The "pro-drug" RibCys decreases the mutagenicity of high-LET radiation in cultured mammalian cells.

We are carrying out studies aimed at reducing the mutagenic effects of high-LET 56Fe ions and 12C ions (56Fe ions, 143 keV/microm; 12C ions, 100 keV/microm) with certain drugs, including RibCys [2-(R,S)-D-ribo-(1',2',3',4'-tetrahydroxybutyl)-thiazolidine-4(R)-carboxylic acid]. RibCys, formed by condensation of L-cysteine with D-ribose, is designed so that the sulfhydryl amino acid L-cysteine is released intracellularly through nonenzymatic ring opening and hydrolysis leading to increased levels of glutathione (GSH). RibCys (4 or 10 mM), which was present during irradiation and for a few hours after, significantly decreased the yield of CD59- mutants induced by radiation in AL human-hamster hybrid cells. RibCys did not affect the clonogenic survival of irradiated cells, nor was it mutagenic itself. These results, together with the minimal side effects reported in mice and pigs, indicate that RibCys may be useful, perhaps even when used prophylactically, in reducing the mutation load created by high-LET radiation in astronauts or other exposed individuals.

Radiation risk to low fluences of alpha particles may be greater than we thought.

Based principally on the cancer incidence found in survivors of the atomic bombs dropped in Hiroshima and Nagasaki, the International Commission on Radiation Protection (ICRP) and the United States National Council on Radiation Protection and Measurements (NCRP) have recommended that estimates of cancer risk for low dose exposure be extrapolated from higher doses by using a linear, no-threshold model. This recommendation is based on the dogma that the DNA of the nucleus is the main target for radiation-induced genotoxicity and, as fewer cells are directly damaged, the deleterious effects of radiation proportionally decline. In this paper, we used a precision microbeam to target an exact fraction (either 100% or < or =20%) of the cells in a confluent population and irradiated their nuclei with exactly one alpha particle each. We found that the frequencies of induced mutations and chromosomal changes in populations where some known fractions of nuclei were hit are consistent with non-hit cells contributing significantly to the response. In fact, irradiation of 10% of a confluent mammalian cell population with a single alpha particle per cell results in a mutant yield similar to that observed when all of the cells in the population are irradiated. This effect was significantly eliminated in cells pretreated with a 1 mM dose of octanol, which inhibits gap junction-mediated intercellular communication, or in cells carrying a dominant negative connexin 43 vector. The data imply that the relevant target for radiation mutagenesis is larger than an individual cell and suggest a need to reconsider the validity of the linear extrapolation in making risk estimates for low dose, high linear-energy-transfer (LET) radiation exposure.

Large-mutation spectra induced at hemizygous loci by low-LET radiation: evidence for intrachromosomal proximity effects.

A mathematical model is used to analyze mutant spectra for large mutations induced by low-LET radiation. The model equations are based mainly on two-break misrejoining that leads to deletions or translocations. It is assumed, as a working hypothesis, that the initial damage induced by low-LET radiation is located randomly in the genome. Specifically, we analyzed data for two hemizygous loci: CD59- mutants, mainly very large-scale deletions (>3 Mbp), in human-hamster hybrid cells, and data from the literature on those HPRT- mutants which involve at least deletion of the whole gene, and often of additional flanking markers (approximately 50-kbp to approximately 4.4-Mbp deletions). For five data sets, we estimated f, the probability that two given breaks on the same chromosome will misrejoin to make a deletion, as a function of the separation between the breaks. We found that f is larger for nearby breaks than for breaks that are more widely separated; i.e., there is a "proximity effect". For acute irradiation, the values of f determined from the data are consistent with the corresponding break misrejoining parameters found previously in quantitative modeling of chromosome aberrations. The value of f was somewhat smaller for protracted irradiation than for acute irradiation at a given total dose; i.e., the mutation data show a decrease that was smaller than expected for dose protraction by fractionation or low dose rate.

Gamma-ray mutagenesis studies in a new human-hamster hybrid, A(L)CD59(+/-), which has two human chromosomes 11 but is hemizygous for the CD59 gene.

Kraemer, S. M., Vannais, D. B., Kronenberg, A., Ueno, A. and Waldren, C. A. Gamma-Ray Mutagenesis Studies in a New Human-Hamster Hybrid, A(L)CD59(+/-), which has Two Human Chromosomes 11 but is Hemizygous for the CD59 Gene. Radiat. Res. 156, 10-19 (2001). We have developed a human-CHO hybrid cell line, named A(L)CD59(+/-), which has two copies of human chromosome 11 but is hemizygous for the CD59 gene and the CD59 cell surface antigen that it encodes. Our previous studies used the A(L) and A(L)C hybrids that respectively contain one or two sets of CHO chromosomes plus a single copy of human chromosome 11. The CD59 gene at 11p13.5 and the CD59 antigen encoded by it are the principal markers used in our mutagenesis studies. The hybrid A(L)CD59(+/-) contains two copies of human chromosome 11, only one of which carries the CD59 gene. The incidence of CD59 (-) mutants (formerly called S1(-)) induced by (137)Cs gamma rays is about fivefold greater in A(L)CD59(+/-) cells than in A(L) cells. Evidence is presented that this increase in mutant yield is due to the increased induction of certain classes of large chromosomal mutations that are lethal to A(L) cells but are tolerated in the A(L)CD59(+/-) hybrid. In addition, significantly more of the CD59 (-) mutants induced by (137)Cs gamma rays in A(L)CD59(+/-) cells display chromosomal instability than in A(L) cells. On the other hand, the yield of gamma-ray-induced CD59 (-) mutants in A(L)CD59(+/-) cells is half that of the A(L)C hybrid, which also tolerates very large mutations but has only one copy of human chromosome 11. We interpret the difference in mutability as evidence that repair processes involving the homologous chromosomes 11 play a role in determining mutant yields. The A(L)CD59(+/-) hybrid provides a useful new tool for quantifying mutagenesis and shedding light on mechanisms of genetic instability and mutagenesis.

The role of glutathione in the toxicity of smoke condensates from cigarettes that burn or heat tobacco.

Inhalation of cigarette smoke aerosol via active smoking is associated with the development of pulmonary inflammation. The cytotoxic potential of cigarette smoke has been hypothetically related to development of pulmonary inflammation since the release of intracellular contents from dead and dying cells has been reported to induce inflammatory foci. In this study, cigarette smoke condensates (CSCs) were prepared from Kentucky 1R4F reference cigarettes and cigarettes that primarily heat tobacco (Eclipse). The two CSCs were then compared for their ability to induce killing in human-hamster A(L) hybrid cells. CSCs prepared from Eclipse were much less cytotoxic than those prepared from reference cigarettes. At 60 microg CSC/ml culture medium, survival for CSC from Eclipse cigarettes was approximately 70% compared with 1% for CSC from burned K1R4F cigarettes. The observed reduction in CSC-Eclipse cytotoxicity toward these mammalian cells is consistent with the previously published observation of a 30% decline in pulmonary white cell count and 40% reduction in visual bronchitis index in human smokers who switched to Eclipse for 2 months. Results with N-acetylcysteine and buthionine-S-R-sulfoximine indicate that glutathione markedly reduces the cytoxicity of both CSCs.

An aberrant form of CD59 derived from HeLa cells.

We isolated a CD59 cDNA from a HeLa cell library which encoded a mutated form of CD59, having a single base substitution (G to T) that changed Arg55 to Met. Since this mutation occurred in the vicinity of the putative active site of CD59, we expressed the aberrant form of the protein in Chinese hamster ovary cells in order to test for effects upon function. We found that the mutation did not influence complement inhibitory activity of CD59. However, the epitopes recognised by the function-blocking CD59 monoclonal antibodies BRIC229 and YTH 53.1 were significantly affected. The G to T substitution caused loss of an Mnl I restriction site which permitted PCR-RFLP analysis. All of 52 human subjects studied, and our in-house HeLa cells, were homozygous for the normal CD59 sequence, indicating that the altered sequence was not due to normal variation in the general population. Therefore this mutation probably arose spontaneously in the HeLa cell line used to generate the commercially obtained cDNA library.

Induction of a bystander mutagenic effect of alpha particles in mammalian cells.

Ever since the discovery of X-rays was made by Röntgen more than a hundred years ago, it has always been accepted that the deleterious effects of ionizing radiation such as mutation and carcinogenesis are attributable mainly to direct damage to DNA. Although evidence based on microdosimetric estimation in support of a bystander effect appears to be consistent, direct proof of such extranuclear/extracellular effects are limited. Using a precision charged particle microbeam, we show here that irradiation of 20% of randomly selected A(L) cells with 20 alpha particles each results in a mutant fraction that is 3-fold higher than expected, assuming no bystander modulation effect. Furthermore, analysis by multiplex PCR shows that the types of mutants induced are significantly different from those of spontaneous origin. Pretreatment of cells with the radical scavenger DMSO had no effect on the mutagenic incidence. In contrast, cells pretreated with a 40 microM dose of lindane, which inhibits cell-cell communication, significantly decreased the mutant yield. The doses of DMSO and lindane used in these experiments are nontoxic and nonmutagenic. We further examined the mutagenic yield when 5-10% of randomly selected cells were irradiated with 20 alpha particles each. Results showed, likewise, a higher mutant yield than expected assuming no bystander effects. Our studies provide clear evidence that irradiated cells can induce a bystander mutagenic response in neighboring cells not directly traversed by alpha particles and that cell-cell communication process play a critical role in mediating the bystander phenomenon.

Mutant yields and mutational spectra of the heterocyclic amines MeIQ and PhIP at the S1 locus of human-hamster AL cells with activation by chick embryo liver (CELC) co-cultures.

Cooking meat and fish at high temperature creates heterocyclic amines (HA) including 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Several HA are mutagens in the Ames' S9/Salmonella assay. While PhIP is a substantial Ames' test mutagen, it is 1000-fold less active than the extraordinarily potent MeIQ. In contrast, MeIQ is significantly less mutagenic than PhIP in several mammalian cell assays, especially in repair-deficient Chinese hamster ovary (CHO) cells. HA are suspect human carcinogens on the basis of (i) epidemiological evidence, (ii) induction of tumors in rodents and monkeys, (iii) DNA adduct formation and (iv) mutagenic capacity. In this study, MeIQ and PhIP were significant mutagens at the S1 locus of co-cultivated human/hamster hybrid AL cells following metabolic activation by beta-napthoflavone (betaNF)-induced chick embryonic liver cultures (CELC). MeIQ was more mutagenic than PhIP in the CELC+AL cell assay. The mutant response curves increase with dose and then plateau (PhIP), or decrease (MeIQ). The inflections in these response curves coincide with dose-dependent decreases in cytochrome CYP1A1 activity. Molecular analysis of S1- mutants indicates that a substantial fraction, >65%, of the mutations induced by PhIP are deletions of 4.2 to 133 (Mbp); half are larger than 21 Mbp. Mutations induced by MeIQ were smaller, most (56%) being less than 5.7 Mbp. When appropriate metabolic activation is combined with a target locus, which can detect both small and large chromosomal mutations, both MeIQ and PhIP are significant mutagens and clastogens in repair proficient mammalian cells.

Antigen S1, encoded by the MIC1 gene, is characterized as an epitope of human CD59, enabling measurement of mutagen-induced intragenic deletions in the AL cell system.

S1 cell membrane antigen is encoded by the MIC1 gene on human chromosome 11. This antigen has been widely used as a marker for studies in gene mapping or in analysis of mutagen-induced gene deletions/mutations, which utilized the human-hamster hybrid cell-line, AL-J1, carrying human chromosome 11. Evidence is presented here which identifies S1 as an epitope of CD59, a cell membrane complement inhibiting protein. E7.1 monoclonal antibody, specific for the S1 determinant, was found to react strongly with membrane CD59 in Western blotting, and to bind to purified, urinary form of CD59 in ELISAs. Cell membrane expression of S1 on various cell lines always correlated with that of CD59 when examined by immunofluorescent staining. In addition, E7.1 antibody inhibited the complement regulatory function of CD59. Identification of S1 protein as CD59 has increased the scope of the AL cell system by enabling analysis of intragenic mutations, and multiplex PCR analysis of mutated cells is described, showing variable loss of CD59 exons.

Analysis of mutant quantity and quality in human-hamster hybrid AL and AL-179 cells exposed to 137Cs-gamma or HZE-Fe ions.

We measured the number of mutants and the kinds of mutations induced by 137Cs-gamma and by HZE-Fe (56Fe [600 MeV/amu, LET = 190 KeV/micrometer) in standard AL human hamster hybrid cells and in a new variant hybrid, AL-179. We found that HZE-Fe was more mutagenic than 137Cs-gamma per unit dose (about 1.6 fold), but was slightly less mutagenic per mean lethal dose, DO, at both the S1 and hprt- loci of AL cells. On the other hand, HZE-Fe induced about nine fold more complex S1- mutants than 137Cs-gamma rays, 28% vs 3%. 137Cs-gamma rays induced about twice as many S1- mutants and hprt-mutants in AL-179 as in AL cells, and about nine times more of the former were complex, and potentially unstable kinds of mutations.

Mutagenic effects of a single and an exact number of alpha particles in mammalian cells.

One of the main uncertainties in risk estimation for environmental radon exposure using lung cancer data from underground miners is the extrapolation from high- to low-dose exposure where multiple traversal is extremely rare. The biological effects of a single alpha particle are currently unknown. Using the recently available microbeam source at the Radiological Research Accelerator Facility at Columbia University, we examined the frequencies and molecular spectrum of S1- mutants induced in human-hamster hybrid (A(L)) cells by either a single or an exact number of alpha particles. Exponentially growing cells were stained briefly with a nontoxic concentration of Hoechst dye for image analysis, and the location of individual cells was computer-monitored. The nucleus of each cell was irradiated with either 1,2,4, or 8 alpha particles at a linear energy transfer of 90 keV/microm consistent with the energy spectrum of domestic radon exposure. Although single-particle traversal was only slightly cytotoxic to A(L) cells (survival fraction approximately 0.82), it was highly mutagenic, and the induced mutant fraction averaged 110 mutants per 10(5) survivors. In addition, both toxicity and mutant induction were dose-dependent. Multiplex PCR analysis of mutant DNA showed that the proportion of mutants with multilocus deletions increased with the number of particle traversals. These data provide direct evidence that a single a particle traversing a nucleus will have a high probability of resulting in a mutation and highlight the need for radiation protection at low doses.

A low, adaptive dose of gamma-rays reduced the number and altered the spectrum of S1- mutants in human-hamster hybrid AL cells.

We examined the effects of a low, adaptive dose of 137Cs-gamma-irradiation (0.04 Gy) on the number and kinds of mutants induced in AL human-hamster hybrid cells by a later challenge dose of 4 Gy. The yield of S1- mutants was significantly less (by 53%) after exposure to both the adaptive and challenge doses compared to the challenge dose alone. The yield of hprt- mutants was similarly decreased. Incubation with cycloheximide (CX) or 3-aminobenzamide largely negated the decrease in mutant yield. The adaptive dose did not perturb the cell cycle, was not cytotoxic, and did not of itself increase the mutant yield above background. The adaptive dose did, however, alter the spectrum of S1- mutants from populations exposed only to the adaptive dose, as well as affecting the spectrum of S1- mutants generated by the challenge dose. The major change in both cases was a significant increase in the proportion of complex mutations compared to small mutations and simple deletions.

Mutant quantity and quality in mammalian cells (AL) exposed to cesium-137 gamma radiation: effect of caffeine.

We examined the effect of caffeine (1,3,7-trimethylxanthine) on the quantity and quality of mutations in cultured mammalian AL human-hamster hybrid cells exposed to 137Cs gamma radiation. At a dose (1.5 mg/ml for 16 h) that reduced the plating efficiency (PE) by 20%, caffeine was not itself a significant mutagen, but it increased by approximately twofold the slope of the dose-response curve for induction of S1- mutants by 137Cs gamma radiation. Molecular analysis of 235 S1- mutants using a series of DNA probes mapped to the human chromosome 11 in the AL hybrid cells revealed that 73 to 85% of the mutations in unexposed cells and in cells treated with caffeine alone, 137Cs gamma rays alone or 137Cs gamma rays plus caffeine were large deletions involving millions of base pairs of DNA. Most of these deletions were contiguous with the region of the MIC1 gene at 11p13 that encodes the S1 cell surface antigen. In other mutants that had suffered multiple marker loss, the deletions were intermittent along chromosome 11. These "complex" mutations were rare for 137Cs gamma irradiation (1/63 = 1.5%) but relatively prevalent (23-50%) for other exposure conditions. Thus caffeine appears to alter both the quantity and quality of mutations induced by 137Cs gamma irradiation.

Heavy ion mutagenesis: linear energy transfer effects and genetic linkage.

We have characterized a series of 69 independent mutants at the endogenous hprt locus of human TK6 lymphoblasts and over 200 independent S1-deficient mutants of the human x hamster hybrid cell line AL arising spontaneously or following low-fluence exposures to densely ionizing Fe ions (600 MeV/amu, linear energy transfer = 190 keV/microns). We find that large deletions are common. The entire hprt gene (> 44 kb) was missing in 19/39 Fe-induced mutants, while only 2/30 spontaneous mutants lost the entire hprt coding sequence. When the gene of interest (S1 locus = M1C1 gene) is located on a nonessential human chromosome 11, multilocus deletions of several million base pairs are observed frequently. The S1 mutation frequency is more than 50-fold greater than the frequency of hprt mutants in the same cells. Taken together, these results suggest that low-fluence exposures to Fe ions are often cytotoxic due to their ability to create multilocus deletions that may often include the loss of essential genes. In addition, the tumorigenic potential of these HZE heavy ions may be due to the high potential for loss of tumor suppressor genes. The relative insensitivity of the hprt locus to mutation is likely due to tight linkage to a gene that is required for viability.

Molecular analysis of mutagenesis by high LET radiation.

Mutation induction by high linear energy transfer [LET] alpha particles and gamma-rays was scored in the human hamster hybrid [AL] cells. Southern blotting technique was used to analyse the molecular changes in the DNA from both the HGPRT- and S1- mutants. Dose dependent mutagenesis in the AL cells irradiated with the charged particles was higher by almost 20 fold at the S1 than the corresponding HGPRT locus. Southern analysis of the mutants induced by the high LET particles showed mostly multilocus deletion at both the HGPRT and S1 genes.

Megabase pair deletions in mutant mammalian cells following exposure to amsacrine, an inhibitor of DNA topoisomerase II.

Amsacrine, [4'-(9-acridinylamino)-methanesulfon-m-auisidide], belongs to the class of cancer chemotherapeutic agents that target DNA topoisomerase II. We show that, over its cytotoxic range, amsacrine is a potent mutagen of the S1 phenotype in the AL (human x hamster) hybrid cell line. By contrast, amsacrine induction of the HPRT- phenotype in AL cells is at least two decades less frequent and is not concentration dependent. Such differential mutation frequencies are hypothesized to reflect the concomitant loss of essential genes neighboring the hprt locus. It may be that some amsacrine cytotoxicity is due to the inactivation of essential genes by large deletions. The AL mutation system is well suited for the detection and mapping of mutations which are large deletions because its MIC1 locus, which controls the expression of the selectable cell surface antigen S1, is on a single human chromosome. This human chromosome 11 is in addition to the genome of the Chinese hamster ovary cell and is basically nonessential. Since there are no sister human chromosomes in AL cells, deletions which extend beyond the MIC1 locus may be conveniently and unambiguously mapped. We have detected the presence or absence of 9 different chromosome 11 markers in 48 S1- mutants cloned from amsacrine-treated cultures. We find that almost all (92%) of the mutants have deletions of at least 1.5-2 megabase pairs in length. The distribution of marker loss frequencies flanking the MIC1 locus does not appear symmetric with respect to distance from that locus. We speculate that amsacrine-induced deletions are mediated by a series of subunit exchanges between overlapping topoisomerase II dimers at the bases of replicons or larger chromosomal structures such as replicon clusters or chromosome minibands.

Identification of class-mu glutathione transferase genes GSTM1-GSTM5 on human chromosome 1p13.

The GSTM1, GSTM2, GSTM3, GSTM4, and GSTM5 glutathione transferase genes have been mapped to human chromosome 1 by using locus-specific PCR primer pairs spanning exon 6, intron 6, and exon 7, as probes on DNA from human/hamster somatic cell hybrids. For GSTM1, the assignment was confirmed by Southern blot hybridization to a pair of 12.5/2.4-kb HindIII fragments. The GSTM1-specific primer pairs can be used to identify individuals carrying non-null GSTM1 alleles. The organization of these five genes was confirmed by the isolation of a yeast artificial chromosome clone (GSTM-YAC2) that contains all five genes. With this clone, the location of the GSTM1-GSTM5 gene cluster on chromosome 1 was confirmed by fluorescence in situ hybridization. Both regional assignment using the fractional length method and examination of probe signal with reference to R-banded chromosomes induced by BrdU places the gene cluster in or near the 1p13.3 region. The close physical proximity of the GSTM1 and GSTM2 loci, which share 99% nucleotide sequence identity over 460 nucleotides of 3'-untranslated mRNA, suggests that the GSTM1-null allele may result from unequal crossing-over.

Chrysotile fiber is a strong mutagen in mammalian cells.

Although chrysotile asbestos is a proven human carcinogen, several studies have concluded that these fibers are not mutagenic to cultured mammalian cells. We show here, on the other hand, that when tested using the AL cell system that detects both intragenic and multilocus mutations, chrysotile is indeed mutagenic and comparable in strength to that of gamma-rays. Southern analysis of the induced mutants shows that the majority contains large deletions ranging in size from a few thousand to several million base pairs. Results of our study demonstrate that, while chrysotile may be less durable in vivo than the amphibole fibers such as crocidolites and amosites, it can effectively create genetic damage involved in the cancer process.

The use of human repetitive DNA to target selectable markers into only the human chromosome of a human-hamster hybrid cell line (AL)

We used the plasmid BLUR-8 that contains an 800-base pair (bp) sequence of human repetitive Alu DNA in a cotransfection protocol to target the plasmids pSV2neo or EBO-pcD-leu-2 (hygro) into a single site of the sole human chromosome, number 11, of a Chinese hamster-human hybrid cell line (AL). The neo and hygro plasmids confer resistance to the antibiotics G418 and hygromycin, respectively. Of the 33 cotransfected clones with single-site insertions, 1/13 without BLUR-8 and 6/20 with BLUR-8 were only in human chromosome 11. A frequency of insertion of 1/13 is not different than expected by chance (rho = 0.3512). On the other hand, the probability that 6/20 insertions, as seen with BLUR-8, occurred by chance is low (rho = 0.0003). We suggest that the human DNA sequences contained in BLUR-8 targeted insertions into only the human chromosome.

A single base change at a splice acceptor site leads to a truncated CAD protein in Urd-A mutant Chinese hamster ovary cells.

We have previously reported the isolation and characterization of mutant Chinese hamster ovary (CHO-K1) cells of the Urd-A complementation group, which require uridine for growth, are deficient in the activities of the first three enzymes of de novo UMP biosynthesis, and produce markedly reduced amounts of a truncated form of the multifunctional protein CAD, which contains these three enzyme activities. We report here that a single base change of G to A at a highly conserved RNA splice acceptor site is responsible for the phenotype of this mutant. In addition to a small amount of apparently normal CAD mRNA, this mutation causes production of two alternative forms of CAD mRNA in the mutant, one that includes the intron just prior to the mutation and one that excludes the exon just after the mutation. The affected splice site is located at the intron-exon boundary just preceding the exon that encodes the beginning of the aspartate transcarbamylase (ATCase) domain of the CAD protein. Both intron inclusion and exon exclusion during RNA processing introduce a translation stop codon upstream of the region encoding this domain, resulting in the production of the truncated CAD protein seen in the Urd-A mutant. This mutation also results in markedly decreased levels of CAD mRNA and protein in the mutant.