Calf Thymus DNA Samples Research Articles

Novel inexpensive ‘turn-on’ fluorescent biosensor for the sensitive detection of DNA damage induced by epirubicin

For several decades, DNA has been well-known as the cellular target for various cytotoxic anticancer drugs. An active research area between chemistry, medicine and molecular biology is studying mechanism of interaction of drug molecules with DNA. In this study, we introduce a novel inexpensive and simple fluorescent biosensor to directly quantitate the DNA damage induced by epirubicin, an anticancer agent, and investigate the underlying molecular mechanisms of damage. The proposed fluorescent biosensor is established using terbium chloride. In damaged DNA, there are ssDNA regions facing the damaged nucleobases. In the damaged DNA, the terbium biosensor coordinates directly with the unpaired nucleobases accompanied by enhancement of its fluorescence, generating a measureable signal proportionate to the amount of damage. Moreover, dithiothreitol is used in this study to asses any possible DNA damage mechanisms that may occur other than the DNA oxidative damage that is commonly induced by various anthracyclines. In this work, the kinetics of the damage induced in DNA by epirubicin in absence and presence of dithiothreitol were compared using the proposed Tb3+ fluorescence biosensor. The results were confirmed using MALDI-TOF mass spectrometric analysis. It revealed that in absence of dithiothreitol, epirubicin induces oxidative DNA damage due to the interaction with reactive oxygen species causing strand breaks where the mass spectra show a significant reduction of the molecular-ion-peak of DNA, and appearance of peaks of smaller m/z indicating the DNA strand-breaks formation. While, in presence of dithiothreitol, epirubicin covalently binds to DNA to form DNA adducts with m/z values larger than the molecular-ion-peak of DNA target. Furthermore, the potency of epirubicin was examined on calf-thymus natural DNA samples with the proposed fluorescence biosensor. Whilst the interaction mechanism of epirubicin with DNA in presence of dithiothreitol shows slower DNA damage kinetics, but it has more potent damaging effect on DNA than in the case when no dithiothreitol is added. This introduces a novel alternative anticancer therapy that requires further investigation. Moreover, the low cost of the proposed fluorescent biosensor allows it to be an automatic simple process for the investigation of any DNA-drug interactions without the need to couple it with any other analytical method.

Sensitive spectrofluorimetric and mass spectroscopic methods for the determination of nucleic acid damage induced by photosensitized anti-inflammatory drugs: Comparative study

Anti-inflammatory drugs are reported to induce changes in nucleic-acids upon UV-irradiation. Such changes have the potential to cause apoptosis, carcinogenesis, and mutagenesis. In this work, the kinetics of the damage induced in DNA by some anti-inflammatory drugs were compared after UV-irradiation. Five commonly used anti-inflammatory drugs; diclofenac, ketoprofen, leflunomide, piroxicam and tolmetin, were studied. Simple, sensitive and eco-friendly methods for the analysis of DNA-damage were proposed including absorption spectroscopy, MALDI-TOF mass spectrometry and fluorescence using TbCl3. Results show that all drugs induced DNA-damage after UV-irradiation. Absorption spectroscopy results demonstrated hyperchromic shift in the absorption band characteristic to DNA, indicating distortion of the double-strand. Mass spectra showed a significant decrease of the molecular-ion-peak of DNA, together with peaks of smaller m/z that indicated the formation of DNA strand-breaks. TbCl3 fluorescence was observed to increase with incubation time of each drug with DNA, indicating the presence of more single-stranded regions in DNA due to damage. TbCl3 fluorescence was used to obtain the kinetics of the induced damage. Results show that DNA-damage occurred via photoinduced oxidative mechanism. Also, the potency of the studied drugs was examined on calf-thymus real DNA samples using TbCl3 fluorescence with ketoprofen and leflunomide being the most photogenotoxic anti-inflammatory drugs.

Monitoring DNA adducts in human blood samples using magnetic Fe3O4@graphene oxide as a nano-adsorbent and mass spectrometry

DNA adducts usually occur at a level of 0.1–1 adducts per 108 unmodified DNA bases, which can cause many adverse health effects if not normally repaired. Using microgram amounts of DNA to detect DNA adducts remains challenging due to the extremely low levels. Commercial solid phase extraction is the most widely used method for extracting DNA adducts from biological samples, but it is time consuming and costly, and the hydrophobic interactions between various alkyl-bonded phases and analytes typically lack selectivity when used to extract DNA adducts. Therefore, an effective method for DNA adducts analysis based on Fe3O4@graphene oxide (GO) nano-adsorbent and ultra-performance liquid chromatography-triple quadrupole mass spectrometry detection was developed in this study, in which the selectivity may also be increased by π-π stacking interactions between GO and adduct molecules. Several variables, including the extraction time, extraction pH, nano-sorbent amount and elution solvent were optimized, to achieve the best extraction efficiency using the proposed method. A small amount of Fe3O4@GO (2.5 mg) exhibited a good adsorption performance for the model DNA adducts, and butanol containing 4% NH3·H2O showed good elution effects. Under the optimized conditions, satisfying recoveries (78–107%) from calf thymus DNA samples were achieved, and 2 adducts were detected in only 2 μg of blood DNA. This method can be used as an effective strategy for DNA adduct analysis, and can be extended to other biological samples.

Fabrication of Cu−CeO2 Coated Multiwall Carbon Nanotube Composite Electrode for Simultaneous Determination of Guanine and Adenine

AbstractA copper nano particles and cerium (IV) oxide modified carbon nanotube based composite on glassy carbon electrode (Cu−CeO2/MWCNT/GCE) was fabricated for simultaneous determination of guanine and adenine. The surface morphology, chemistry and conductance of the prepared electrodes were characterized by scanning electron microscopy (SEM), energy dispersion X‐ray (EDX), X‐Ray photoelectron spectroscopy (XPS) and electrochemical impedance spectroscopy (EIS). The Cu−CeO2/MWCNT/GCE improved electrochemical behaviour of guanine and adenine compared to other electrodes. The modified electrode was also used for individual and simultaneous determination of guanine and adenine. Under optimized conditions, the calibration curves were obtained linearly in the range of 0.20 to 6.00 μM for the guanine and 0.10 to 8.0 μM for the adenine by differential pulse voltammetry. The limits of detection of guanine and adenine were calculated as 0.128 and 0.062 μM, respectively. Interferences studies were also performed in the presence of inorganic and organic compounds. Moreover, the determination of guanine and adenine contents were carried out in a calf thymus DNA sample by the developed method with satisfactory results.

Immobilization-free programmable hairpin probe for ultrasensitive electronic monitoring of nucleic acid based on a biphasic reaction mode.

This work designs a novel programmable hairpin probe (PHP) for the immobilization-free electrochemical detection of nucleic acid by coupling polymerase/nicking-induced isothermal signal amplification strategy with a biphasic reaction mode for the first time. The designed PHP (including a target-recognition region, a template sequence for enzymatic reaction and an inactivated anti-streptavidin aptamer) could program multiple isothermal reactions in the solution phase accompanying in situ amplified detectable signal at the electrode surface by the labeled ferrocene tag on the PHP. Upon addition of target analyte into the detection solution, target DNA initially hybridized with the recognition region on the PHP. Replication-induced strand-displacement generated an activated anti-streptavidin aptamer with the assistance of polymerase. Then, the polymerase/nicking enzymes could cleave and polymerize repeatedly the replication product, thus resulting in the formation of numerous template-complementary DNA initiator strands. The released initiator strands could retrigger the programmable hairpin probe to produce a large number of activated anti-streptavidin aptamers, which could be captured by the immobilized streptavidin on the electrode, thus activating the electrical contact between the labeled ferrocene and the electrode. Going after the aptamers, the carried ferrocene could produce the in situ amplified electronic signal within the applied potentials. Under optimal conditions, the electrochemical signal increased with the increasing target DNA concentration in the dynamic range from 5 fM to 10 pM with a detection limit (LOD) of 2.56 fM at the 3sblank criterion. Importantly, the methodology with high specificity was also validated and evaluated by assaying 6 target DNA-spiked human serum and calf thymus DNA samples, and the recoveries were 95-110%. Further work for the programmable hairpin probe could be even utilized in a real world sample to detect miRNA-21 at femtomol level.

Synthesis of ionic liquids coated nanocrystalline zeolite materials and their application in the simultaneous determination of adenine, cytosine, guanine, and thymine

In this study, ionic liquids coated nanocrystalline zeolite based inorganic-organic hybrid materials were synthesized. Nanocrystalline ZSM-5 zeolite was prepared under hydrothermal condition in the presence of propyltriethoxysilane as an additive in the synthesis composition of conventional ZSM-5. Ionic liquids (methylimidazolium chloride and 1-butyl-3-methylimidazolium chloride) were coated on the surface of nanocrystalline ZSM-5 by the post synthesis method. For comparative study, ionic liquids coated conventional ZSM-5 based inorganic-organic hybrid materials were also prepared. Ionic liquids coated nanocrystalline ZSM-5/ZSM-5 modified electrodes were constructed for the simultaneous determination of all four DNA bases such as adenine, cytosine, guanine, and thymine. Difference in the electro-catalytic activity of the modified electrode is explained with the help of textural properties, conductivity, and density functional theory. The analytical performance of the proposed method was demonstrated in the simultaneous determination of all four DNA bases in calf thymus DNA sample.

Ionic liquids coated Fe3O4 based inorganic–organic hybrid materials and their application in the simultaneous determination of DNA bases

Ionic liquids (ILs) coated Fe3O4 based inorganic–organic hybrid materials (represented as Fe3O4/ILs) were synthesized. ILs such as methylimidazolium chloride ([Hmim][Cl]) and 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) were investigated. For comparative study, quaternary ammonium salts such as choline chloride, cetyltrimethylammonium bromide [C16H33N(CH3)3][Br], and trimethylstearylammonium chloride [C18H37N(CH3)3][Cl] were also investigated. Materials were characterized by X-ray diffraction, nitrogen sorption, Fourier transform infrared and scanning/transmission electron microscopy. Electrochemical sensors based on Fe3O4/ILs modified glassy carbon electrodes were fabricated for the simultaneous determination of all four DNA bases. The electrochemical behavior of DNA bases was investigated in detail. Various reaction parameters such as effect of scan rate, number of electrons involved in the rate determining step, electron transfer coefficient, surface adsorbed concentration, and the electrode reaction standard rate constant were investigated. Catalytic activity obtained at various Fe3O4/ILs modified electrodes was explained using DFT calculation. The analytical performance of the sensor was demonstrated in the simultaneous determination of guanine, adenine, thymine, and cytosine in calf thymus DNA sample.

Simultaneous detection of guanine, adenine, thymine, and cytosine at polyaniline/MnO2 modified electrode

In this study, PANI/MnO2 nanocomposite was synthesized by the reaction of polyaniline (PANI) and KMnO4 in the aqueous medium. Material was characterized by X-ray diffraction, nitrogen sorption, scanning electron microscopy, transmission electron microscopy, thermogravimetric analysis, and FT-IR. PANI/MnO2 modified electrode was investigated in the simultaneous detection of all four DNA bases (guanine, adenine, thymine, and cytosine) using cyclic and differential pulse voltammetries. pH was optimized to obtain the best peak potential separation and response current. This enabled us for the simultaneous voltammetric determination of all four DNA bases at physiological condition (pH 7). PANI/MnO2 nanocomposite exhibited high-current sensitivity for these analytes compared to MnO2 and PANI modified electrodes. Under the optimum conditions, PANI/MnO2 exhibited low detection limit, good sensitivity, and wide linear range for the simultaneous detection of G, A, T, and C. Moreover, the proposed method was successfully applied in the determination of G, A, T, and C contents in a calf thymus DNA sample with satisfactory results. The reliability and stability of the modified electrode provide a good possibility for applying the technique in the routine analysis for a selected class of electroactive organic/bio-molecules.

Detection of DNA Strand Breaks in Gamma-Irradiated Lymphocytes Using Surface Plasmon Resonance

Surface plasmon resonance (SPR) is a sensitive, rapid, simple and low cost method for detection of biological molecules. In this study, SPR technology with alkaline phosphatase as a probe was utilized to measure DNA strand breaks induced by (60)Co gamma rays. The doses were from 0.01-10 Gy with a dose rate of 0.1 Gy/min. The results demonstrate that the SPR technology can be used to estimate strand breaks of calf thymus DNA. SPR signals of the calf thymus DNA samples increased with increasing gamma ray doses and the relationship of y = sqrt (3297x + 582.6) (r = 0.99) between the SPR signal and gamma dose was obtained. Estimation of DNA strand breaks in irradiated lymphocytes by SPR also demonstrated an increase in SPR signal with increasing dose and the exponential relationship of y = 169.43 × (1 - exp(-0.89x)) (r = 0.93) was obtained. The initial yield of the SPR signal is about 150.79 mdeg · Gy(-1) and compared to the sensitivity of 0.05 Gy achieved by the neutral single cell gel electrophoresis (SCGE), the SPR-based assay of DNA strand breaks was found to be more sensitive (0.02 Gy). We therefore propose that SPR technology with alkaline phosphatase as the probe is a sensitive, simple and quick method for detection of DNA strand breaks in gamma-irradiated lymphocytes.

An approach based on ultra-high pressure liquid chromatography–tandem mass spectrometry to quantify O6-methyl and O6-carboxymethylguanine DNA adducts in intestinal cell lines

O6-methylguanine (O6-MeG) and O6-carboxymethylguanine (O6-CMG) are characteristic promutagenic and toxic DNA adducts formed by nitrosated glycine derivates and N-nitrosopeptides. Since endogenous nitrosation has been hypothesised as a plausible origin for the association between red and processed meat intake and colorectal cancer, a highly sensitive, fast and specific quantitative assay is needed to correlate the dose of individual DNA adducts with the effects of food consumption and individual digestive and metabolic processes. An ultra-high pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) assay for quantitation of O6-MeG and O6-CMG, using the deuterated analogues as internal standards (ISTD), was developed. Samples of calf thymus DNA containing O6-MeG and O6-CMG were purified by acid hydrolysis and solid phase extraction prior to quantification by UHPLC–MS/MS in the selected reaction monitoring mode. The method was successfully validated in terms of repeatability (RSD<10%), reproducibility (RSD<15%) and linearity (99.9%) by incubating 0.1mg calf thymus DNA with the known N-nitroso compound potassium diazoacetate (KDA). The limit of quantitation was 30fmolmg−1 DNA for O6-MeG or 1 adduct per 108 nucleotides and 50fmolmg−1 DNA for O6-CMG or 1.7 adducts per 108 nucleotides. Subsequently, the method was applied to human colon carcinoma cell lines, Caco-2 and HT-29, treated with KDA to illustrate its capability to quantify O6-MeG and O6-CMG DNA adducts using biological relevant models in vitro. This method will support further research to unravel the mechanistic basis of endogenous nitrosation processes upon consumption of red and processed meat products.

ChemInform Abstract: Purine 5′,8‐Cyclonucleoside Lesions: Chemistry and Biology

AbstractReview: 76 refs.

Purine 5′,8-cyclonucleoside lesions: chemistry and biology

5',8-Cyclo-2'-deoxyadenosine and 5',8-cyclo-2'-deoxyguanosine in their 5'R and 5'S diastereomeric forms are tandem-type lesions observed among the DNA modifications and identified in mammalian cellular DNA in vivo. These lesions result from the chemistry of the C5' radicals generated by the attack of HO˙ radicals to 2-deoxyribose units. Quantitative determination of these lesions in biological samples as biomarkers of free radical damage is a challenge. Results reported for irradiated samples of calf thymus DNA have been critically reviewed, underlining the need of further research for the potential involvement of these lesions in human health (76 references).

X-ray induced damage in DNA monitored by X-ray photoelectron spectroscopy

In this work, the chemical changes in calf thymus DNA samples were analyzed by X-ray photoelectron spectroscopy (XPS). The DNA samples were irradiated for over 5 h and spectra were taken repeatedly every 30 min. In this approach the X-ray beam both damages and probes the samples. In most cases, XPS spectra have complex shapes due to contributions of C, N, and O atoms bonded at several different sites. We show that from a comparative analysis of the modification in XPS line shapes of the C 1s, O 1s, N 1s, and P 2p peaks, one can gain insight into a number of reaction pathways leading to radiation damage to DNA.

Anomalous electric birefringence behavior of sonicated DNA fragments as observed in reversing-pulse transients and steady-state sign reversal: A multicomponent approach

Anomalous electric birefringence signals of a sonicated and column-fractionated medium-size calf thymus DNA sample (bp = 570) in Na + solutions were measured at 7 °C. The reversing-pulse electric birefringence (RPEB) signal pattern was theoretically calculated in the low electric field region for two axially symmetric models coexisting in equilibrium in solution. The RPEB theory is based on the electric dipole moment due to ion-fluctuation along the longitudinal direction and the electric polarizability anisotropy (Δ α′), together with various electric and optical parameters assigned to the models. An analytical method was developed for the steady-state birefringence of the two-component system in a wide range of electric fields. The NaDNA samples exhibit complex RPEB patterns mixed with negative- and positive-going profiles. An experimental RPEB signal of NaDNA at an absorbance ( A 260) of 8 was fitted to theoretical curve at weak electric fields. The anomalous RPEB signal was attributed to the component 2, which shows a dip in the buildup and another in the reverse processes with a positive sign and a larger relaxation time. For the component 1, a normal DNA profile with negative sign is associated with a narrow dip in the reverse and a faster relaxation time in the decay signal. The field-strength dependence of observed steady-state birefringence δ(∞) could be fitted for NaDNA at A 260 = 8 by the SUSID orientation function with saturated ionic and electronic moments. An apparent positive maximum and the sign reversal in δ(∞) at weak electric fields is an interplay between the positive component 2 with positive optical factor Δ g and negative Δ α′ and the negative component 1 with negative Δ g and positive Δ α′. Possible conformation of two DNA components involved in solution was estimated.

Speciation studies of cis-platin adducts with DNA nucleotides via elemental specific detection (P and Pt) using liquid chromatography-inductively coupled plasma-mass spectrometry and structural characterization by electrospray mass spectrometry

As the cis-platinum (cis-Pt) antitumoral effect in mammals seems to be related to its binding to DNA components, experiments with in vitro incubation of the individual DNA nucleotides with cis-Pt and analysis of the products by electrospray mass spectrometry (ESI-Q-TOF) are described here. The only detectable complex of such binding has been the one formed by a cis-Pt molecule bound to two adjacent guanines (m/z 921), as confirmed by collisional induced dissociation. The separation of the cis-Pt adducts from the unreacted nucleotides has been conducted by high-performance liquid chromatography coupled on-line with inductively coupled plasma mass spectrometry (HPLC-ICP-MS), monitoring 31P and 195Pt. Two different chromatographic columns have been evaluated for this purpose: a RP-amide-C16 and a narrow-bore C8. Best separation characteristics for the four nucleotides of DNA (coming from adenine, thymine, cytosine and guanine nucleobases) and the formed cis-Pt adduct were obtained for the C8 column using a mobile phase containing 60 mM ammonium acetate (pH = 5.8) and 7.5% MeOH. This HPLC-ICP-MS method allowed an easy separation and detection of free nucleotides (by monitoring P) from the synthesized adduct (containing P and Pt in the same molecule). Quantitative capabilities of the proposed hybrid method, by monitoring 31P and 195Pt, have been compared by analysing the cis-Pt adduct formed by the oligonucleotide of sequence 5′-TCCGGTCC-3′ after incubation with cis-Pt and enzymatic hydrolysis. Final application of this methodology to commercially available calf thymus DNA samples has been also satisfactorily accomplished.

High-Performance Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry for the Detection and Quantitation of Benzo[a]pyrene−DNA Adducts

A method, using HPLC combined with electrospray tandem mass spectrometry (ES-MS/MS), was developed and validated to detect and quantify the major DNA adduct resulting from exposure to the ultimate tumorigenic benzo[a]pyrene (BP) metabolite, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Calf thymus DNA was reacted with BPDE, digested enzymatically to nucleosides, and the major DNA adduct, 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-BPDE), was purified by HPLC. Similar procedures were applied to prepare dG-BPDE-d8 from [1,2,3,4,5,6,11,12-(2)H8]BPDE for use as an internal standard. The HPLC-ES-MS/MS method was validated using a mixture of hydrolyzed salmon testis DNA (82 microg) and 10 pg dG-BPDE (analogous to 6.9 adducts/10(8) nucleotides). The results indicated an inter- and intraday accuracy of 99-100% and precision of 1.6-1.7% (relative standard deviation). When applied to a calf thymus DNA sample modified in vitro with [1,3-(3)H]BPDE, the method gave a value very similar to those obtained by radiolabeling, (32)P-postlabeling, and immunoassay. HPLC-ES-MS/MS analysis of hepatic DNA from mice treated intraperitoneally with 0.5 and 1.0 mg of [7,8-(3)H]BP gave values comparable to those determined by 32P-postlabeling and immunoassay. Lung DNA from mice fed a 0.3% coal tar diet (containing approximately 2 mg BP/g coal tar) for one month had 0.6 +/- 0.04 dG-BPDE adducts/10(8) nucleotides. This value is much lower than the 102 +/- 14 total DNA adducts/10(8) nucleotides determined by 32P-postlabeling, which suggests that dG-BPDE makes only a minor contribution to the DNA adducts formed in lung tissue of mice administered coal tar. The HPLC-ES-MS/MS method was used to assess human lung DNA samples for the presence of dG-BPDE. Based upon a limit of detection of 0.3 dG-BPDE adducts/10(8) nucleotides, when using 100 microg of DNA, dG-BPDE was detected in only 1 out of 26 samples. These observations indicate that HPLC-ES-MS/MS is suitable to assess the contribution of BP to DNA damage caused by exposures to polycyclic aromatic hydrocarbon (PAH) mixtures. The results further suggest that dG-BPDE may contribute only a small fraction of the total DNA adducts detected by other DNA adduct methodologies in individuals exposed to PAHs.

Analysis of DNA adducts of acetaldehyde by liquid chromatography–mass spectrometry

A highly sensitive method using liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) was developed for the analysis of DNA adducts of acetaldehyde (AA). AA, which is the primary oxidative metabolite of ethanol, is considered to possess carcinogenic activity. AA reacts with the exocyclic amino group of guanine in DNA to form N 2-ethylguanine (Et-Gua) and 1, N 2-propanoguanine (Pr-Gua) adducts. With the present method, such adducts were detected as the base forms from DNA chains using depurination in the pretreatment process. In our measurement with LC–ESI-MS, the limits of detection (LODs) of the Et-Gua and Pr-Gua adducts of the base forms were 3.0·10 −10 and 1.0·10 −9 M, respectively, and the LODs are about two orders of magnitude lower than those of the nucleoside forms. Calf thymus DNA samples treated with AA and NaBH 3CN were analyzed by this method. Et-Gua was clearly detected and, in the absence of NaBH 3CN, Pr-Gua was detected predominantly. Furthermore, the method was also applied to study whether or not these two adducts are formed in DNA of cultured HL-60 cells during exposure to AA for 24 h. Pr-Gua was clearly detected and traces of Et-Gua were also detected in the DNA of the cells. Although the sensitivity of this method is lower by at least one order of magnitude than the 32P-postlabeling assay, currently the most sensitive method, our method does not involve complex enzymatic reactions for the postlabeling and the use of troublesome radioactive materials. Furthermore, it enables structural identification of guanine adducts. The present method would be a useful tool for studies of Et-Gua and Pr-Gua adducts in connection with carcinogenesis.

Inter-laboratory Validation of Procedures for Measuring 8-oxo-7,8-dihydroguanine/8-oxo-7,8-dihydro-2′-deoxyguanosine in DNA

The aim of ESCODD, a European Commission funded Concerted Action, is to improve the precision and accuracy of methods for measuring 8-oxo-7,8-dihydroguanine (8-oxoGua) or the nucleoside (8-oxodG). On two occasions, participating laboratories received samples of different concentrations of 8-oxodG for analysis. About half the results returned (for 8-oxodG) were within 20% of the median values. Coefficients of variation (for three identical samples) were commonly around 10%. A sample of calf thymus DNA was sent, dry, to all laboratories. Analysis of 8-oxoGua/8-oxodG in this sample was a test of hydrolysis methods. Almost half the reported results were within 20% of the median value, and half obtained a CV of less than 10%. In order to test sensitivity, as well as precision, DNA was treated with photosensitiser and light to introduce increasing amounts of 8-oxoGua and samples were sent to members. Median values calculated from all returned results were 45.6 (untreated), 53.9, 60.4 and 65.6 8-oxoGua/10 6 Gua; only seven laboratories detected the increase over the whole range, while all but one detected a dose response over two concentration intervals. Results in this trial reflect a continuing improvement in precision and accuracy. The next challenge will be the analysis of 8-oxodG in DNA isolated from cells or tissue, where the concentration is much lower than in calf thymus DNA.

Ultratrace determination of adenine in the presence of copper by adsorptive stripping voltammetry

An electrochemical stripping procedure for ultra-trace measurements of adenine is described based on the adsorption of the adenine–copper complex on a static mercury drop electrode. Cyclic voltammetry was used to characterize the interfacial and redox behavior. Optimum experimental conditions were found when using a 0.005 M NaOH solution containing 0.4 ppm of copper, an accumulation potential of −0.30 V, a scan rate of 100 mV s −1 and a linear scan mode. There is a linear response to adenine concentration in the range of 0.1–1.0 ppb and the detection limit for 6 min accumulation time was of 4.0 ppt (3.0×10 −11 M). Proper conditions for measuring the adenine in presence of guanine, thymine and cytosine were also investigated. The method was applied for the determination of adenine in a sample of single-stranded calf thymus DNA.

Interactions of two cytotoxic organotin(IV) compounds with calf thymus DNA.

The reactions with DNA of two antitumor active organotin(IV) compounds, the dimer of bis[(di-n-butyl 3,6-dioxaheptanoato)tin] (C(52)H(108)Sn(4)O(1) x 2H(2)O), compound 1, and tri-n-butyltin 3,6,9-trioxodecanoate (C(19)H(40)SnO(5) x 1/2H(2)O), compound 2, were analysed by circular dichroism, DNA melting experiments and gel mobility shift assays. It is found that both complexes modify only slightly the B-type circular dichroism spectroscopy (CD) spectrum of calf thymus DNA. On the other hand, both complexes were found to affect significantly the parameters of the thermally induced helix-to-coil transition. Addition of 1 or 2 to calf thymus DNA samples does not favor DNA renaturation after melting ruling out formation of interstrand crosslinks. Moreover, the effects of both compounds on plasmid DNA gel mobility were investigated. From the analysis of the present results it is inferred that both organotin(IV) compounds do interact with DNA, probably at the level of the phosphate groups.