Alkali-Labile Sites

Of wider significance was the generation of 2-phenylethyl radical by oxyhemoglobin-mediated oxidation of phenelzine (2-phenylethylhydrazine), which was shown to be more efficient in promoting alkali-labile sites than in producing direct DNA strand scission (Fig. 6) [25]. 

Single and double strand breaks and alkali labile sites... 

Figure 4.1 The comet assay. A single-cell suspension is embedded in agarose on a slide. Cells are then subject to lysis followed by electrophoresis. If present, damaged DNA migrates out of the nucleoid structure during electrophoresis to producing a characteristic comet shape. Double-strand breaks are revealed under neutral conditions, whereas alkali conditions additionally show single-strand breaks and alkali labile sites. Image analysis of stained DNA is used to quantitate the amount of damaged DNA in the comet tail.

DIA, DNA strand breaks/alkali-labile sites, Syrian hamster embryo cells + NT 200 Parent Casto (1979)... 

DIH, DNA strand breaks, alkali labile site, human bronchial epithelial + NT 200 Chang et al. (1990)... 

Methyl methanesulfonate induced DNA single-strand breaks and alkali-labile sites in human lymphocytes in vitro. It induced unscheduled DNA synthesis in human epidermal keratinocytes and in oral epithelial and fibroblast cell cultures. Methyl methanesulfonate induced gene mutations in human lymphoblasts at the hprt locus and sister chromatid exchanges and micronuclei in HepG2 human liver cells in vitro. 

Schutte, H.H., van der Schans, G.P. Lohman, P.H.M. (1988) Comparison of induction and repair of adducts and of alkali-labile sites in human lymphocytes and granulocytes after exposure to ethylating agents. Mutat. Res., 108,23-37... 

Besides the reactions leading to the alkali-labile sites, there are free-radical reactions that lead immediately to strand breaks. These strand breaks can be assayed by centrifuging the irradiated DNA (after denaturation by heat and formaldehyde).238 The unwinding of the bro-... 

Dizdaroglu M, Schulte-Frohlinde D, von Sonntag C (1977) y-Radiolysis of DNA in oxygenated aqueous solution. Structure of an alkali-labile site. Z Naturforsch 32c 1021—1022 Dizdaroglu M, Gajewski E, Reddy P, Margolis SA (1989) Structure of a hydroxyl radical induced DNA-protein cross-link involving thymine and tyrosine in nucleohistone. Biochemistry 28 3625-3628... 

Alkali-labile sites (ALS) may contain an AP such as 2-dRL or certain damaged bases that are released from the sugar moiety upon treatment with alkali (OH" or an organic base such as piperidine). Subsequent to this, a strand break is induced, and this procedure is often used to detect damaged bases within DNA. As the mechanism of the decomposition of 2-dRL by alkali is concerned, it is been suggested that the carbonyl function at C(l ) acidifies H2 and deprotonation [reaction (6)] leads to a (3-elimination of the phosphate group [reaction (7)]. 

Upon attack at C(2 ) and in the presence of 02, the sugar moiety is mainly modified into an erythrose unit (Sugiyama et al. 1993) which is an alkali-labile site and undergoes a retroaldol reaction after deprotonation of H4 [reaction (45)], but a Z-form type structure favors C(2 )-hydroxylation. 

Sugiyama M, Ando A, Nakao K, et al. 1989. Influence of vitamin B2 on formation of chromium(V), alkali-labile sites, and lethality of sodium chromate(VI) in Chinese hamster V-70 cells. Cancer Res 49 6180-6184. 

Sugiyama M, Tsuzuki K, Haramaki N. 1993. Influence of o-phenanthroline on DNA single -strand breaks, alkali-labile sites, glutathione reductase, and formation of chromium(V) in Chinese hamster V-79 cells treated with sodium chromate(VI). Arch Biochem Biophys 305(2) 261-266. 

This method allows the detection of single- and double-stranded breaks and alkali labile sites. These breaks can be a result of radiation, direct and indirect mutagens, alkylation agents, intercalating agents, oxidative DNA damage, and incomplete DNA repair. 

DNA primary damage DNA single and double strand-breaks, alkali-labile sites, incomplete excision repair sites, DNA-DNA and DNA-protein crosslinks. 

Assay Limitations and Interpretation. Incorporation of BrdU itself can contribute to SCE, because it results in single strand-breaks and alkali-labile sites. Immunofluorescence methods that only necessitate small doses of BrdU for SCE visualization yield a lower SCE background than other techniques (Wilson and Thompson 2007). A method using Biotin-dUTP instead of BrdU was recently reported to overcome this technical issue (Wojcik et al. 2004). 

In addition to the differing affinity of metals towards DNA, the steric structure of DNA bases is crucially important in all reactions with metals. In addition, an indirect mechanism of interaction with DNA is that of reactive oxygen spedes (ROS) and free radical generation, as well as depurina-tion which, in the cell, is detectable as alkali-labile sites. The existence of apurinic sites eventually can lead to strand breaks via cleavage of AP endonucleases. 

As alluded to above, the major precursors to OH radical induced ssb are the C-4 radical and the C-5 radicd resulting from H-atom abstraction of H-4 and H-5 /H-5 respectively [2]. From irradiation of plasmid DNA in aqueous solution under aerobic conditions, it is estimated that the probability of prompt ssb formation on interaction with the OH radical is -13% [88]. To measure prompt ssb it is essential to irradiate and subsequently maintain the DNA at 4 °C during electrophoresis to avoid the formation of heat-labile sites. The yield of heat labile sites is -30% of the yield of prompt ssb [63]. Further, DNA should be retained at near neutral pH to avoid the formation of alkali-labile sites. For instance, if C-1 radicals are produced in DNA by OH radicals or direct effects, these C-1 radicals lead to abasic sites with the formation of deoxyribonolactone, an alkali-labile site [89,90]. In the presence of oxygen, the C-1 radical forms a peroxyl radical adduct which decomposes into a carbocation at C-1 and the superoxide radical anion. 

---