Bosutinib, an SRC inhibitor, induces caspase-independent cell death associated with permeabilization of lysosomal membranes in melanoma cells.

BACKGROUND: SRC kinase (SRC proto-oncogene, non-receptor tyrosine kinase) is a promising target for the treatment of solid cancers including human melanoma. Bosutinib (Bosu), a SRC inhibitor, has already been applied to the treatment of human chronic myelogenous leukemia and also has been assessed its safety in dogs. AIM: The aim of this study was to clarify a novel anti-tumour mechanism of Bosu in canine and human melanoma cells. MATERIALS AND METHODS: The canine and human melanoma cells were treated with Bosu and its effects were evaluated by the cell viability, the protein expression levels such as caspase-3 and LC3, Annexin V/Propidium iodide staining, and confocal immunostaining. RESULTS: Bosu induced the massive caspase-independent cell death, and blocked autophagy flux, which resulted from lysosomal dysfunction. Lysosomal dysfunction caused by Bosu was due to lysosomal membrane permeabilization (LMP), which resulted in the release of lysosomal hydrolases including cathepsin B. CONCLUSION: Our data suggest that Bosu induces the cell death through induction of LMP in melanoma cells and is a promising therapeutic agent for treatment of melanoma in both dogs and humans.

Associations of single nucleotide polymorphisms in precursor-microRNA (miR)-125a and the expression of mature miR-125a with the development and prognosis of autoimmune thyroid diseases.

It is important to search the biomarker to predict the development and prognosis of autoimmune thyroid diseases (AITDs) such as Hashimoto's disease (HD) and Graves' disease (GD). MicroRNA (miR) bind directly to the 3' untranslated region of specific target mRNAs to suppress the expression of proteins, promote the degradation of target mRNAs and regulate immune response. miR-125a is known to be a negative regulator of regulated upon activation normal T cell expressed and secreted (RANTES), interleukin (IL)-6 and transforming growth factor (TGF)-ß; however, its association with AITDs remains unknown. To clarify the association between AITDs and miR-125a, we genotyped the rs12976445 C/T, rs10404453 A/G and rs12975333 G/T polymorphisms in the MIR125A gene, which encodes miR-125a, using direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods in 155 patients with GD, 151 patients with HD and 118 healthy volunteers. We also examined the expression of miR-125a in peripheral blood mononuclear cells (PBMCs) from 55 patients with GD, 79 patients with HD and 38 healthy volunteers using quantitative real-time PCR methods. We determined that the CC genotype and C allele of the rs12976445 C/T polymorphism were significantly more frequent in patients with HD compared with control subjects (P < 0·05) and in intractable GD compared with GD in remission (P < 0·05). The expression of miR-125a was correlated negatively with age (P = 0·0010) and down-regulated in patients with GD compared with control subjects (P = 0.0249). In conclusion, miR-125a expression in PBMCs and the rs12976445 C/T polymorphism were associated with AITD development and prognosis.

Differences in detecting blasts between ADVIA 2120 and Beckman-Coulter LH750 hematology analyzers.

The complete blood count is often used as a screening tool to detect hematologic abnormalities in the peripheral blood. In patients with suspected or known leukemia, blast cells serve as an indicator of disease pathology. We compared the analytical performance of the Siemens ADVIA 2120 to the Beckman-Coulter LH750 in the detection of blasts. In the study, 390 blood samples were analyzed from a general hospital population, which included oncology patients. The presence of blasts, as indicated by the analyzers' flags, was compared with the results of a manual differential (regarded as the reference method). The ADVIA 2120 demonstrated 100% sensitivity at detecting blasts compared with 62% using the LH750. This improved sensitivity came at the expense of a lower specificity (49% vs. 86%). The effect of the false-positive and false-negative samples on the laboratory's manual review was partially (false-positive) or completely (false-negative) mitigated by triggering of other criteria. Detecting blasts in the peripheral blood depends on the performance characteristics of the hematology analyzer, in conjunction with the stringency of the laboratory's manual review criteria.

Translesional synthesis past acetylaminofluorene-derived DNA adducts catalyzed by human DNA polymerase kappa and Escherichia coli DNA polymerase IV.

Human DNA polymerase kappa (pol kappa) has a sequence significantly homologous with that of Escherichia coli DNA polymerase IV (pol IV). We used a truncated form of human pol kappa (pol kappaDeltaC) and full-length pol IV to explore the miscoding properties of these enzymes. Oligodeoxynucleotides, modified site-specifically with N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-AF), were used as DNA templates in primer extension reactions that included all four dNTPs. Reactions catalyzed by pol kappaDeltaC were partially blocked one base prior to dG-AAF or dG-AF, and also opposite both lesions. At higher enzyme concentrations, a significant fraction of primer was extended. Analysis of the fully extended reaction product revealed incorporation of dTMP opposite dG-AAF, accompanied by much smaller amounts of dCMP, dAMP, and dGMP and some one- and two-base deletions. The product terminating 3' to the adduct site contained AMP misincorporated opposite dC. On templates containing dG-AF, dAMP, dTMP, and dCMP were incorporated opposite the lesion in approximately equal amounts, together with some one-base and two-base deletions. Steady-state kinetics analysis confirmed the results obtained from primer extension reactions catalyzed by pol kappa. In contract, primer extension reactions catalyzed by pol IV were blocked effectively by dG-AAF and dG-AF. At high concentrations of pol IV, full-length products were formed containing primarily one- or two-base deletions with dCMP, the correct base, incorporated opposite dG-AF. The miscoding properties of pol kappa observed in this study are consistent with mutational spectra observed when plasmid vectors containing dG-AAF or dG-AF are introduced into simian kidney cells [Shibutani, S., et al. (2001) Biochemistry 40, 3717-3722], supporting a model in which pol kappa plays a role in translesion synthesis past acetylaminofluorene-derived lesions in mammalian cells.

Identification and quantification of tamoxifen-DNA adducts in the liver of rats and mice.

A new HPLC gradient system was developed for (32)P-postlabeling analysis to identify and quantify hepatic tamoxifen-DNA adducts of rats and mice treated with tamoxifen. Four stereoisomers of alpha-(N(2)-deoxyguanosinyl)tamoxifen (dG(3')(P)-N(2)-TAM), alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen (dG(3')(P)-N(2)-N-desmethyl-TAM), and alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide (dG(3')(P)-N(2)-TAM N-oxide) were prepared by reacting either alpha-acetoxytamoxifen, alpha-acetoxy-N-desmethyltamoxifen or alpha-acetoxytamoxifen N-oxide with 2'-deoxyguanosine 3'-monophosphate, and used as standard markers for (32)P-postlabeling/HPLC analysis. Our HPLC gradient system can separate the above 12 nucleotide isomers as nine peaks; six peaks representing two each trans epimers (fr-1 and fr-2) of dG(3')(P)-N(2)-TAM, dG(3')(P)-N(2)-N-desmethyl-TAM and dG(3')(P)-N(2)-TAM N-oxide, and three peaks representing a mixture of two cis epimers (fr-3 and fr-4) of nucleotides. Tamoxifen was given to female F344 rats and DBA/2 mice by gavage at doses of 45 mg/kg/day and 120 mg/kg/day, respectively, for 7 days. Totally 15 and 17 tamoxifen-DNA adducts were detected in rats and mice, respectively; among them 13 adducts were observed in both rats and mice. trans-dG-N(2)-TAM (fr-2) and trans-dG(3')(P)-N(2)-N-desmethyl-TAM (fr-2) were two major adducts in both animals. Except for these two adducts, trans-dG-N(2)-TAM N-oxide (fr-2) was the third abundant adduct that accounted for 6.4% of the total adducts in mice, while this accounted for only 0.3% in rats. A trans-isomer (fr-1) and cis-isomers (fr-3 and -4) of dG(3')(P)-N(2)-TAM, dG(3')(P)-N(2)-N-desmethyl-TAM and dG(3')(P)-N(2)-TAM N-oxide were also detected as minor adducts in both animals except for cis-form of dG-N(2)-TAM N-oxide in rats. Although the administered dose for rats was 2.7-fold less than that for mice, the total adduct level of rats (216 adducts/10(8) nucleotides) were 3.8-fold higher than mice (56.2 adducts/10(8) nucleotides). Thus, these three types of tamoxifen adducts accounted for 95.0 and 92.5% of the total DNA adducts of the rats and mice, respectively. The formation of tamoxifen adducts primarily resulted from alpha-hydroxylation of tamoxifen.

Mechanism of lower genotoxicity of toremifene compared with tamoxifen.

An increased incidence of endometrial cancer has been reported in breast cancer patients taking tamoxifen (TAM) and in healthy women participating in the TAM chemoprevention trials. Because TAM-DNA adducts are mutagenic and detected in the endometrium of women treated with TAM, TAM adducts are suspected to initiate the development of endometrial cancer. Treatment with TAM has been known to promote hepatocarcinoma in rats, but toremifene (TOR), a chlorinated TAM analogue, did not. TAM adducts are primarily formed via sulfonation of the alpha-hydroxylated TAM metabolites. To explore the mechanism of the lower genotoxicity of TOR, the formation of DNA adducts induced by TOR metabolites was measured using (32)P-postlabeling/ high-performance liquid chromatography analysis and compared with that of TAM metabolites. When alpha-hydroxytoremifene was incubated with DNA, 3'-phosphoadenosine 5'-phosphosulfate, and either rat or human hydroxysteroid sulfotransferase, the formation of DNA adducts was two orders of magnitude lower than that of alpha-hydroxytamoxifen. alpha-hydroxytoremifene was a poor substrate for rat and human hydroxysteroid sulfotransferases. In addition, the reactivity of alpha-acetoxytoremifene, a model activated form of TOR, with DNA was much lower than that of alpha-acetoxytamoxifen. Thus, TOR is likely to have lower genotoxicity than TAM. TOR may be a safer alternative by avoiding the development of endometrial cancer.

Translesional synthesis on a DNA template containing N2-methyl-2'-deoxyguanosine catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I.

Formaldehyde is produced in most living systems and is present in the environment. Evidence that formaldehyde causes cancer in experimental animals infers that it may be a carcinogenic hazard to humans. Formaldehyde reacts with the exocyclic amino group of deoxyguanosine, resulting in the formation of N2-methyl-2'-deoxyguanosine (N2-Me-dG) via reduction of the Schiff base. The same reaction is likely to occur in living cells, because cells contain endogenous reductants such as ascorbic acid and gluthathione. To explore the miscoding properties of formaldehyde-derived DNA adducts a site-specifically modified oligodeoxynucleotide containing a N2-Me-dG was prepared and used as the template in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. The primer extension reaction was slightly stalled one base before the N2-Me-dG lesion, but DNA synthesis past this lesion was readily completed. The fully extended products were analyzed to quantify the miscoding specificities of N2-Me-dG. Preferential incorporation of dCMP, the correct base, opposite the lesion was observed, along with small amounts of misincorporation of dTMP (9.4%). No deletions were detected. Steady-state kinetic studies indicated that the frequency of nucleotide insertion for dTMP was only 1.2 times lower than for dCMP and the frequency of chain extension from the 3'-terminus of a dT:N2-Me-dG pair was only 2.1 times lower than from a dC:N2-Me-dG pair. We conclude that N2-Me-dG is a miscoding lesion capable of generating G-->A transition mutations.

Influence of flanking sequence context on the mutagenicity of acetylaminofluorene-derived DNA adducts in mammalian cells.

Site-specifically modified oligodeoxynucleotides were used to explore the influence of neighboring base sequence context on the mutagenic potential of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-AF) in mammalian cells. Oligodeoxynucleotides ((5)(')TCCTCCTNXNCTCTC, where X is dG-AAF, dG-AF, or dG and N is C, A, G, or T) with different bases flanking the lesion were incorporated into a single-strand shuttle plasmid vector and used to establish the mutational frequency and specificity of dG-AAF and dG-AF adducts in simian kidney (COS-7) cells. Vectors containing dG-AAF promote preferential incorporation of dCMP at the site of the lesion; misincorporation of dAMP and dTMP also was observed. Mutational frequencies range from 11 to 23%. High mutational frequencies (18-23%) were observed when G or T was positioned 5' to dG-AAF and a lower frequency (11%) when C was 5' to the lesion. dCMP was predominantly incorporated opposite the dG-AF adduct when C, A, or T was 5' to the lesion; dAMP and dTMP were misincorporated at a frequency of 2-4%. With G 5' to the lesion, the overall mutational frequency for dG-AF ranged between 11 and 70%; the highest value occurred when C was the 3' flanking base, and the predominant mutation event was G --> T transversion (59%). We conclude from these experiments that dG-AAF and dG-AF promote G --> T transversions and G --> A transitions in mammalian cells. The mutational frequency and specificity of dG-AF vary significantly, depending on the nature of the bases flanking the lesion.

Miscoding potential of the N2-ethyl-2'-deoxyguanosine DNA adduct by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I.

Acetaldehyde, a major metabolite of ethanol, reacts with dG residues in DNA, resulting in the formation of the N(2)-ethyl-2'-deoxyguanosine (N(2)-Et-dG) adduct. This adduct has been detected in lymphocyte DNA of alcohol abusers. To explore the miscoding property of the N(2)-Et-dG DNA adduct, phosphoramidite chemical synthesis was used to prepare site-specifically modified oligodeoxynucleotides containing a single N(2)-Et-dG. These N(2)-Et-dG-modified oligodeoxynucleotides were used as templates for primer extension reactions catalyzed by the 3' --> 5' exonuclease-free (exo(-)) Klenow fragment of Escherichia coli DNA polymerase I. The primer extension was retarded one base prior to the N(2)-Et-dG lesion and opposite the lesion; however, when the enzyme was incubated for a longer time or with increased amounts of this enzyme, full extension occurred. Quantitative analysis of the fully extended products showed the preferential incorporation of dGMP and dCMP opposite the N(2)-Et-dG lesion, accompanied by a small amounts of dAMP and dTMP incorporation and one- and two-base deletions. Steady-state kinetic studies were also performed to determine the frequency of nucleotide insertion opposite the N(2)-Et-dG lesion and chain extension from the 3' terminus from the dN.N(2)-Et-dG (N is C, A, G, or T) pairs. These results indicate that the N(2)-Et-dG DNA adduct may generate G --> C transversions in living cells. Such a mutational spectrum has not been detected with other methylated dG adducts, including 8-methyl-2'-deoxyguanosine, O(6)-methyl-2'-deoxyguanosine, and N(2)-methyl-2'-deoxyguanosine. In addition, N(2)-ethyl-2'-deoxyguanosine triphosphate (N(2)-Et-dGTP) was efficiently incorporated opposite a template dC during DNA synthesis catalyzed by the exo(-) Klenow fragment. The utilization of N(2)-Et-dGTP was also determined by steady-state kinetic studies. N(2)-Et-dG DNA adducts are also formed by the incorporation of N(2)-Et-dGTP into DNA and may cause mutations, leading to the development of alcohol- and acetaldehyde-induced human cancers.

Mutagenic properties of estrogen quinone-derived DNA adducts in simian kidney cells.

DNA damage caused by catechol estrogens has been shown to play an etiologic role in tumor formation. Catechol estrogens are reactive to DNA and form several DNA adducts via their quinone forms. To explore the mutagenic properties of 2-hydroxyestrogen-derived DNA adducts in mammalian cells, N(2)-(2-hydroxyestrogen-6-yl)-2'-deoxyguanosine and N(6)-(2-hydroxyestrogen-6-yl)-2'-deoxyadenosine adducts induced by quinones of 2-hydroxyestrone, 2-hydroxyestradiol, or 2-hydroxyestriol were incorporated site-specifically into the oligodeoxynucleotides ((5)(')TCCTCCTCXCCTCTC, where X is dG, dA, 2-OHE-N(2)-dG, or 2-OHE-N(6)-dA). The modified oligodeoxynucleotides were inserted into single-stranded phagemid vectors followed by transfection into simian kidney (COS-7) cells. Preferential incorporation of dCMP, the correct base, was observed opposite all 2-OHE-N(2)-dG adducts. Only targeted G --> T transversions were detected; the highest mutation frequency (18.2%) was observed opposite the 2-OHE(2)-N(2)-dG adduct, followed by 2-OHE(1)-N(2)-dG (4.4%) and 2-OHE(3)-N(2)-dG (1.3%). When 2-OHE-N(6)-dA adducts were used, preferential incorporation of dTMP, the correct base, was observed. Targeted mutations representing A --> T transversions were detected, accompanied by small numbers of A --> G transitions. The highest mutation frequencies were observed with 2-OHE(1)-N(6)-dA and 2-OHE(3)-N(6)-dA (14.5 and 14.1%, respectively), while 2-OHE(2)-N(6)-dA exhibited a mutation frequency of only 6.0%. No mutations were detected with vectors containing unmodified oligodeoxynucleotides. Thus, 2-OHE quinone-derived DNA adducts are mutagenic, generating primarily G --> T and A --> T mutations in mammalian cells. The mutational frequency varied depending on the nature of the 2-OHE moiety.

Identification of hepatic tamoxifen-DNA adducts in mice: alpha-(N(2)-deoxyguanosinyl)tamoxifen and alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide.

Tamoxifen-DNA adducts detected in the liver of mice treated with tamoxifen have not yet been identified. In the present study a new type of tamoxifen-DNA adduct, four stereoisomers of alpha-(N:(2)-deoxyguanosinyl)tamoxifen N:-oxide 3'-monophosphate (dG(3'P)-N:(2)-TAM N:-oxide) were prepared as standard DNA adducts by reacting 2'-deoxyguanosine 3'-monophosphate with trans-alpha-acetoxytamoxifen N:-oxide in addition to four stereoisomers of alpha-(N:(2)-deoxyguano- sinyl)tamoxifen 3'-monophosphate (dG(3'P)-N:(2)-TAM) that was reported previously. Liquid chromatography-electrospray ionization-mass spectrometry of the reaction products gave the most abundant ion at m/z 731 ([M - H](-)), which corresponded to dG(3'P)-N:(2)-TAM N:-oxide. The modified products digested by alkaline phosphatase corresponded to the isomers of dG-N:(2)-TAM N:-oxide whose structures were identified previously by mass spectrometry and nuclear magnetic resonance. Using these standard markers, we analyzed the hepatic DNA adducts of female DBA/2 mice treated with tamoxifen at a dosage of 120 mg/kg/day for 7 days by (32)P-post-labeling coupled with an HPLC/radioactive detector. Mixtures of eight isomers of dG(3'P)-N:(2)-TAM and dG(3'P)-N:(2)-TAM N-oxide were separated into six peaks, since each of the cis epimers were not separated under the present HPLC conditions. Nine adducts were detected in all liver samples of mice. An epimer of trans-dG(3'P)-N:(2)-TAM was detected as the principal DNA adduct at a level of 29.0 adducts/10(8) nucleotides, which accounted for 53.3% of the total tamoxifen-DNA adducts. Lesser amounts of cis-dG(3'P)-N:(2)-TAM (2.8%) were also observed. An epimer of the trans-dG(3'P)-N:(2)-TAM N:-oxide (3.9 adducts/10(8) nucleotides) was detected as the third biggest adduct (7.2% of the total). The cis-dG(3'P)-N:(2)-TAM N:-oxide (0.4 adducts/10(8) nucleotides) accounted for 0.7% of the total. Thus, dG(3'P)-N:(2)-TAM and dG(3'P)-N:(2)-TAM N:-oxide were identified in tamoxifen-treated mouse liver.

Identification of tamoxifen-DNA adducts induced by alpha-acetoxy-N-desmethyltamoxifen.

Treatment with tamoxifen increased the risk of endometrial cancers in breast cancer patients and women participating in the chemoprevention study. In our laboratory, tamoxifen-DNA adducts, including alpha-(N(2)-deoxyguanosinyl)tamoxifen (dG-N(2)-TAM), were detected in the endometrium of women taking tamoxifen [Shibutani, S., et al. (1999) Chem. Res. Toxicol. 12, 646-653]. On the basis of recent animal studies, deoxyguanosinyl-N-desmethyltamoxifen (dG-N-desmethylTAM) adducts are also suspected to be formed in the liver. In the study presented here, we synthesized alpha-acetoxy-N-desmethyltamoxifen as a model activated metabolite of N-desmethyltamoxifen. The overall yield of alpha-acetoxy-N-desmethyltamoxifen from alpha-hydroxytamoxifen was approximately 42%. alpha-Acetoxy-N-desmethyltamoxifen was highly reactive to 2'-deoxyguanosine, as was similarly observed for tamoxifen alpha-sulfate. The two reaction products were identified as a mixture of epimers of the trans form or cis form of alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen (dG-N(2)-N-desmethylTAM) by mass and proton magnetic resonance spectroscopy. In addition, the trans and cis forms of dG 3'-monophosphate-N(2)-N-desmethylTAM were prepared as standard markers for (32)P-postlabeling/HPLC analysis. Using this technique, dG-N(2)-N-desmethylTAM adducts were detected in calf thymus DNA reacted with alpha-acetoxy-N-desmethyltamoxifen.

Identification of tamoxifen-DNA adducts in the endometrium of women treated with tamoxifen.

The risk of developing endometrial cancer increases significantly for women treated with tamoxifen (TAM); the present study was designed to investigate the mechanism of this carcinogenic effect. Endometrial tissue was obtained from 16 women treated for varying lengths of time with TAM and from 15 untreated control subjects. DNA was analyzed with a (32)P-post-labeling/HPLC on-line monitoring assay capable of detecting 2.5 adducts/10(10) nucleotides. Using this sensitive and specific assay, TAM-DNA adducts were detected in eight women. The major adducts found were trans and cis epimers of alpha-(N(2)-deoxyguanosinyl) tamoxifen (dG-N(2)-TAM); levels ranged between 0.2-12 and 1.6-8.3 adducts/10(8) nucleotides, respectively. There was marked inter-individual variation in the relative amounts of cis and trans adducts present. Low levels (0.74-1.1 adducts/10(8) nucleotides) of trans and cis forms of dG-N(2)-TAM N-oxide were detected in one patient. DNA adducts derived from 4-hydroxytamoxifen quinone methide were not observed. We conclude from this analysis that trans and cis dG-N(2)-TAMs accumulate in significant amounts in the endometrium of many, but not all, women treated with this drug. The level of adducts found, coupled with the previous demonstration of their mutagenicity [Cancer Res., 59, 2091, 1999], suggest that a genotoxic mechanism may be responsible for TAM-induced endometrial cancer.

Excision of tamoxifen-DNA adducts by the human nucleotide excision repair system.

The antiestrogen tamoxifen is used in the treatment of breast cancer and has recently been recommended as a chemopreventive drug for women at high risk for breast cancer. However, women treated with the drug have an increased incidence of endometrial cancer. It has been suggested that this endometrial cancer might result from mutagenic DNA adducts, which are formed by electrophilic tamoxifen species generated by metabolic activation of the drug. Because the frequency of damage-induced mutations is strongly dependent on the repairability of the lesion, we investigated the repair of the major tamoxifen-DNA adducts by the human nucleotide excision repair system. Using the reconstituted human excision repair system and synthetic DNA substrates, we found that the four types of tamoxifen-DNA adducts detected in the endometrium were repaired with moderate to poor efficiency by nucleotide excision repair. It is concluded that individual variations in repair capacity may play a role in the development of tamoxifen-induced endometrial cancer.

Comparison of the mutagenic properties of 8-oxo-7,8-dihydro-2'-deoxyadenosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine DNA lesions in mammalian cells.

The comparative mutagenicity of 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was explored using simian kidney (COS-7) cells. Oligodeoxynucleotides ¿5'-TCCTCCT- G(1)X(2)CCTCTC or 5'-TCCTCCTX(1)G(2)CCTCTC (X = dA, dG, 8-oxodA or 8-oxodG) containing 8-oxodA or 8-oxodG positioned within codon 60 or 61 of the non-coding strand of human c-Ha-ras1 gene were inserted into a single-stranded phagemid shuttle vector. The vector was replicated in COS-7 cells and the progeny plasmids were used to transform Escherichia coli DH10B. The transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequence analysis to establish the mutation frequency and specificity. When 8-oxodA was positioned at X(1), targeted A(oxo)-->C transversions were detected; the mutation frequency was 1.2%. When 8-oxodA was positioned at X(2), one targeted mutant among 416 colonies screened (an A(oxo)-->G transition) was detected. Thus, the mutation frequency and spectrum of 8-oxodA depend on the sequence context of the lesion. The mutation frequency of 8-oxodG at X(1) and X(2) was 5.2 and 6.8%, respectively. G(oxo)-->T transversions dominated the spectrum, accompanied by small numbers of G(oxo)-->A transitions and G(oxo)-->C transversions. We conclude that 8-oxodA has mutagenic potential in mammalian cells, generating A-->C transversions. However, when tested under similar conditions, the mutation frequency of 8-oxodA is at least four times lower than that of 8-oxodG.