Joseph A. Aguilera

Variation of Single-Strand Break Yield with Scavenger Concentration for Plasmid DNA Irradiated in Aqueous Solution

We have measured the yield of single-strand breaks (SSBs), induced by 137Cs gamma radiation as assayed by agarose gel electrophoresis, for three plasmids and SV40 DNA irradiated in aerobic aqueous solution. DNA SSBs are caused mainly by the hydroxyl radical under these conditions. To characterize the reactivity of DNA with the hydroxyl radical, we investigated the variation of the G value for SSBs [G(SSB)] with the concentration of added hydroxyl radical scavengers. We find that simple competition kinetics does not describe our results, but that a nonhomogeneous kinetics model is in good agreement. At a DNA concentration of 50 micrograms cm-3, G(SSB) for the direct effect is about 1 x 10(-5) mumol J-1 for the DNA substrates studied. This is equivalent to 2 x 10(-10) SSB Gy-1 Da-1. Estimates of the efficiency of SSB induction per OH. radical interaction with DNA (0.32-0.44) reveal that all plasmids are essentially equal in reactivity.

EGFR regulates the side population in head and neck squamous cell carcinoma.

Objective: To identify the presence of side population (SP) cells in established head and neck squamous carcinoma cell (HNSCC) lines and to determine the role of EGFR in the regulation of the side population of these cells.

Polyamine-Induced Compaction and Aggregation of DNA- A Major Factor in Radioprotection of Chromatin under Physiological Conditions

Spermine at physiological levels and ionic strength induces compaction and aggregation of SV40 DNA and minichromosomes resulting in marked radioprotection of the DNA against gamma-ray-induced formation of single-strand breaks. This phenomenon, termed the PICA effect, results in yields of single-strand breaks in DNA and minichromosomes comparable to those found with intact cells and is considered to be a major mechanism responsible for radioprotection of cellular DNA.

DNA strand-break yields after post-irradiation incubation with base excision repair endonucleases implicate hydroxyl radical pairs in double-strand break formation

PURPOSE To determine the increases in SSB and DSB yields after post gamma-irradiation incubation of plasmid DNA with the Escherichia coli base excision repair endonucleases formamidopyrimidine-DNA N-glycosylase (FPG) and endonuclease III (endo III). MATERIALS AND METHODS Aqueous solutions of plasmid DNA were irradiated with 137Cs gamma-rays in the presence of 10(-4) - 10(-1) mol dm(-3) formate. After irradiation, aliquots were treated with FPG and/or endo III. SSB and DSB yields were then determined using gel electrophoresis. RESULTS Both SSB and DSB yields were found to increase after enzyme incubation, with the increase in the DSB yield being approximately equal to the square of the increase in the SSB yield. The correlation between the increases in the SSB and DSB yields was unaffected by the scavenger concentration during irradiation. CONCLUSION Under the conditions used, the majority of DSB appear to be formed from two hydroxyl radical attacks.Purpose : To determine the increases in SSB and DSB yields after post gamma-irradiation incubation of plasmid DNA with the Escherichia coli base excision repair endonucleases formamidopyrimidineDNA N -glycosylase (FPG) and endonuclease III (endo III). Materials and methods : Aqueous solutions of plasmid DNA were irradiated with 137 Cs gamma-rays in the presence of 10 -4 -10 -1 mol dm -3 formate. After irradiation, aliquots were treated with FPG and/or endo III. SSB and DSB yields were then determined using gel electrophoresis. Results : Both SSB and DSB yields were found to increase after enzyme incubation, with the increase in the DSB yield being approximately equal to the square of the increase in the SSB yield. The correlation between the increases in the SSB and DSB yields was unaffected by the scavenger concentration during irradiation. Conclusion : Under the conditions used, the majority of DSB appear to be formed from two hydroxyl radical attacks.

Yield of strand breaks as a function of scavenger concentration and LET for SV40 irradiated with 4He ions.

We have measured by gel electrophoresis the yields of single- and double-strand breaks (SSBs and DSBs) induced in aqueous solutions of SV40 DNA and the SV40 minichromosome by 137Cs gamma rays (mean LET 0.3 keV micron-1) and 4He ions (mean LETs 85, 102, and 152 keV microns-1). DNA SSBs are caused mainly by the hydroxyl radicals under these conditions and are reduced in yield as either the hydroxyl radical scavenger concentration or the LET is increased (over the range studied). The G(SSB) for 4He ion irradiation is less by a factor of up to 10 than the G(SSB) for gamma irradiation, depending upon the scavenger concentration. The difference in the yields of SSBs agrees well with the difference in the yields of hydroxyl radicals for the radiations in question. In contrast, the yields of DSBs are similar for gamma and 4He ion irradiation over much of the range of scavenging capacity studied. However, at the highest scavenger concentrations the yields of DSBs are greater for 4He ion irradiation. In addition, the yields of DSBs remain almost constant with increasing LET (over the range studied). Therefore the relative yield of DSBs per SSB increases with increasing LET, supporting the hypothesis that increasing LET leads to an increased clustering of damage in DNA.

Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines

OBJECTIVES Evaluate the cytotoxicity and genotoxicity of short- and long-term e-cigarette vapor exposure on a panel of normal epithelial and head and neck squamous cell carcinoma (HNSCC) cell lines. MATERIALS AND METHODS HaCaT, UMSCC10B, and HN30 were treated with nicotine-containing and nicotine-free vapor extract from two popular e-cigarette brands for periods ranging from 48 h to 8 weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion, and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. RESULTS E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapor nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. CONCLUSION E-cigarette vapor, both with and without nicotine, is cytotoxic to epithelial cell lines and is a DNA strand break-inducing agent. Further assessment of the potential carcinogenic effects of e-cigarette vapor is urgently needed.

Variation of single-strand break yield with scavenger concentration for the SV40 minichromosome irradiated in aqueous solution.

We have measured the yield of single-strand breaks (SSBs) induced in aerobic aqueous solution by 137Cs gamma irradiation for the SV40 minichromosome as measured by agarose gel electrophoresis. Under these conditions, DNA SSBs are caused mainly by the hydroxyl radical. To characterize the reactivity of the SV40 minichromosome with the hydroxyl radical and to compare its behavior with that of naked DNA, we examined the variation of the G value for SSB formation, G(SSB), with the concentration of added hydroxyl radical scavengers. We find that simple competition kinetics is not applicable, but that a nonhomogeneous kinetics model is in much better agreement. Estimates of the efficiency of SSB induction per OH radical interaction with the SV40 minichromosome (0.04-0.05) indicate that this substrate is about five times more radioresistant than naked DNA at scavenging capacities < 10(8) s-1. At a DNA concentration of 50 micrograms ml-1, G(SSB) for the direct effect in the minichromosome is about 1 x 10(-5) mumol J-1 (2 x 10(-10) SSB Gy-1 Da-1), essentially equal to that for naked DNA.

Effect of polyamine-induced compaction and aggregation of DNA on the formation of radiation-induced strand breaks: Quantitative models for cellular radiation damage

The yield of DNA single-strand breaks, G(SSB), upon gamma irradiation of SV40 DNA and SV40 minichromosomes in aqueous solution under aerobic conditions was determined at physiological ionic strength in the presence of various potential radioprotective agents. Putrescine (PUT), spermidine (SPD), glutathione, trans-4,5-dihydroxy-1,2-dithiane, 2-mercaptoethyl disulfide and cystamine, all at 0.1-10 mM, spermine (SPM, 0.1-1 mM) and WR-33278 (WRSSWR, 0.1-2 mM) lowered G(SSB) of SV40 DNA. These results were expected from the ability of these agents to scavenge OH radical in the bulk solution. However, SPD, above 10 mM, and SPM and WRSSWR, each above 2 mM, produced dramatic radioprotection attributed to polyamine-induced compaction and aggregation of the DNA (PICA effect). The DNA of SV40 minichromosomes was inherently less radiosensitive and was subject to a PICA effect at lower polyamine concentrations, i.e. approximately 5 mM SPD, approximately 0.6 mM SPM and approximately 0.5 mM WRSSWR. The PICA effect decreased G(SSB) for SV40 DNA and minichromosomes by one to two orders of magnitude, depending upon the scavenging capacity of the medium. The final yields were similar for SV40 DNA and minichromosomes and were comparable to the corresponding yield determined for cells. Results for the yield of double-strand breaks indicated that the yield of double-strand breaks, G(DSB), for DNA and minichromosomes is subject to a PICA effect by SPM and SPD comparable to that measured for G(SSB). Values of G(SSB) for SV40 DNA and minichromosomes subjected to the PICA effect were well approximated by calculations based upon a 30-nm cylinder assumed to model their condensed states. The results indicate that a major fraction of the formation of SSBs in condensed DNA and minichromosomes results from nonscavengeable radical intermediates. Minichromosomes subjected to the PICA effect of 2 mM SPM were protected against formation of radiation-induced SSBs 1.5-fold by 20 mM DTT but 5-fold by 10 mM DTT plus 10 mM WR-1065 relative to 2 mM SPM alone. Thus WR-1065 is capable of providing marked protection of compacted and aggregated minichromosomes, a protection ascribed to the chemical repair of DNA radicals by WR-1065.

DNA strand break yields after post-high LET irradiation incubation with endonuclease-III and evidence for hydroxyl radical clustering

Purpose : To determine the increase in single- (SSB) and double-strand break (DSB) yields after post-high LET irradiation incubation of plasmid DNA with the endonuclease-III (endo-III) of Escherichia coli. Materials and methods : Plasmid DNA in aerobic aqueous solution was irradiated with one of five radiation types: 137 Cs γ-rays (LET ~0.3keV μ m -1) , 244 Cm α -particles (140-190 keV μ m -1) , 4 He ions (97 keV μ m -1) , 56 Fe ions (143keV μ m -1) or 197 Au ions (1440keV μ m -1) . The irradiated samples were then incubated with endo-III. SSB and DSB yields were quantified by agarose gel electrophoresis. Results : Endo-III incubation produced an increase in the SSB and DSB yields. The increases were in general lower after the high LET irradiation than after γ-irradiation. This may reflect inhibition of the activity of endo-III by the nearby DNA damage expected from high LET radiation. It can be shown that even if the activity of endo remains unchanged, significantly lower increases in SSB and DSB yields would still be expected. Conclusion : The results provide evidence for clustered DNA damage after high LET irradiation.

Nuclear Thiols: Technical Limitations on the Determination of Endogenous Nuclear Glutathione and the Potential Importance of Sulfhydryl Proteins

Significant discrepancies were found between the values for glutathione levels determined by the Tietze enzymatic assay and those measured by labeling with monobromobimane followed by HPLC analysis when these methods were applied to proliferating and quiescent cells of the 66 murine mammary tumor line depleted of glutathione by buthionine sulfoximine or to nuclei prepared from these cells by permeabilization with Nonident detergent. The probable origin of the discrepancy was traced to the presence of acid-soluble sulfhydryl proteins in the extracts which are thought to lead to erroneous values in the Tietze assay method. Using the monobromobimane-HPLC method it was found that the low-molecular-weight thiol levels in nuclei prepared by detergent permeabilization equilibrate in less than 1 min with the permeabilizing medium, indicating that (i) endogenous nuclear glutathione levels cannot be determined reliably using conventional methods of cellular disruption and (ii) the endogenous nuclear glutathione level is likely to be the same as the cytoplasmic value. The levels of protein sulfhydryl associated with the nuclear preparations were found to be of the same magnitude as the cytoplasmic GSH level and must therefore be considered a potentially significant source of thiol capable of repairing DNA radicals.