Induced Mutation Frequency Research Articles

Review Article

Review Article

Frameshift mutations induced by the acridine mustard ICR-191 in embryos and in the adult gill and hepatopancreas of rpsL transgenic zebrafish

To determine whether frameshift mutations can be detected in rpsL transgenic zebrafish ( Brachydanio rerio), embryos, and adult fish were treated with 6-chloro-9-[3-(2-chloroethylamino)-propylamino]-2-methoxyacridine (ICR-191). Embryos exposed to 0, 10, or 20 μM ICR-191 in a water bath for 18 h exhibited induced mutant frequencies (MFs) of 14 × 10 −5, 16 × 10 −5, and 25 × 10 −5, respectively. Only embryos exposed to 20 μM ICR-191 showed a significant increase in MF. The mutational spectra differed between the control and ICR-191-treated groups and single G:C pair insertions, which are a marked characteristic of ICR-191 mutagenesis, were observed in both 10 and 20 μM-treated embryos. In adult fish treated with 1 μM ICR-191 in a water bath for 18 h, a significant increase in MFs was observed in both gill (12 × 10 −5 and 44 × 10 −5 in control and treated fish, respectively), and hepatopancreas (5 × 10 −5 and 29 × 10 −5, respectively) 2 weeks after exposure. Sequence analysis showed that 58% of mutations in gill and 94% of mutations in hepatopancreas were single G:C pair insertions, which is typical of mutations induced by ICR-191. Additionally, these mutations occurred predominantly at a single site (CC sequence at bps 140–141) in the rpsL gene. Three weeks after exposure, however, the increased MFs and prominent mutational spectra of ICR-treated fish were undetectable. These findings suggest that using our protocols the rpsL transgenic zebrafish mutation assay is more effective for adult fish than for embryos, but that frameshift mutations can be detected in both embryos and adults at appropriate sampling times after treatment with ICR-191.

Qualitatively and quantitatively similar effects of active and passive maternal tobacco smoke exposure on in utero mutagenesis at the HPRT locus.

BackgroundInduced mutagenesis in utero is likely to have life-long repercussions for the exposed fetus, affecting survival, birth weight and susceptibility to both childhood and adult-onset diseases, such as cancer. In the general population, such exposures are likely to be a consequence of the lifestyle choices of the parents, with exposure to tobacco smoke one of the most pervasive and easily documented. Previous studies attempting to establish a direct link between active smoking and levels of somatic mutation have largely discounted the effects of passive or secondary exposure, and have produced contradictory results.MethodsData from three studies of possible smoking effects on in utero mutagenesis at the HPRT locus were compiled and reanalyzed, alone and in combination. Where possible, passive exposure to environmental tobacco smoke was considered as a separate category of exposure, rather than being included in the non-smoking controls. Molecular spectra from these studies were reanalyzed after adjustment for reported mutation frequencies from the individual studies and the entire data set.ResultsA series of related studies on mutation at the X-linked HPRT locus in human newborn cord blood samples has led to the novel conclusion that only passive maternal exposure to tobacco mutagens has a significant effect on the developing baby. We performed a pooled analysis of the complete data from these studies, at the levels of both induced mutation frequency and the resulting mutational spectrum.ConclusionOur analysis reveals a more commonsensical, yet no less cautionary result: both active maternal smoking and secondary maternal exposure produce quantitatively and qualitatively indistinguishable increases in fetal HPRT mutation. Further, it appears that this effect is not perceptibly ameliorated if the mother adjusts her behavior (i.e. stops smoking) when pregnancy is confirmed, although this conclusion may also be affected by continued passive exposure.

Overproduction of DNA polymerase eta does not raise the spontaneous mutation rate in diploid human fibroblasts

Telomerase-immortalized lines of diploid xeroderma pigmentosum variant (XP-V) fibroblasts (XP115LO and XP4BE) were complemented for constitutive or regulated expression of wild-type human DNA polymerase eta (hpol eta). The ectopic gene was expressed from a retroviral LTR at a population average of 34- to 59-fold above the endogenous (mutated) mRNA and high levels of hpol eta were detected by immunoblotting. The POLH cDNA was also cloned downstream from an ecdysone-regulated promoter and transduced into the same recipient cells. Abundance of the wild-type mRNA increased approximately 10-fold by addition of ponasterone to the culture medium. Complemented cell lines acquired normal resistance to the cytotoxic effects of UVC, even in the presence of 1 mM caffeine. They also tolerated higher levels of UVC-induced template lesions during nascent DNA elongation when compared to normal fibroblasts (NHF). UVC-induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase ( HPRT) locus were measured in the XP115LO + XPV cell line overproducing hpol eta constitutively (E. Bassett, N.M. King, M.F. Bryant, S. Hector, L. Pendyala, S.G. Chaney, M. Cordeiro-Stone, The role of DNA polymerase eta in translesion synthesis past platinum–DNA adducts in human fibroblasts, Cancer Res. 64 (2004) 6469–6475). Induced mutation frequencies were significantly reduced, even below those observed in NHF; however, the average mutation frequency in untreated cultures was about three-fold higher than in the isogenic vector-control cell line. In this study, spontaneous HPRT mutation frequencies were measured at regular intervals, as isogenic fibroblasts either lacking or overproducing hpol eta were expanded for 100 population doublings. The mutation rates estimated from these results were not significantly increased in XP115LO cells expressing abnormal levels of hpol eta, relative to the cells lacking this specialized polymerase. These findings suggest that diploid human fibroblasts with normal DNA repair capacities and intact checkpoints are well protected against the potential mutagenic outcome of overproducing hpol eta, while still benefiting from accurate translesion synthesis of UV-induced pyrimidine dimers.

Extreme cytotoxicity and susceptibility to hprt mutagenesis in Ku-deficient xrs-6 cells treated with bleomycin in plateau phase

In an attempt to determine the possible role of Ku-dependent end joining in mutagenesis resulting from DNA double-strand breaks, mutations induced by bleomycin at the hprt locus in plateau phase normal CHO-K1 and Ku-deficient xrs-6 cells were examined. Plateau phase xrs-6 cells were 500-fold more sensitive to chronic bleomycin treatment than were CHO-K1 cells. XRCC4-deficient XR-1 cells were approximately 100-fold and DNA-PKcs-deficient XR-C1 and V-3 cells 15- to 30-fold more sensitive than CHO-K1 cells. These hypersensitivities are much greater than those previously reported for acute treatments with bleomycin or ionizing radiation. While the induced mutation frequencies at comparable levels of survival were slightly lower in xrs-6 cells, mutations were induced by bleomycin at much lower concentrations in xrs-6 than in CHO-K1 cells. For both cell lines bleomycin treatment resulted in a marked increase in the incidence of complete hprt deletions, while point mutations in hprt cDNA were rare. The results suggest that bleomycin-induced double-strand breaks tend to generate very large deletions in both cell lines and that this effect occurs at much lower levels of double-strand breaks in Ku-deficient than in normal cells.

Dose-Rate Effect on Transgenerational Mutation Frequencies in Spermatogonial Stem Cells of the Medaka Fish

The estimation of transgenerational genetic risk of radiation exposure to non-human species is crucial for the protection of ecosystems. Here we determined the frequency of specific-locus mutations at the five pigmentation loci in medaka spermatogonial stem cells after gamma irradiation at 0.03 cGy/min and 95 cGy/min. At each total dose, the mutation frequency was significantly lower in the 0.03-cGy/min group than in the 95-cGy/min group, suggesting a dose-rate effect. The ratio of the induced mutation frequency at 0.03 cGy/min to that at 95 cGy/min was approximately 0.42 from 0 to 1.9 Gy and approximately 0.33 from 1.9 to 4.75 cGy. In the mouse, this ratio is estimated to be 0.33 (Russell and Kelly, Proc. Natl. Acad. Sci. USA 79, 542-544, 1982). It is thus possible that the magnitude of the dose-rate effect on transgenerational mutation frequencies is comparable between mouse and medaka spermatogonia, suggesting similar dose-rate effects among vertebrates.

The role of DNA polymerase eta in translesion synthesis past platinum-DNA adducts in human fibroblasts.

Cisplatin, a widely used chemotherapeutic agent, has been implicated in the induction of secondary tumors in cancer patients. This drug is presumed to be mutagenic because of error-prone translesion synthesis of cisplatin adducts in DNA. Oxaliplatin is effective in cisplatin-resistant tumors, but its mutagenicity in humans has not been reported. The polymerases involved in bypass of cisplatin and oxaliplatin adducts in vivo are not known. DNA polymerase eta is the most efficient polymerase for bypassing platinum adducts in vitro. We evaluated the role of polymerase eta in translesion synthesis past platinum adducts by determining cytotoxicity and induced mutation frequencies at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus in diploid human fibroblasts. Normal human fibroblasts (NHF1) were compared with xeroderma pigmentosum variant (XPV) cells (polymerase eta-null) after treatment with cisplatin. In addition, XPV cells complemented for polymerase eta expression were compared with the isogenic cells carrying the empty expression vector. Cytotoxicity and induced mutagenicity experiments were measured in parallel in UVC-irradiated fibroblasts. We found that equitoxic doses of cisplatin induced mutations in fibroblasts lacking polymerase eta at frequencies 2- to 2.5-fold higher than in fibroblasts with either normal or high levels of polymerase eta. These results indicate that polymerase eta is involved in error-free translesion synthesis past some cisplatin adducts. We also found that per lethal event, cisplatin was less mutagenic than UVC. Treatment with a wide range of cytotoxic doses of oxaliplatin did not induce mutations above background levels in cells either expressing or lacking polymerase eta, suggesting that oxaliplatin is nonmutagenic in human fibroblasts.

The potent colon carcinogen, 1,2-dimethylhydrazine induces mutations primarily in the colon

1,2-Dimethylhydrazine (DMH) is a potent colon carcinogen that is commonly used as an initiator in studies of the effects of diet on colon cancer. Previous studies have shown that although this compound produces multiple tumors in the colons in most individuals of every species tested, it is, at best, marginally mutagenic in the bone marrow (micronuclei) and small intestine (Dlb-1 mutations). Here we report its mutagenicity in the primary target tissue, the colonic epithelium, by means of the Muta™Mouse cII assay, an assay for intragenic mutations in a lambda shuttle vector that is integrated into the genome of these mice. Animals were treated with 0, 10, 20, or 30 mg/ml of DMH, either as a single injection or as multiple weekly injections, and mutations were measured in both the small intestine and colon. In the small intestine, there was an increase in mutant frequency following a single injection of DMH, but this was significant only at 30 mg/kg [induced mutant frequency (MF) = 18 × 10 −5 mutants/plaque]. In the colon, following a single treatment of DMH, there was a significant increase in mutant frequency at doses of 20 and 30 mg/kg (induced MF = 17 × 10 −5 and 23 × 10 −5 mutants/plaque, respectively). Following ten injections of 20 mg/kg of DMH, there was a greater than ten-fold increase in mutations in the colon (MF = 275 × 10 −5 mutants/plaque) than the small intestine (MF = 25 × 10 −5 mutants/plaque). These results show that DMH, under the conditions typically used for dietary studies, induces large numbers of mutations in the tissue in which it induces most cancers.

The mutagenic effects of 7,12-dimethylbenz[ a]anthacene, 3-methylcholanthrene and benzo[ a]pyrene to the developing Syrian hamster fetus measured by an in vivo/in vitro mutation assay

The transplacental mutagenicity of three polycylic aromatic hydrocarbons, 7,12-dimethylbenz[ a]anthacene (DMBA), 3-methylcholanthrene (MC) and benzo[ a]pyrene (BP), was measured by an in vivo/in vitro mutation assay. Fetal sensitivity and dose-response characteristics with regard to transplacental mutagenesis by these compounds have never been quantified. In the current experiment, pregnant Syrian hamsters were exposed to these compounds at day 12 of gestation. Twenty-four hours later the fetuses were removed and their cells were allowed a 5-day expression time in culture. They were then seeded for colony formation and also for mutation selection by diphtheria toxin. DMBA at 0.2 mmol/kg (51.3 mg/kg) had an induced mutant frequency of 1.56×10 −4 mutants per surviving cell. This was 598 times the historical control. DMBA at 0.2 mmol/kg was 3.6 times more potent than the highly mutagenic positive control, ethylnitrosourea, at 1 mmol/kg. DMBA also caused a dose-dependent increase in cloning efficiency, which was highly correlated with mutation rate. BP and MC were less effective than DMBA, causing increased mutations that were 31.6 and 17.7 times the historical control, respectively, and for neither was there any correlation of mutation rate with cloning efficiency. The special effectiveness of DMBA as a transplacental mutagen may relate to its ability to cause increased cell division and fixation of DNA lesions as mutations.

Mutation frequencies in murine keratinocytes as a function of carcinogenic status.

A link between genetic abnormalities and carcinogenesis is well established. It follows that a correlation exists between mutation frequency and malignant progression. We have determined the spontaneous and DNA damage-induced mutation frequencies for a series of cell lines derived from SENCAR mouse keratinocytes at various stages of malignant progression. Nontumorigenic mouse keratinocytes (3PC), papillomas (MT1/2), squamous-cell carcinomas (CH72), and spindle-cell carcinomas (CH72T4) were transfected with damaged or undamaged shuttle vectors containing a supF mutation reporter gene. The plasmid mutation frequencies were determined by blue/white screening. The spontaneous plasmid mutation frequency of the squamous-cell carcinoma line was slightly higher than the mutation frequencies of the other cell lines tested. The DNA damage induced by triplex-directed psoralen crosslinks increased the mutation frequencies sixfold to eighteenfold in all cell lines tested, with no significant differences among the cell lines. Sequence analyses revealed that the spindle-cell carcinoma line had a different spontaneous mutation spectrum from the other cell lines. DNA damage-induced mutations were predominantly point mutations at the triplex-duplex junction in all of the cell lines tested, as expected. These data suggested that a strong mutator phenotype was not required for progression to an advanced malignant phenotype in our model system.

In vivo mutation in gene A of splenic lymphocytes from phiX174 transgenic mice.

Single-burst analysis was applied to a forward assay for gene A mutation in splenic lymphocytes of phiX174 transgenic mice for the purpose of optimizing analytical parameters for identifying in vivo mutations. The effect of varying the cutoff value for an in vivo burst on induced mutant frequency, fold increase, and the significance of the difference between control and N-ethyl-N-nitrosourea (ENU)-treated mice was calculated by two different methods. The plating density was reduced to an average of less than 10 background mutant plaques per aliquot in order to separate in vitro bursts. The spectrum of mutations contributing < 60 plaques per aliquot from control animals was not significantly different from the control spectra from E. coli or transgenic phiX174 cells in culture. The mutant spectra from ENU-treated animals was highly different between mutant bursts of > 80 plaques per aliquot compared to mutations contributing < 60 plaques per aliquot (P < 0.000001), the former fitting the spectrum expected for ENU-induced mutations. The latter spectrum was also different from control animals and E. coli (P < 0.000001), suggesting the difference was caused by ex vivo mutation. With the mutations found in this study, the total number of reported target sites for gene A is now 33. The results support the interpretation that, in contrast to results for the lacI transgene, 100% of mutants isolated in gene A from control animals and cells were fixed in E. coli. We attribute the difference between the two genes to hot-spot sites for mutation in gene A and to a testable hypothesis that the mosaic plaque assay for the lacI transgene underestimates the frequency of ex vivo mutants.

化学物質によるｉｎ ｖｉｔｒｏ及びｉｎ ｖｉｖｏにおける突然変異の定量的解析に関する研究

The mutagenicities of methyl, ethyl, isopropyl, and butyl methanesulfonates and the corresponding alkylderivatives of N-nitrosoureas were examined using E. coli Hs30R strain deficient in the excision repair system. Their mutagenic capacities were standardized by converting the experimentally obtained mutation frequencies into those per unit of the concentration-time integrated dose with various dimensions, μM×h, mM×h, and M×h. The mutation frequencies under various conceptual exposure conditions were computed by using those at unit integrated dose, on the assumption that the dose-response relation is linear on a log-log scale.An attempt was made to establish a procedure for kinetic formulation of the mutagenic chemical modification of the mutational target(s). Using the proposed kinetic formulation, a quantitative analysis was made of the temperature dependence of mutation frequency in terms of the activation energies for mutagenic modification induced in the cell by mutagens. E. coli Hs30R strain was treated with alkyl methanesulfonates and N-alkyl-N-nitrosoureas at 37, 25, and 15°C for 1 hour and the induced mutation frequencies were normalized in terms of the concentration-time integrated dose. The plot of the normalized mutation frequency versus the reciprocal of the reaction temperature gave a straight line which corresponds to the Arrehnius plot, enabling us to estimate the apparent activation energy of the initial chemical event leading to mutation.In order to study the mutagenic interaction between two chemicals, an analytical method for the classification of the mutagenic interactions as “equivalent” or “independent” is proposed. The mutation frequencies induced by simultaneous (combined) treatments of Salmonella typhimurium TA100 with two simple alkylating agents were compared with those induced by separate treatments with the two mutagens. Furthermore, the comparison was made of the frequencies of micronucleated reticulocytes (MNRETs) produced in ddY mice treated with model chemicals such as alkylating agents, spindle poisons, and an oxidizing agent. The results indicate that the analytical method proposed here is appropriate to evaluate the combined effects of chemicals both in bacterial mutagenesis and micronucleus induction.

Response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine and its role in mutagenesis.

To understand the response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and its role in human mutagenesis. The response of the human REV3 gene to MNNG was measured in human 293 cells and FL cells by RT-PCR. By using antisense technology, mutation analysis at HPRT locus (on which lesion-targeted mutation usually occurs) was conducted in human transgenic cell line FL-REV3(-) by 8-azaguanine screening, and mutation occurred on undamaged DNA template was detected by using a shuttle plasmid pZ189 as the probe in human transgenic cell lines 293-REV3(-) and FL-REV3(-). The blockage effect of REV3 was measured by combination of reverse transcription-polymerase chain reaction to detect the expression of antisense REV3 RNA and Western blotting to detect the REV3 protein level. The human REV3 gene was significantly activated by MNNG treatment, as indicated by the upregulation of REV3 gene expression at the transcriptional level in MNNG-treated human cells, with significant increase of REV3 expression level by 0.38 fold, 0.33 fold and 0.27 fold respectively at 6 h, 12 h and 24 h in MNNG-treated 293 cells (P<0.05); and to 0.77 fold and 0.65 fold at 12 h and 24 h respectively in MNNG-treated FL cells (P<0.05). In transgenic cell line (in which REV3 was blocked by antisense REV3 RNA), high level of antisense REV3 RNA was detected, with a decreased level of REV3 protein. MNNG treatment significantly increased the mutation frequencies on undamaged DNA template (untargeted mutation), and also at HPRT locus (lesion-targeted mutation). However, when REV3 gene was blocked by antisense REV3 RNA, the MNNG-induced mutation frequency on undamaged DNA templates was significantly decreased by 3.8 fold (P<0.05) and 5.8 fold (P<0.01) respectively both in MNNG-pretreated transgenic 293 cells and FL cells in which REV3 was blocked by antisense RNA, and almost recovered to their spontaneous mutation levels. The spontaneous HPRT mutation was disappeared in REV3-disrupted cells, and induced mutation frequency at HPRT locus significantly decreased from 8.66 x 10(-6) in FL cells to 0.14 x 10(-6) in transgenic cells as well (P<0.01). The expression of the human REV3 can be upregulated at the transcriptional level in response to MNNG. The human REV3 gene plays a role not only in lesion-targeted DNA mutagenesis, but also in mutagenesis on undamaged DNA templates that is called untargeted mutation.

Mutation induction with UVB in mouse skin epidermis is suppressed in acute high-dose exposure

The time and dose dependence of ultraviolet B (UVB)-induced mutant frequency (MF) in skin epidermis and dermis was studied with transgenic Muta™ mice harboring λgt10 lacZ shuttle vector. Mutants of the lacZ transgene appearing in these tissues after 0.5 kJ/m 2 UVB irradiation were fully expressed in 3–7 days, and the frequencies of those fully expressed mutants were maintained for at least the following 3 weeks. These fully expressed MFs increased dose-dependently, with the initial slope for the epidermis four times larger than that for dermis. Surprisingly, in epidermis, an inhibition of the dose-dependent mutation induction was evident after irradiation above 0.5 kJ/m 2 UVB, lowering the increment more than eight-fold, while such suppression was not observed in dermis. This anticarcinogenic epidermal response disappeared with dose fractionation when the fractions were delivered at 4-week intervals, but not when delivered every day, showing that the induced mutation suppression is maintained under continual repetitive exposure, without which it expires within 4 weeks. These results suggest that repetition of heavy sun exposure at long intervals, e.g. recreational sunbathing every summer, is more likely to cause skin cancer than every day continual exposure even if the total UV doses are the same.

Vitamin C Prevents DNA Mutation Induced by Oxidative Stress

The precise role of vitamin C in the prevention of DNA mutations is controversial. Although ascorbic acid has strong antioxidant properties, it also has pro-oxidant effects in the presence of free transition metals. Vitamin C was recently reported to induce the decomposition of lipid hydroperoxides independent of metal interactions, suggesting that it may cause DNA damage. To directly address the role of vitamin C in maintaining genomic integrity we developed a genetic system for quantifying guanine base mutations induced in human cells under oxidative stress. The assay utilized a plasmid construct encoding the cDNA for chloramphenicol acetyl transferase modified to contain an amber stop codon, which was restored to wild type by G to T transversion induced by oxidative stress. The mutation frequency was determined from the number of plasmids containing the wild type chloramphenicol acetyl transferase gene rescued from oxidatively stressed cells. Cells were loaded with vitamin C by exposing them to dehydroascorbic acid, thereby avoiding transition metal-related pro-oxidant effects of ascorbic acid. We found that vitamin C loading resulted in substantially decreased mutations induced by H(2)O(2). Depletion of glutathione led to cytotoxicity and an increase in H(2)O(2)-induced mutation frequency; however, mutation frequency was prominently decreased in depleted cells preloaded with vitamin C. The mutation results correlated with a decrease in total 8-oxo-guanine measured in genomic DNA of cells loaded with vitamin C and oxidatively stressed. These findings directly support the concept that high intracellular concentrations of vitamin C can prevent oxidation-induced mutations in human cells.

A role for p53 in the frequency and mechanism of mutation.

The tumor suppressor protein, p53, is often referred to as the guardian of the genome. When p53 function is impaired, its ability to preserve genomic integrity is compromised. This may result in an increase in mutation on both a molecular and chromosomal level and contribute to the progression to a malignant phenotype. In order to study the effect of p53 function on the acquisition of mutation, in vitro and in vivo models have been developed in which both the frequency and mechanism of mutation can be analyzed. In human lymphoblastoid cells in which p53 function was impaired, both the spontaneous and induced mutant frequency increased at the autosomal thymidine kinase (TK) locus. The mutant frequency increased to a greater extent in cell lines in which p53 harbored a point mutation than in those lines in which a "null" mutation had been introduced by molecular targeting or by viral degradation indicating a possible "gain-of-function" associated with the mutant protein. Further, molecular analysis revealed that the loss of p53 function was associated with a greater tendency towards loss-of-heterozygosity (LOH) within the TK gene that was due to non-homologous recombination than that found in wild-type cells. Most data obtained from the in vivo models uses the LacI reporter gene that does not efficiently detect mutation that results in LOH. However, studies that have examined the effect of p53 status on mutation in the adenine phosphoribosyl transferase (APRT) gene in transgenic mice also suggest that loss of p53 function results in an increase in mutation resulting from non-homologous recombination. The results of these studies provide clear and convincing evidence that p53 plays a role in modulating the mutant frequency and the mechanism of mutation. In addition, the types of mutation that occur within the p53 gene are also of importance in determining the mutant frequency and the pathways leading to mutation.

The kinase inhibitor STI571 reverses the Bcr–Abl induced point mutation frequencies observed in pre-leukemic P190 Bcr–Abl transgenic mice

Chronic myelogenous leukemia (CML) and 25% of adult onset acute lymphoblastic leukemia (ALL) are associated with the expression of Bcr–Abl, a constitutively activated protein tyrosine kinase. Bcr–Abl associated leukemias are characterized by a high degree of chromosomal and genomic instability. It is unclear if the phenotype of genomic instability is a primary consequence of Bcr–Abl expression or if it is acquired secondarily. We have attempted to answer this question in previous studies by measuring the frequency of point mutations in double heterozygote transgenic mice derived from mating homozygous P190 Bcr–Abl transgenic mice (line 623) and the Big Blue Mice ® (Stratagene). Our results showed a 2–3-fold increase in the point mutation frequency in pre-leukemic (i.e. about 100 days before the onset of leukemia) P190 mice, compared to control mice (C57/BL6). In the present report, we extended these prior studies to ascertain if Bcr–Abl induced point mutations is a reversible phenotype. Pre-leukemic P190 Bcr–Abl/Big Blue double homozygous and C57/BL6 control mice were injected with the c-Abl specific kinase inhibitor STI571 for 10 consecutive days. We observed a decrease in the Bcr–Abl induced mutation frequencies in spleen and kidney tissue from mice treated with STI571. These results confirm that Bcr–Abl can directly and reversibly induce an increase in point mutation frequencies that could contribute to the genomic instability observed in Bcr–Abl positive leukemias.

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/cm 2 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/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 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 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.

Inhibition of DNA adduct formation and mutagenic action of 3-amino-1-methyl-5h-pyrido[4,3-b]indole by chlorophyllin-chitosan in rpsL transgenic mice.

We have studied the inhibitory effect of chlorophyllin‐chitosan (Chl‐Chi) complex, an insoluble form of chlorophyllin, on the DNA adduct formation and mutagenesis by a heterocyclic food mutagen‐carcinogen, 3‐amino‐l‐methyl‐5H‐pyrido[4,3‐b]indole (Trp‐P‐2), in mice carrying the E. coli rpsL gene as a mutagenesis reporter. Upon administration of a diet containing 0.002% or 0.01% Trp‐P‐2, DNA adducts were formed in various tissues in a dose‐dependent manner, with the maximum level observed in the liver. Addition of 3% Chl‐Chi to the diet reduced the Trp‐P‐2 adduct by up to 90%. The rpsL mutant frequencies increased significantly in both the liver and spleen upon administration of a 0.01% Trp‐P‐2 diet. Addition of Chl‐Chi to the diet decreased these induced mutant frequencies to the background level. No harmful effect of Chl‐Chi was detected during these experiments. The results show that Chl‐Chi may be a candidate chemopreventive agent against the genotoxic action of Trp‐P‐2, and possibly also other aromatic carcinogens in the diet.

Mutation frequency and type during ageing in mouse seminiferous tubules

Mutations arise in the germline by errors of replication, recombination and repair, and the movement of transposable elements. Transgenic mice bearing reporter genes such as lacZ have proven useful for measurements of spontaneous and induced mutation frequencies, as well as studies of the effects of ageing. In this study, testicular DNA from lacZ transgenic mice was examined for age-related effects on mutation frequency and type. The recovered transgene was tested for simple substitutions and rearrangements including transposition of endogenous mobile elements. There was no evidence for either an age-related accumulation of mutations, or for the insertion of retrotransposons into the lacZ reporter gene in the testis. We conclude that the frequency of retrotransposition of several mouse mobile elements into the lacZ reporter gene is less than 3.73×10 −8. This is significantly less than the known frequency of ∼7% of all spontaneous mutations in the mouse being due to retrotransposition of these elements.