Synthesis of novel DNA cross-linking antitumour agents based on polyazamacrocycles

We are seeking to develop more effective alkylating agents as antitumour agents. In previous work conformationally restricted nitrogen mustards were synthesised containing piperidine or pyrrolidine rings. The free bases were designed to be bifunctional alkylating agents via aziridinium ion formation and the effects of varying the distances between the two alkylating sites were studied. Some efficient cross-linkers of naked DNA were prepared but few of these compounds exhibited significant cytotoxicity in human tumour cells in vitro. We have extended this work by making tri- and tetra-azamacrocyclic compounds containing two to four potential alkylating sites. Most of these compounds were powerful DNA alkylating agents and showed cytotoxicity (IC(50) values 6-100microM) comparable with chlorambucil (45microM) and melphalan (8.5microM). In particular the cyclen derivative 2a was more than 10(4) times more effective at cross-linking DNA (2a XL(50)<10nM) than chlorambucil (XL(50) 100microM), and showed significant cytotoxicity in human tumour cells in vitro.     

Non-random intrachromosomal distribution of chromatid aberrations induced by X-rays,alkylating agents and ethanol in Vicia faba

A reconstructed karyotype of Vicia faba with all chromosomes individually distinguishable was treated with triethylene melamine (TEM), cytostasan (CYT) (a new benzimidazol nitrogen mustard), mitomycin C (MI), ethanol (EA) and X-rays. The distribution within chromosomes of induced chromatid abberations was non-random for all agents. The number of segments involved in aberration clustering corresponded to the number of sites representing constitutive heterochromatin, or the regions immediately adjacent to these, as evidenced by the position of Giemsa marker bands. Which of these potential regions of aberration clustering reacted with preferential involvement in aberrations was, in part at least, dependent upon the inducing agent used. It is argued that this may be due to differences in the base composition and/or molecular conformation of heterochromatic regions. Unexpectedly, the distribution pattern of chromatid aberrations induced by mitomycin C was found to be different from those after treatment with the alkylating agents TEM and cytostasan although mitomycin C is assumed to induce aberrations via alkylation. If mitomycin C-induced aberrations are indeed due to alkylation, this indicates that different alkylating agents do not necessarily result in identical patterns of abberation clustering. The other two alkylating agents and ethanol resulted in similar patterns of preferential distribution of abberations. X-Ray induced chromatid aberrations also showed a non-random intrachromosomal distribution, but the clustering was less pronounced than after treatment with the chemical agents.     

Cell Hybridization Study of Resistance to Alkylating Agents

5-Aziridinyl-2,4-Dinitrobenzamide (CB 1954) has been reported to be a highly selective inhibitor of the Walker tumour, with a therapeutic index of 60 (refs. 1 and 2). This compound, however, differs from other tumour inhibitory alkylating agents in that it is monofunctional and fails to inhibit the growth of several animal tumours which respond to difunctional alkylating agents. Compounds closely related in structure to CB 1954 are either much less active or inactive against the Walker tumour³. The structural specificity and biological properties of CB 1954 indicate that its mechanism of action is different from that of the tumour inhibitory difunctional alkylating agents. Whereas the latter are thought to be cytotoxic primarily as a result of their reaction with DNA, CB 1954 may interfere with a specific stage of purine biosynthesis². We have shown by cell hybridization that, unlike resistance to a difunctional alkylating agent, cellular resistance to CB 1954 is lost on fusion with a sensitive cell.     

Autologous tumour-specific cytotoxic T-cell clone established from tumour-infiltrating lymphocytes (TIL) of malignant ascites in the absence of recombinant interleukin 2(rIL2): Activation by autologous tumour cell alone

Mixtures of tumour infiltrating lymphocytes (TIL) and tumour cells collected from malignant ascites of a patient with pancreatic cancer were cultured using a microplate without recombinant interleukin 2(rIL2). TIL rapidly proliferated from 21–51 days after the initiation of culture in 20 out of 30 wells tested. Cytotoxicity was examined in 5 out of the 20 TIL-growing wells. One CD8⁺TIL (well-1) displayed autologous tumour-specific cytotoxicity. Repeated stimulation with autologous tumour cells, in the absence of rIL2, was required for further propagation in long-term (60 days) culture of TIL. Four clones were established from well-1 by limiting dilution without rIL2. Surface phenotypes of the 4 clones were the same as those of well-1, i.e., CD8⁺, CD16, CD25⁺, HLA-DR⁺. And autologous tumour cells were required for continuous proliferation of these CD8⁺ T-cell clones. Both well-1 and the 4 clones displayed similar degrees of cytotoxicity restricted to autologous tumour cells. These results indicate that TIL from malignant ascites may contain precursor cytotoxic T-lymphocytes (CTL) sensitizedin vivo to autologous tumour cells, and that TIL are able to grow for several weeks or more with substantial increases in cell numbers in the absence of rIL2.     

The detection and occurrence of multiple forms of D-ribose 5-phosphate ketol isomerase

The alkylating thiovinyl fragment released from S-(1,2-dichlorovinyl)-l-[³⁵S]-cysteine by a lyase reacted with about 12% of the amino acid residues in poly-l-lysine and about 6% in poly-l-arginine. The reaction of alkylating fragment with DNA was considerably reduced through complex formation of DNA with these polypeptides. When (alkylating fragment)-treated DNA interacted with either normal or (alkylating fragment)-treated polypeptides, the products had an abnormal biphasic melting profile. The first (lower) T_m is apparently due to (alkylating fragment)-substituted regions of DNA which are not complexed with polypeptide and have, like (alkylating fragment)-substituted DNA, a higher T_m than free, native DNA. A second, much higher T_m is due to (alkylating fragment)-substituted regions of DNA which are complexed with polypeptide. These complexes were, however, less stable and had lower T_m values than those prepared from normal, native DNA. The implications of complex formation with respect to susceptibility of tissues and species to toxic agents are discussed.     

Neuroendocrine cells within colorectal tumours induced by dimethylhydrazine

Summary Colorectal adenocarcinomas were induced in male Wistar rats, by weekly subcutaneous administration of 1,2-dimethylhydrazine, classified according to the degree of differentiation and submitted to immunocytochemistry for the peptides cholecystokinin (CCK), gastrin, gastric inhibitory polypeptide (GIP), glucagon, neurotensin, pancreatic polypeptide (PP), peptide YY (PYY), somatostatin and vasoactive intestinal polypeptide (VIP) and the biogenic monoamine 5-hydroxytryptamine. Well- or moderately well-differentiated adenocarcinomas comprised 46% of the tumour population, only 4% were poorly-differentiated adenocarcinomas, and the remaining 50% possessed a mixture of these two morphologies. Glucagon, PYY and 5-hydroxytryptamine immunoreactive cells were frequently observed within well- or moderately well-differentiated tumours and within such regions of tumours possessing a mixed morphological pattern. The tumours contained no cells immunoreactive for any of the peptides not normally located within the colorectum, nor did they contain cells immunoreactive for somatostatin and VIP, although known positive controls did stain. Poorly-differentiated tumours and portions of tumours of mixed type, were consistently negative. 5-hydroxytryptamine was the most frequently located of the three antigens, being detected in 87% of the moderately well-differentiated tumours and 32% of the tumours with mixed morphologies. 11% of moderately well-differentiated tumours possessed 5-hydroxytryptamine positive cells in such profusion that they contributed significantly to the tumour mass. The distribution of glucagon-and PYY-immunoreactive cells was similar, although they occurred with a lower frequency, presumably corresponding to their lower numbers within the normal colorectal mucosa. Additionally, these two peptide immunoreactivities were colocalized in the majority of cells, although some cells contained only one antigen. The immense numbers of cells immunoreactive for peptides and monoamine in a significant proportion of colorectal adenocarcinomas suggests that they have arisen from multipotential endodermal stem cells within the tumours and are not part of the normal epithelial population being engulfed as the tumour grows.     

Analysis of macromolecule alkylation in tissues of intact and tumor carrying mice]

Alkylation DNA reparation kinetics and the disintegration of alkylated RNA, proteins and lipids in liver, spleen and brain of intact and 22A hepatomic mice after a injection of 1-14C-nitrosomethylurea at a therapeutic dose are studied. The tissue studied are different in their macromolecules and lipids alkylation, in DNA reparation and RNA, protein and lipid degradation rates. Possible correlation between the time of the occurrence of DNA damages and the frequency of tumour emergence in different tissues is discussed. It is found that normal cells eliminate more rapidly degraded RNA, protein and lipid molecules and more rapidly repair DNA damages as compared with 22A hepatoma cells. It is suggested to be due to more rapid macromolecule metabolism in normal cells which specifies a selective sensitivity of tumour cells to alkylating agents and nitrosoalkylureas. The time of the occurrence of damages induced with alkylating agents and nitrosomethylureas is supposed to be a critical parameter in processes resulting in the selective sensitivity of normal and tumour cells.     

Effect of alkylating agents on the expression of inducible genes of Escherichia coli

Increasing doses of alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine, diethyl sulphate and ethylmethane sulphonate cause an inhibition of the expression of the recA and sfiA genes of wild-type Escherichia coli. This behaviour was not observed in a lexA56 mutant which has a defective LexA repressor that is unable to bind to the SOS operator. Furthermore, an ada-1 mutant showed the same behaviour as the wild-type strain indicating that the adaptive proteins are not responsible for the inhibition of recA and sfiA at high doses of alkylating agents. These results suggest that the inhibitory effect of these alkylating agents may be found in the interaction between the LexA repressor and the control regions of sfiA and recA. On the other hand, high doses of either UV light or mitomycin C produced only a slight decrease in the induction of recA and sfiA, whereas bleomycin had no effect. The fact that a repressor structurally related to LexA repressor, such as LacI protein, showed the same behaviour as the LexA repressor when a Lac+ strain was treated with alkylating agents, suggests that these compounds can modify the binding abilities of repressors to DNA, producing a limited or even abolished release of repressors, and so decreasing the expression of inducible genes.     

Crystal structure of the human O(6)-alkylguanine-DNA alkyltransferase

The mutagenic and carcinogenic effects of simple alkylating agents are mainly due to O⁶-alkylation of guanine in DNA. This lesion results in transition mutations. In both prokaryotic and eukaryotic cells, repair is effected by direct reversal of the damage by a suicide protein, O⁶-alkylguanine-DNA alkyltransferase. The alkyltransferase removes the alkyl group to one of its own cysteine residues. However, this mechanism for preserving genomic integrity limits the effectiveness of certain alkylating anticancer agents. A high level of the alkyltransferase in many tumour cells renders them resistant to such drugs. Here we report the X-ray structure of the human alkyltransferase solved using the technique of multiple wavelength anomalous dispersion. This structure explains the markedly different specificities towards various O⁶-alkyl lesions and inhibitors when compared with the Escherichia coli protein (for which the structure has already been determined). It is also used to interpret the behaviour of certain mutant alkyltransferases to enhance biochemical understanding of the protein. Further examination of the various models proposed for DNA binding is also permitted. This structure may be useful for the design and refinement of drugs as chemoenhancers of alkylating agent chemotherapy.     

Exploring molecular mechanisms in chemically induced cancer: complementation of mammalian DNA repair defects by a prokaryotic gene

Exposure of man to chemical agents can occur intentionally, as in the treatment of disease, or inadvertently because the environment contains a wide range of synthetic or naturally occurring chemicals. The alkylating agents are a diverse group of compounds (Fig. 1) and comprise a good example of such xenobiotics, since much is known about their occurrence, and their biological effects include carcinogenicity, mutagenicity, toxicity and teratogenicity. Exposure to potentially carcinogenic alkylating agents such as nitrosamines may occur occupationally, from cigarette smoke, from certain foodstuffs and even endogenously through the ingestion of the appropriate precursor chemicals.¹ At the other extreme, the cytotoxic effects of agents such as the chloroethylating nitrosamides or mustards have been exploited in the design of certain antitumour drugs.² The effectiveness of antitumour agents and the other, mostly adverse, biological effects of alkylating agents have been ascribed to their ability to damage cellular macromolecules, in particular DNA. This review concentrates on investigations carried out over the past two years on the role of DNA damage in carcinogenesis, but we shall see how recent advances in this area of research have also led to a better understanding of the mechanisms of the cytotoxic effects of alkylating antitumour agents.     

Alkylation resistance of E. coli cells expressing different isoforms of human alkyladenine DNA glycosylase (hAAG)

The alkyladenine DNA glycosylase (AAG) has been cloned from mouse and humans. AAG knock out mouse cells are sensitized to a variety of alkylating and cross-linking agents suggesting AAG is active on a variety of substrates. In humans, two isoforms have been characterized that are generated by alternative splicing and contain either exon 1a or 1b (hAAG1 or hAAG2). In this study, we examine the ability of the both known isoforms of human AAG (hAAG) to contribute to survival of Escherichia coli from treatments with simple alkylating agents and cross-linking alkylating agents. Our results show that hAAG is effective at repairing methyl lesions when expressed in E. coli, but is unable to afford increased resistance to alkylating agents producing larger alkyl lesions such as ethyl lesions or lesions produced by the cross-linking alkylating agents N,N'-bis-chloroethyl-N-nitrosourea (BCNU), N-(2-chloroethyl)-N-nitrosourea (CNU) or mitomycin C. In the case of CNU, expression of hAAG causes increased sensitivity rather than resistance, suggesting deleterious effects of hAAG activity. We also demonstrate that there are no apparent differences between the two isoforms of hAAG when recovery from damage produced by all alkylating agents is tested.     

Alkylating agent and chromatin structure determine sequence context-dependent formation of alkylpurines

We determined the adduct maps of S(N)1 and S(N)2 alkylating agents in cultured human cells (in vivo) and in vitro to probe DNA-protein interactions along sequences of the promoter and exon 1 of the Fragile-X mental retardation 1 (FMR1) gene. Using ligation-mediated polymerase chain reaction (LMPCR), we compared the piperidine-sensitive alkylpurines sites generated by treating cultured cells (in vivo) and naked DNA (in vitro) with S(N)1 (N-methyl-N-nitrosourea, N-nitroso(acetoxymethyl)methylamine and 1-methyl-3-nitro-1-nitrosoguanidine) and S(N)2 alkylating agents (dimethyl sulfate (DMS), methane sulfonic acid methyl ester, iodo methane, diethyl sulfate, methane sulfonic acid ethyl ester and iodo ethane). The FMR1 promoter has four sites where DNA-protein interactions are observed. In these regions, the S(N)1 methylating agent reactions produced only hypo-reactive sites. In contrast, iodoalkane S(N)2 alkylating agents (MeI and EtI) reactions generated only hyper-reactive sites. Although there are hyper-reactive sites for the other S(N)2 reagents, the hyper-reactive site at +14 on the FMR1 map is more pronounced for the sulfate and sulfonate-derived alkylating agents than for the iodoalkanes. However, DMS modification in the presence of methyl sulfone, a compound that does not alkylate DNA, eliminates the hyper-reactive site observed at +14. This suggests that the electron-rich oxygen atoms of the sulfate and sulfonate-derived S(N)2 alkylating agent structure position the alkylating moiety to the neighboring N-7-guanine position to favor alkyl transfer to the guanine. Using KMnO(4) to probe for single-strand DNA, an unpaired cytosine base was detected at the 5'-side of the hyper- reactive guanine base at position +14, consistent with the formation of a local DNA single-strand bulge. In conclusion, we show that the sequence context-dependent formation of alkylpurines is determined by the chemical nature of the alkylating agent, the DNA sequence context, chromatin structure, and the presence of other non-reactive molecules that can inhibit alkylation.     

Carbinolamines and related structures--potential alkylating metabolites of clinically active anticancer drugs

The precise biochemical mechanism by which a number of clinically-active anticancer compounds function remains unclear. Among these are procarbazine (NSC-77213), cyclophosphamide (NSC-26271), streptozotocin (NSC-85998), dacarbazine (NSC-45388), and hexamethylmelamine (NSC-13875). In all cases, there is an N-methyl or N-alkyl substituent which can be or has been shown to generate carbinolamine-like intermediates as a result of oxidative metabolism. Such intermediates can react with amines, imines, sulfhydryls and similar functional groups to form covalent linkages. Thus, carbinolamine metabolites of these clinically-active compounds are proposed as the active agents capable of altering covalently nucleic acids and proteins. It is this alkylating property that may be responsible for these compounds adversely effecting the mitosis of neoplastic cells. Thus, a unifying hypothesis is proposed whereby metabolic hydroxylation of various miscellaneous anticancer agents is the basis for biological activity. In essence, therefore, three broad classes of alkylating agents may be perceived: (1) the classical alkylators such as the nitrogen and sulfur mustards and the sulfonates, (2) bioreductive alkylating agents, and (3) biooxidative alkylating agents such as the carbinolamines. Though the chemical spectrum of each category may be highly diverse, nevertheless, all function as alkylating agents.     

Sequence-dependent formation of alkyl DNA adducts: A review of methods, results, and biological correlates

Understanding the influence of the DNA sequence on chemical-DNA interactions may provide insight into the processes of chemical carcinogenesis and mutagenesis. This article provides a brief overview of studies and methods devoted to examining the distribution of DNA adducts produced by alkylating agents. Particular emphasis is placed on discussion of DNA adducts generated by simple alkylating agents and the role that their distribution may play in the generation of mutational hotspots.     

Genetic heterogeneity of heat shock protein synthesis as a factor determining the resistance of mammalia to stressors

The relationship between the levels of 70 kDa family heat shock protein (Hsp) synthesis and lymphocyte sensitivity to stressors was investigated. Lymphocyte cultivation in mitogen deprived culture medium and (or) the cell treatment with alkylating agents have been used as a stress challenge. On the model of two inbred murine strains genetically contrasting by the sensitivity to alkylating agents we succeeded in demonstration that the basic level of Hsp synthesis depends on genotype. The quantity Hsp mRNA, as well as the intracellular level of the proteins were significantly higher in BALB/c than in C57BL/6 mice. The mice characterized by higher Hsp levels demonstrated higher resistance to alkylating agent action. The induction of surplus amount of Hsp by heat shock increased the cell resistance to the alkylating agent melphalan. Lymphocyte isolated from high Hsp producers, BALB/c mice, were more resistant to apoptotic signals induced by mitogen deprivation.     

Genetic heterogeneity of heat shock protein synthesis as a factor determining the resistance to stressors in mammalia

The relationship between the levels of 70 kDa family heat shock protein (Hsp) synthesis and lymphocyte sensitivity to stressors was investigated. Lymphocyte cultivation in mitogen deprived culture medium and/or the cell treatment with alkylating agents have been used as a stress challenge. Model experiments with two inbred murine strains genetically contrasting by the sensitivity to alkylating agents demonstrated that the basic level of Hsp synthesis depends on genotype. The quantity Hsp70 mRNA, as well as intracellular level of the proteins, in BALB/c was significantly higher than those in C57BL/6 mice. The mice, which were characterized by higher Hsp levels, demonstrated higher resistance to alkylating agent action. The induction of surplus amount of Hsp by heat shock increased the cell resistance to an alkylating agent melphalan. Lymphocyte isolated from high Hsp producers BALB/c mice were more resistant to apoptotic signals induced by mitogen deprivation.     

Mutation Induction by Difunctional Alkylating Agents in NEUROSPORA CRASSA

The genetic characterization of ad-3 mutants of Neurospora crassa induced by two carcinogenic difunctional akylating agents, 1,2,4,5-diepoxypentane (DEP) and 1,2,7,8-diepoxyoctane (DEO), has shown that point mutations at the ad-3B locus have similar complementation patterns. In addition to the induction of point mutations, DEP induces a low frequency (7.5%) of multilocus deletions, whereas DEO induces an extremely high frequency (42.0%). The distribution of the different classes of ad-3 mutants and the frequency of multilocus deletion mutants among DEP-induced mutants are not significantly different from those induced by the monofunctional alkylating agents EI, EMS and ICR-177 at comparable forward-mutation frequencies. Moreover, the frequencies of DEP-induced ad-3B mutants showing allelic completion or having nonpolarized complementation patterns are similar to those of ad-3B mutants induced by monofunctional agents. It is suggested, therefore, that the mechanism of mutation-induction by DEP in N. crassa is similar to that of monofunctional alkylating agents. Mutation-induction by DEO probably results both from the mechanism of action of monofunctional alkylating agents and from inter-strand cross-linkage of the DNA molecular by the two functional epoxy groups.     

The alkaline single cell electrophoresis assay with eight mouse organs: results with 22 mono-functional alkylating agents (including 9 dialkyl N-nitrosoamines) and 10 DNA crosslinkers

The genotoxicity of 22 mono-functional alkylating agents (including 9 dialkyl N-nitrosoamines) and 10 DNA crosslinkers selected from IARC (International Agency for Research on Cancer) groups 1, 2A, and 2B was evaluated in eight mouse organs with the alkaline single cell gel electrophoresis (SCGE) (comet) assay. Groups of four mice were treated once intraperitoneally at the dose at which micronucleus tests had been conducted, and the stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow were sampled 3, 8, and/or 24 h later. All chemicals were positive in the SCGE assay in at least one organ. Of the 22 mono-functional alkylating agents, over 50% were positive in all organs except the brain and bone marrow. The two subsets of mono-functional alkylating agents differed in their bone marrow genotoxicity: only 1 of the 9 dialkyl N-nitrosoamines was positive in bone marrow as opposed to 8 of the 13 other alkylating agents, reflecting the fact that dialkyl N-nitrosoamines are poor micronucleus inducers in hematopoietic cells. The two groups of mono-functional alkylating agents also differ in hepatic carcinogenicity in spite of the fact that they are similar in hepatic genotoxicity. While dialkyl N-nitrosoamines produce tumors primarily in mouse liver, only one (styrene-7,8-oxide) out of 10 of the other type of mono-functional alkylating agents is a mouse hepatic carcinogen. Taking into consideration our previous results showing high concordance between hepatic genotoxicity and carcinogenicity for aromatic amines and azo compounds, a possible explanation for the discrepancy might be that chemicals that require metabolic activation show high concordance between genotoxicity and carcinogenicity in the liver. A high percent of the 10 DNA crosslinkers were positive in the SCGE assay in the gastrointestinal mucosa, but less than 50% were positive in the liver and lung. In this study, we allowed 10 min alkali-unwinding to obtain low and stable control values. Considering that DNA crosslinking lesions can be detected as lowering of not only positive but also negative control values, low control values by short alkali-treatment might make it difficult to detect DNA crosslinking lesions. In conclusion, although both mono-functional alkylating agents and DNA crosslinkers are genotoxic in mouse multiple organs, the genotoxicity of DNA crosslinkers can be detected in the gastrointestinal organs even though they were given intraperitoneally followed by the short alkali-treatment.     

The Walker 256 carcinoma: a cell type inherently sensitive only to those difunctional agents that can form DNA interstrand crosslinks.

The Walker 256 rat tumour has been maintained in vivo for over 60 years and until recently was used as a primary screen for new antitumour agents. This screen was particularly useful in identifying difunctional alkylating agents as potentially useful anticancer agents and it would seem that the Walker tumour is composed of cells sensitive towards this type of agent. A cell line (WS) established from the Walker tumour retained the sensitivity of the tumour towards difunctional agents and we have examined its phenotype in comparison to a derived, resistant, cell line (WR). The response of WR cells to a range of cytotoxic agents was similar to other established cell lines whilst WS cells were much more sensitive only towards difunctional reacting agents. There were no significant differences in the binding of these agents to the DNA of WS or WR cells. All the agents towards which WS cells showed sensitivity were, without exception, capable of reacting with DNA in Walker cells and forming DNA-DNA interstrand crosslinks. WS cells were not sensitive to busulphan, BCNU, CCNU or Me-CCNU but these agents did not produce interstrand crosslinks in the DNA of either WS or WR cells. Thus WS cells are intrinsically sensitive to specific DNA damage and this is probably a DNA interstrand crosslink. Hybrid cells produced by fusion of WS with WR cells lacked the inherent sensitivity of the WS cells towards cisplatin; sensitivity was therefore a recessive characteristic. Transfection of WS cells with human DNA also gave rise to 2 cisplatin-resistant clones, although it could not be ascertained if these clones were true transfectants or revertants. The survival of these resistant clones, after treatment with cisplatin, was about the same as WR cells a finding which would be consistent with complementation by a transferred gene or reversion of a single gene defect in WS cells. In their sensitivity only to difunctional compounds and lack of an apparent DNA excision repair defect the phenotype of Walker cells strongly resembles those cells from human patients suffering from Fanconi's anaemia and also of yeast snm1 mutant cells. The mechanisms giving rise to this failure to tolerate specific DNA damage (which seems to involve the inability to recover from the initial inhibition of DNA synthesis and may involve a single defect of a gene involved in the late steps of crosslink repair), do not involve drug uptake, drug binding to DNA, cell size, cell doubling time or DNA excision repair.     

Mutagenic activity of antibiotics alone and in conjunction with alkanesulfonates

Differential and combined effects of 0.25 and 0.50% antibiotics (ampicillin, neomycin, furadentine) and alkylating agents (ethyl methanesulfonate, methyl ethanesulfonate, methyl methanesulfonate) were assayed on Phaseolus vulgaris L. (2 n = 22) at the M₂ generation for chlorophyll mutations. The general types scored were Albino, Xantha, Virescens and Maculata. Yellowish-green leaves having red mid-veins and veinlets were observed only amongst the progeny raised after treatment with 0.25% ethyl methanesulfonate or 0.25% methyl ethanesulfonate + 0.25% ampicillin. The frequency of chlorophyll mutation after combined treatments in general was higher than after differential treatments. Methyl methanesulfonate among alkanesulfonates and neomycin among antibiotics induced higher frequencies of chlorophyll mutations. No chlorophyll mutant was produced by ampicillin.Although antibiotics induced a lower frequency of chlorophyll mutation than alkylating agents, the frequency and pattern of spectra of chlorophyll mutants showed an action of antibiotics in inducing mutation similar to that of alkylating agents. Therefore, it is considered that antibiotics are potential mutagens.