Human lymphocytes exposed to low doses of ionizing radiations become refractory to high doses of radiation as well as to chemical mutagens that induce double-strand breaks in DNA

Human lymphocytes exposed to low doses of ionizing radiation from incorporated tritiated thymidine or from X-rays become less susceptible to the induction of chromatid breaks by high doses of X-rays. This response can be induced by 0.01 Gy (1 rad) of X-rays, and has been attributed to the induction of a repair mechanism that causes the restitution of X-ray-induced chromosome breaks. Because the major lesions responsible for the induction of chromosome breakage are double-strand breaks in DNA, attempts have been made to see if the repair mechanism can affect various types of clastogenic lesions induced in DNA by chemical mutagens and carcinogens. When cells exposed to 0.01 Gy of X-rays or to low doses of tritiated thymidine were subsequently challenged with high doses of tritiated thymidine or bleomycin, which can induce double-strand breaks in DNA, or mitomycin C, which can induce cross-links in DNA, approximately half as many chromatid breaks were induced as expected. When, on the other hand, the cells were challenged with the alkylating agent methyl methanesulfonate (MMS), which can produce single-strand breaks in DNA, approximately twice as much damage was found as was induced by MMS alone. The results indicate that prior exposure to 0.01 Gy of X-rays reduces the number of chromosome breaks induced by double-strand breaks, and perhaps even by cross-links, in DNA, but has the opposite effect on breaks induced by the alkylating agent MMS. The results also show that the induced repair mechanism is different from that observed in the adaptive response that follows exposure to low doses of alkylating agents.     

Adaptive response to alkylating agents in the Drosophila sex-linked recessive lethal assay

The adaptive response to alkylating agents was studied in Drosophila assays under various treatment procedures. Pre-treatment of males as well as treatment of females with low doses of EMS (0.05-0.1 mM) did not affect sex-linked recessive lethal (SLRL) rates induced by high doses of this mutagen (10 mM, various feeding duration) in mature sperm cells. Pre-treatment of males with a low dose of MMS (0.1 mM) enhanced mutagenesis induced by the high dose of EMS (10 mM) at different stages of spermatogenesis, the observed effects exceeding the additive action of both mutagens. On the contrary, larval pre-treatment with the adaptive dose of EMS (0.05 mM) resulted in resistance of their germ cells to higher doses of EMS (1 mM). Specifically, offspring production increased while dominant lethality in F(1) as well SLRL frequency in F(2) was significantly reduced as compared with the effects of larval exposure to the challenge dose. Under the conditions tested, the adaptive response of germ cells to alkylating agents was demonstrated in larvae, but not in adult flies.     

Germline mutation induction at mouse repeat DNA loci by chemical mutagens

Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of male mice exposed to two monofunctional alkylating agents, ethylnitrosourea (ENU) and isopropyl methanesulfonate (iPMS), and a topoisomerase II inhibitor, etoposide. Pre-meiotic exposure to the alkylating agents resulted in a highly significant increase in ESTR mutation rate, but did not alter post-meiotically exposed cells. Pre-meiotic mutation induction by ENU and iPMS was linear within the interval of doses from 12.5 to 25mg/kg and reached a plateau at higher concentrations. Paternal exposure to etoposide resulted in ESTR mutation induction at meiotic stages but did not affect post- or pre-meiotic cells. The pattern of ESTR mutation induction after pre-meiotic and meiotic exposure to chemical mutagens was similar to that previously obtained by various traditional approaches for monitoring germline mutation in mice. The results of this study show that ESTR loci provide a new efficient experimental system for monitoring the genetic effects of chemical mutagens, capable of detecting increases in mutation rates at low doses of exposure.     

The modification of induced genetic change in yeast by an amino acid analogue

Summary Treatment of diploid yeast cultures with the amino acid analogue, para-fluorophenylalanine (PFPA), at concentrations which caused inhibition of growth, resulted in up to 5 fold increases in the frequency of mitotic gene conversion at two different heteroallelic loci. With haploid yeast cultures, growth in PFPA increased the rate of forward mutation to canavanine resistance by at least 2 fold.Growth of diploids in PFPA prior to exposure to the deaminating agent nitrous acid, the cross-linking agent mitomycin C, the alkylating chemical ethylmethanesulphonate (EMS) and UV light resulted in significant changes in the potency of these diverse mutagens to induce intragenic recombination. For all four mutagens, increased frequencies of gene convertants/viable cell were observed in those cultures which had been exposed to the amino acid analogue prior to mutagen treatment. In haploid WT yeast cells, amino acid analogue incorporation resulted in an enhanced frequency of UV induced forward mutation to canavanine resistance whilst in a DNA repair deficient rad 6 mutant this interaction between UV and PFPA was abolished.The results have been interpreted on the basis of incorporation of the analogue into enzymes involved with DNA replication with a consequent loss of fidelity of such enzymes and increased errors in base incorporation.     

Sensitivity of a S. cerevisiae RAD27 deletion mutant to DNA-damaging agents and in vivo complementation by the human FEN-1 gene

We have investigated the sensitivity to DNA-damaging agents of a strain of Saccharomyces cerevisiae containing a deletion of the RAD27 gene. The mutant strain is sensitive to a number of alkylating agents that modify DNA at a variety of positions, including one that produces primarily phosphotriesters. In contrast, the mutant strain is not sensitive to the oxidizing agent hydrogen peroxide. The introduction of a plasmid containing the FEN-1 gene (the human ortholog of the RAD27 gene) can substantially complement the sensitivity to alkylating agents observed in the mutant strain.     

Repair of DNA containing O6-alkylguanine.

O6-Alkylguanines, important DNA adducts formed by alkylating agents, can lead to mutations and to cell death unless repaired. The major pathway of repair involves the transfer of the alkyl group from the DNA to a cysteine acceptor site in the protein O6-alkylguanine-DNA alkyltransferase. The alkyltransferase brings about this transfer without need for cofactors and the DNA is restored completely by the action of a single protein, but the cysteine acceptor site is not regenerated and the number of O6-alkylguanines that can be repaired is equal to the number of active alkyltransferase molecules. The alkylated form of the protein is unstable in mammalian cells and is degraded rapidly. Cloning of the cDNAs for the alkyltransferase proteins from bacteria, yeast, and mammals indicates a significant similarity, particularly in the region surrounding the cysteine acceptor site. There is a major difference in the regulation of the alkyltransferase between mammalian cells and certain bacteria, where it is induced as part of the adaptive response to alkylating agents. Regulation of the content of alkyltransferase in mammalian cells differs with species and cell type and, in some cases, the level of the protein is increased by exposure to alkylating agents or X rays. A significant fraction of human tumor cell lines do not express the alkyltransferase gene and, thus, are much more sensitive to mutagenesis and killing by alkylating agents. The frequency of primary tumor cells that lack alkyltransferase protein is not yet clear. However, it is known that the level of alkyltransferase in tumors is a significant factor in resistance to both methylating agents and bifunctional chloroethylating agents. Inactivation of the alkyltransferase, which can be brought about by pretreatment with an alkylating agent or by exposure to O6-benzylguanine (a powerful nontoxic inhibitor), sensitizes tumor cells to these chemotherapeutic alkylating agents and may prove a useful therapeutic strategy.     

Hypersensitivity to very-low single radiation doses: Its relationship to the adaptive response and induced radioresistance

There is now little doubt of the existence of radioprotective mechanisms, or stress responses, that are upregulated in response to exposure to small doses of ionizing radiation and other DNA-damaging agents. Phenomenologically, there are two ways in which these induced mechanisms operate. First, a small conditioning dose (generally below 30 cGy) may protect against a subsequent, separate, exposure to radiation that may be substantially larger than the initial dose. This has been termed the adaptive response. Second, the response to single doses may itself be dose-dependent so that small acute radiation exposures, or exposures at very low dose rates, are more effective per unit dose than larger exposures above the threshold where the induced radioprotection is triggered. This combination has been termed low-dose hypersensitivity (HRS) and induced radioresistance (IRR) as the dose increases. Both the adaptive response and HRS/IRR have been well documented in studies with yeast, bacteria, protozoa, algae, higher plant cells, insect cells, mammalian and human cells in vitro, and in studies on animal models in vivo. There is indirect evidence that the HRS/IRR phenomenon in response to single doses is a manifestation of the same underlying mechanism that determines the adaptive response in the two-dose case and that it can be triggered by high and low LET radiations as well as a variety of other stress-inducing agents such as hydrogen peroxide and chemotherapeutic agents although exact homology remains to be tested. Little is currently known about the precise nature of this underlying mechanism, but there is evidence that it operates by increasing the amount and rate of DNA repair, rather than by indirect mechanisms such as modulation of cell-cycle progression or apoptosis. Changed expression of some genes, only in response to low and not high doses, may occur within a few hours of irradiation and this would be rapid enough to explain the phenomenon of induced radioresistance although its specific molecular components have yet to be identified.     

A single-molecule PCR approach to the measurement of induced expanded simple tandem repeat instability in vitro

Sensitive and precise models are needed to identify potential genotoxicity at environmentally relevant doses of mutagens. The size length alterations in expanded simple tandem repeat (ESTR) loci have been used as a biomarker of genetic instability caused by a variety of agents in the mouse germline. The mechanisms operating in both spontaneous and induced instability are poorly understood. We have developed a single-molecule polymerase chain reaction (SM-PCR) method to investigate mutation at the mouse ESTR locus Ms6-hm in the murine C3H/10T1/2 embryonic cell line. Growth of cells to levels of high cell density induced increased ESTR instability, with mutation frequencies 5.1-fold (+/-2.8) over sub-confluent cultures. Accordingly, cell cultures were maintained at sub-confluent levels for further investigations of the induction of ESTR mutation by genotoxic agents. Treatment with the DNA alkylating agent N-nitroso-N-ethylurea (ENU) resulted in a 1.94-fold (+/-1.1) increase in mutation frequency, similar to responses measured previously in the germline in vivo. Therefore, mutagen exposure can also affect somatic (non-meiotic) rapidly dividing mouse cells. This SM-PCR approach eliminates the requirement of sub-cloning individual treated cells, thereby, reducing the time needed to screen for ESTR mutation, and will be a very useful tool for future investigations into the mechanisms involved in ESTR mutation.     

Comparison of the levels of chromosome aberration and sister chromatid exchange induced by chemical mutagens in vitro

Dose dependencies of the induction of sister chromatid exchanges (SCEs) and chromosome aberrations were studied under in vivo exposure of mouse bone marrow cells to 5 alkylating agents. The efficacy of the induction of SCEs for all the substances was 20 to 60 times higher than that of the induction of chromosome aberrations. It was demonstrated that SCEs induced by chemical mutagens in vivo and in vitro are more sensitive tests than chromosome aberrations.     

The use of yeast cultures for the detection of environmental mutagens using a fluctuation test.

A microbial fluctuation test, modified for the detection of environmental mutagens has been evaluated using a number of strains of the yeast Saccharomyces cerevisiae. Auxotrophic diploid cultures of yeast which produce prototrophic colonies by both mitotic gene conversion and mutation have been extensively utilized for the detection and evaluation of chemicals showing genetic activity. A number of the yeast strains utilized were shown to be suitable for use in the fluctuation test although the time scales of the experiments were considerably extended (up to 16 days) compared to those involving bacteria. The yeast strains respond to doses of mutagens at least a 100-fold lower than that required in a conventional short exposure treat and plate experiment. In experiments involving the induction of mitotic gene conversion at the tryptophan-5 and histidine-4 loci in the fluctuation test significant increases in prototrophic cells were produced in the presence of the insecticide Lindex (0.05 microng/ml), the preservative Thiomersal (0.0001 microng/ml), a mahogany hair dye (0.01 microng/ml), the herbicide Paraquat (0.02 microng/ml) and the alkylating agent ethyl methane sulphonate (0.1 microng/ml). The results demonstrate that the fluctuation test provides an extremely sensitive assay for the detection of chemicals which show genetic activity in yeast at non-toxic concentrations.     

A novel role for DNA photolyase: binding to DNA damaged by drugs is associated with enhanced cytotoxicity in Saccharomyces cerevisiae.

DNA photolyase binds to and repairs cyclobutane pyrimidine dimers induced by UV radiation. Here we demonstrate that in the yeast Saccharomyces cerevisiae, photolyase also binds to DNA damaged by the anticancer drugs cis-diamminedichloroplatinum (cis-DDP) and nitrogen mustard (HN2) and by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Surprisingly, mutations in photolyase were associated with resistance of yeast cells to cis-DDP, MNNG, 4-nitroquinoline oxide (4NQO), and HN2. Transformation of yeast photolyase mutants with the photolyase gene increased sensitivity to these agents. Thus, while the binding of photolyase to DNA damaged by UV radiation aids survival of the cell, binding to DNA damaged by other agents may interfere with cell survival, perhaps by making the lesions inaccessible to the nucleotide excision repair system.     

Signaling of DNA damage is not sufficient to induce p53 response: (re)activation of wt p53 protein strongly depends on cellular context

It is generally accepted that exposure of cells to a variety of DNA-damaging agents leads to up-regulation and activation of wild-type (wt) p53 protein. We investigated the (re)-activation of p53 protein in two human cancer cell lines in which the gene for this tumor suppressor is not mutated: HeLaS(3) cervix carcinoma and MCF-7 breast cancer cells, by induction via different genotoxic and cytotoxic stimuli. Treatment of human cells with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or different anti-cancer drugs resulted in a strong DNA damage as evidenced by Comet assay and a marked increase in site-specific phosphorylation of H2AX. Unlike in MCF-7 cells, in HeLaS(3) cells the expression of p53 protein did not increase after MNNG treatment despite a strong DNA damage. However, other agents for example doxorubicin markedly induced p53 response in HeLaS(3) cells. After exposure of these cells to MNNG, the ATM-dependent effector proteins Chk2 and NBS1 were phosphorylated, thereby evidencing that MNNG-induced DNA breakage was recognized and properly signaled. In HeLaS(3) cells wt p53 protein is not functional due to E6-mediated targeting for accelerated ubiquitylation and degradation. Therefore, the activation of a p53 response to genotoxic stress in HeLaS(3) cells seems to depend on the status of E6 oncoprotein. Indeed, the induction of p53 protein in HeLaS(3) cells in response to distinct agents inversely correlates with the cellular level of E6 oncoprotein. This implicates that the capability of different agents to activate p53 in HeLaS(3) cells primarily depends on their inhibitory effect on expression of E6 oncoprotein.     

X-ray-sensitive mutants of Chinese hamster ovary cell line. Isolation and cross-sensitivity to other DNA-damaging agents

A standard technique of microbial genetics, which involves the transfer of cells from single colonies by means of sterile toothpicks, has been adapted to somatic cell genetics. Its use has been demonstrated in the isolation of X-ray-sensitive mutants of CHO cells. 9000 colonies have been tested and 6 appreciably X-ray-sensitive mutants were isolated. (D10 values 5-10-fold of wild-type D10 value.) A further 6 mutants were obtained which showed a slight level of sensitivity (D10 values less than 2-fold of wild-type D10 value). The 6 more sensitive mutants were also sensitive to bleomycin, a chemotherapeutic agent inducing X-ray-like damage. Cross-sensitivity to UV-irradiation and treatment with the alkylating agents, MMS, EMS and MNNG, was investigated for these mutants. Some sensitivity to these other agents was observed, but in all cases it was less severe than the level of sensitivity to X-irradiation. Each mutant showed a different overall response to the spectrum of agents examined and these appear to represent new mutant phenotypes derived from cultured mammalian cell lines. One mutant strain, xrs-7, was cross-sensitive to all the DNA-damaging agents, but was proficient in the repair of single-strand breaks.     

Adaptive response and induced resistance

Cellular stress responses are upregulated following exposure to radiation and other DNA-damaging agents. Therefore radiation response can be dose dependent so that small acute exposures (and possibly exposures at very low dose rates?) are more lethal per unit dose than larger exposures above a threshold (typically 10-40 cGy) where induced radioprotection is triggered. We have termed these interlinked phenomena low-dose hypersensitivity (HRS) and induced radioresistance (IRR) as the dose increases. HRS/IRR has been recorded in cell-survival studies with yeast, bacteria, protozoa, algae, higher plant cells, insect cells, mammalian and human cells in vitro, and in studies on animal normal-tissue models in vivo. There is indirect evidence that cell survival-related HRS/IRR in response to single doses is a manifestation of the same underlying mechanism that determines the well-known adaptive response in the two-dose case and that it can be triggered by high- and low-LET radiations as well as a variety of other stress-inducing agents such as hydrogen peroxide and chemotherapeutic agents. Little is currently known about the precise nature of this underlying mechanism, but there is evidence that it operates by increasing the amount and rate of DNA repair, rather than by indirect mechanisms such as modulation of cell-cycle progression or apoptosis. Changed expression of some genes, only in response to low and not high doses, may occur within a few hours of irradiation and this would be rapid enough to explain the phenomenon of induced radioresistance although its specific molecular components have yet to be identified. Net cancer risk is a balance between cell transformation and cell kill. Our known low-dose cell-survival responses suggest that lethality may more than compensate for transformation at low radiation doses. However, adaptive reduction in sensitivity to radio-mutation has also been reported, which implies the existence also of enhanced mutation following very low single doses. So far this has not been confirmed, but provided the trigger dose for mutational protection was lower than the trigger dose for protection against cytotoxicity, cell killing would still dominate over at least the first 10 cGy of low-LET exposure. This would lead to a non-linear, threshold, dose-risk relationship and even provide some explanation for anecdotal reports of apparent 'health promoting' effects and lowered cancer risk from very low exposure to ionising radiation.     

Heritable and cancer risks of exposures to anticancer drugs: inter-species comparisons of covalent deoxyribonucleic acid-binding agents

In the past years, several methodologies were developed for potency ranking of genotoxic carcinogens and germ cell mutagens. In this paper, we analyzed six sub-classes of covalent deoxyribonucleic acid (DNA) binding antineoplastic drugs comprising a total of 37 chemicals and, in addition, four alkyl-epoxides, using four approaches for the ranking of genotoxic agents on a potency scale: the EPA/IARC genetic activity profile (GAP) database, the ICPEMC agent score system, and the analysis of qualitative and quantitative structure-activity and activity-activity relationships (SARs, AARs) between types of DNA modifications and genotoxic endpoints. Considerations of SARs and AARs focused entirely on in vivo data for mutagenicity in male germ cells (mouse, Drosophila), carcinogenicity (TD₅₀s) and acute toxicity (LD₅₀s) in rodents, whereas the former two approaches combined the entire database on in vivo and in vitro mutagenicity tests. The analysis shows that the understanding and prediction of rank positions of individual genotoxic agents requires information on their mechanism of action. Based on SARs and AARs, the covalent DNA binding antineoplastic drugs can be divided into three categories. Category 1 comprises mono-functional alkylating agents that primarily react with N7 and N3 moieties of purines in DNA. Efficient DNA repair is the major protective mechanism for their low and often not measurable genotoxic effects in repair-competent germ cells, and the need of high exposure doses for tumor induction in rodents. Due to cell type related differences in the efficiency of DNA repair, a strong target cell specificity in various species regarding the potency of these agents for adverse effects is found. Three of the four evaluation systems rank category 1 agents lower than those of the other two categories. Category 2 type mutagens produce O-alkyl adducts in DNA in addition to N-alkyl adducts. In general, certain O-alkyl DNA adducts appear to be slowly repaired, or even not at all, which make this kind of agents potent carcinogens and germ cell mutagens. Especially the inefficient repair of O-alkyl—pyrimidines causes the high mutational response of cells to these agents. Agents of this category give high potency scores in all four expert systems. The major determinant for the high rank positions on any scale of genotoxic of category 3 agents is their ability to induce primarily structural chromosomal changes. These agents are able to cross-link DNA. Their high intrinsic genotoxic potency appears to be related to the number of DNA cross-links per target dose unit they can induce. A confounding factor among category 3 agents is that often the genotoxic endpoints occur closed to or toxic levels, and that the width of the mutagenic dose range, i.e., the dose area between the lowest observed effect level and the LD₅₀, is smaller (usually no more than 1 logarithmic unit) than for chemicals of the other two categories. For all three categories of genotoxic agents, strong correlations are observed between their carcinogenic potency, acute toxicity and germ cell specificity.     

Carcinogens can induce homologous recombination between duplicated chromosomal sequences in mouse L cells.

The ability of a series of DNA-damaging agents to induce homologous intrachromosomal recombination between duplicated genes in the chromosome of mouse cells was investigated. The target cells were the thymidine kinase-deficient mouse L-cell strain 333M, which contains a single integrated copy of a plasmid with two herpes simplex virus thymidine kinase (Htk) genes, each containing an 8-base-pair XhoI linker inserted at a unique site. Expression of a functional Htk enzyme requires a productive recombinational event between the two nonfunctional genes. The spontaneous rate of recombination in this strain is 3 per 10(6) cells per generation. The agents tested represent physical carcinogens (UV and ionizing radiation), a simple alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), an alkylating cross-linking agent (mitomycin C), and a reactive metabolite of a polycyclic aromatic hydrocarbon ((+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [BPDE] ). The background frequency of tk+ recombinants in the untreated population averaged 18 X 10(-6) +/- 5 X 10(-6). Ionizing radiation had little or no effect on recombination; exposure to mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, BPDE, or UV, at doses that lowered the survival to between 90 and 10% of the control, caused a dose-dependent increase in frequency of recombinants, reaching 50 X 10(-6) to 100 X 10(-6). No tk+ cells could be generated with a control cell line that contained only one mutant copy of the Htk gene. Molecular hybridization analysis showed that 85 to 90% of the tk+ recombinants retained the Htk gene duplication, consistent with nonreciprocal transfer of wild-type genetic information, gene conversion. In the rest, only a single copy of the Htk gene remained, reflecting a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids. Each recombinant tested contained an XhoI-resistant (wild-type) Htk gene.     

Formation and fate of cross-links induced by polyfunctional anticancer drugs in yeast

Summary A method to detect low levels of interstrand cross-links in DNA of Saccharomyces cerevisiae is described. Isopycnic ultracentrifugation of alkali-treated, unpurified Eaton press homogenates allows the detection of less than one cross-link per yeast chromosome. Efficient separation of single-and double-stranded DNA requires low cell density and addition of glycerol during homogenization. Using a yeast strain defective in excision repair, a dose dependent formation of interstrand cross-links after treatment of cells with biological doses of nitrogen mustard. Triaziquone and Chloramubil could be demonstrated. The most powerful of these alkylating agents is Triaziquone: half of the DNA molecules are shown to be cross-linked after a 12 min exposure to 9×10^-9 g/ml of the drug. The cross-linking reaction continues after excessive alkylating agent is removed. After having reached a maximum the fraction continues after excessive alkylating agent is removed. After having reached a maximum the fraction of renaturable DNA decreases upon further incubation. The speed of this after-reaction depends on temperature: 48 h after the end of treatment renaturability of DNA has almost completely disappeared when cells are kept at 36° C.     

Induction of homologous recombination following in utero exposure to DNA-damaging agents

Much of our understanding of homologous recombination, as well as the development of the working models for these processes, has been derived from extensive work in model organisms, such as yeast and fruit flies, and mammalian systems by studying the repair of induced double strand breaks or repair following exposure to genotoxic agents in vitro. We therefore set out to expand this in vitro work to ask whether DNA-damaging agents with varying modes of action could induce somatic change in an in vivo mouse model of homologous recombination. We exposed pregnant dams to DNA-damaging agents, conferring a variety of lesions at a specific time in embryo development. To monitor homologous recombination frequency, we used the well-established retinal pigment epithelium pink-eyed unstable assay. Homologous recombination resulting in the deletion of a duplicated 70 kb fragment in the coding region of the Oca2 gene renders this gene functional and can be visualized as a pigmented eyespot in the retinal pigment epithelium. We observed an increased frequency of pigmented eyespots in resultant litters following exposure to cisplatin, methyl methanesulfonate, ethyl methanesulfonate, 3-aminobenzamide, bleomycin, and etoposide with a contrasting decrease in the frequency of detectable reversion events following camptothecin and hydroxyurea exposure. The somatic genomic rearrangements that result from such a wide variety of differently acting damaging agents implies long-term potential effects from even short-term in utero exposures.