32P-postlabeling high-performance liquid chromatography (32P-HPLC) adapted for analysis of 8-hydroxy-2'-deoxyguanosine.

8-Hydroxy-2'-deoxyguanosine (8-OH-dG) is a promutagenic lesion in DNA caused by reactive oxygen species. It normally exists at a level of 0.1-1 per 10(5) 2'-deoxyguanosines (dG). To analyze the lesion in easily obtainable biological samples, a very sensitive analytical method is required. The method should also handle the problem with potential oxidation of dG to 8-OH-dG during workup and analysis. 32P-postlabeling high-performance liquid chromatography (32P-HPLC) is an analytical method previously used to analyze lipophilic DNA adducts at levels as low as 1 per 10(9) normal nucleotides when analyzing microgram amounts of DNA. This method was adapted for analysis of 8-OH-dG. The aim was to develop an analytical method that provided a high sensitivity and good reproducibility, prevented oxidation of dG present in samples to 8-OH-dG, was capable of analyzing DNA from very small samples and still offered high sample throughput and ease of use. In analysis of calf thymus DNA, the method had a detection limit of 0.1 8-OH-dG per 10(5) dG when 1 microgram of DNA was used. The standard deviation of repeated analyses of the same sample was +/-10% and the result corresponded well with the established analytical method using HPLC with electrochemical detection. 32P-HPLC is sensitive enough to enable analysis of low levels of 8-OH-dG in biological samples such as small volumes of blood, needle biopsies and tissue swabs. It also substantially reduces oxidation of dG to 8-OH-dG during sample workup and analysis.     

A two-site enzyme-linked immunosorbent assay for cytokeratin 8.

A monoclonal enzyme-linked immunosorbent assay (ELISA) was developed for the determination in biological fluids of cytokeratin 8, a potential marker for malignant diseases. Two monoclonal antibodies (MAbs), TS 3 and TS 4, with different epitope specificity, were selected from 4 cytokeratin-8 reactive antibodies. TS 3 was used for coating and TS 4 as HRP-conjugate, respectively. Antibodies were selected with the aim of optimizing the discriminatory capacity between cytokeratin 8 levels in sera from healthy persons and from patients with malignant diseases. In sera from healthy individuals the mean value was determined to be 3.1 +/- 2.3 ng/ml with an upper cut-off level of 7.8 ng/ml (+ 2 SD) using purified cytokeratin 8 as standard. Sera from patients with colon cancer and pancreatic cancer were found to have significantly elevated levels, showing a 4- to more than 10-fold increase compared with the normal level. In patients with ovarian cancer no significant elevation was seen. Cytokeratin 8 monitoring may be of value for patients with colon and pancreatic cancer.     

Method for the determination of 4-Demethoxydaunorubicin,its quinone and hydroquinone metabolites in human plasma and urine by high-performance liquid chromatography

Summary 4-Demethoxydaunorubicin (4-DMDNR) is a new orally active analogue of daunorubicin (DNR). We have developed a high-performance liquid chromatography (HPLC) method capable of separating and identifying 4-DMDNR, five possible fluorescent quinone metabolites and three possible non-fluorescent hydroquinone metabolites. Methods are described for high-yield synthesis of reference metabolites. The limit of detection of the fluorescence assay was less than 1 ng/ml after extraction of 1 ml plasma or urine with chloroform/propan-2-ol (2:1), with coefficients of variation in k' (HPLC column capacity factors) of less than 3% throughout the day. Efficiency of the extraction method described exceeded 80% in control experiments. Blood and urine samples were analysed from four cancer patients who had received 50 mg/m² orally as three divided doses every 8 h. A typical urinary profile of the drug and its metabolites was: parent drug, 13%; 4-demethoxydaunorubicinol (4-DMDNOL), 80%; 4-DMDNR 7-hydroxyaglycone, 4% and 4-DMDNOL 7-hydroxyaglycone, 3%. 4-DMDNOL was the major metabolite detected in plasma. A further metabolite identified as the 7-deoxyaglycone of 4-DMDNOL was detected in plasma of two patients at concentrations equal to or greater than the parent drug. In the other two patients no trace of the metabolite was detected.     

An enzyme-linked immunosorbent assay for the determination of src-family tyrosine kinase activity in breast cancer

Summary An enzyme-linked immunosorbent assay is described for the determination of protein tyrosine kinase activity originating from the presence of src-like tyrosine kinases in biological samples. In this assay a peptide derived from p34^cdc2, cdc2(6–20)NH₂, is coupled to the wells of a maleic anhydride-activated microtiter plate. This particular peptide has been described as an efficient and specific substrate for protein tyrosine kinases belonging to the src family kinases (Chenget al., J. Biol. Chem. 267 (1992) 9248–9256). After incubation of the coated substrate with sample and ATP, the amount of phosphorylated tyrosyl residues is determined with phosphotyrosine specific antibodies and a secondary peroxidase-labeled antibody. The assay appears to be very sensitive and is linear with sample protein concentration and phosphorylation time. Intra-assay variation is < 5%. whereas day-to-day variation is < 10%.The results of the assay have been compared with an ELISA in which the broad-specificity tyrosine kinase substrate poly(GluNa,Tyr)4:1 was coated. The results of both assays in 27 cytosolic breast cancer samples correlated very well (r = 0.94), in accordance with the predominant expression of src kinase activity in breast cancers (Ottenhoff-Kalffet al., Cancer Res. 52 (1992), 4773–4778).The present assay provides an easy, reproducible, and quick alternative for the usual radioactive methods used for the determination of src-kinase activities including immunecomplex kinase assay and TCA-precipitation assays. It allows the determination of src-like activities in human tumors for routine diagnostic purposes.     

One-step monoclonal antibody based enzyme-linked immunosorbent assay for direct determination of cortisol in serum

Monoclonal antibodies to cortisol have obvious potential advantages as starting materials for assay systems to detect their levels in body fluids. This is very important for monitoring pituitary gland and adrenal functions. To develop a one-step competitive heterogeneous enzyme-linked immunosorbent assay (ELISA), a monoclonal anti-cortisol antibody was generated using a reasonably designed haptenic derivative. Cortisol-3-O-carboxymethyloxime was coupled to carrier protein bovine serum albumin (BSA) to enhance its immunogenicity. Spleen cells were prepared from a BALB/c mouse, which had repeatedly been immunized with a conjugate of cortisol-3-O-carboxymethyloxime-bovine serum albumin (cortisol-3-O-CMO-BSA), to be fused with SP2/0 myeloma cells. After one fusion experiment, four hybridoma clones secreting a practical antibody were established. One of the resulting monoclonal antibodies, 2C9D11B5, showed an affinity constant (Ka) of 1.4 × 10(10) M(-1) for cortisol and provided a practical calibration curve (limit of detection [LOD], 0.26 ng per assay) in this ELISA system employing cortisol-21-hemisuccinate-horseradish peroxidase (cortisol-21-HS-HRP) as a tracer. Cross-reactivities with related C-21 steroids were acceptably low: 11-deoxycortisol (3.5%), cortisone (0.47%), corticosterone (<0.01%), progesterone (<0.01%), 17-hydroxyprogesterone (1.2%), 6-hydroxycortisol (7.6%), and tetrahydrocortisol (<0.01%). The intra-assay and inter-assay coefficient of variations (CVs) ranged from 4.3% to 9.2% and 3.8% to 10.4 %, respectively. The analytical recoveries were 92.3% to 116.3%. Serum cortisol levels of healthy volunteers were determined after chilled acetone, stripped to be 292.76 ± 201.38 ng/mL (n=5), which are in the reference range.     

Sensitive detection of 8-hydroxy-2'-deoxyguanosine in DNA by 32P-postlabeling assay and the basal levels in rat tissues

Oxidative damage from reactive oxygen species including freeradicals has been considered to play a vital role in many degenerativediseases and measurement of 8-hydroxy-2'-deoxyguanosine (Oh⁸dG)in tissue DNA has been used as a benchmark for oxidative DNAdamage. We report here an ultrasensitive ³²P-postlabeling methodto detect and quantitate Oh⁸dG in DNA and have determined basallevels of Oh⁸dG in rat tissues. The method is comprised of DNAdigestion to 3'-monophosphates, 5'-³²P-labeling, conversionto 5'-monophosphates and separation by a 2-directional PEI-cellulose TLC (Dl = 1.5 M formic acid; and D2 = 0.6 M ammoniumformate, pH 6.0). Under these conditions, all radioactive contaminantswere either removed from the chromatogram (normal nucleotidesand ³²Pi) or remained at the origin (ATP and other contaminants),while Oh⁸dG migrated in the middle of the chromatogram. Calfthymus DNA incubated with ascorbic acid and H₂O₂ produced predominantlyone spot under the chromatography conditions used; a chromatographicallyidentical spot was also detected in untreated DNA, but at amuch lower level (125 ± 40 Oh⁸dG/10⁶ nucleotides). Achromatographically identical spot was also found in dGp incubatedwith ascorbic acid and H₂O₂, but not with dAp, dCp or dTp. Whenapplied to rat tissue DNA, the assay readily permitted detectionof Oh⁸dG in the liver, lung, kidney, heart, brain, spleen, intestinesand mammary epithelial cells of 3-month old female Sprague-Dawleyrats. The tissue Oh⁸dG levels were found in the range of 87± 29 to 133 ± 49 per 10⁶ nucleotides, with liverand heart being the highest and kidney and brain the lowestThese values are in the vicinity to those found by gas chromatography/massspectrometry but 10–50 times higher than those reportedby HPLC-electrochemical detection. Because of its high sensitivity(<1 Oh⁸dG per 10^5–6 nucleotides) to detect Oh⁸dG usingnanogram quantity of DNA digest, the ³²P-postlabeling methodis likely to be valuable in quantitating Oh⁸dG in human tissuebiopsies.     

Quantitative immunoanalysis of promutagenic 8-hydroxy-2'-deoxyguanosine in oxidized DNA

The modified DNA base 8-hydroxyguanine has been implicated inspontaneous mutagenesis, carcinogenesis and cellular aging.Polyclonal antibodies specific for the 8-hydroxy-2'-deoxyguanosinemoiety in oxidized DNA were used for sensitive detection andquantitation of this biomarker of oxidative damage to cellularDNA. The analysis was performed with immunoslot blot assay (ISB)of oxidized DNA modified in vitro with methylene blue plus lightand upon H₂O₂ treatment of cultured human cells. The level of8-OHdG in DNA exposed to 90 and 120 min light in the presenceof 100 µM methylene blue showed 15.96 ± 2.4 and22.65 ± 3.65 pmol/µg DNA compared to 0.107 ±0.024 pmol/µg in commercial calf thymus DNA control. Inherentdamage, due to cellular endogenous oxidation of DNA, increasedsignificantly upon inhibition of catalase activity in humancells with 10 mM azide. The damage increased further on exposureof azide-treated cells to H₂O₂. The amounts of 8-OHdG followingtreatment of cells with 10 and 100 µM H₂O₂ were determinedto be 205 ± 42 and 333 ± 17.5 pmol/µg DNArespectively. Very low but quantifiable antibody binding wasseen with the ‘control unoxidized’ human nuclearDNA. This DNA, obtained under controlled conditions to restrictthe induction of 8-OHdG during isolation, provides a backgroundlevel of 0.022 ± 0.005 pmol 8-OHdG/µg DNA. Thequantitative assessment of 8-OHdG by ISB assay, with fmol sensitivityand direct analysis using unhydrolyzed DNA, should prove a highlyvaluable alternative to currently used approaches to detecting8-OHdG in enzymatic DNA hydrolysates.     

An enzyme-linked immunosorbent assay for hypoxia marker binding in tumours.

An enzyme-linked immunosorbent assay (ELISA) has been developed for measuring the in vivo binding of a hexafluorinated 2-nitroimidazole (CCI-103F) in tumour tissue biopsies. The binding of CCI-103F is believed to reflect the presence of hypoxia in tumours. The ELISA provides a sensitive and convenient method of measuring CCI-103F binding which does not require the injection of radioactive reagents. The ELISA is based on reagents prepared from synthetic antigens formed by the reductive activation and binding of CCI-103F to proteins in novel test tube experiments. Calibration of the ELISA involved comparing the ELISA with the radioactivity contained either in protein-CCI-103F adducts formed in vitro with tritiated CCI-103F or in tissues isolated from a tumour-bearing dog which had been injected with tritium-labelled CCI-103F. The two approaches to calibration are compared. The scope and limitation of the ELISA for measuring the binding of CCI-103F is discussed and an example of the application of the ELISA to measuring changes in tumour hypoxia in canine patients undergoing fractionated radiation therapy is presented.     

8-hydroxy-2'-deoxyguanosine in cervical cells: correlation with grade of dysplasia and human papillomavirus infection

In this study, the 8-hydroxy-2'-deoxyguanosine (8-OHdG) level was assessed in human cervical cells by an immunoperoxidase method and was related to the presence of human papillomavirus (HPV) infection and precancerous lesions. After optimizing the immunohistochemical method of detecting oxidative DNA damage in whole cells, we have used this technique to estimate the oxidative damage in cervical cells collected during a routine PAP test. The analysis of variance (ANOVA) of the data from human samples showed significant differences in the 8-OHdG content among normal, low-grade and high-grade squamous intraepithelial lesion (SIL, HGSIL and LGSIL, respectively; P < 0.001). In the comparison of the three groups, statistically significant differences were detected between normal SIL and HGSIL (P < 0.001) and between LGSIL and HGSIL (P = 0.003), whereas no statistically significant difference was found between normal SIL and LGSIL (P = 0.1). Grouping observations by HPV status, no significant difference was detected in 8-OHdG levels between HPV(+) and HPV(-) subjects (P = 0.8). The polytomous and proportional odds models, extensions of the logistic regression analysis, showed that the effect of 8-OHdG levels in rising the risk of dysplasia was roughly constant through SIL grades. In conclusion, the immunoperoxidase method, applied to single human cervical cells, provides clear evidence that significant differences exist in 8-OHdG content between normal and dysplastic cells and that oxidative DNA damage might play an important role in cervical carcinogenesis.     

Radiation-induced formation of 8-hydroxy-2'-deoxyguanosine and its prevention by scavengers

Reactive oxygen species (ROS) induce 8–hydroxy–2'-deoxyguanosine(8-OHdG) formation, which has been proposed as a key biomarkerrelevant to carcinogenesis. 8-OHdG has been induced in a numberof different ways, most often without knowledge of the specifictype and amount of ROS generated. We have measured 8-OHdG formationin calf thymus DNA exposed to ionizing radiation under conditionsgenerating either hydroxyl radicals (OH·), superoxideanions (O2^–) or both. Additionally, we investigated therelationship between the scavenger effect of the drug 5-aminosalicylicacid (5-ASA) and increasing OH· exposure toward 8-OHdGformation. The effect of this drug was compared to those ofthe physiological scavengers ascorbate and reduced glutathione(GSH). We found that OH· generated 8-OHdG in a dose-dependentmanner, whereas O₂^– did not cause 8-OHdG formation. 5-ASA,ascorbate and GSH all acted as hydroxyl radical scavengers,although with different concentration-effect curves, emphasizingthe importance of using relevant pharmaco-/physiological concentrationsin studies focusing on therapeutic applications of scavengers.The scavenger effect of 5-ASA at concentrations     

Rapid and sensitive enzyme-linked immunosorbent assay for the microsomal epoxide hydrolase

A rapid and sensitive indirect enzyme-linked immunosorbent assay(ELISA) was developed for micrsomal epoxide hydrolase of ratliver. The assay, which is easily and readily performed, issignificantly more sensitive than most enzymatic epoxide hydrolaseassays routinely used and electroimmunoassays previously developed.The limit of sensitivity of the ELISA is between 2–5 ngof microsomal epoxide hydrolase. Using the ELISA microsomalepoxide hydrolases of mouse and rat liver were shown to be antigenicallyvery similar, while microsomal epoxide hydrolases of guineapig, monkey and human liver are antigenically distinct fromthose of rat and mouse. The ELISA developed here is capableof detecting microsomal epoxide hydrolase of rat and mouse livereven when significant enzymatic activity is lost. These resultsindicate that the antigenic sites recognized by the antibodiesused are distinct from the catalytic site of the epoxide hydrolase.Approximately 1.9% of rat microsomal protein was quantifiedas microsomal epoxide hydrolase by the ELISA. Low levels ofmicrosomal epoxide hydrolase were also detected in rat livercytosol (0.02% of the cytosolic protein) demonstrating thatmicrosomal epoxide hydrolase is not totally membrane bound orthat an immunologically related protein occurs in the cytosolof normal rat liver. The ELISA developed here will be valuablein investigating further the role of microsomal epoxide hydrolase.     

Dimethylarsinic acid induces 8-hydroxy-2'-deoxyguanosine formation in the kidney of NCI-Black-Reiter rats

Dirnethylarsenic peroxyl radical [(CH(3))(2)AsOO] has been postulated to be responsible for DNA damage induced by dimethylarsinic acid (DMA). In an effort to elucidate the possible mechanism of tumor-inducing potential of DMA, an experiment was designed to investigate the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a specific marker of oxidative base damage in the kidney tissues of NCI-Black Reiter (NBR) rats. Animals were divided into four groups and administered the vehicle - saline, 5, 10 and 20 mg/kg body weight respectively of DMA by gavage, once a day, 5 days a week, for a period of 4 weeks. DMA induced increase of 8-OHdG levels in the kidney of the rats treated, with the highest level at the dose of 10 mg/kg body weight. Analysis of the kidney for cell proliferation employing PCNA-positive index showed greater proliferation in the tissues of treated rats. However, DMA did not have any influence on apoptosis in this regimen. Histopathological examination of the kidney selections revealed the presence of vacuolated degeneration and dilation of the proximal tubule cells in two groups (10 and 20 mg/kg body weight). This study provides evidence to substantiate the role of DMA in inducing oxidative DNA damage in the kidney.     

Peroxidative in vitro metabolism of diethylstilbestrol induces formation of 8-hydroxy-2'-deoxyguanosine

The generation of superoxide and hydroxyl radicals is knownto be implicated in the hydroxylation of 2'-deoxyguanosine (dG)at the C-8 position and of guanine base residues in DNA. Itwas also shown previously that in the presence of horseradishperoxidase, hydrogen peroxide and Fe³⁺-EDTA complex, diethylstilbestrol(DES) induces single strand breaks in DNA, caused by the productionof superoxide anion (O^–) and hydroxyl (OH) radicals. Bymeans of high-pressure liquid chromatography and electrochemicaldetection a strong indication is adduced that dG is oxidizedto 8-hydroxy- 2'-deoxyguanosine during peroxidative in vitrometabolism of DES, which might be at the basis of DES inducedcell transformation.     

Possible effect of infection with liver fluke (Opisthorchis viverrini) on the monitoring of urine by enzyme-linked immunosorbent assay for human exposure to aflatoxins.

Several laboratories have initiated studies to assess human exposure to aflatoxin at an individual level by measuring aflatoxin metabolites in the urine by immunoassay. The fact that the antibodies recognize a variety of metabolites, albeit with differing affinities, means that any environmental factor that modifies the pattern of urinary metabolites associated with a given exposure could affect quantification in immunoassay. We have examined two such possible effects: (i) the pattern of metabolites after a dose of 14C-aflatoxin B1 in rats and (ii) the pattern of metabolites in hamsters and humans with and without exposure to liver flukes. We found no dose-related effect on the pattern of urinary metabolites over a 250-fold range, but there was a significant increase in the proportion of water-soluble aflatoxin metabolites in hamsters infected with liver fluke over that in uninfected animals. In human urine samples, there also appeared to be a difference in the metabolites in individuals infected with liver fluke from those in uninfected persons. These observations are relevant to both mechanistic and monitoring aspects of research into aflatoxins.     

Rapid assay of N-butyl-N-(3-carboxypropyl)nitrosamine in rat organs and urine by high-performance liquid chromatography after derivatization

The concentration of N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), which is the major metabolite of the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), was measured in the urine, thymus, liver, kidney, and bladder of rats orally administered with BBN. Since BCPN is a carboxylic acid, it forms an ester with 9-anthryldiazomethane (ADAM), which is a fluorescent labeling agent highly sensitive to carboxylic acids. Thus, BCPN and ADAM were reacted at 40 degrees for 1 hr, and the resulting ester was separated and measured by high-performance liquid chromatography (HPLC) with a reverse-phase type column. The range of measurement was 0 to 40 micrograms/ml, and the coefficient of variation (CV) was 3.8%. When 0.025% BBN was given orally to rats in tap water, the BCPN concentration in the urine was very high at 220 micrograms/ml, while it was 0.15 microgram/100 mg in the wet tissues of the thymus, 0.35 microgram/100 mg in the liver, 0.40 microgram/100 mg in the kidney, and 1.2 microgram/100 mg in the bladder. The BCPN concentration in the bladder, in which tumors are induced by the administration of BBN, was thus higher than those in the other organs.     

Improved high-performance liquid chromatography assay of doxorubicin: Detection of circulating aglycones in human plasma and comparison with thin-layer chromatography

Summary We compared doxorubicin and metabolite pharmacokinetic data obtained from thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) assay of plasma samples from six patients who had been treated with doxorubicin. Duplicate 1-ml samples were extracted with chloroform: isopropanol (1:1) and assayed using a sensitive HPLC system incorporating a dual pump gradient with tetrahydrofuran as the mobile phase and fluorescence detection. Duplicate 1-ml samples from the same specimens were assayed using a modification of a previously described TLC assay. Areas under the curve for doxorubicin by HPLC (3.36±2.30 M · h) and TLC (4.16±2.50 M · h) were not significantly different (P=0.5). Terminal half-life of doxorubicin by HPLC (28.0±6.98 h) and TLC (23.2±7.8) (P=0.29) and the calculated total-body clearances by HPLC (0.55±0.29 l/min) and TLC (0.45±0.23) (P=0.55) were not significantly different. Areas under the curve for doxorubicinol by HPLC (2.75±1.4 M · h) and TLC (2.53±7.1 M · h) (P=0.73) showed no significant differences. HPLC detected a mixed 7-deoxydoxorubicinol aglycone-doxorubicin aglycone peak, 7-deoxydoxorubicin aglycone, and two nonpolar, unidentified metabolites. TLC detected the following aglycone metabolites: doxorubicin aglycone, doxorubicinol aglycone, 7-deoxydoxorubicinol aglycone, an unidentified polar metabolite, and several unidentified nonpolar metabolites. From these data we conclude that HPLC and TLC detect concentrations of doxorubicin and doxorubicinol from human plasma equally well to concentrations of 7.0 nM (4 pmol injected doxorubicin). Aglycones do circulate in human plasma at concentrations above the detection limits of both assays. Doxorubicinol aglycone, which is detected by TLC but not by HPLC, may be formed from artifactual breakdown of doxorubicinol during TLC development. Unidentified nonpolar compounds seen on HPLC and TLC may represent further doxorubicin metabolism than previously described.     

Development of an enzyme-linked immunosorbent assay for the detection of human calretinin in plasma and serum of mesothelioma patients

Background  

Calretinin is one of the well-established immunohistochemical markers in the diagnostics of malignant mesothelioma (MM). Its utility as a diagnostic tool in human blood, however, is scarcely investigated. The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for human calretinin in blood and to assess its usefulness as a potential minimally invasive diagnostic marker for MM.     

[32P]ATP mediates formation of 8-hydroxy-2'-deoxyguanosine from 2'- deoxyguanosine, a possible problem in the 32P-postlabeling assay

8-Hydroxy-2'-deoxyguanosine (8-OH-dG) is a biomarker for oxidative stress on DNA, a common lesion in mammalian cells. A correlation between increased levels of 8-OH-dG and diseases like diabetes, infections and cystic fibrosis has been found in humans. 8-OH-dG levels have been shown to be decreased by antioxidants, an indication of the importance of dietary habits. 8-OH-dG is used as a biomarker for oxidative stress in vivo as well as in vitro and is suggested to be a mutagenic DNA lesion. Different methods are used for the analyses of 8- OH-dG, i.e. GC-MS, HPLC-EC and 32P-postlabeling. The most commonly used method is HPLC-EC. In the analysis of 8-OH-dG, the work-up procedure for DNA, as well as the preparation for analysis, are of critical importance as there is a risk for auto-oxidation of deoxyguanosine (dG), which would result in false high background levels and low sensitivity in analysis. 32P-Postlabeling has recently been applied to the analysis of 8-OH-dG and has shown to be a very sensitive method for the detection of DNA adducts. It is shown here that after extrapolation to normal 32P-postlabeling conditions, [32P]ATP generated 8-OH-dG to levels of 25 8-OH-dG/10(5) dG. [32P]ATP mediated the formation of 8-OH- dG from dG in a dose-dependent manner at all dose levels (0.13-12 microCi). The reaction occurred immediately and increased with time in a linear dose-response fashion. At high doses (6.0 and 12 microCi) the dose-response declined after 24 h, which indicates a possible decomposition or rearrangement of 8-OH-dG. A repeated experiment with 5 microCi [32P]ATP during 2 h resulted in a linear formation of 8-OH-dG and a level of 19 8-OH-dG/10(5) dG. The results indicated that awareness of the auto-oxidation generated by 32P[ATP] in the postlabeling assay is of utmost importance and that dG must be separated before 32P-postlabeling of 8-OH-dG.      

Comparison of high-performance liquid chromatography and bioassay of amphotericin B in serum

Summary. For the determination of amphotericin B in serum a high-performance liquid chromatography (HPLC) method using simple protein precipitation and an isocratic mobile phase as well as a plate diffusion bioassay are described. Using 5-fluorocytosine-resistant strain of Candida albicans and peptone in medium as an antagonist of 5-fluorocytosine allows simple measurement of amphotericin B both in the absence and in the presence of 5-fluorocytosine. The correlation coefficient ( r ) between bioassay and HPLC runs is 0.88. Generally, the biological determination gives slightly higher values of amphotericin B than the HPLC method. Both methods are useful for amphotericin B drug monitoring.
Zusammenfassung. Es werden für die Bestimmung von Amphotericin B im Serum eine HPLC-Methode, die eine einfache Proteinfällung und eine isokratische mobile Phase verwendet, und ein Bioassay mittels eines Plattendiffusionstests beschrieben. Die Verwendung eines 5-Fluorcytosin-resistenten Stammes von Candida albicans und Pepton im Medium erlauben die Bestimmung von Amphotericin B sowohl in Abwesenheit als auch in Anwesenheit von 5-Fluorcytosin in einfacher Weise. Die Korrelation zwischen Bioassay und der HPLC-Methode beträgt r = 0.88. Im allgemeinen ergibt die biologische Methode etwas höhere Werte im Vergleich zur HPLC-Bestimmung. Beide Methoden sind für die Bestimmung von Amphotericin-B-Serumspiegeln brauchbar.     

5-Aminolevulinic acid mediates the in vivo and in vitro formation of 8-hydroxy-2'-deoxyguanosine in DNA

5-Aminolevulinic acid (ALA), a heme precursor accumulated inchemical and inborn porphyrias, may behave as an endogenouspro-oxidant In chronically treated rats (40 mg ALA/kg body wtevery 2 days for 15 days) the steady-state level of 8-hydroxy-2'-deoxyguanosine(8-OHdG) in liver DNA (94.5 ± 233 residues/106 dG) was4.5 times higher than in non-treated rats (21 ± 7.5 residues/10⁶ dG). In vitro exposure of calf thymus DNA to ALA (0.05-5mM) in the presence of 10 uM Fe²+ caused the formation of 8-OHdG.The amount of 8-OHdG rose from 135 ± 15 residues/10⁶dG in the control system to 1140 ± 150 residues/106 dGafter incubation with 5 mM ALA and 10 µM Fe²1. Diethylenetriaminepentaaceticacid (5 mM) or mannitol (100 mM) inhibited the formation of8-OHdG by 63 and 69% respectively, evidencing the involvementof both H2O2 and HO in this process. Hydrogen peroxide (100µM) or Fe²+ alone did not cause DNA oxidation. The presentdata support the hypothesis that ALA-generated reactive oxygenspecies can oxidize DNA and may be involved in the developmentof primary liver cell carcinoma in individuals with symptomaticacute intermittent porphyria.