonated purine radical. The electron transfer competes with the fast protonation of the purine radical anion: A-. + py----A + py.- and A-. + H2O in equilibrium AH. respectively. The electron transfer is found to have a diffusion-controlled reaction rate constant of approximately 1.2 X 10(10) for TMP and 3.5 X 10(9) dm3 mol-1 s-1 for UMP.

60Co gamma-rays induce predominantly C/G to G/C transversions in double-stranded M13 DNA.

Upon irradiation with gamma rays of an oxygenated aqueous solution of double-stranded M13 DNA, a very specific mutation spectrum was found with respect to both the type and the positions in the DNA sequence. Of the 23 mutations, which were sequenced, 16 represent a C/G to G/C transversion. A C/G to T/A transition was found once and a G/C to T/A transversion twice. The remaining 4 mutations are frameshifts, 2 are identical and formed by the insertion of a G/C basepair; the other 2 mutations are due to a duplication of 10 basepairs situated at different positions but with a remarkable homology in base sequence. Fourteen mutations, including the 2 duplications are found in the neighbourhood of a TGCT/ACGA sequence.

Fast protonation of adenosine and of its radical anion formed by hydrated electron attack; a nanosecond optical and dc-conductivity pulse radiolysis study.

The study of the reaction of the hydrated electron with adenosine by optical and dc-conductivity pulse radiolysis on nano- and microsecond timescales has been carried out in an attempt to answer the question whether the electron adduct radical becomes protonated or not. The following conclusions have been reached: (1) the reaction of the hydrated electron with adenosine is followed by a water-mediated protonation, which must be complete with 5 ns; (2) no spectral indication of a further protonation of the protonated electron adduct of adenosine of 2'-deoxyadenosine has been found between 40 and 5000 ns; (3) the equilibrium reaction between radiation produced H3O+ and adenosine with a pKa of 3.5 plays an important role in the kinetics of the conductivity transients.

DNA damage induced by reduced nitroimidazole drugs.

Five nitroimidazole drugs were reduced electrolytically and by gamma-radiolysis at fast (300 mumoles or 100% per hr) and slow (3-9 mumoles or 1-3% per hr) reduction rates in the presence of Escherichia coli DNA and single stranded or double stranded DNA from the bacteriophage phi X174. The degree of DNA damage depends upon the rate of drug reduction, where slow reduction produces more damage than fast reduction. The efficiency of damage produced is in the order metronidazole greater than ornidazole greater than azomycin greater than misonidazole greater than benznidazole which reveals a linear correlation between the one-electron reduction potential (E17) and the negative logarithm of the concentration of reduced drug at which 37% of the original DNA activity remains. Damage is not influenced by the presence of O2 at least between about 1-100 ppm. We suggest the protonated one-electron nitro radical anion as a possible candidate for the active damaging species and explain the basis of the relative cytotoxicity of these drugs under conditions of hypoxia.

The biological activity of single-stranded phi X174 DNA, modified by N-hydroxy-2-aminofluorene, is inhibited by guanine imidazole ring-opening of the major, non-lethal aminofluorene-DNA adduct.

The major aminofluorene-DNA derivative, found in the liver of rats after administration of the hepatocarcinogen N-acetyl-2-aminofluorene and identified as N-(deoxyguanosin-8-yl)-2-aminofluorene (dGuo-C8-AF), was introduced in different amounts in single-stranded phi X174 DNA by reacting the DNA with tritium labeled N-hydroxy-2-aminofluorene. The modified DNA was subsequently incubated in 0.1 M NaOH at 37 degrees C for increasing periods of time to convert the dGuo-C8-AF residues into their guanine imidazole ring-opened forms. The degree of conversion was determined by measuring the amount of residual N-(guanin-8-yl)-2-aminofluorene in trifluoroacetic acid hydrolyzates of the alkali-treated DNA by h.p.l.c. In addition, the effect of ring opening on the biological activity of the DNA was monitored by transfecting the DNA to Escherichia coli wild-type spheroplasts. The results indicate that the major aminofluorene-DNA adduct formed initially, which contributes little to inactivation, becomes lethal when its guanine imidazole ring is opened.

Effect of radiation-induced reduction of nitroimidazoles on biologically active DNA.

Radiation-chemical reductions have been carried out with several nitroimidazoles. Reduction of these drugs in the presence of single-stranded phi chi 174 DNA causes extensive lethal damage. However, relatively stable (end) products, do not contribute to the damage, although glyoxal is potentially toxic. This demonstrates that a short-lived intermediate in the reduction process is responsible. Further, the quantity of damage in the DNA depends on both dose (reduction)-rate and also the nature of the drug.

Radiation damage to phi X174 DNA and biological effects.

Dilute aqueous solutions of biologically active DNA can serve as a simplified model system of the cell. As a biological endpoint the survival of the DNA (after transfection to E. coli spheroplasts) is used. Damage in the DNA, irradiated in water with gamma rays, can be ascribed to reactions with primary waterradicals. By introducing additives in such solutions, which will scavenge the primary waterradicals, competition between a scavenger and DNA for such radicals can be studied. Comparison of different additives makes it possible to decide whether a compound behaves like a simple scavenger, radiosensitizer or like a radioprotector. In this context work has been done with the electron-affinic radiosensitizers metronidazole, misonidazole and nifuroxime. We have found that these wellknown cellular sensitizers do not enhance the inactivation of biologically active DNA. They act as simple competitive scavenger for waterradicals. However, if besides a sensitizer a trace of a metalloporphyrin containing compound (e.g. cyt. c) is present during irradiation an enhanced DNA inactivation, which can be interpreted as sensitization, is observed. Without sensitizer metalloporphyrins induce an enhanced protection of DNA. Apart from these effects the consequences of both chemical-(sulphydryl) and enzymatic-(excision; recombination) repair has been studied. It has been found that sulphydryl compounds are able to react with DNA radicals, modifying the radiation damage in such a way that e.g. breaks are prevented. Further in double-stranded DNA a considerable amount of OH and also H radical damage appeared to be reparable by the excision-repair mechanism. However, post-replication repair had only very small or no effect on the amount of damage.

Electrolytic reduction of nitroheterocyclic drugs leads to biologically important damage in DNA.

The effect of electrolytic reduction of nitroimidazole drugs on biologically active DNA was studied. The results show that reduction of the drugs in the presence of DNA affects inactivation for both double-stranded (RF) and single-stranded phi X174 DNA. However, stable reduction products did not make a significant contribution to the lethal damage in DNA. This suggests that probably a short-lived intermediate of reduction of nitro-compounds is responsible for damage to DNA.

By-pass of the major aminofluorene-DNA adduct during in vivo replication of single- and double-stranded phi X174 DNA treated with N-hydroxy-2-aminofluorene.

To examine the effects of aminofluorene-DNA adduct formation on the biological activity of DNA, single-stranded (ss) phi X174 DNA and phi X174 replicative form (RF) DNA were modified to different extents with 3H-labeled N-hydroxy-2-aminofluorene and subsequently transfected to Escherichia coli spheroplasts with different repair capabilities. When the fraction of active ss phi X174 DNA molecules was measured as a function of the mean number of adducts per molecule, exponential survival curves were obtained from which it could be deduced that in wild-type, uvrA- and recA- cells at least 86%, and in uvrC- cells at least 82% of the introduced adducts do not cause inactivation. In the case of RF DNA the survival curves are non-exponential, but they nevertheless show that an exceptionally high number of adducts per RF molecule must be introduced to destroy its biological activity. On average 52 adducts per RF molecule were needed to reduce the survival to 37%, irrespective of whether wild-type, uvrA- or recA- cells were used. On the other hand, the survival of the uvrC- cells was considerably lower, but even in these cells a majority of the adducts is not lethal. By h.p.l.c. analysis of the modified DNA after hydrolysis with trifluoroacetic acid, 81 and 84% of the adducts in ss- and RF DNA, respectively, could be identified as N-(guanin-8-yl)-2-aminofluorene. The results strongly indicate that this type of major modification product is very frequently by-passed during replication of both single- and double-stranded DNA. The results together with the data obtained by sucrose gradient analysis both before and after an alkali treatment and those obtained by h.p.l.c. analysis suggest that inactivation of ssDNA is mainly due to minor modifications such as secondary lesions consisting of chain breaks and alkali-labile sites together with unidentified interaction products.

Radiation-induced strand breaks in phi X174 replicative form DNA: an improved experimental and theoretical approach.

To determine the yield of radiation-induced single-strand, double-strand and potential breaks (breaks which are converted into actual breaks by alkali or heat treatment) oxygenated aqueous solutions of phi X174 supercoiled circular double-stranded (RFI) DNA were irradiated with increasing doses of gamma-irradiation and subjected to electrophoresis on agarose gels both before and after heat treatment. A complete separation was obtained of RFI, RFII (relaxed circle due to one or more single-strand breaks) and RFIII (linear DNA due to one double-strand break). A computer-assisted spectrophotometric procedure was developed, which enabled us to measure very accurately the amount of DNA present in the three DNA fractions. The quantitative changes of each fraction of DNA with dose could be fitted to a straightforward statistical model, which described the dose-dependent formation of the different types of breaks and from which the D37-values of single-strand, potential single-strand and double-strand breaks could be calculated to be 0.42 +/- 0.02, 1.40 +/- 0.25 and 57 +/- 36 Gy respectively. Potential double-strand breaks were not formed significantly under our conditions. In addition the maximum distance between two independently introduced single-strand breaks in opposite strands resulting in a double-strand break could be determined. The values before and after heat treatment are shown to be 29 +/- 6 and 102 +/- 13 nucleotides, respectively.

Adduct formation is involved in radiosensitization, mediated by cytochrome c, of phi X174 DNA by misonidazole.

Radiosensitization by misonidazole of biologically active phi X174 DNA, mediated by cytochrome c, is most probably at least partly due to formation of an adduct between sensitizer and DNA, which can be removed from the DNA by a mild alkaline treatment thereby restoring the activity of the DNA.

Reaction rate of OH radicals with phi X174 DNA: influence of salt and scavenger.

A derivation is given for the dependence of the rate constant of the reaction of OH radicals with a spherical macromolecule on the rate by which such radicals are scavenged by the medium. Experiments were carried out with oxygenated solutions of dilute single-stranded phi X174 DNA at 10(-4)M NaCl (large reaction radius of DNA) or at 10(-4)M NaCl + MgCl2 (small reaction radius) with t-butanol as a scavenger. The results of these experiments cannot be described by simple second-order competition, but can be explained by the predicted dependence of the rate constant of the reaction OH + DNA on the concentration of t-butanol. Furthermore, the results show that only part of the reactions of OH radicals with phi X174 DNA leads to DNA inactivation, and that even at zero scavenger concentration OH radicals are scavenged by other molecules than DNA, presumably impurities remaining even after careful purification of the DNA.

Contrasting effects of cytochrome C on the radiosensitivity of single-stranded phi X174 DNA in the presence of misonidazole or phenol under anoxia.

The effect of misonidazole and cytochrome c on biologically active DNA was studied. In a solution of single-stranded phi X174 DNA the presence of both sensitizer and cytochrome c is needed to evoke radiosensitization. However, sensitizer or cytochrome c alone protects the DNA. Cytochrome c offers even an extra protection. The sensitization is dependent on the concentration of misonidazole. This finding opens up the possibility of studying sensitizers in a relatively simple system.

Repair of damage in double-stranded phi X174 (RF) DNA due to radiation-induced water radicals.

Experiments in which the yields of radiation-induced OH and H radicals were varied, showed that both types of water radicals inactivate phi X174 RF DNA to about the same extent as measured by transfection of the (irradiated) DNA to E. coli wild-type spheroplasts. On the other hand, using spheroplasts prepared from E. coli strains, deficient in one of the proteins involved in excision DNA repair (uvrA- or uvrC-) or in post-replication repair (recA-), clear differences between damage originating from OH or H radical attack were found. Part of the radiation damage due to H radicals appeared to be repairable by an uvrA-gene-dependent repair mechanism, whereas this repair pathway does not play an important role in the case of OH radical damage. The reverse applies to uvrC-gene-dependent repair, which only affects OH radical damage (obtained under anoxic conditions), but has no influence on damage due to H radicals. Irradiation of double-stranded phi X174 (RF) DNA in the presence of oxygen however, yields damage--due to OH radicals only--which appeared not to be sensitive to either uvrC- or uvrA-gene-dependent repair. Furthermore, post-replication repair (recA) has only very little effect on the amount of inactivation by H or OH radicals, when irradiation is carried out under anoxic conditions. We did not find significant inactivation due to hydrated electrons, whether the biological activity was determined by use of wild-type spheroplasts or of strains deficient in excision or post-replication repair proteins.