Molecular analysis of hprt mutations induced by chromium picolinate in CHO AA8 cells

Chromium picolinate (CrPic) is a popular dietary supplement, marketed to the public for weight loss, bodybuilding, and control of blood sugar. Recommendations for long-term use at high dosages have led to questions regarding its safety. Previous studies have reported that CrPic can cause chromosomal aberrations and mutations. The purpose of the current work was to compare the mutagenicity of CrPic as a suspension in acetone versus a solution in DMSO, and to characterize the hprt mutations induced by CrPic in CHO AA8 cells. Treatments of 2% acetone or 2% DMSO alone produced no significant increase in 6-thioguanine (6-TG)-resistant mutants after 48 h exposures. Mutants resistant to 6-TG were generated by exposing cells for 48 h to 80 microg/cm(2) CrPic in acetone or to 1.0mM CrPic in DMSO. CrPic in acetone produced an average induced mutation frequency (MF) of 56 per 10(6) surviving cells relative to acetone solvent. CrPic in acetone was 3.5-fold more mutagenic than CrPic in DMSO, which produced an MF of 16.2. Characterization of 61 total mutations in 48 mutants generated from exposure to CrPic in acetone showed that base substitutions comprised 33% of the mutations, with transversions being predominant; deletions made up 62% of the mutations, with one-exon deletions predominating; and 1-4 bp insertions made up 5% of the characterized mutations. CrPic induced a statistically greater number of deletions and a statistically smaller number of base substitutions than have been measured in spontaneously generated mutants. These data confirm previous studies showing that CrPic is mutagenic, and support the contention that further study is needed to verify the safety of CrPic for human consumption.     

Chromium(III) tris(picolinate) is mutagenic at the hypoxanthine (guanine) phosphoribosyltransferase locus in Chinese hamster ovary cells

Chromium trispicolinate (CrPic) is a popular dietary supplement that is not regulated by the Food and Drug Administration. We are using this compound as a bio-available model to explore the role of Cr(III) in Cr(VI)-induced cancers. The ability of CrPic to cause mutations at the hypoxanthine (guanine) phosphoribosyltransferase (hprt) locus of CHO AA8 cells has been measured after a 48 h exposure. The highest dose tested was 80 μg/cm² CrPic, which, if fully soluble, would be equivalent to 1 mM or 0.44 mg/ml CrPic, and would correspond to 1 mM Cr(III) or 52 μg/ml Cr(III). This exposure resulted in 68±16% cell survival based on 48 h cell counts, and 24±11% survival by 7-day colony formation. Exposure of CHO cells to CrPic produced a statistically significant increase in 6-thioguanine (6-TG)-resistant cells over the dose range tested. The 80 μg/cm² CrPic exposure resulted in an average induced mutation frequency (MF) of 58 per 10⁶ surviving cells, or an average 40-fold increase in hprt mutants relative to untreated cells. An equivalent dose of 3 mM Pic was highly cytotoxic and did not yield hprt mutants. The dose range of 0.375–1.5 mM Pic produced a slight increase in hprt mutants, but the increase was not statistically significant. An equivalent dose of 1 mM chromic chloride yielded an induced MF of 9 per 10⁶ surviving cells, or a 10-fold increase in mutants with cell survivals of >100%. The coordination of Cr(III) with picolinic acid may make the metal more genotoxic than other forms of Cr(III). In light of the current results and the known ability of Cr(III) and CrPic to accumulate in tissues, as well as the growing evidence of Cr(III) involvement in Cr(VI)-induced cancers, we caution against ingestion of large doses of CrPic for extended periods.     

Chromium(III) tris(picolinate) is mutagenic at the hypoxanthine (guanine) phosphoribosyltransferase locus in Chinese hamster ovary cells.

Chromium trispicolinate (CrPic) is a popular dietary supplement that is not regulated by the Food and Drug Administration. We are using this compound as a bio-available model to explore the role of Cr(III) in Cr(VI)-induced cancers. The ability of CrPic to cause mutations at the hypoxanthine (guanine) phosphoribosyltransferase (hprt) locus of CHO AA8 cells has been measured after a 48 h exposure. The highest dose tested was 80 microg/cm(2) CrPic, which, if fully soluble, would be equivalent to 1mM or 0.44 mg/ml CrPic, and would correspond to 1mM Cr(III) or 52 microg/ml Cr(III). This exposure resulted in 68+/-16% cell survival based on 48 h cell counts, and 24+/-11% survival by 7-day colony formation. Exposure of CHO cells to CrPic produced a statistically significant increase in 6-thioguanine (6-TG)-resistant cells over the dose range tested. The 80 microg/cm(2) CrPic exposure resulted in an average induced mutation frequency (MF) of 58 per 10(6) surviving cells, or an average 40-fold increase in hprt mutants relative to untreated cells. An equivalent dose of 3mM Pic was highly cytotoxic and did not yield hprt mutants. The dose range of 0.375-1.5mM Pic produced a slight increase in hprt mutants, but the increase was not statistically significant. An equivalent dose of 1mM chromic chloride yielded an induced MF of 9 per 10(6) surviving cells, or a 10-fold increase in mutants with cell survivals of >100%. The coordination of Cr(III) with picolinic acid may make the metal more genotoxic than other forms of Cr(III). In light of the current results and the known ability of Cr(III) and CrPic to accumulate in tissues, as well as the growing evidence of Cr(III) involvement in Cr(VI)-induced cancers, we caution against ingestion of large doses of CrPic for extended periods.     

Lack of mutagenicity of chromium picolinate in the hypoxanthine phosphoribosyltransferase gene mutation assay in Chinese hamster ovary cells

Chromium picolinate (CrPic, Chromax) is a dietary supplement that is stable and more bioavailable than other commercially available forms of chromium. Chromium supplementation is known to enhance the action of insulin, particularly in insulin resistance and type 2 diabetes mellitus. A previous study reported that CrPic produced increases in mutations of the hypoxanthine phosphoribosyltransferase (Hprt) gene in Chinese hamster ovary (CHO) cell mutation tests. This study, however, evaluated CrPic produced by the testing laboratory and used an atypical 48 h exposure period for this test system. The current study evaluated the mutagenic potential of the most widely utilized commercial form of CrPic in CHO/Hprt mutation tests following International Conference on Harmonisation (ICH) Guidelines (+/-S9 metabolic activation with a 5h exposure) in addition to repeating the test with a 48 h exposure period -S9 activation. CrPic was suspended in dimethyl sulfoxide (DMSO) up to a concentration of 50 mg/mL; exposures were conducted under conditions in which precipitate was not evident and under conditions in which some precipitate of CrPic was visually evident in the cell culture medium at the highest concentrations (500 microg/mL). The concentrations evaluated for mutagenicity ranged from 15.6 to 500 microg/mL (+S9 and -S9) for the 5 h exposure and 31.3-500 microg/mL for the 48 h exposure (-S9). Only a slight degree of cytotoxicity was seen in the standard tests up to the limit of solubility in the medium. Toxicity, i.e., cloning efficiency < or =50% of the solvent control, but no mutagenic increases were observed at 500 microg/mL following a 48 h exposure period. The results of these studies showed that CrPic was non-mutagenic in two independent CHO/Hprt assays and in an assay using a 48 h exposure period.     

Mutational specificity of thymine deprivation-induced mutation in the lacI gene of Escherichia coli

LacI⁻ mutants obtained following 2 and 6 h of thymine deprivation were cloned and sequenced. The mutational spectra recovered were dissimilar. After 2 h of starvation the majority of mutations were base substitutions, largely G: C→C: G transversions. Frameshift mutations but not deletions were observed. In contrast, following 6 h of starvation, with the exception of the G: C→C: G transversion, all possible base substitutions were recovered. Moreover, several deletions but no frameshift events were observed. The differences in the mutational spectra recovered after two periods of thymine deprivation highlight the role of altered nucleotide pools and the potential influence of DNA replication mechanisms.     

A comparison of the genotoxicity of ethylnitrosourea and ethyl methanesulfonate in lacZ transgenic mice (Muta™Mouse)

We compared the induction of gene mutations and chromosomal aberrations by ethylating agents in lacZ transgenic mice (Muta™Mouse). Chromosomal aberrations were detected by the peripheral blood micronucleus assay. Gene mutations were detected in the lacZ transgene. A small amount of blood was sampled from a tail vessel during the expression time for fixation of gene mutations in vivo; this enabled us to detect and compare clastogenicity and gene mutations in the identical mouse. Single intraperitoneal injections of ENU (50–200 mg/kg) and EMS (100–400 mg/kg) strongly induced micronucleated reticulocytes (MN) detectable in peripheral blood 48 h after treatment. The maximum MN frequencies induced were 6.6% and 3.3% for ENU (100 mg/kg) and EMS (400 mg/kg), respectively (the control value was 0.3%). lacZ mutant frequency (MF) was analyzed in bone marrow and liver 7 days after treatment. Spontaneous MFs were 2.0–4.6x10⁻⁶. MF in bone marrow was increased by ENU to 3.4x10⁻⁵ at 200 mg/kg and induced by EMS to 1.8x10⁻⁵ at 400 mg/kg. In liver, however, both chemicals at their highest doses induced only slight increases in MF. The induction of both micronuclei and lacZ mutations in bone marrow by both ENU and EMS correlated better with O⁶-ethylguanine adducts than with N7-ethylguanine adducts. The mutants (19 for ENU and 12 for EMS) were subjected to DNA sequence analysis. Among EMS-induced mutations, 75% were GC to AT transitions, which were probably caused by O⁶-ethylguanine. Among ENU-induced mutations, in contrast, 40% occurred as AT base pair substitutions (6 AT to TA transversions and 2 AT to GC transitions) (no such mutations were induced by EMS). These results, together with the known reactivity of ENU to thymine suggest that thymine adducts play a significant role in the ENU mutagenesis.     

A newly established GDL1 cell line from gpt delta mice well reflects the in vivo mutation spectra induced by mitomycin C

In order to create a novel in vitro test system for detection of large deletions and point mutations, we developed an immortalized cell line. A SV40 large T antigen expression unit was introduced into fibroblasts derived from gpt delta mouse lung tissue and a selected clone was established as the gpt delta L1 (GDL1) cell line. The novel GDL1 cells were examined for mutant frequencies (MFs) and for molecular characterization of mutations induced by mitomycin C (MMC). The GDL1 cells were treated with MMC at doses of 0.025, 0.05, and 0.1 microg/mL for 24h and mutations were detected by Spi- and 6-thioguanine (6-TG) selections. The MFs of the MMC-treated cells increased up to 3.4-fold with Spi- selection and 3.5-fold with 6-TG selection compared to MFs of untreated cells. In the Spi- mutants, the number of large (up to 76 kilo base pair (kbp)) deletion mutations increased. A majority of the large deletion mutations had 1-4 base pairs (bp) of microhomology in the deletion junctions. A number of the rearranged deletion mutations were accompanied with deletions and insertions of up to 1.1 kbp. In the gpt mutants obtained from 6-TG selection, single base substitutions of G:C to T:A, tandem base substitutions occurring at the 5'-GG-3' or 5'-CG-3' sequence, and deletion mutations larger than 2 bp were increased. We compared the spectrum of MMC-induced mutations observed in vitro to that of in vivo using gpt delta mice, which we reported previously. Although a slight difference was observed in MMC-induced mutation spectra between in vitro and in vivo, the mutations detected in vitro included all of the types of mutations observed in vivo. The present study demonstrates that the newly established GDL1 cell line is a useful tool to detect and analyze various mutations including large deletions in mammalian cells.     

Effect of caloric restriction on Hprt lymphocyte mutation in aging rats

Caloric restriction (CR) reduces tumor incidence and retards aging in laboratory animals, including non-human primates. Because of the relationships among mutation, disease susceptibility, and aging, we investigated whether or not CR affects the accumulation of somatic cell mutations in aging animals. Starting at approximately 2 months of age, male CD rats (Harlan Sprague-Dawley-derived) were placed on different levels of dietary intake: ad libitum (AL) feeding, and 90% (10% CR), 75% (25% CR) and 60% (40% CR) of the total calories consumed by AL animals. At 3, 6, 12, and 24 months after the beginning of CR, Hprt mutant frequencies (MFs) were determined. The MFs measured in spleen lymphocytes from AL and CR rats sacrificed at 3 months of dietary restriction were similar for all dietary groups. However, the MFs at 6, 12, and 24 months of CR were significantly higher in AL-fed rats compared with animals on 40% CR: (4.5+/-0.4)x10(-6) versus (3.3+/-0.3)x10(-6) (P=0.032) in 6 months CR rats; (10.3+/-2.3)x10(-6) versus (7.3+/-1.2)x10(-6) in 12 months CR rats (P=0.04), and (18.3+/-3.2)x10(-6) versus (7.8+/-1.0)x10(-6) (P=0.001) in 24 months CR rats. In addition, rats receiving 25% CR for 24 months had a MF, (10.7+/-2.0)x10(-6), between the 40% CR and AL rats. Multiplex PCR of the Hprt gene in mutant clones from 12 and 24 months 40% CR rats and the corresponding AL rats detected deletions in 42% of CR mutants and 19% of AL mutants. Because of the difference in Hprt MF in the two groups, the estimated MF associated with deletions in CR rats was similar to the deletion MF in AL rats. This observation implies that the lower MF in CR rats is due to a reduction in smaller Hprt mutations (i.e. base substitutions and frameshifts). The pattern of smaller Hprt mutations from AL rats suggests that many were produced by reactive oxygen species (ROS). The results indicate that CR reduces the accumulation of spontaneous somatic cell mutation in aging rats, especially those caused by base substitutions and frameshifts.     

Mutagenicity in lung of Big Blue mice and induction of tandem-base substitutions in Salmonella by the air pollutant peroxyacetyl nitrate (PAN): predicted formation of intrastrand cross-links

Peroxyacetyl nitrate (PAN) is a ubiquitous air pollutant formed from NO₂ reacting with acetoxy radicals generated from ambient aldehydes in the presence of sunlight and ozone. It contributes to eye irritation associated with photochemical smog and is present in most urban air. PAN was generated in a chamber containing open petri dishes of Salmonella TA100 (gas-phase exposure). After subtraction of the background mutation spectrum, the spectrum of PAN-induced mutants selected at 3.1-fold above the background mutant yield was 59% GC→TA, 29% GC→AT, 2% GC→CG, and 10% multiple mutations — primarily GG→TT tandem-base substitutions. Using computational molecular modeling methods, a mechanism was developed for producing this unusual tandem-base substitution. The mechanism depends on the protonation of PAN near the polyanionic DNA to release NO₂⁺ resulting in intrastrand dimer formation. Insertion of AA opposite the dimerized GG would account for the tandem GG→TT transversions. Nose-only exposure of Big Blue^® mice to PAN at 78 ppm (near the MTD) was mutagenic at the lacI gene in the lung (mutant frequency ±S.E. of 6.16±0.58/10⁵ for controls versus 8.24±0.30/10⁵ for PAN, P=0.016). No tandem-base mutations were detected among the 40 lacI mutants sequenced. Dosimetry with ³H-PAN showed that 24 h after exposure, 3.9% of the radiolabel was in the nasal tissue, and only 0.3% was in the lung. However, based on the molecular modeling considerations, the labeled portion of the molecule would not have been expected to have been bound covalently to DNA. Our results indicate that PAN is weakly mutagenic in the lungs of mice and in Salmonella and that PAN produces a unique signature mutation (a tandem GG→TT transversion) in Salmonella that is likely due to a GG intrastrand cross-link. Thus, PAN may pose a mutagenic and possible carcinogenic risk to humans, especially at the high concentrations at which it is present in some urban environments.     

DNA base sequence changes induced by diethyl sulfate in postmeiotic male germ cells of Drosophila melanogaster

Summary The DNA base sequence changes induced by diethyl sulfate (DES) were analyzed in postmeiotic male germ cells of Drosophila melanogaster. 31 transmissible vermilion mutants were recovered in F₁ and F₂ generations, with a frequency of 2.6 × 10^–4 for the F₁, and of 1.8–13 × 10^–4 for the F₂. The results show that DES induces both base pair substitutions (93%) and deletions (7%). In accord with its relatively high ability to alkylate oxygens in DNA, the most frequent type of sequence alteration among the basepair changes are GC-AT transitions, accounting for 73% of mutations, followed by transversions AT-TA (10%). DES also induced AT-GC transitions and AT-CG transversions. Both induced deletions were intralocus deletions, not occurring between basepair repeats. No influence of neighboring bases on the mutation position was found.     

The mutagenic properties of BrdUTP in a random mutagenesis process

To explore the mutagenic properties of the nucleotide analogue bromodeoxyuridine triphosphate (BrdUTP), the wild type α-amylase (xamy) gene from Xanthomonas campestris pv. campestris 8004 was used as a mutational target. It was mutated using PCR techniques to partially replace deoxythymidine triphosphate (dTTP) with BrdUTP. A total of 18 mutants were selected for DNA sequencing from the mutagenesis libraries by their ability to hydrolyze the starch. The results showed that 70% of the total mutations were single base-pair substitutions; BrdUTP also induced deletion and insertion mutation types. Among single base-pair substitutions, the predominant mutation type is transition (84%), but three kinds of transversions (16%) were also detected. It thus mainly induces A:T → G:C and T:A → C:G transitions. This result indicated that when bromouracil is present as a deoxyribonucleoside triphosphate substrate it mainly paired with dAMP, and when it is present as a template base it could pair with free dGTP. Three mutational hot spots induced by BrdUTP were revealed in this work.     

Mutational specificity of ethyl methanesulfonate in excision-repair-proficient and -deficient strains of Drosophila melanogaster

Summary The vermilion gene was used as a target to determine the mutational specificity of ethyl methanesulfonate (EMS) in germ cells of Drosophila melanogaster. To study the impact of DNA repair on the type of mutations induced, both excision-repair-proficient (exr ⁺) and excision-repair-deficient (exr ^–) strains were used for the isolation of mutant flies. In all, 28 mutants from the exr ⁺ strain and 24 from the exr ^– strain, were characterized by sequence analysis. In two mutants obtained from the exr ⁺ strain, small deletions were observed. All other mutations were caused by single base-pair changes. In two mutants double base-pair substitutions had occurred. Of the mutations induced in the exr ⁺ strain, 22 (76%) were GCAT transitions, 3 (10%) ATTA transversions, 2 (6%) GCTA transversions and 2 (6%) were deletions. As in other systems, the mutation spectrum of EMS in Drosophila is dominated by GCAT transitions. Of the mutations in an exr ^– background, 12 (48%) were GCAT transitions, 7 (28%) ATTA transversions, 5 (20%) GCTA transversions and 1 (4%) was a ATGC transition. The significant increase in the contribution of transversion mutations obtained in the absence of an active maternal excision-repair mechanism, clearly indicates efficient repair of N-alkyl adducts (7-ethyl guanine and 3-ethyl adenine) by the excision-repair system in Drosophila germ cells.     

Total gene deletions and mutant frequency of the HPRT gene as indicators of radiation exposure in Chernobyl liquidators

This study was conducted to determine the utility of deletion spectrum and mutant frequency (MF) of the hypoxanthine phosphoribosyl transferase gene (HPRT) as indicators of radiation exposure in Russian Liquidators who served in 1986 or 1987 in the clean up effort following the nuclear power plant accident at Chernobyl. HPRT MF was determined using the cloning assay for 117 Russian Controls and 122 Liquidators whose blood samples were obtained between 1991 and 1998. Only subjects from whom mutants were obtained for deletion analysis are included. Multiplex PCR analysis was performed on cell extracts of 1080 thioguanine resistant clones from Controls and 944 clones from Liquidators. Although the deletion spectra of Liquidators and Controls were similar overall, the Liquidator deletion spectrum was heterogeneous over time. Most notable, the proportion of total gene deletions was higher in 1991–1992 Liquidators than in Russian Controls (χ²=10.5, p=0.001) and in 1993–1994 Liquidators (χ²=8.3, p=0.004), and was marginally elevated relative to 1995–1996 Liquidators (χ²=3.3, p=0.07). This type of mutation has been highly associated with radiation exposure. Total gene deletions were not increased after 1992. Band shift mutations were also increased in the 1991–1992 Liquidators but were associated with increased MF of both Liquidators and Controls (p=0.009), not with increased MF in 1991–1992 Liquidators (p=0.7), and hence are not believed to be associated with radiation exposure. Regression analysis demonstrated that relative to Russian Controls HPRT MF was elevated in Liquidators overall when adjusted for age and smoking status (37%, p=0.0001), and also was elevated in Liquidators sampled in 1991–1992 (72%, p=0.0076), 1993–1994 (22%, p=0.037), and 1995–1996 (62%, p=0.0001). In summary, HPRT MF was found to be the more sensitive and persistent indicator of radiation exposure, but the specificity of total gene deletions led to detection of probable heterogeneity of radiation exposure within the exposed population.     

Molecular characterization of Rifr mutations in Pseudomonas aeruginosa and Pseudomonas putida

The rpoB gene encoding for β subunit of RNA polymerase is a target of mutations leading to rifampicin resistant (Rif^r) phenotype of bacteria. Here we have characterized rpoB/Rif^r system in Pseudomonas aeruginosa and Pseudomonas putida as a test system for studying mutational processes. We found that in addition to the appearance of large colonies which were clearly visible on Rif selective plates already after 24 h of plating, small colonies grew up on these plates for 48 h. The time-dependent appearance of the mutant colonies onto selective plates was caused by different levels of Rif resistance of the mutants. The Rif^r clusters of the rpoB gene were sequenced and analyzed for 360 mutants of P. aeruginosa and for 167 mutants of P. putida. The spectrum of Rif^r mutations characterized for P. aeruginosa grown at 37 °C and that characterized for P. putida grown at 30 °C were dissimilar but the differences almost disappeared when the mutants of both strain were isolated at the same temperature, at 30 °C. The strong Rif^r phenotype of P. aeruginosa and P. putida was accompanied only with substitutions of these residues which belong to the putative Rif-binding pocket. Approximately 70% of P. aeruginosa mutants, which were isolated at 37 °C and expressed weak Rif^r phenotype, contained base substitutions in the N-terminal cluster of the rpoB gene. The differences in the spectra of mutations at 30 °C and 37 °C can be explained by temperature-sensitive growth of several mutants in the presence of rifampicin. Thus, our results imply that both the temperature for the growth of bacteria and the time for isolation of Rif^r mutants from selective plates are critical when the rpoB/Rif^r test system is employed for comparative studies of mutagenic processes in Pseudomonas species which are conventionally cultivated at different temperatures.     

Saccharomyces cerevisiae pol30 (Proliferating Cell Nuclear Antigen) Mutations Impair Replication Fidelity and Mismatch Repair

To understand the role of POL30 in mutation suppression, 11 Saccharomyces cerevisiae pol30 mutator mutants were characterized. These mutants were grouped based on their mutagenic defects. Many pol30 mutants harbor multiple mutagenic defects and were placed in more than one group. Group A mutations (pol30-52, -104, -108, and -126) caused defects in mismatch repair (MMR). These mutants exhibited mutation rates and spectra reminiscent of MMR-defective mutants and were defective in an in vivo MMR assay. The mutation rates of group A mutants were enhanced by a msh2 or a msh6 mutation, indicating that MMR deficiency is not the only mutagenic defect present. Group B mutants (pol30-45, -103, -105, -126, and -114) exhibited increased accumulation of either deletions alone or a combination of deletions and duplications (4 to 60 bp). All deletion and duplication breakpoints were flanked by 3 to 7 bp of imperfect direct repeats. Genetic analysis of one representative group B mutant, pol30-126, suggested polymerase slippage as the likely mutagenic mechanism. Group C mutants (pol30-100, -103, -105, -108, and -114) accumulated base substitutions and exhibited synergistic increases in mutation rate when combined with msh6 mutations, suggesting increased DNA polymerase misincorporation as a mutagenic defect. The synthetic lethality between a group A mutant, pol30-104, and rad52 was almost completely suppressed by the inactivation of MSH2. Moreover, pol30-104 caused a hyperrecombination phenotype that was partially suppressed by a msh2 mutation. These results suggest that pol30-104 strains accumulate DNA breaks in a MSH2-dependent manner.     

Kinds and spectrum of mutations induced by 1-nitrosopyrene adducts during plasmid replication in human cells.

1-Nitropyrene has been shown in bacterial assays to be the principal mutagenic agent in diesel emission particulates. It has also been shown to be mutagenic in human fibroblasts and carcinogenic in animals. To investigate the kinds of mutations induced by this carcinogen and compare them with those induced by a structurally related carcinogen, (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetra-hydrobenzo [a]pyrene (BPDE) (J.-L. Yang, V. M. Maher, and J. J. McCormick, Proc. Natl. Acad. Sci. USA 84:3787-3791, 1987), we treated a shuttle vector with tritiated 1-nitrosopyrene (1-NOP), a carcinogenic mutagenic intermediate metabolite of 1-nitropyrene which forms the same DNA adduct as the parent compound, and introduced the plasmids into a human embryonic kidney cell line, 293, for DNA replication to take place. The treated plasmid, pZ189, carrying a bacterial suppressor tRNA target gene, supF, was allowed 48 h to replicate in the human cells. Progeny plasmids were then rescued, purified, and introduced into bacteria carrying an amber mutation in the beta-galactosidase gene in order to detect those carrying mutations in the supF gene. The frequency of mutants increased in direct proportion to the number of DNA-1-NOP adducts formed per plasmid. At the highest level of adduct formation tested, the frequency of supF mutants was 26 times higher than the background frequency of 1.4 X 10(-4). DNA sequencing of 60 unequivocally independent mutant derived from 1-NOP-treated plasmids indicated that 80% contained a single base substitution, 5% had two base substitutions, 4% had small insertions or deletions (1 or 2 base pairs), and 11% showed a deletion or insertion of 4 or more base pairs. Sequence data from 25 supF mutants derived from untreated plasmids showed that 64% contained deletions of 4 or more base pairs. The majority (83%) of the base substitution in mutants from 1-NOP-treated plasmids were transversions, with 73% of these being G . C --> T . A. This is very similar to what we found previously in this system, using BPDE, but each carcinogen produced its own spectrum of mutations. Of the five hot spots for base substitution mutations produced in the supF gene with 1-NOP, two were the same as seen with BPDE-treated plasmids. However, the three other hot spots were cold spots for BPDE-treated plasmids. Conversely, four of the other five hot spots seen with BPDE-treated plasmids were cold spots for 1-NOP-treated plasmids. Comparison of the two carcinogens for the frequency of supF mutants induced per DNA adduct showed that 1-NOP-induced adducts were 3.8 times less than BPDE adducts. However, the 293 cell excised 1-NOP-induced adducts faster than BPDE adducts.     

HGPRT mutation induction by N-ethyl-N-nitrosourea as measured by 6-thioguanine resistance is higher in male than in female Syrian hamster fetuses

BACKGROUND: The consequences of mutations in embryonic and fetal cells are serious and contribute to high prenatal sensitivity to mutagenic agents. An understanding of the factors that influence the yield of such mutations is important for management of adverse effects of perinatal exposures. Resistance to 6-thioguanine (6-TG) can be utilized to study mutational events at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. HGPRT is X-linked and recessive. According to the Lyon hypothesis, male cells have only one X-chromosome and female cells randomly inactivate the second X-chromosome. This leads to the prediction that X-linked genes should be equally sensitive to the mutagenic effects of toxicants in male and female fetuses. METHODS: We tested this supposition by in utero exposure of Syrian hamster fetuses to N-ethyl-N-nitrosourea (ENU) at day 12 of gestation. ENU is a strong carcinogen and mutagen. HGPRT mutations were detected by selection with 6-TG. RESULTS: Surprisingly. the male cells had 4 to 5 times more 6-TG mutants than female cells, in two separate experiments (p<0.001). Ouabain resistance, reflecting a co-dominant autosomal locus, was used as a control, and we found that there was no significant difference between male and female cells (p=0.549). CONCLUSIONS: Possible reasons for the sex difference in mutations include escape of the second X-chromosome from inactivation in some of the female cells, or higher mutability in male cells. In any event, there is a gender difference in vulnerability to mutation of an X-linked gene that has previously not been appreciated, and that may be relevant to toxicological studies of such genes. HGPRT is frequently used to monitor mutagenic events in human fetuses.     

Comprehensive identification of mutations induced by heavy‐ion beam irradiation in Arabidopsis thaliana

Heavy‐ion beams are widely used for mutation breeding and molecular biology. Although the mutagenic effects of heavy‐ion beam irradiation have been characterized by sequence analysis of some restricted chromosomal regions or loci, there have been no evaluations at the whole‐genome level or of the detailed genomic rearrangements in the mutant genomes. In this study, using array comparative genomic hybridization (array‐CGH) and resequencing, we comprehensively characterized the mutations in Arabidopsis thaliana genomes irradiated with Ar or Fe ions. We subsequently used this information to investigate the mutagenic effects of the heavy‐ion beams. Array‐CGH demonstrated that the average number of deleted areas per genome were 1.9 and 3.7 following Ar‐ion and Fe‐ion irradiation, respectively, with deletion sizes ranging from 149 to 602 180 bp; 81% of the deletions were accompanied by genomic rearrangements. To provide a further detailed analysis, the genomes of the mutants induced by Ar‐ion beam irradiation were resequenced, and total mutations, including base substitutions, duplications, in/dels, inversions, and translocations, were detected using three algorithms. All three resequenced mutants had genomic rearrangements. Of the 22 DNA fragments that contributed to the rearrangements, 19 fragments were responsible for the intrachromosomal rearrangements, and multiple rearrangements were formed in the localized regions of the chromosomes. The interchromosomal rearrangements were detected in the multiply rearranged regions. These results indicate that the heavy‐ion beams led to clustered DNA damage in the chromosome, and that they have great potential to induce complicated intrachromosomal rearrangements. Heavy‐ion beams will prove useful as unique mutagens for plant breeding and the establishment of mutant lines.     

Evaluation of the database on mutant frequencies and DNA sequence alterations of vermilion mutations induced in germ cells of Drosophila shows the importance of a neutral mutation detection system

The vermilion gene in Drosophila has extensively been used for the molecular analysis of mutations induced by chemicals in germ cells in vivo. The gene is located on the X-chromosome and is a useful target for the study of mutagenesis since all types of mutations are generated. We have critically evaluated this system with respect to sensitivity for mutation induction and selectivity for different types of mutations, using a database of more than 600 vermilion mutants induced in postmeiotic male germ cells by 18 mutagens. From most of these mutants the mutation has been analysed. These data showed 336 base substitutions, 96 intra-locus DNA rearrangements and 78 multi-locus deletions (MLD). Mutants containing a MLD were either heterozygous sterile or homozygous and hemizygous lethal. The distribution of both basepair (bp) changes and intra-locus rearrangements over the coding region of the vermilion gene was uniform with no preferences concerning 5' or 3' regions, certain exons, splice sites, specific amino acid changes or nonsense mutations. Possible hotspots for base substitutions seem to be related to the type of DNA damage rather than to the vermilion system. Gene mutations other than bp changes were examined on sequence characteristics flanking the deletion breakpoints. Induction frequencies of vermilion mosaic mutants were, in general, higher than those of vermilion complete mutants, suggesting that persistent lesions are the main contributors to the molecular spectra. Comparison of induction frequencies of vermilion mutants and sex-linked recessive lethal (SLRL) mutants for the 18 mutagens showed that the sensitivity of the vermilion gene against a mutagenic insult is representative for genes located on the X-chromosome. The effect of nucleotide excision repair (NER) on the formation of SLRL mutants correlated with an increase of transversions in the vermilion spectra under NER deficient conditions. Furthermore, the clastogenic potency of the mutagens, i.e., the efficiency to induce chromosomal-losses vs. SLRL forward mutations, shows a positive correlation with the percentage of DNA deletions in the molecular spectra of vermilion mutants.