The mutagenic spectrum of acridine-linked aniline nitrogen mustards in AS52 cells: implications of DNA targeting with high selectivity for adenine or guanine bases.

The mutational spectra generated in AS52 cells at the gpt gene locus by aniline mustards were studied by the isolation of resistant clones and sequencing of the altered gene. A set of four aniline mustards (both mono- and bifunctional) linked to a DNA-affinic 9-aminoacridine (9-AA) carrier was used, together with the untargeted mustards chlorambucil (CHL) and its half-mustard, and the DNA binding carrier, 9-AA. Both 9-AA and CHL were weak cytotoxins, with the DNA-targeted mustards being markedly (10-40-fold) more dose potent, and the bifunctional ones somewhat more toxic than the monofunctional ones. 9-AA produced a different spectrum of mutations to the spontaneous background, with more minor addition events and less base pair substitutions, and showing for the first time that frameshift events so characteristic of 9-AA in bacteria or bacteriophage also occur in mammalian cells. The mutational spectra of the DNA-targeted mustards were quite different both from this and from the lesions caused by the untargeted mustards, which cause largely transition mutations at AT sites (despite a clear preference for formation of N(7)-guanine adducts). There were very few transition mutations, suggesting that the initial O(6)-alkylguanine/O(4)-alkylthymine lesions considered to give rise to these are relatively rare. There was also a lower incidence of complete deletions, usually attributed to DNA cross-links. For the short chain length targeted mustards, which form initial stable adducts largely (95%) at guanine N(7) sites, base pair substitution mutations, predominantly transversions, involved AT and GC base pairs equally. In contrast, the longer chain length targeted mustards, which form >90% of initial adducts at adenine N(1) sites, generated also formed transversion mutations, but these overwhelmingly (24/27) involved AT base pairs.

Mutations induced by some DNA minor groove binding alkylators in AS52 Chinese hamster cells.

Nitrogen mustards are commonly used in cancer chemotherapy. They interact with DNA at electronegative sites, primarily forming N7 guanine mono-adducts and interstrand cross-links. Targeting nitrogen mustards to DNA by attachment of a DNA minor groove binding carrier such as the bisbenzimidazoles Hoechst 33258 (pibenzimol) or Hoechst 33342 (HOE) makes it possible to direct DNA alkylation to more specific stretches of DNA. We have performed a detailed molecular analysis of 6-thioguanine resistant clones arising in Chinese hamster AS52 cells after treatment with HOE, in comparison with a mono- and bifunctional pair of bisbenzimidazole-targeted nitrogen mustards (MGBs). HOE showed no significant ability to induce 6-thioguanine resistant mutants, possibly because drug-treated cells are highly susceptible to apoptosis within very short times. Neither of the MGBs caused the rapid cell death seen with the bisbenzimidazole. However, both MGBs were weaker mutagens than previously found for undirected mustards in the same system, an effect that we suggest could relate to greater structure-directed binding to less mutable DNA sites in the minor groove. Additionally, the nature of some of the mutants suggested there may be a small component of topo I and/or II-mediated events in the mutagenicity of the MGBs. Both MGBs showed high activity in causing deletion mutations, which may be due to errors in attempted repair of the complex lesions formed by minor groove targeted alkylators.

Amsacrine-induced mutations in AS52 cells.

Amsacrine is an acridine-derived inhibitor of topoisomerase II that intercalates into DNA. We performed a detailed molecular analysis of 6-thioguanine (6-TG)-resistant mutant colonies arising in AS52 cells following Amsacrine treatment. AS52 cells carry a single copy of the bacterial gpt gene, functionally expressed using the SV40 early promoter and stably integrated into the Chinese hamster ovary genome. A 1-hr treatment with 0.1 to 0.5 microM Amsacrine was both cytotoxic and mutagenic, resulting in an average mutant frequency (MF) of 143 x 10(6) at 0.5 microM. Fifty independent 6-TG-resistant colonies were isolated for further study. These clones were initially characterised by PCR to estimate the relative proportion of putative point mutants and deletions or rearrangements; then a subset of mutants was further characterised by Southern blotting, Northern blotting, and DNA sequence analysis. Total deletion of the gpt gene sequences was found in 1 (2%) of the mutants, and 7 (14%) of the mutant clones had altered PCR patterns, suggesting complex deletions or rearrangements. The remaining 42 (84%) mutants had a wild-type PCR profile. Of these, 21 mutants were further analysed by Southern blotting. Interestingly, Southern blotting revealed genomic deletions/rearrangements in 12 of 21 mutants with a wild-type PCR profile. These deletions/rearrangements were further shown to affect gpt gene expression. The remaining nine mutants with a wild-type PCR profile were sequenced. Four of these mutants had mutations in the gpt structural gene. Overall, genomic deletions/rearrangements were observed in 12/21 independent mutants subjected to PCR and Southern blotting. Thus, deletions/rearrangements were the most common mutation observed following Amsacrine treatment of AS52 cells.

Comparative mutational spectra of the nitrogen mustard chlorambucil and its half-mustard analogue in Chinese hamster AS52 cells.

Nitrogen mustards play an important role in current cancer chemotherapy. The most effective antitumour agents are those carrying two alkylating functions, probably through their ability to form interstrand cross-links in DNA. Such lesions appear to create more of a block in DNA replication and are more difficult to repair than are most monoadducts. Although there were early reports that monofunctional drugs were more mutagenic than the bifunctional drugs, this has not been formally proved using structurally related drugs in a mutagenicity assay capable of detecting a range of different events. We have studied both the mutagenic potency and spectrum of events caused by treatment with the clinical agent, chlorambucil, compared with its half-mustard analogue, in Chinese hamster ovary (CHO)-AS52 cells. Although both drugs caused comparable increases in mutation frequency at doses killing 90% of cells (from around 9x10-6 to around 9x10-5 mutant cells), the nature of events differed significantly between the drugs. By far the majority of mutations caused by the half-mustard were transversion mutations, and almost all of these could be interpreted in relation to the DNA adducts that are known to be formed. In contrast, the majority of chlorambucil-induced mutations were major deletions, and point mutations were only identified from a few clones. Parallel micronucleus assays verified that chlorambucil has a stronger ability to break chromosomes than the half-mustard. These two drugs are thought to form similar monoadducts, but only the full mustard can form interstrand cross-links. The data suggest that DNA cross-links, although only a minor fraction of the total lesions, dominate the mutagenic spectrum and lead to gross changes at the chromosome level that can not be readily associated with individual lesions produced by the drug.

Induction of mitotic crossing-over by the topoisomerase II poison DACA (N-[2-dimethylamino)ethyl]acridine-4-carboxamide) in Saccharomyces cerevisiae.

The antitumor agent DACA (N-[2-dimethylamino)ethyl]acridine-4-carboxamide) a new DNA intercalating topoisomerase II poison, was distinguishable from clinical topoisomerase poisons (amsacrine, daunorubicin, doxorubicin and etoposide) in its induction of aberrant colonies in the yeast Saccharomyces cerevisiae D5. It was not only more recombinogenic, but was recombinogenic at non-toxic drug concentrations. DACA at 680 microM (2-h exposure time), induced 1.2% aberrant colonies of which 0.32% were mitotic crossing-over events. The presence of the rad52 mutation abolished mitotic crossing-over and greatly increased drug toxicity. The concentration for 50% inhibition of survival of the rad52 mutant was 100 microM, as compared with 4900 microM for the wild-type. Drug toxicity was marginally increased by the presence of rad3 and rad18 mutations. Rad3 mutations increased the incidence of crossing-over events but had little effect on other mutagenic or recombinogenic events. In contrast, the rad18 mutation increased the incidence of all types of aberrant colonies. The inclusion of hydroxyurea and caffeine, as non-specific repair inhibitors, caused weak and strong inhibition, respectively, of all types of aberrant colonies. Inclusion of the protein-synthesis inhibitor cycloheximide reduced mitotic cross-over but had little effect on the incidence of other aberrations. It is concluded that DACA induces lesions which are repaired by a recombinational repair pathway involving the RAD52 product, and that RAD3 and RAD18 products are each involved in the generation of recombinational events.

Modulation of mutagenic properties in a series of DNA-directed alkylating agents by variation of chain length and alkylator reactivity.

Four series of aniline mustards linked to a DNA-affinic acridine chromophore by alkyl chains of varying length (2-5 carbon atoms) have been studied for their mutagenic properties, as estimated in four strains of Salmonella typhimurium and in Saccharomyces cerevisiae strain D5. The four series have very different mustard reactivities, as determined by the aniline link group (-O-, -CH2-, -S- or -SO2-). Some of the derived compounds cause frameshift mutagenesis which can be detected in TA98 and also "petite" mutagenesis activity, neither of which occur to significant extents with the parent mustards or with 9-aminoacridine. None of the derived compounds are as effective as the parent mustards in mitotic crossing-over, nor do they show ability for frameshift mutagenesis in S. typhimurium TA1977 which is typical of acridines. Some of the compounds have comparable frameshift activity to compounds such as ICR-191, but appear to have a different base-pair preference. The results indicate clear structure-activity relationships for the spectrum of mutagenic activity, which relate to both chain length and alkylator reactivity, for these compounds.

Validation of the crisis triage rating scale for psychiatric emergencies.

Using a sample of 500 emergency psychiatric patients at Victoria Hospital in London, Ontario, this study replicated part of the research on the Crisis Triage Rating Scale (CTRS) conducted by Bengelsdorf, Levy, Emerson and Barile in 1984. The relationship between the suggested CTRS cut-off score and the decision whether or not to hospitalize the patient was studied, independently of these scores. The relative contribution of each of the subscales (Dangerousness, Support System and Ability to Cooperate) to this decision was also determined. The results of this study suggest that using a cut-off score of 9, the easily administered Crisis Triage Rating Scale could be an additional assessment aid in determining whether patients require emergency hospital admission to a psychiatric unit.

Variation in the mutagenic potential of acridines in Salmonella typhimurium, with stage of the culture.

The mutagenic potential of proflavine differed by up to two orders of magnitude in three strains of Salmonella typhimurium carrying the frameshift marker hisC3076, depending upon the stage of the culture. In cultures at a specific stage, proflavine showed decreased mutagenesis in a recA as compared with a wildtupe or uvrB derivative. The related compound 9-aminoacridine also showed marked variation in mutagenic potential with the stage of the bacterial culture, but was equally mutagenic in all three strains, at least in log phase cells. Within the literature, there are conflicting data regarding the mutagenicity of acridines, and the contribution of repair enzymes to this event. We suggest that many of the reported differences can be related to the variations in bacterial growth conditions in different laboratories, and urge authors to supply more details of these.

Structure-activity relationships for the mutagenic activity of tricyclic intercalating agents in Salmonella typhimurium.

A total of 25 different tricyclic DNA-intercalating chromophores bearing a common -CONH(CH2)2N(CH3)2 solubilizing sidechain have been compared with the 'classical' frameshift mutagen 9-aminoacridine for their ability to induce revertants in Salmonella typhimurium strain TA1537 (sensitive to frameshift mutation by acridine mutagens). The compounds showed varying levels of activity in this strain. For the fused linear and fused angular tricyclics, activity varied from zero to similar levels to 9-aminoacridine, but with no discernable relationship between activity and either structure or the measured physico-chemical properties. However, the '2-1' tricyclic compounds had essentially no mutagenic activity. Since several of these compounds have high in vivo antitumor activity, this is useful knowledge.

The mutagenic effects of diacridines and diquinolines in microbial systems.

Two series of difunctional DNA-intercalating agents (diacridines and diquinolines) were tested for mutagenic properties in Salmonella typhimurium strain TA1537, and for 'petite' mutagenesis activity in Saccharomyces cerevisiae, and also compared in terms of their structural, lipophilic and DNA-binding properties. Diacridines with only a short chain length were monointercalators, while those with an alkyl linker chain longer than C6 were bisintercalators. Although the bisintercalators especially bound very tightly to DNA, none of these compounds was as effective a frameshift mutagen in TA1537 as the parent chromophore 9-aminoacridine. However, the two (monointercalating) diacridines of shortest chain length were still able to cause frameshifts, and this ability returned (albeit weakly) in the bisintercalators of longest chain length. Although 9-aminoacridine showed no ability for 'petite' mutagenesis, the diacridines of longer chain length were very effective in causing this mitochondrial event. In the quinoline series, both the parent chromophore (4-aminoquinoline) and all the diquinolines were weak monointercalators. None of these compounds showed any ability for frameshift mutagenesis, although some were very weak mitochondrial mutagens. It is concluded that linking two acridines produces compounds whose mutagenic properties might have been predicted from our current knowledge of the parent molecules. However, despite a similar ability to intercalate DNA, the diquinolines show no resemblance to acridines in their mutagenic properties.

Protection by inhibitors of multidrug resistance against mitochondrial mutagenesis in Saccharomyces cerevisiae.

A number of inhibitors thought to act on the drug efflux mechanism of multidrug-resistant cells have been tested for their ability to inhibit the induction of respiration-deficient (petite) colonies of the yeast Saccharomyces cerevisiae by mitochondrial mutagens. The mutagens tested were 3,6-diamino-9-(4-[(methylsulphonyl)aminophenyl]amino) acridine (an antitumour compound related to both amsacrine and proflavine), ethidium bromide, quinolinium dibromide (NSC 176319, a non-intercalative DNA binding antileukaemia agent) and rhodamine 123. The inhibitors tested included verapamil, perhexiline, chlorpromazine, trifluoperazine, reserpine, chloroquine, quinacrine, tamoxifen, clomiphene, cyclosporin A, valinomycin, amphotericin B and Tween 80. Several of these agents protected against mitochondrial mutagenesis, the most active being verapamil, reserpine, chloroquine, cyclosporin A and Tween 80. The correspondence between activity against multidrug resistance and activity in the yeast system strongly implies some degree of similarity in mechanisms for drug efflux from multidrug-resistant cells and drug uptake into the mitochondria of yeast. Agents protecting against the uptake of drugs into mitochondria of mammalian cells may have use in minimizing the long-term toxicity of anticancer drugs mediated by mitochondrial drug retention.

'Petite' mutagenesis and mitotic crossing-over in yeast by DNA-targeted alkylating agents.

Although the biological properties (cytotoxicity, mutagenicity and carcinogenicity) of alkylating agents result from their bonding interactions with DNA, such compounds generally do not show any special binding affinity for DNA. A series of acridine-linked aniline mustards of widely-varying alkylator reactivity have been designed as DNA-directed alkylating agents. We have considered whether such DNA targeting has an effect on mutagenic properties by evaluating this series of drugs in comparison with their untargeted counterparts for toxic, recombinogenic and mutagenic properties in Saccharomyces cerevisiae strain D5. The simple untargeted aniline mustards are effective inducers of mitotic crossing-over in this strain, but resemble other reported alkylators in being rather inefficient inducers of the "petite" or mitochondrial mutation in yeast. However, the majority of the DNA-targeted mustards were very efficient petite mutagens, while showing little evidence of mitotic crossing-over or other nuclear events. The 100% conversion of cells into petites and the lack of a differential between growing and non-growing cells are similar to the effects of the well characterised mitochondrial mutagen ethidium bromide. These data suggest very different modes of action between the DNA-targeted alkylators and their non-targeted counterparts.

Complexation of fibronectin with tissue transglutaminase.

Previous work [Lorand, L., Dailey, J. E., & Turner, P. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1057-1059] showed that fibronectin might serve as a specific carrier for transglutaminases accidentally discharged from erythrocytes or other cells into plasma. In the present study we examined the association of these proteins in purified systems. Complexation was readily demonstrable by nondenaturing electrophoresis, using dansylcadaverine-dependent activity staining as well as immunoblotting procedures, and also by HPLC gel filtration. The results indicate a stoichiometry of 2:1 for the binding of the human erythrocyte transglutaminase (80K) to human plasma fibronectin (440K). The attachment is noncovalent in nature and does not involve cross-linking of the proteins either to themselves or to each other. Binding occurs in the absence of Ca2+, suggesting that a domain on the transglutaminase molecule other than the catalytic site is needed for complexation with fibronectin. Limited proteolysis with chymotrypsin for delineating the relevant region in fibronectin yielded two gelatin- (collagen) binding fragments (56K and 46K), each displaying affinity for transglutaminase. Moreover, these fragments--like intact fibronectin--bound erythrocyte transglutaminase and gelatin simultaneously in ternary complexes.

'Petite' mutagenesis by anticancer drugs in Saccharomyces cerevisiae.

The mitochondria of cancer cells are potential targets for chemotherapy. Drugs which primarily affect mitochondrial DNA can be screened using a 'petite' mutagenesis assay in Saccharomyces cerevisiae. We have used this approach to estimate the antimitochondrial effects of a range of current clinical and experimental antitumour drugs with varying modes of action. Of agents currently in the clinic, the antimetabolites 5-fluorouracil and methotrexate were extremely effective in inducing this respiratory defect, providing cells were growing during treatment. Adriamycin, BCNU, bleomycin, methyl CCNU, cis-platinum, chlorambucil, daunomycin, nitracine, nitrogen mustard and hycanthone were also weakly effective 'petite' mutagens, in either growing or non-growing conditions. None of the currently used agents but some experimental drugs induced high numbers of 'petite' mutants during growing or non-growing conditions. To date, where such agents have been tested clinically, they have proved either ineffective or very toxic. It is possible that antimitochondrial effects on non-proliferating cellular tissues such as the heart might cause unacceptable toxicity and preclude the clinical use of such agents. For those agents effective against proliferating cells, the mitochondria could be an important target for chemotherapy in some cell types. This type of drug appears relatively uncommon in the clinic at present. The 'petite' mutagenesis assay could be more widely used as a screen to optimize this property in development of analogues of current clinical agents, or in developing new types of anticancer drug.

Mitotic crossing-over by anticancer drugs in Saccharomyces cerevisiae strain D5.

Treatment with an anticancer drug causing mitotic crossing-over could lead to expression of recessive genes, previously masked in a heterozygote. Used clinically, such drugs might cause an increased risk of cancer in cases of familial tumours, such as Wilm's tumour or retinoblastoma. Potentially, novel forms of drug resistance could also be unmasked by such a recombinogenic event. We have estimated the extent of this potential problem in current clinical drugs by comparing a range of antitumour agents for ability to cause mitotic crossing-over in Saccharomyces cerevisiae strain D5. We have compared these data with ability to cause an increase in total aberrant colonies in the same experiments. Although many of the agents known to cause point mutation also have some ability for mitotic crossing-over, there are also point mutagens which have little recombinogenic potential. Conversely, some effective recombinogens appear to be either very specific or rather ineffective point mutagens. Although the most generally effective agents in the present experiments were alkylating agents, several other types of drug including DNA-cutting agents, topoisomerase inhibitors, other DNA-binding drugs and antimetabolites may stimulate mitotic crossing-over. None of the mitotic inhibitors or the DNA minor groove binding drugs tested caused recombinogenic events. It would seem that the ability to induce mitotic crossing-over is an important endpoint in its own right. Assays for this event might provide an important complement to other assays commonly required for registration of new pharmaceuticals.

Fibronectin as a carrier for the transglutaminase from human erythrocytes.

Nondenaturing electrophoresis was used to demonstrate that, immediately upon exposure to plasma, the transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) from erythrocytes undergoes a significant shift in mobility. The plasma effect shows saturable characteristics and depends entirely on the presence of fibronectin in plasma, indicative of complex formation between this protein and transglutaminase. The results suggest a specific carrier function for fibronectin that might be of physiological importance in determining the fate of a tissue transglutaminase accidentally discharged into plasma.

Anaerobic incubation and mutagenicity of nitracrine analogues in Salmonella typhimurium.

Nitracrine [1-nitro-9-(dimethylaminopropylamino)-acridine] is a clinical antitumour agent with hypoxia-selective cytotoxicity and radiosensitizing activity. Developments in tumour therapy using either nitracrine itself or some of its analogues under development are likely to be targeted at anaerobic regions of tumours. We have investigated the effects of anaerobiosis on mutagenic activity of nitracrine and a small series of analogues in three derivatives of Salmonella typhimurium frameshift strain hisC3076. Nitracrine-induced mutagenicity was apparently enhanced by a period of anaerobic incubation followed by an aerobic period, with maximal mutagenic effectiveness (as revertants/nmol) being seen at 24 h anaerobiosis. However, the maximal number of nitracrine-induced revertants was not increased by anaerobic incubation, and the relationship between mutagenicity and toxicity remained similar. Comparisons of the effects of anaerobiosis on mutagenicity of bacterial strains with different DNA repair capacities suggested that anaerobiosis was not simply depressing 'SOS' repair. Comparisons of nitracrine with four of its analogues showed that this effect was not a universal characteristic of either acridine derivatives or of nitracrine analogues. Rather, the dose displacements obtained paralleled hypoxic cell selective cytotoxicity in mammalian cells. If this result extends to other compounds, experiments of this nature using the bacterial strains could provide a novel and useful screening system to identify hypoxia-selective cytotoxic anticancer drugs of potential clinical value.

Frameshift mutagenesis by nitracrine analogues in wild-type, uvrB, polA and recA strains of Salmonella typhimurium, with and without plasmid pKM101.

The mutagenic potential of 9-[(3-dimethylaminopropyl)amino]-acridine and its 1-, 2-, 3- and 4-nitro derivatives was studied in several strains of Salmonella typhimurium carrying the frameshift marker hisC3076. The strains all carried deep rough (rfa) mutations, and were either wild-type with respect to DNA repair capacity or carried recA, uvrB, polA1 or polA3 (amber) mutations. Derivatives with and without plasmid pKM101 were also studied. The des-nitro compound resembled 9 aminoacridine and other simple intercalating compounds. Both toxicity and mutagenesis were apparently unaffected by the uvrB and recA mutations or by the presence of plasmid pKM101. However, mutagenicity was reduced by the polA1 mutation, and virtually eliminated by the polA3 mutation. The drug was substantially more toxic in the latter, slightly more toxic in the former, of these polA- strains. Plasmid pKM101 enhanced mutagenesis and protected from toxicity in both polA1- and polA3- strains, although it did not restore either of these parameters to the level in the wild-type strain. The 2-nitro compound was generally similar to the des-nitro compound, except that it was considerably more toxic and apparently non-mutagenic in the recA-bearing strain. By contrast, mutagenicity of the 3- and 4-nitro compounds was enhanced by the uvrB mutation and by the presence of the plasmid. These compounds were highly toxic but non-mutagenic in the recA- strain, and showed some increased toxicity in polA1- and polA3- strains. The 1-nitro compound has been previously found to cross-link DNA. Unlike well-characterised cross-linkers such as mitomycin C it was highly mutagenic in the uvrB- strain, and this mutagenesis was enhanced by plasmid pKM101, but eliminated by the recA mutation. At high doses, where the drug was completely toxic towards uvrB- or recA-carrying strains, it became mutagenic in the DNA-repair-proficient strains. This 'high-dose' mutagenesis was enhanced by plasmid pKM101, but reduced by the polA1 mutation and almost eliminated by the polA3 mutation. Although there are several possible interpretations of these data, they are compatible with the suggestion that the lesion induced by high doses (but not by low doses) of nitracrine is a cross-link, but that this is not the major mutagenic lesion.