Induction Of Alkali-labile Sites Research Articles

Differences in activation of G2/M checkpoint in keratinocytes after genotoxic stress induced by hydrogen peroxide and ultraviolet a radiation

Long-wave ultraviolet radiation (UVA) may cause extensive DNA damage via reactive oxygen species (ROS). In this study we examined whether UVA- and H2O2-mediated DNA damage have equivalent effects on the induction of G2/M phase checkpoint and cell cycle progression in a transformed keratinocyte cell line HaCaT. By employing single cell gel electrophoresis (comet assay) we determined the equipotent doses of UVA and H2O2 with respect to the induction of alkali-labile sites (an indicator of oxidative DNA decay). However, in contrast to H2O2 which caused a pronounced G2/M cell cycle arrest 24h after treatment, UVA irradiation did not affect cell cycle progression. Increasing UVA doses up to 150 kJ/m2 did not affect cell cycle and proliferation whereas increasing H2O2 concentrations caused a cell cycle block or cell death. Cytometric analysis revealed that G2/M cell cycle arrest took place beyond the cyclin B1 restriction point. We conclude that the DNA damage induced by UVA is easily repaired and does not perturb cell growth, whereas the H2O2-induced damage leads ultimately to cell cycle arrest or cell death.

Role of G-->T transversions in the mutagenicity of alkylperoxyl radicals: induction of alkali-labile sites in bacteriophage M13mp19.

The mutagenicity of peroxyl radicals, ubiquitous products of lipid peroxidation, was assessed using an in vitro M13 forward mutational assay. Single-stranded M13mp19 plasmids were incubated with a range of concentrations of the azo initiator 2,2'-azobis(2-amidinopropane) hydrochloride, and then transfected into competent, SOS-induced Escherichia coli JM105 cells. Incubation with peroxyl radicals produced a concentration-dependent decrease in phage survival, with a 500 microM concentration of the azo initiator reducing the transfection efficiency by more than 90% while inducing a corresponding 6-fold increase in lacZ alpha mutation frequencies. Peroxyl radical-induced mutagenesis was completely prevented by the peroxyl radical scavenger Trolox. Automated DNA sequence analysis of the lacZ alpha gene of 100 peroxyl radical-induced mutants revealed that the most frequent sequence changes were base pair substitutions (92/95), with G-->T transversions predominating (73/92). Alkaline treatment prior to transfection diminished the mutagenicity of damaged plasmids to a level resembling that of unmodified DNA. While abasic sites might account for the sensitivity to alkaline cleavage, the possibility that unidentified nonabasic alkaline-labile lesions also contribute to peroxyl radical mutagenesis cannot be excluded. Collectively, these findings raise the possibility that DNA damage caused by a major class of endogenous radicals contributes to one of the most common spontaneous mutational events, the G-->T transversion.

Comparison of induction and repair of adducts and of alkali-labile sites in human lymphocytes and granulocytes after exposure to ethylating agents

A comparative study has been made of the induction and repair of adducts and alkali-labile sites in the DNA of human lymphocytes and granulocytes exposed to the ethylating agents N-ethyl- N-nitrosourea (ENU) and diethyl sulphate (DES). To evaluate these damages, the human blood cells were treated with highly 3H-labelled ENU and DES, and the resulting 3H-ethyl adducts were analysed via HPLC. Alkali-labile sites introduced in the DNA during treatment with non-radioactive ENU and DES were detected by alkaline elution with fluorometric quantitation of the DNA in the eluted fractions. All known adducts induced by ENU and DES could be detected by the HPLC methods applied. Furthermore, these adducts were separated from a number of unidentified compounds, because of the improved resolution on the columns used. Most of the adducts were rather persistent during a subsequent incubation period of up to 20 h after treatment, but some partly disappeared (7-ethyladenine and 3-ethyladenine). The induction of alkali-labile sites in lymphocytes and granulocytes was very similar, but the kinetics of the removal of these sites appeared to be quite different. In granulocytes there was hardly any repair, whereas in lymphocytes, particularly after ENU treatment, a substantial and relatively fast repair was observed. Induction of alkali-labile sites in human lymphocytes and granulocytes occurred also at 0°C; the data suggest that this kind of damage is not a result of enzymic repair processes. A comparison of the induction and the repair of alkali-labile sites in lymphocytes and granulocytes with those of the various ethyl adducts did not give a clue as to the identity of the adduct that could be responsible for the lability towards alkali.

A comparative study of dimethylhydrazine regioisomers and the methylazoxymethanol metabolite of 1,1- and 1,2-dimethylhydrazine in relation to transformation in human fibroblasts

Comparative analysis of the cytotoxicity, transformation efficiency, induction of alkali labile sites (ALS) and DNA methylation in human foreskin fibroblasts was carried out with two dimethylhydrazine (DMH) regio-isomers (1,1-DMH and 1,2-DMH) and the acetate (A) derivative of the metabolite methylazoxymethanol (MAM) of 1,2-DMH. Effective ED 50 cytotoxic doses for MAMA, 1,1-DMH and 1,2-DMH were 0.056, 6.83 and 6.30 mM, respectively. MAMA and 1,1-DMH were more effective transformers than 1,2-DMH. However, methylation of purines accounted for less than 1% of the total radiolabel associated with DNA for all 3 agents. 1,2-DMH, 1,1-DMH and MAMA induced O 6MeGua/N 7MeGua ratio of 0.04, 0.32 and 0.18, respectively. Only MAMA induced measurable alkali labile lesions at transforming doses. These results suggest that other mechanisms may play a role in the initiation of transformation events by hydrazine analogues.

Sequence specific damage of DNA induced by reducing sugars.

Reducing sugars induced alkali-labile sites in DNA. The DNA reacted with D-fructose 6-phosphate or D-fructose in the presence of Cu2+ was cleaved by the treatment with aqueous piperidine at 90 degrees C for 30 min. Alkali-labile sites were induced frequently at the pyrimidine residues, especially at the pyrimidine residues in pyrimidine-purine (5'----3') sequences. Transition metal ions such as Cu2+ and oxygen radicals such as hydrogen peroxide were possibly involved in the induction of alkali-labile sites.

Effects of sulphydryl compounds on the radiation damage in biologically active DNA.

The effect of sulphydryl compounds on the induction of alkali-labile sites and on the contribution of such sites to the inactivation of single-stranded phi X174 DNA was studied. Cysteamine is capable of reacting with DNA radicals, thereby modifying the radiation damage in such a way that the induction of immediate and latent breaks is reduced. This depends on the pH of the solution. With cysteine only, a pH dependent protection, against lethal alkali-labile potential breaks could be observed. The damage other than breaks is not influenced by the presence of sulphydryl compounds.