Cadmium(II), unlike nickel(II), inhibits 8-oxo-dGTPase activity and increases 8-oxo-dG level in DNA of the rat testis, a target organ for cadmium(II) carcinogenesis. (25/1399)

8-Oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) is an enzyme which prevents incorporation into DNA of promutagenic 8-oxo-2'-deoxyguanosine (8-oxo-dG) from a deoxynucleotide pool damaged by endogenous oxidants. Its inhibition may thus be carcinogenic. We previously found that Cd(II) inhibited 8-oxo-dGTPase in both cell free systems and cultured cells. To verify this finding in a relevant animal model, we investigated the effects of Cd(II) on cellular 8-oxo-dGTPase activity and nuclear DNA 8-oxo-dG levels in the rat testis, a target organ for Cd(II) carcinogenesis. Ni(II), which does not induce testicular tumors in rats and is a weaker in vitro inhibitor of 8-oxo-dGTPase than Cd(II), was investigated as a comparison. Male F344/NCr rats were given a single s.c. dose of 20 micromol Cd(II) acetate, 90 micromol Ni(II) acetate or 180 micromol sodium acetate (controls) per kg body wt and killed 2, 8, 24 or 48 h later (three rats/time point). Cd(II) caused a gradual decrease in testicular 8-oxo-dGTPase activity with time. It became significant only after 8 h post-injection (P < 0.05) and resulted in a final 50% loss of the enzyme activity at 48 h (P < 0. 01). Although the results for Ni(II) at 8 h and later were apparently lower than the controls, the decrease did not reach statistical significance. Treatment of rats with Cd(II) led to an early and progressive increase (from 130% at 2 h to 200% at 48 h versus the controls) of the 8-oxo-dG level in testicular DNA (P < 0. 05 or better). Ni(II) acetate also tended to raise the testicular 8-oxo-dG level, but the increase was transient, with an apparent maximum at 8 h, and did not approach statistical significance (P < 0. 2). Thus, Cd(II), unlike Ni(II), is able to inhibit 8-oxo-dGTPase activity and to raise 8-oxo-dG levels in rat testicular DNA. However, the time course of both effects indicates that 8-oxo-dGTPase inhibition is most likely not the sole cause of the increase in 8-oxo-dG.  (+info)

Exposure to hexavalent chromium does not increase 8-hydroxydeoxyguanosine levels in Korean chromate pigment workers. (26/1399)

This study was performed to determine whether chromium exposure increased 8-hydroxydeoxyguanosine levels in respiratory epithelial and white blood cells of chromate pigment workers. The subjects of this study were 22 chromium pigment workers and 16 controls in a chromate pigments factory. To estimate the level of exposure, hexavalent chromium concentrations in the factory air were measured. Chrominum concentrations of venous blood and spot urine, and 8-hydroxydeoxyguanosine levels in DNA extracted from sputum and white blood cells were determined. Correlation coefficients were calculated between them and their statistical significance was tested. Hexavalent chromium concentration in the factory air ranged from below limit of detection to 0.5150 mg/m3. Chromium levels in blood and 8-hydroxydeoxyguanosine levels in DNA extracted from venous blood and sputum were not statistically different between the two groups. Urine chromium level was significantly higher among workers. Among the correlation coefficients between blood chromium concentration, urine chromium concentration, blood 8-OH-dG level, and sputum 8-OH-dG level, none was statistically significant for workers, controls, and total subjects. Duration of employment did not show any significant correlation with those four variables, either. These results suggest that neither the hydroxyl radical nor 8-hydroxydeoxyguanosine is formed by the reduction of hexavalent chromium, or that one or both of these is formed and then effectively removed by oxygen free radical scavengers or 8-hydroxydeoxyguanosine repair enzymes. Since increased exposure to hexavalent chromium did not result in increased 8-hydroxydeoxyguanosine levels, it is unlikely that hexavalent chromium induces lung cancer through 8-hydroxydeoxyguanosine formation.  (+info)

Generation of reactive oxygen species and 8-hydroxy-2'-deoxyguanosine formation from diesel exhaust particle components in L1210 cells. (27/1399)

The generation of the reactive oxygen species during the interaction of diesel exhaust particles (DEP) with NADPH-cytochrome P450 reductase (P450 reductase) was investigated by electron spin resonance using the spin-trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). Addition of DEP extract to an incubation mixture of mouse lung microsomes in the presence of NADPH resulted in a time-dependent NADPH oxidation and acetylated-cytochrome c reduction. Using purified P450 reductase as the enzyme source, superoxide radicals which were detected as the spin adduct (DMPO-OOH) while metabolized by P450 reductase were dependent upon both DEP and enzyme concentrations. The ELISA method using a specific monoclonal antibody revealed that DEP produced 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is formed from deoxyguanosine in DNA by hydroxyl radicals, in the culture medium of L1210 cells. Active oxygen scavengers such as superoxide dismutase and catalase effectively blocked the formation of 8-OHdG in culture medium, and deferoxamine, which inhibits hydroxyl radicals production by chelating iron, was also effective in inhibiting the DEP-produced 8-OHdG formation. These results indicate that DEP components produce 8-OHdG through the hydroxyl radical formation via superoxide by redox cycling of P450 reductase.  (+info)

Oxidative damage to plant DNA in relation to growth conditions. (28/1399)

In this study we investigated the level of 8-oxo-2'-deoxyguanosine (8-oxodG) in DNA of Cardamine pratensis plants subjected to different growth conditions trying to answer the question whether factors like light and water accessibility or low temperature may have an impact on the total DNA oxidative damage. The level of this modified nucleoside was determined using HPLC coupled to UV absorbance and electrochemical detection (HPLC-UV-EC). We did not observe any statistically significant differences in 8-oxodG level between DNA of etiolated and light exposed plants as well as between DNA of regularly watered and drought-subjected plants. In contrast, we have shown that chilling (1 degree C for 28 h) brings about the increase of 8-oxodG level in DNA.  (+info)

Oxidative damage to the c-fos gene and reduction of its transcription after focal cerebral ischemia. (29/1399)

We investigated oxidative damage to the c-fos gene and to its transcription in the brain of Long-Evans rats using a transient focal cerebral ischemia and reperfusion (FCIR) model. We observed a significant (p < 0.001) increase in the immunoreactivity to 8-hydroxy-2'-guanine (oh8G) and its deoxy form (oh8dG) in the ischemic cortex at 0-30 min of reperfusion in all 27 animals treated with 15-90 min of ischemia. Treatment with a neuronal nitric oxide synthase (nNOS) inhibitor, 3-bromo-7-nitroindazole (60 mg/kg, i.p.), abolished the majority but not all of the oh8G/oh8dG immunoreactivity. Treatment with RNase A reduced the oh8G immunoreactivity, suggesting that RNA may be targeted. This observation was further supported by decreased levels of mRNA transcripts of the c-fos and actin genes in the ischemic core within 30 min of reperfusion using in situ hybridization. The reduction in mRNA transcription occurred at a time when nuclear gene damage, detected as sensitive sites to Escherichia coli Fpg protein in the transcribed strand of the c-fos gene, was increased 13-fold (p < 0.01). Our results suggest that inhibiting nNOS partially attenuates FCIR-induced oxidative damage and that nNOS or other mechanisms induce nuclear gene damage that interferes with gene transcription in the brain.  (+info)

Direct demonstration in synthetic oligonucleotides that N,N'-bis(2-chloroethyl)-nitrosourea cross links N1 of deoxyguanosine to N3 of deoxycytidine on opposite strands of duplex DNA. (30/1399)

The sequence specificity and covalent structure of the lesion caused by the DNA interstrand cross-linking reaction of N,N'-bis(2-chloroethyl)-nitrosourea (BCNU) were investigated using synthetic oligonucleotides. The efficiency of interstrand cross-linking was found to parallel the efficiency of monoadduct formation, preferring deoxyguanosine-deoxycytidine-rich duplexes and, particularly, runs of deoxyguanosine. No explicit sequence specificity was observed. Enzymatic digestion of purified, interstrand cross-linked DNA returned primarily the unmodified deoxynucleosides, along with 1-[N3-deoxycytidyl]-2-[N1-deoxyguanosyl]ethane. This substance was characterized by comparison of its mass spectrum, high-pressure liquid chromatography retention time, and UV spectrum to an authentic standard prepared by chemical synthesis. These studies provide the first direct evidence that BCNU has no strong sequence preference for interstrand cross-linking and that substance 4, which has been previously isolated from BCNU-treated DNA, derives from alkylation on opposite strands of DNA. The lack of sequence preference and lesion structure together suggest that one source of BCNU interstrand cross-links is linkage of deoxyguanosine and deoxycytidine partners from a single bp.  (+info)

Mutagenesis of the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine DNA adduct in mammalian cells. Sequence context effects. (31/1399)

Site-specifically modified oligodeoxynucleotides were used to investigate the mutagenic properties of a major cooked food mutagen-derived DNA adduct, N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (dG-C8-PhIP). dG-C8-PhIP-modified oligodeoxynucleotides were prepared by reacting an oligodeoxynucleotide containing a single dG (5'-TCCTCCTXGCCTCTC, where X = C, A, G, or T) with N-acetoxy-PhIP. The unmodified and dG-C8-PhIP-modified oligomers were inserted into single-stranded phagemid vectors. These single-stranded vectors were transfected into simian kidney (COS-7) cells. The progeny plasmid obtained was used to transform Escherichia coli DH10B. When dC was at the 5'-flanking position to dG-C8-PhIP, preferential incorporation of dCMP, the correct base, was observed opposite the dG-C8-PhIP. Targeted G --> T transversions were detected, along with lesser amounts of G --> A transitions and G --> C transversions. No mutations were detected for the unmodified vector. The influence of sequence context on the dG-C8-PhIP mutation frequency and spectrum was also explored. When the dC 5'-flanking base was replaced by dT, dA, or dG, the mutational spectra were similar to that observed with dC-flanking base. Higher mutational frequencies (28-30%) were observed when dC or dG was 5' to dG-C8-PhIP. A lower mutational frequency (13%) was observed when dA was at the 5' to the lesion. Single-base deletions were detected only when dG or dT flanked the adduct. We conclude that dG-C8-PhIP is mutagenic, generating primarily G --> T transversions in mammalian cells. The mutational frequency and specificity of dG-C8-PhIP vary depending on the neighboring sequence context.  (+info)

Human RNase H-mediated RNA cleavage from DNA-RNA duplexes is inhibited by 6-deoxythioguanosine incorporation into DNA. (32/1399)

Mercaptopurine and thioguanine are anticancer and immunosuppressive agents that exert their primary cytotoxic effects via incorporation of deoxythioguanosine (dG(s)) into DNA, but the precise mechanism(s) by which this causes cytotoxicity remains unknown. We initially determined that the level of dG(s) incorporation into DNA of human T- and B-lineage leukemia cell lines did not correlate significantly with the extent of cytotoxicity (IC(50)), except that there was no cytotoxicity in the absence of dG(s) incorporation. To elucidate biological processes perturbed by dG(s) incorporation into DNA, we chemically synthesized oligodeoxyribonucleotides containing a single dG(s) (11 mer and 19 mer), which decreased the melting temperature (T(m)) of DNA-DNA duplexes without major structural changes, as evidenced by circular dichroism spectra. Using nuclear extracts from human lymphoblastic leukemia cells (CCRF-CEM, NALM6, and Molt4), we documented that dG(s) incorporation into the DNA strand of DNA-RNA heteroduplexes significantly inhibited human RNase H-catalyzed RNA cleavage (80-90% inhibition) and that a similar inhibition was evident with bacterial RNase H. These data provide the first evidence that thiopurines inhibit the function of RNase H, indicating that their mechanism of cytotoxicity may involve interference with this component of the replication machinery.  (+info)