Mutagenesis In Mammalian Cells Research Articles

Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells

Here we report the development of a baculovirus-based delivery system that enables the efficient incorporation of unnatural amino acids into proteins in mammalian cells. We have exploited the large cargo-capacity (>30 kb) and stability of the double-stranded DNA genome of baculovirus to deliver to a variety of cell types all of the components required to genetically incorporate novel amino acids. These include the engineered tRNA/aminoacyl-tRNA synthetase pair and the nonsense mutant of the target gene. Mammalian cell transduction efficiency of baculovirus was significantly improved by incorporating genetic elements from mammalian viruses. Two polyspecific tRNA/aminoacyl-tRNA synthetase pairs were inserted into this expression system, enabling the site-specific incorporation of a variety of unnatural amino acids with novel chemical and biological properties into proteins.

Highly efficient, in vivo optimized, archaeal endonuclease for controlled RNA splicing in mammalian cells

ARCHAEA-ExPRESs is an mRNA modification technology that makes use of components derived from the Archaeon Methanocaldococcus jannaschii, namely the tRNA splicing endonuclease (MJ-EndA) and its natural substrate, the bulge-helix-bulge (BHB) structure (1). These components can perform both cis- and trans-splicing in cellular and animal models and may provide a convenient way to modulate gene expression using components independent of cellular regulatory networks. To use MJ-EndA in stable expression mammalian systems, we developed variants characterized by high efficiency and sustainable in vivo activity. The MJ-EndA variants were created by the introduction of proper localization signals followed by mutagenesis and direct selection in mammalian cells. Of note, enzyme selection used an in vivo selection method based on puromycin resistance conferred to cells by BHB-mediated intron splicing from an out-of-frame puromycin N-acetyl transferase (PAC) gene. This approach yielded several endonuclease variants, the best of which showed 40-fold higher activity compared to the parental enzyme and stable processing of 30% of the target mRNA. Notably, these variants showed complete compatibility with long-term expression in mammalian cells, suggesting that they may be usefully applied in functional genomics and genetically modified animal models.

DNA-Reactive Protein Monoepoxides Induce Cell Death and Mutagenesis in Mammalian Cells

Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to act by cross-linking cellular biomolecules, their exact mechanisms of action have not been established. In cells, these compounds form a mixture of DNA lesions, including nucleobase monoadducts, interstrand and intrastrand cross-links, and DNA-protein cross-links (DPCs). Interstrand DNA-DNA cross-links block replication and transcription by preventing DNA strand separation, contributing to toxicity and mutagenesis. In contrast, potential contributions of drug-induced DPCs are poorly understood. To gain insight into the biological consequences of DPC formation, we generated DNA-reactive protein reagents and examined their toxicity and mutagenesis in mammalian cells. Recombinant human O(6)-alkylguanine DNA alkyltransferase (AGT) protein or its variants (C145A and K125L) were treated with 1,2,3,4-diepoxybutane to yield proteins containing 2-hydroxy-3,4-epoxybutyl groups on cysteine residues. Gel shift and mass spectrometry experiments confirmed that epoxide-functionalized AGT proteins formed covalent DPC but no other types of nucleobase damage when incubated with duplex DNA. Introduction of purified AGT monoepoxides into mammalian cells via electroporation generated AGT-DNA cross-links and induced cell death and mutations at the hypoxanthine-guanine phosphoribosyltransferase gene. Smaller numbers of DPC lesions and reduced levels of cell death were observed when using protein monoepoxides generated from an AGT variant that fails to accumulate in the cell nucleus (K125L), suggesting that nuclear DNA damage is required for toxicity. Taken together, these results indicate that AGT protein monoepoxides produce cytotoxic and mutagenic DPC lesions within chromosomal DNA. More generally, these data suggest that covalent DPC lesions contribute to the cytotoxic and mutagenic effects of bis-electrophiles.

117 Non-B DNA structure-induced genetic instability in mammalian cells

Repetitive regions of genomic DNA can adopt a number of different structures, e.g. Z-DNA and they are at risk for increased genetic instability and disease development [As reviewed in (Zhao, Bacolla, Wang, & Vasquez, 2010)]. We found Z-DNA structure is intrinsically mutagenic in cultured mammalian cells and in chromosomes of transgenic mice (Wang Carbajal, Vijg, DiGiovanni, & Vasquez, 2008). Our recent data showed CG14 repeats, a model Z-DNA-forming sequence, could stall DNA polymerase progress in mammalian COS-7 cells, while AT14 repeats that does not form Z-DNA conformation, did not affect replication. These data suggested a replication-dependent model of Z-DNA-induced mutagenesis in mammalian cells. In addition, we also found that Z-DNA caused mutation and DNA breakage in the absence of DNA replication, and the spectrum of mutation from unreplicated system tended to have more large deletions than replicated DNA, suggested a replicated-independent pathway of Z-DNA-induced genetic instability (Wang, Christensen, & Vasquez, 2004). Several DNA repair proteins were found to be involved in Z-DNA induced mutagenesis, but in different ways: adding XPA protein back in, otherwise, deficient XP12RO cells increased Z-DNA-induced mutation frequency by −50%, while expressing MSH2 protein in Hec59 cells reduced Z-DNA-induced mutation frequency by −25%. Interestingly, cytosine methylation in CpG repeats induced higher frequencies of deletions and rearrangements with a broader mutant size distribution than that found with unmethylated CpG repeats. We discovered that unusual structural features of the methylated CpG sequences precluded local nucleosome assembly leading to genetic instability. Thus, non-B DNA-induced mutation appears to be a very complex process where DNA replication, repair and epigenetic modification are all involved.

Targeted Gene Modification of Hematopoietic Progenitor Cells in Mice Following Systemic Administration of a PNA-peptide Conjugate

Hematopoietic stem cell (HSC) gene therapy offers promise for the development of new treatments for a variety of hematologic disorders. However, efficient in vivo modification of HSCs has proved challenging, thus imposing constraints on the therapeutic potential of this approach. Herein, we provide a gene-targeting strategy that allows site-specific in vivo gene modification in the HSCs of mice. Through conjugation of a triplex-forming peptide nucleic acid (PNA) to the transport peptide, antennapedia (Antp), we achieved successful in vivo chromosomal genomic modification of hematopoietic progenitor cells, while still retaining intact differentiation capabilities. Following systemic administration of PNA-Antp conjugates, sequence-specific gene modification was observed in multiple somatic tissues as well as within multiple compartments of the hematopoietic system, including erythroid, myeloid, and lymphoid cell lineages. As a true functional measure of gene targeting in a long-term renewable HSC, we also demonstrate preserved genomic modification in the bone marrow and spleen of primary recipient mice following transplantation of bone marrow from PNA-Antp-treated donor mice. Our approach offers a minimally invasive alternative to ex vivo gene therapy, by eliminating the need for the complex steps of stem cell mobilization and harvesting, ex vivo manipulation, and transplantation of stem cells. Therefore, our approach may provide new options for individualized therapies in the treatment of monogenic hematologic diseases such as sickle cell anemia and thalassemia.

Safety Study of Culinary-Medicinal Royal Sun Agaricus, Agaricus brasiliensis S. Wasser et al. KA21 (Higher Basidiomycetes) Assessed by Prokaryotic as well as Eukaryotic Systems

As part of the safety evaluation of culinary-medicinal Royal Sun Agaricus, Agaricus brasiliensis KA21, for human consumption, we performed the bacterial reverse mutation test, the mouse micronucleus test, and mouse lymphoma test using A. brasiliensis KA21 as the test substance. The reverse mutation test utilized five bacterial strains, including Salmonella typhimurium TA100, TA1535, TA98, and TA1537, and Escherichia coli WP2 uvrAO. For the micronucleus test we used mice. For the mouse lymphoma test, we used one of the most commonly used mammalian cell mutagenesis systems; the L5178YTK +/- mouse lymphoma-TK assay detects the mutations at the thymidine kinase locus caused by base-pair changes, frameshift, and small deletions. All the tests were conducted according to the guidelines for genotoxicity testing of drugs by the Ministry of Health, Labor, and Welfare, Japan. In the bacterial reverse mutation test, no toxicity was observed up to a dose of 5,000 μg/plate. In the mouse micronucleus test, no toxicity was noted up to a dose of 1 g/kg body weight. In the mouse lymphoma test, frequency of the mutation was equal both in the presence or absence of KA21. Supporting the long history of human consumption of A. brasiliensis, the data shown in this study strongly indicate the safety of this mushroom.

Validation-based insertional mutagenesis identifies lysine demethylase FBXL11 as a negative regulator of NFκB

We describe a highly efficient use of lentiviral validation-based insertional mutagenesis (VBIM) to generate large populations of mammalian cells in which a strong promoter is inserted into many different genomic loci, causing greatly increased expression of downstream sequences. Many different selections or screens can follow, to isolate dominant mutant clones with a desired phenotypic change. The inserted promoter can be excised or silenced at will, to prove that the insertion caused the mutation. Cloning DNA flanking the insertion site identifies the locus precisely. VBIM virus particles are pseudotyped with VSV G protein, allowing efficient infection of most mammalian cell types, including non-dividing cells, and features are included that give high yields of stable virus stocks. In several different selections, useful mutants have been obtained at frequencies of approximately 10(-6) or higher. We used the VBIM technique to isolate mutant human cells in which the F-box leucine-rich protein 11 (FBXL11), a histone H3K36 demethylase, is shown to be a negative regulator of NFkappaB. High levels of FBXL11 block the ability of NFkappaB to bind to DNA or activate gene expression, and siRNA-mediated reduction of FBXL11 expression has the opposite effects. The H212A mutation of FBXL11 abolishes both its histone H3K36 demethylase activity and its ability to inhibit NFkappaB. Thus, we have used a powerful tool for mutagenesis of mammalian cells to reveal an aspect of the complex regulation of NFkappaB-dependent signaling.

Gene transcription increases DNA damage-induced mutagenesis in mammalian stem cells

DNA damage-induced mutations in actively transcribed genes in stem cells underlie genetic diseases including cancer. Here we investigated whether transcription affects DNA damage-induced gene mutations in mouse embryonic stem cells. To this aim we developed cell lines in which transcription of an Hprt minigene reporter, located at a different genomic positions, is regulated by the tTA2 Tetracycline-controlled transactivator. This allows detection of mutagenic events at both Hprt and tTA2 using a single selection. We found that UV-C and benzo[a]pyrenediolepoxide induced significantly more mutations at the Hprt minigene when the gene was transcribed. The transcription-associated increase in UV-C-induced mutagenesis appears independent of the integration site of the Hprt minigene. Molecular analysis of UV-induced Hprt mutants revealed that transcription of damaged DNA enhances the frequency of nucleotide substitutions and triggers the generation of intragenic deletions at the Hprt minigene. We speculate that these deletions are a result of error-prone DNA end-joining of double strand DNA breaks that are generated when replication forks collide with transcription complexes stalled at DNA lesions.

Integration of in vivo and in vitro approaches to characterize the toxicity of Antalarmin, a corticotropin-releasing hormone receptor antagonist

Non-clinical studies were conducted to evaluate the toxicity of Antalarmin, a corticotropin-releasing hormone type 1 receptor antagonist being developed for therapy of stress-related pathologies. Antalarmin was not genotoxic in bacterial mutagenesis assays, mammalian cell mutagenesis assays, or in vivo DNA damage assays. In a 14-day range-finding study in rats, Antalarmin doses ≥500 mg/kg/day (3000 mg/m 2/day) induced mortality. In a 90-day toxicity study in rats, no gross toxicity was seen at doses of 30, 100, or 300 mg/kg/day (180, 600, or 1800 mg/m 2/day, respectively). Antalarmin (300 mg/kg/day) induced mild anemia, increases in serum γ-glutamyl transferase activity, and microscopic hepatic pathology (bile duct hyperplasia and epithelial necrosis, periportal inflammation). Microscopic renal changes (cortical necrosis, inflammation, hypertrophy, nephropathy) were observed in rats at all Antalarmin doses. In a 14-day range-finding study in dogs, Antalarmin doses ≥50 mg/kg/day (1000 mg/m 2/day) induced repeated emesis and bone marrow suppression. In a 90-day toxicity study in dogs, Antalarmin (4, 8, or 16 mg/kg/day (80, 160, or 320 mg/m 2/day, respectively)) induced bone marrow and lymphoid depletion, but no gross toxicity. Comparative in vitro studies using rat, dog, and human neutrophil progenitors demonstrated that canine bone marrow cells are highly sensitive to Antalarmin cytotoxicity, while rat and human bone marrow cells are relatively insensitive. As such, the bone marrow toxicity observed in dogs is considered likely to over-predict Antalarmin toxicity in humans. The hepatic and renal toxicities seen in rats exposed to Antalarmin identify those tissues as the most likely targets for Antalarmin toxicity in humans.

Excision repair is required for genotoxin-induced mutagenesis in mammalian cells

Certain hexavalent chromium [Cr(VI)] compounds are human lung carcinogens. Although much is known about Cr-induced DNA damage, very little is known about mechanisms of Cr(VI) mutagenesis and the role that DNA repair plays in this process. Our goal was to investigate the role of excision repair (ER) pathways in Cr(VI)-mediated mutagenesis in mammalian cells. Repair-proficient Chinese hamster ovary cells (AA8), nucleotide excision repair (NER)-deficient (UV-5) and base excision repair (BER)-inhibited cells were treated with Cr(VI) and monitored for forward mutation frequency at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. BER was inhibited using methoxyamine hydrochloride (Mx), which binds to apurinic/apyrimidinic sites generated during BER. Notably, we found that both NER-deficient (UV-5 and UV-41) and BER-inhibited (AA8 + Mx) cells displayed attenuated Cr(VI) mutagenesis. To determine whether this was unique to Cr(VI), we included the alkylating agent, methylmethane sulfonate (MMS) and ultraviolet (UV) radiation (260 nm) in our studies. Similar to Cr(VI), UV-5 cells exhibited a marked attenuation of MMS mutagenesis, but were hypermutagenic following UV exposure. Moreover, UV-5 cells expressing human xeroderma pigmentosum complementation group D displayed similar sensitivity to Cr(VI) and MMS-induced mutagenesis as AA8 controls, indicating that the genetic loss of NER was responsible for attenuated mutagenesis. Interestingly, Cr(VI)-induced clastogenesis was also attenuated in NER-deficient and BER-inhibited cells. Taken together, our results suggest that NER and BER are required for Cr(VI) and MMS-induced genomic instability. We postulate that, in the absence of ER, DNA damage is channeled into an error-free system of DNA repair or damage tolerance.

Antimutagenic effect of lectin and N-methyl-N’-nitro-N-nitrosoguanidine on induced mutagenesis in mammalian cells in vitro

Factors and ways in which macromolecules influence the mutation process are considered. An antimutagenic effect is demonstrated in a study of the combined influence of lectins and the alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) on mutagenesis in Chinese hamster cells. Thus, in different schemes of treatment of cells with albumin and MNNG, the experimental frequency of mutations induced by the two agents was statistically reliably lower than the theoretically expected level for their independent combined action. The possibility that there exist common targets and mechanisms through which different types of mutagenic agents act on the cell DNA is discussed.

Characterization of carbadox-induced mutagenesis using a shuttle vector pSP189 in mammalian cells

Carbadox, a quinoxaline 1,4-dioxide derivative, is a known mutagen with its functional mechanism yet to be well defined. In the present study we used a shuttle vector assay in vitro to uncover the functional details of carbadox-induced mutagenesis in mammalian cells. The plasmid DNA of a shuttle vector pSP189 was treated with different doses of carbadox at 37 °C for 1 or 2 h with or without the presence of S9. The target gene SupF in the plasmid was sequenced after replication in Vero cells followed by amplification in Escherichia coli MBM7070 to evaluate mutation frequency. DNA sequencing analysis of recovered carbadox-induced mutations revealed 76.3% single base substitution, 7.9% single base insertion, 10.5% single base deletion and 5.3% large fragments deletion. All single base substitutions occurred at G:C base pairs, among which transversion and transition occurred at a 2:1 ratio. The mutations did not occur randomly in the supF gene, but had sequence specificity and hotspots instead: most substitutions were detected at the nucleotide N in a 5′-NNTTNN-3′ sequence; 75% of base insertions were seen in the 5′-TCC-3′ sequence; whereas all large fragments deletions occurred in the 5′-ANGGCCNAAA-3′ sequence. Nucleotide 129, 141 and 155 in the supF gene of plasmid pSP189 were identified as the hotspots for carbadox-induced mutations that accounted for 65% of all single base substitutions. We conclude that carbadox and its metabolites induce sequence-specific DNA mutations at high frequencies, therefore its safe usage in animal husbandry should be seriously considered.

Ochratoxin A-Induced Mutagenesis in Mammalian Cells Is Consistent with the Production of Oxidative Stress

Ochratoxin A (OTA) is a widespread mycotoxin in food and a powerful nephrocarcinogen in rats. The mutagenicity of OTA has been extensively investigated but with conflicting results, thus leaving open the mechanistic question for OTA carcinogenicity. Here, we examined the mutagenicity of OTA by using well-standardized mutation assays such as the hypoxanthine-guanine phosphoribosyltransferase (HPRT) assay in Chinese hamster V79 cells and the thymidine kinase assay in mouse lymphoma LY5178 cells. OTA-induced HPRT mutations were characterized at the molecular level. In V79 cells, OTA produced a dose- and time-related decrease in cell number as a consequence of the transitory cytostatic effect mediated by G2/M cell cycle arrest. In both mutation assays, OTA was weakly mutagenic and this effect was independent of biotransformation. OTA-induced mutations were characterized by point mutations (48%) and a lack of a detectable reverse-transcription polymerase chain reaction product (52%). The pattern of OTA-induced point mutations was similar to that of spontaneous mutants, suggesting that OTA induced an increase of the endogenous oxidative metabolism but not covalent DNA adducts. Our data support a model where OTA is mutagenic via oxidative DNA damage induction.

Genotoxic Mechanisms of Asbestos Fibers: Role of Extranuclear Targets

Asbestos fibers are carcinogenic to both humans and experimental animals. The continued discoveries of exposure routes whereby the general public is exposed to asbestos suggest a long-term, low-dose exposure for a large number of people. However, the mechanisms by which asbestos induces malignancy are not entirely understood. In previous studies, we have shown that asbestos is an effective gene and chromosomal mutagen when assayed using the highly sensitive AL mutation assay and that the mutagenicity is mediated by reactive oxygen species. The objective of the present study is to determine the origin of these radical species, particularly reactive nitrogen species, in fiber mutagenesis. Using the radical probe 5',6'-chloromethyl-2',7'-dihydroxyphenoxazine diacetate to trap reactive radical species, we showed that crocidolite increased the levels of oxyradicals in cytoplasts, in the absence of the nucleus, in a dose-dependent manner, which was reduced significantly by cotreatment with the radical scavenger dimethyl sulfoxide. Treatment of enucleated cells with crocidolite asbestos followed by rescue fusion using karyoplasts from control cells resulted in significant mutant induction, indicating that the nuclear-cytoplasmic interaction is necessary for fiber mutagenesis. Using the fluorescent probe 2,3-diaminonaphthotriazole, crocidolite fibers were shown to induce a dose-dependent increase of nitric oxide production, which was suppressed significantly by concurrent treatment with the nitric oxide synthase inhibitor, NG-methyl-L-arginine (L-NMMA). Similarly, there was a dose-dependent decrease in the mutation yield induced by crocidolites in the presence of graded doses of L-NMMA. These data showed that extranuclear targets play an essential role in the initiation of oxidative damage that mediates fiber mutagenesis in mammalian cells.

Mutagenesis of diploid mammalian genes by gene entrapment

The present study describes a genome-wide method for biallelic mutagenesis in mammalian cells. Novel poly(A) gene trap vectors, which contain features for direct cloning vector–cell fusion transcripts and for post-entrapment genome engineering, were used to generate a library of 979 mutant ES cells. The entrapment mutations generally disrupted gene expression and were readily transmitted through the germline, establishing the library as a resource for constructing mutant mice. Cells homozygous for most entrapment loci could be isolated by selecting for enhanced expression of an inserted neomycin-resistance gene that resulted from losses of heterozygosity (LOH). The frequencies of LOH measured at 37 sites in the genome ranged from 1.3 × 10−5 to 1.2 × 10−4 per cell and increased with increasing distance from the centromere, implicating mitotic recombination in the process. The ease and efficiency of obtaining homozygous mutations will (i) facilitate genetic studies of gene function in cultured cells, (ii) permit genome-wide studies of recombination events that result in LOH and mediate a type of chromosomal instability important in carcinogenesis, and (iii) provide new strategies for phenotype-driven mutagenesis screens in mammalian cells.

Mutations C1157F and C1234W of von Willebrand factor cause intracellular retention with defective multimerization and secretion.

The D3 domain of von Willebrand factor (VWF) is involved in the multimerization process of the protein through the formation of disulfide bridges. We identified heterozygous substitutions, C1157F and C1234W, in the VWF D3 domain in two unrelated families with unclassified and type 2A von Willebrand disease, respectively. VWF was characterized by a low plasmatic level, an abnormal binding to platelet GPIb and a high capacity of secretion from endothelial cells following DDAVP infusion. Using site-directed mutagenesis and expression in mammalian cells, we have investigated the impact of these mutations upon the multimerization, secretion and storage of VWF. Using COS-7 cells both mutated recombinant VWF (rVWF) displayed only lower molecular weight multimers. Pulse-chase analysis and endoglycosidase H digestion experiments showed the intracellular retention of mutated rVWF in pre-Golgi compartments. Study of hybrid rVWF obtained with a constant amount of wild-type (WT) DNA and increasing proportions of mutated plasmids established that both substitutions reduced the release of WT VWF in a dose-dependent manner and failed to form high molecular weight multimers. Using transfected AtT-20 stable cell lines, we observed similar granular storage of the two mutants and WT rVWF. Our data suggest that cysteines 1157 and 1234 play a crucial role in the early step of the folding of the molecule required for a normal transport pathway, maturation and constitutive secretion. In contrast, their substitution does not prevent the storage and inducible secretion of VWF.

Transposons Reanimated in Mice

Transposons have proved valuable in the genetic modification of many organisms but not mammals. New work reported by Ding et al. (2005) in this issue of Cell and by Collier et al. (2005) and Dupuy et al. (2005) in a recent issue of Nature now reveals that insertional mutagenesis in mammalian cells is possible thanks to modified derivatives of the piggyBac and Sleeping Beauty DNA transposons. However, the slow rate of proliferation of DNA transposons suggests that derivatives of the L1 retrotransposon might be more powerful for mutagenesis of germline cells. In either case, the future of transposon-driven genetic analyses of mice and other mammals looks promising.

Mutagenic effect of a keV range N + beam on mammalian cells

The radiobiological effects of a keV (5–20 keV) range nitrogen ion (N +) beam on mammalian cells were studied, particularly with regard to the induction of mutation in the cell genome. The experiment demonstrated that the 20 keV N + beam, which resulted in cell death to a certain extent, induced a 2–3 fold increase in the mutation rates at the CD59 gene locus of the mammalian A L cells as compared to the control. Within certain fluence ranges (0–6 × 10 14 N +/cm 2), the cell survival displayed a down–up–down pattern which is similar to the phenomenon known as ‘hyper-radiosensitivity’ manifested under low-dose irradiation; the CD59 mutation rate firstly showed a gradual rise up to a 3-fold increment above the background level as the ion fluence went up to 4 × 10 14 N +/cm 2, after this peak point however, a downtrend appeared though the ion fluence increased further. It was also observed that the fraction of CD59 mutation bears no proportional relation to ion energy in further experiments of mutation induction by N + beams with the incident energies of 5, 10, 15 and 20 keV at the same fluence of 3 × 10 14 N +/cm 2. Analyses of the deletion patterns of chromosome 11 in CD59 − mutants induced by 5–20 keV N + beams showed that these ions did not result in large-size chromosome deletions in this mammalian cell system. A preliminary discussion, suggesting that the mutagenic effect of such low-energy ion influx on mammalian cells could result from multiple processes involving direct collision of particles with cellular DNA, and cascade atomic and molecular reactions due to plentiful primary and secondary particles, was also presented. The study provided the first glimpse into the roles low-energy ions may play in inducing mutagenesis in mammalian cells, and results will be of much value in helping people to understand the contribution of low-energy ions to radiological effects of various ionising radiations.

Mammalian cell mutagenesis of the DNA adducts of vinyl chloride and crotonaldehyde

Vinyl chloride and crotonaldehyde are known mutagens and carcinogens that, through their reaction with DNA, form specific deoxyguanosine adducts. To investigate the mutagenic potential of a subset of the possible deoxyguanosine lesions, site-specific adducts of vinyl chloride and crotonaldehyde were synthesized, inserted into a shuttle vector, and replicated in mammalian cells. Mutation yields of the DNA adducts of vinyl chloride and crotonaldehyde were found to be 2% and 5-6%, respectively, thus suggesting that these adducts could contribute to the overall genotoxicity and carcinogenicity associated with exposure to these chemicals.

Mutagenicity and DNA repair of glycidamide-induced adducts in mammalian cells

Glycidamide (GA)-induced mutagenesis in mammalian cells is not very well understood. Here, we investigated mutagenicity and DNA repair of GA-induced adducts utilizing Chinese hamster cell lines deficient in base excision repair (BER), nucleotide excision repair (NER) or homologous recombination (HR) in comparison to parent wild-type cells. We used the DRAG assay in order to map pathways involved in the repair of GA-induced DNA lesions. This assay utilizes the principle that a DNA repair deficient cell line is expected to be affected in growth and/or survival more than a repair proficient cell. A significant induction of mutations by GA was detected in the hprt locus of wild-type cells but not in BER deficient cells. Cells deficient in HR or BER were three or five times, respectively, more sensitive to GA in terms of growth inhibition than were wild-type cells. The results obtained on the rate of incisions in BER and NER suggest that lesions induced by GA are repaired by short patch BER rather than long patch BER or NER. Furthermore, a large proportion of the GA-induced lesions gave rise to strand breaks that are repaired by a mechanism not involving PARP. It is suggested that these strand breaks, which might be the results from alkylation of the backbone phosphate, are misrepaired by HR during replication thereby leading to a clastogenic rather than a mutagenic pathway. The type of lesion responsible for the mutagenic effect of GA cannot be concluded from the results presented in this study.