Oxygen is not required for degradation of DNA by glutathione and Cu(II).

Previous studies by others have shown that thiols, such as glutathione, cause cleavage of DNA in the presence of Cu(II) ions and that the hydroxyl radical derived from molecular oxygen is the major cleaving species. In this paper, we present several lines of evidence that strongly suggest that molecular oxygen is not essential for DNA cleavage and that thiyl radicals may also be involved. Indirect evidence is presented to indicate that glutathione may substitute oxygen as an electron acceptor. In addition, DNA degradation occurs to a significant extent under anaerobic conditions and no inhibition of single-strand cleavage of supercoiled plasmid DNA is seen in the presence of superoxide dismutase and catalase. In view of the ubiquitous presence of glutathione, these results could be of interest under certain diseased conditions where copper concentrations are elevated.

Restriction fragment pattern analysis of HPRT mutations induced in rat-liver epithelial cells by alkylating and arylating agents.

Structural alterations in the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in genomic DNA of adult rat-liver (ARL) epithelial cells that were mutated by alkylating and arylating mutagens were studied by restriction enzyme fragment pattern (RFP) analysis. ARL cells were mutated with the direct-acting alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or the activation-dependent arylating agents 7,12-dimethylbenz[a]anthracene (DMBA) and N-2-acetylaminofluorene (AAF). Alterations in the HPRT gene of at least 10 independent 6-thioguanine-resistant (TGr) clones mutated by each chemical were analyzed using 8 different restriction endonucleases; Hind III, EcoRI, BamHI, XbaI, Hae III, XhoI, MspI and PstI, and a full-length HPRT cDNA as a probe in molecular hybridization. Among the 10 MNNG-induced mutants, the RFPs obtained with most endonucleases displayed no changes, while an altered RFP was found in only one mutant using XbaI. None of the 10 DMBA-induced mutants displayed altered RFPs. Restriction analysis of the 10 AAF-induced mutants showed no abnormality in HPRT gene structure in most restriction digests, while altered RFPs were detected in one mutant using MspI and in two mutants with XbaI digestion. Overall, the studies reveal an absence of major DNA sequence changes in 26 of 30 induced mutants although the mutant phenotype of 4 of the TGr clones can be attributed to gross chromosomal changes or a point mutation at the restriction site. The absence of detectable alterations in the RFPs of the majority of the mutants is strongly suggestive of base substitution as the major molecular alteration underlying the mutant phenotype. The HPRT activity of 14 of 30 mutants was at least 5% of the wild-type level, which is consistent with a structural alteration in the gene product expressed as partial activity of the enzyme. Therefore, the data are interpreted as indicating that in the ARL cells, all 3 mutagens induced primarily localized alterations in base sequences in the HPRT gene together with a few mutations involving large sequence changes.

Inhibition of repair of O6-methyldeoxyguanosine and enhanced mutagenesis in rat-liver epithelial cells.

The pro-mutagenicity of chemically-induced methylation of DNA at the O6 position of deoxyguanosine was studied in cultured adult rat liver epithelial cells. To modify the level of O6-methyldeoxyguanosine (O6-medGuo) resulting from exposure to an alkylating agent, partial depletion of the O6-alkylguanine-DNA alkyltransferase (AGT) repair system was produced by pretreatment of ARL 18 cells with a non-toxic dose of exogenous O6-methylguanine (O6-meG). Exposure of cells to 0.6 mM O6-meG for 4 h depleted AGT activity by about 40%. Intact and pretreated cells were exposed to a range of doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was quantified by measurement of 6-thioguanine-resistant mutants. The mutagenicity of MNNG was dose dependent and was greater in O6-meG pretreated cultures than in intact cultures. Immunoslot blot measurement of O6-medGuo employing a mouse monoclonal antibody demonstrated that MNNG produced O6-medGuo and that the intact liver cells were efficient in eliminating this lesion from their DNA. Since depletion of AGT would be expected to affect the rate of elimination of only O6-medGuo, it is concluded that this lesion is highly pro-mutagenic.

Interaction of quercetin with DNA.

In vitro experiments to study interaction of the mutagenic flavonoid quercetin with DNA are described. Calf thymus DNA treated with quercetin for various time periods was subjected to S1 nuclease hydrolysis. Thermal melting profiles of treated DNA were also determined using S1 nuclease. The rate of DNA hydrolyzed after 1 hr of pretreatment with quercetin was found to be only about 50% of that in its absence. However, after 10 and 24 hrs of treatment with the drug, the rate of S1 nuclease hydrolysis was observed to be greater than that of native DNA. Thermal melting profiles of DNA, treated with quercetin for 10 and 24 hrs, indicated a slight decrease in melting temperatures. Gel filtration of native DNA, which had been digested with S1 nuclease after preincubation with quercetin for 24 hrs, indicated the production of various sized degraded molecules. The results suggest that the initial interaction of quercetin with DNA may have a stabilizing effect on its secondary structure, but prolonged treatment leads to an extensive disruption of the double helix.

Interaction of intercalating and non-intercalating agents with DNA: use of hydroxyapatite chromatography and S1 nuclease.

We have used hydroxyapatite (HA) chromatography and S1 nuclease hydrolysis to study the modification in the secondary structure of DNA caused by certain intercalating and non-intercalating ligands. The principal conclusions of HA experiments were as follows: (1) when native DNA, complexed with drugs believed to bind to DNA by intercalation (ethidium bromide, acridine orange, actinomycin D and acriflavin), is chromatographed on HA a lower affinity of DNA for HA is observed; also, the DNA elutes from HA columns as a drug-DNA complex; (ii) ligands that are known to interact with DNA by surface interactions do not show these effects; (iii) it may be possible to quantitate the binding of the intercalating drug to DNA and to determine its degree of binding by HA chromatography. Possibly, intercalation causes a change in the configuration of the sugarphosphate backbone of DNA, resulting in an altered steric orientation or 'burial' of phosphate groups with reduced availability for surface interactions with HA. S1 nuclease was used to determine the thermal melting profiles of DNA complexed with ethidium bromide and acridine orange. The melting profile in both cases was found to be biphasic with considerably reduced denaturation even at 95 degrees C. This is accounted for by the property of intercalating agents of stabilizing the secondary structure of DNA and the reported preference in binding to G-C base pairs.

Degradation of DNA by silicic acid.

S1 nuclease hydrolysis and hydroxyapatite chromatography were used to study the effect of silicic acid on DNA. Native calf thymus DNA was incubated with increasing concentrations of silicic acid (DNA nucleotide/silicic acid molar ratios of 1:0.25, 1:0.5 and 1:1) and subjected to S1 nuclease hydrolysis. An increasing degree of DNA degradation was seen suggesting a destabilization of the secondary structure. A decrease in melting temperature was also observed. Hydroxyapatite chromatography indicated that incubation at the molar ratio of 1:1 resulted in denaturation and degradation of DNA.

Effect of riboflavin and light on the secondary structure of DNA.

S1 nuclease hydrolysis and benzoylated naphthoylated DEAE cellulose (BND-cellulose) chromatography have been used to study the effect of riboflavin and visible light on DNA. Native calf thymus DNA was incubated with riboflavin in the presence of fluorescent light for various time periods and subjected to S1 nuclease hydrolysis. An increasing degree of DNA degradation was seen suggesting a destabilization of the secondary structure. A decrease in melting temperature was also observed. Incubation with riboflavin and illumination caused adherence to BND-cellulose indicating the production of single stranded regions or breaks in the native double stranded molecules. However, when incubation was done in dark and in the presence of triplet excited state quencher, potassium iodide, a reduced adherence of DNA to BND-cellulose was seen. Plasmid pBR322 DNA was also treated with riboflavin under these conditions and subjected to agarose gel electrophoresis. No degradation could be seen in dark incubated and potassium iodide treated samples. These results indicate that the adherence of DNA to BND-cellulose in dark is possibly due to the binding of aromatic residues to the resin suggesting the formation of a complex between riboflavin and DNA.

Effect of alkylation on the secondary structure of DNA.

S1 nuclease hydrolysis and bezoylated naphthoylated DEAE-cellulose (BND-cellulose) chromatography have been used to demonstrate that alkylation of DNA by dimethyl sulfate at neutral pH leads to the production of partially denatured molecules under conditions where no significant depurination occurs. DNA was alkylated with increasing concentrations of the alkylating agent, and subjected to enzymatic degradation and binding to BND cellulose. An increasing degree of DNA hydrolysis and adherence to BND cellulose was seen. On hydroxyapatite chromatography the alkylated DNA still eluted at the position of double-stranded molecules suggesting the presence of partially denatured regions. The presence of salt had a preventive effect on such denaturation.