Circulating tumor DNA association with residual cancer burden after neoadjuvant chemotherapy in triple-negative breast cancer in TBCRC 030.

BACKGROUND: We aimed to examine circulating tumor DNA (ctDNA) and its association with residual cancer burden (RCB) using an ultrasensitive assay in patients with triple-negative breast cancer (TNBC) receiving neoadjuvant chemotherapy. PATIENTS AND METHODS: We identified responders (RCB 0/1) and matched non-responders (RCB 2/3) from the phase II TBCRC 030 prospective study of neoadjuvant paclitaxel versus cisplatin in TNBC. We collected plasma samples at baseline, 3 weeks and 12 weeks (end of therapy). We created personalized ctDNA assays utilizing MAESTRO mutation enrichment sequencing. We explored associations between ctDNA and RCB status and disease recurrence. RESULTS: Of 139 patients, 68 had complete samples and no additional neoadjuvant chemotherapy. Twenty-two were responders and 19 of those had sufficient tissue for whole-genome sequencing. We identified an additional 19 non-responders for a matched case-control analysis of 38 patients using a MAESTRO ctDNA assay tracking 319-1000 variants (median 1000 variants) to 114 plasma samples from 3 timepoints. Overall, ctDNA positivity was 100% at baseline, 79% at week 3 and 55% at week 12. Median tumor fraction (TFx) was 3.7 × 10-4 (range 7.9 × 10-7-4.9 × 10-1). TFx decreased 285-fold from baseline to week 3 in responders and 24-fold in non-responders. Week 12 ctDNA clearance correlated with RCB: clearance was observed in 10 of 11 patients with RCB 0, 3 of 8 with RCB 1, 4 of 15 with RCB 2 and 0 of 4 with RCB 3. Among six patients with known recurrence, five had persistent ctDNA at week 12. CONCLUSIONS: Neoadjuvant chemotherapy for TNBC reduced ctDNA TFx by 285-fold in responders and 24-fold in non-responders. In 58% (22/38) of patients, ctDNA TFx dropped below the detection level of a commercially available test, emphasizing the need for sensitive tests. Additional studies will determine whether ctDNA-guided approaches can improve outcomes.

Determining optimal eluter design by modeling physical dose enhancement in brachytherapy.

PURPOSE: In situ drug release concurrent with radiation therapy has been proposed to enhance the therapeutic ratio of permanent prostate brachytherapy. Both brachytherapy sources and brachytherapy spacers have been proposed as potential eluters to release compounds, such as nanoparticles or chemotherapeutic agents. The relative effectiveness of the approaches has not been compared yet. This work models the physical dose enhancement of implantable eluters in conjunction with brachytherapy to determine which delivery mechanism provides greatest opportunity to enhance the therapeutic ratio. MATERIALS AND METHODS: The combined effect of implanted eluters and radioactive sources were modeled in a manner that allowed the comparison of the relative effectiveness of different types of implantable eluters over a range of parameters. Prostate geometry, source, and spacer positions were extracted from treatment plans used for 125 I permanent prostate implants. Compound concentrations were calculated using steady-state solution to the diffusion equation including an elimination term characterized by the diffusion-elimination modulus (ϕb ). Does enhancement was assumed to be dependent on compound concentration up to a saturation concentration (csat ). Equivalent uniform dose (EUD) was used as an objective to determine the optimal configuration of eluters for a range of diffusion-elimination moduli, concentrations, and number of eluters. The compound delivery vehicle that produced the greatest enhanced dose was tallied for points in parameter space mentioned to determine the conditions under whether there are situations where one approach is preferable to the other. RESULTS: The enhanced effect of implanted eluters was calculated for prostate volumes from 14 to 45 cm3 , ϕb from 0.01 to 4 mm-1 , csat from 0.05 to 7.5 times the steady-state compound concentration released from the surface of the eluter. The number of used eluters (ne ) was simulated from 10 to 60 eluters. For the region of (csat , Φ)-space that results in a large fraction of the gland being maximally sensitized, compound eluting spacers or sources produce equal increase in EUD. In the majority of the remaining (csat , Φ)-space, eluting spacers result in a greater EUD than sources even where sources often produce greater maximal physical dose enhancement. Placing eluting implants in planned locations throughout the prostate results in even greater enhancement than using only source or spacer locations. CONCLUSIONS: Eluting brachytherapy spacers offer an opportunity to increase EUD during the routine brachytherapy process. Incorporating additional needle placements permits compound eluting spacer placement independent of source placement and thereby allowing a further increase in the therapeutic ratio. Additional work is needed to understand the in vivo spatial distribution of compound around eluters, and to incorporate time dependence of both compound release and radiation dose.

Kilovoltage radiosurgery with gold nanoparticles for neovascular age-related macular degeneration (AMD): a Monte Carlo evaluation.

This work uses Monte Carlo radiation transport simulation to assess the potential benefits of gold nanoparticles (AuNP) in the treatment of neovascular age-related macular degeneration with stereotactic radiosurgery. Clinically, a 100 kVp x-ray beam of 4 mm diameter is aimed at the macula to deliver an ablative dose in a single fraction. In the transport model, AuNP accumulated at the bottom of the macula are targeted with a source representative of the clinical beam in order to provide enhanced dose to the diseased macular endothelial cells. It is observed that, because of the AuNP, the dose to the endothelial cells can be significantly enhanced, allowing for greater sparing of optic nerve, retina and other neighboring healthy tissue. For 20 nm diameter AuNP concentration of 32 mg g(-1), which has been shown to be achievable in vivo, a dose enhancement ratio (DER) of 1.97 was found to be possible, which could potentially be increased through appropriate optimization of beam quality and/or AuNP targeting. A significant enhancement in dose is seen in the vicinity of the AuNP layer within 30 µm, peaked at the AuNP-tissue interface. Different angular tilting of the 4 mm beam results in a similar enhancement. The DER inside and in the penumbra of the 4 mm irradiation-field are almost the same while the actual delivered dose is more than one order of magnitude lower outside the field leading to normal tissue sparing. The prescribed dose to macular endothelial cells can be delivered using almost half of the radiation allowing reduction of dose to the neighboring organs such as retina/optic nerve by 49% when compared to a treatment without AuNP.

Radiosensitizer-eluting nanocoatings on gold fiducials for biological in-situ image-guided radio therapy (BIS-IGRT).

Image-guided radiation treatments (IGRT) routinely utilize radio-opaque implantable devices, such as fiducials or brachytherapy spacers, for improved spatial accuracy. The therapeutic efficiency of IGRT can be further enhanced by biological in situ dose painting (BIS-IGRT) of radiosensitizers through localized delivery within the tumor using gold fiducial markers that have been coated with nanoporous polymer matrices loaded with nanoparticles (NPs). In this work, two approaches were studied: (i) a free drug release system consisting of Doxorubicin (Dox), a hydrophilic drug, loaded into a non-degradable polymer poly(methyl methacrylate) (PMMA) coating and (ii) poly(d,l-lactic-co-glycolic acid) (PLGA) NPs loaded with fluorescent Coumarin-6, serving as a model for a hydrophobic drug, in a biodegradable chitosan matrix. Temporal release kinetics measurements in buffer were carried out using fluorescence spectroscopy. In the first case of free Dox release, an initial release within the first few hours was followed by a sustained release over the course of the next 3 months. In the second platform, release of NPs and the free drug was controlled by the degradation rate of the chitosan matrix and PLGA. The results show that dosage and rate of release of these radiosensitizers coated on gold fiducials for IGRT can be precisely tailored to achieve the desired release profile for radiation therapy of cancer.

Detection of hotspot mutations and polymorphisms using an enhanced PCR-RFLP approach.

Ethidium gel-based PCR-RFLP is widely used, and is perhaps the simplest method for detection of known mutations in cancer-related genes and for genotyping a wide range of other human diseases. However, its application is limited by the fact that it can only detect mutant alleles that are present in more than 5-10% of wild-type alleles. Here we present a method that allows a 1-2 order enhancement in the sensitivity of the widely used PCR-RFLP without substantially increasing the effort and cost associated with it. This method is a modification to our previously reported amplification via primer ligation at the mutation (APRIL-ATM) method, which utilizes ligation of a primer at a restriction site formed by a mutation, followed by a ligation-mediated PCR amplification which amplifies only the mutation-containing DNA molecules. By combining this method with the artificial introduction of restriction sites during PCR, we demonstrate that assays can be designed and validated for detecting hot-spot mutations in codons 273, 158, and 248 of the TP53 gene (p53) and potentially for most mutations of interest. This approach is validated by using samples where the mutation was artificially introduced at these p53 positions. The increased sensitivity offered by the method further allows us to rapidly screen for low frequency polymorphisms in pooled DNA samples. The frequency of an MSH2 missense polymorphism (965G>A) was quantified in pooled genomic DNA samples from 205 and 221 U.S. and Polish colorectal cancer patients, respectively, and an equal number of ethnicity-matched controls. The data revealed a 3-5% prevalence of this polymorphism in the patient and the control populations. Individual sequencing of all 852 patient samples demonstrated an excellent agreement among the two independent approaches. The present enhanced PCR-RFLP reduces the effort involved in high throughput polymorphism studies and promises to find applications in genotyping and association studies involving low frequency polymorphisms and mutations.

Reproducible and inexpensive probe preparation for oligonucleotide arrays.

We present a new protocol for the preparation of nucleic acids for microarray hybridization. DNA is fragmented quantitatively and reproducibly by using a hydroxyl radical-based reaction, which is initiated by hydrogen peroxide, iron(II)-EDTA and ascorbic acid. Following fragmentation, the nucleic acid fragments are densely biotinylated using a biotinylated psoralen analog plus UVA light and hybridized on microarrays. This non-enzymatic protocol circumvents several practical difficulties associated with DNA preparation for microarrays: the lack of reproducible fragmentation patterns associated with enzymatic methods; the large amount of labeled nucleic acids required by some array designs, which is often combined with a limited amount of starting material; and the high cost associated with currently used biotinylation methods. The method is applicable to any form of nucleic acid, but is particularly useful when applying double-stranded DNA on oligonucleotide arrays. Validation of this protocol is demonstrated by hybridizing PCR products with oligonucleotide-coated microspheres and PCR amplified cDNA with Affymetrix Cancer GeneChip microarrays.

Highly selective isolation of unknown mutations in diverse DNA fragments: toward new multiplex screening in cancer.

Cancer research would greatly benefit from technologies that allow simultaneous screening of several unknown gene mutations. Lack of such methods currently hampers the large-scale detection of genetic alterations in complex DNA samples. We present a novel mismatch-capture methodology for the highly efficient isolation and amplification of mutation-containing DNA from diverse nucleic acid fragments of unknown sequence. To demonstrate the potential of this method, heteroduplexes with a single A/G mismatch are formed via cross-hybridization of mutant (T-->G) and wild-type DNA-fragment populations. Aldehydes are uniquely introduced at the position of mismatched adenines via the Escherichia coli glycosylase, MutY. Subsequent treatment with a biotinylated hydroxylamine results in highly specific and covalent biotinylation of the site of mismatch. For PCR amplification, synthetic linkers are then ligated to the DNA fragments. Biotinylated DNA is then isolated and PCR amplified. Mutation-containing DNA fragments can subsequently be sequenced to identify type and position of mutation. This method correctly detects a single T-->G transversion introduced into a 7-kb plasmid containing full-length cDNA from the p53 gene. In the presence of a high excess wild-type DNA (1:1000 mutant:normal plasmids) or in the presence of diverse DNA fragment sizes, the DNA fragments containing the mutation are readily detectable and can be isolated and amplified. The present Aldehyde-Linker-Based Ultrasensitive Mismatch Scanning has a current limit of detection of one base substitution in 7 Mb of DNA and increases the limit for unknown mutation scanning by two to three orders of magnitude. Homozygous and heterozygous p53 regions (G-->T, exon 4) from genomic DNA are also examined, and correct identification of mutations is demonstrated. This method should allow large-scale detection of genetic alterations in cancer samples without any assumption as to the genes of interest.

Novel non-isotopic detection of MutY enzyme-recognized mismatches in DNA via ultrasensitive detection of aldehydes.

A highly sensitive method to detect traces of aldehyde-containing apurinic/apyrimidinic (AP) sites in nucleic acids has been developed. Based on this method, a novel approach to detect DNA base mismatches recognized by the mismatch repair glycosylase MutY is demonstrated. Open chain aldehydes generated in nucleic acids due to spontaneous depurination, DNA damage or base excision of mismatched adenine by MutY are covalently trapped by a new linker molecule [fluorescent aldehyde-reactive probe (FARP), a fluorescein-conjugated hydroxylamine derivative]. DNA containing AP sites is FARP-trapped, biotinylated and immobilized onto neutravidin-coated microplates. The number of FARP-trapped aldehydes is then determined via chemiluminescence using a cooled ICCD camera. AP sites induced in plasmid or genomic calf thymus DNA via mild depurination or by simple incubation at physiological conditions (pH 7, 37 degreesC) presented a linear increase in chemiluminescence signal with time. The procedure developed, from a starting DNA material of approximately 100 ng, allows detection of attomole level (10(-18) mol) AP sites, or 1 AP site/2 x 10(7) bases, and extends by 1-2 orders of magnitude the current limit in AP site detection. In order to detect MutY-recognized mismatches, nucleic acids are first treated with 5 mM hydroxylamine to remove traces of spontaneous aldehydes. Following MutY treatment and FARP-labeling, oligonucleotides engineered to have a centrally located A/G mismatch demonstrate a strong chemiluminescence signal. Similarly, single-stranded M13 DNA that forms mismatches via self-complementation (average of 3 mismatches over 7429 bases) and treated with MutY yields a signal approximately 100-fold above background. No signal was detected when DNA without mismatches was used. The current development allows sensitive, non-isotopic, high throughput screening of diverse nucleic acids for AP sites and mismatches in a microplate-based format.

Fluorescent labelling of closely-spaced aldehydes induced in DNA by bleomycin-Fe(III).

PURPOSE: To test the ability of FARPhC and FARP, two novel fluorescent reagents, to label aldehyde-containing sites (principally abasic sites) generated in DNA by the radiomimetic drug bleomycin, and to use fluorescent energy transfer from FARPhc (donor) to FARP (acceptor) to quantitate such closely-spaced sites. MATERIALS AND METHODS: FARPhc, 7-hydroxycoumarin-3-carboxylic acid (((((amino-oxymethyl) carbonyl) hydrazino) carbonylethyl) amide) was synthesized with a protocol similar to the one recently reported for FARP (a fluorescein-based probe). RESULTS: Both FARPhc and FARP form stable oxime bonds with the open-chain aldehydes generated upon acidic depurination of DNA. Plasmid DNA exposed to bleomycin-Fe(III)-ascorbate undergoes extensive strand breakage, and upon subsequent reaction with FARPhc and/or FARP it becomes fluorescently labelled, indicating the generation of aldehyde-containing sites. The binding of the probes to calf thymus or plasmid DNA results in significant fluorescent energy transfer among closely-spaced fluorophores, as revealed by the fluorescence increase following digestion of fluorescently labelled samples with nuclease P1. The fluorescent quenching is most evident when both FARPhc and FARP are used simultaneously to trap aldehyde sites. When single-stranded oligonucleotides engineered to contain either one or two closely spaced bleomycin binding sites are exposed to bleomycin and then fluorescently labelled, the oligonucleotides demonstrate significantly increased fluorescent energy transfer with two binding sites indicating a dependence of aldehyde site generation and clustering on the local sequence of a single strand. CONCLUSIONS: A new detection method for DNA damage induced by bleomycin following fluorescent labelling of aldehyde group-containing sites (FLAGS) and their clustering via fluorescent energy transfer is demonstrated. The method is applicable to any form of DNA. This work may lead to a general approach for the quantification of multiply damaged sites in DNA, a subset of DNA lesions that may have major biological significance.

Fluoresceinated phosphoethanolamine for flow-cytometric measurement of lipid peroxidation.

A new lipophilic fluorescein probe (fluor-DHPE) has been identified that can assay lipid peroxidation in mammalian cells on a cell-by-cell or selected-cell-subpopulation basis by flow cytometry. Application of this approach requires that the fluorescent probe be nonexchangeable among cells. Fluorescein is an appropriate fluorophore, since its fluorescence matches the specifications of common flow cytometers and the compound loses its fluorescence upon reaction with peroxyl radicals. Upon examination of four lipophilic derivatives of fluorescein, fluor-DHPE was found to be the only probe that was nonexchangeable among labeled and unlabeled rat RBC for at least 24 h. The exposure of fluor-DHPE-labeled RBC to benzoyl peroxide followed by mixing the sample with RBC unexposed to peroxide led to a decrease in fluorescence. Furthermore, the flow cytometer could clearly select the subpopulation of cells undergoing lipid peroxidation from those cells that were not. Fluor-DHPE-labeled-RBC obtained from rats and exposed to cumene hydroperoxide also displayed a gradual decrease in fluorescence. This decrease was preventable by either regulation of the vitamin E content in the animal diet or in vitro supplementation of cells with vitamin E. We conclude that fluor-DHPE is a stable and nonexchangeable probe for monitoring lipid peroxidation in cell subpopulations by flow cytometry.

Fluorescent labelling of abasic sites: a novel methodology to detect closely-spaced damage sites in DNA.

PURPOSE: To test the ability of a new fluorescent reagent to label abasic sites in DNA and to use fluorescent energy transfer as a measure of closely-spaced abasic sites in DNA. MATERIALS AND METHODS: A fluorescein-conjugated hydroxylamine derivative (FARP, 5-(((2-(carbohydrazino)-methyl)thio)acetyl)aminofluorescein, aminooxyacetyl hydrazide) that reacts covalently with open chain aldehydes in DNA has been synthesized. Upon depurination of plasmid DNA and reaction with FARP a stable oxime bond is formed between FARP and the generated open chain aldehydes. RESULTS: By independently quantitating the generated abasic sites, it is shown that most of the generated abasic sites under acidic conditions become fluorescently labelled. The limit of sensitivity with the fluorometer used is approximately 90 femtomole abasic sites, corresponding to 1 abasic site per 17000 base pairs. DNA can be fluorescently labelled over a wide range of FARP:base pair ratios following different extent of depurination, and fluorescent loadings of 1 FARP:20000 base pairs up to 1 FARP:10 base pairs are demonstrated. The heavily labelled samples display significant fluorescence quenching due to the proximity of abasic sites labelled with FARP, that undergo fluorescence energy transfer. Treatment of heavily labelled DNA samples with nuclease P1 results in an increase in fluorescence due to the release of the fluorescent labels in the solution. The relative increase in fluorescence is a quantitative measure of the proximity of labelled abasic sites. Furthermore it is shown that if only 1% of DNA contains abasic sites generated in close proximity (within 10-20 base pairs or less of each other) the resulting quenching is significant enough to detect, even if the rest of the DNA contains isolated abasic sites. CONCLUSIONS: The present approach constitutes a novel fluorescence-based method to detect abasic sites in nucleic acids and demonstrates the feasibility of detecting the presence of closely-spaced damage sites in DNA via fluorescence energy transfer. The technique also comprises a general and convenient method to fluorescently label nucleic acids without introducing strand breaks as a result of the labelling procedure.

5-[125I]iodo-2'-deoxyuridine in the radiotherapy of brain tumors in rats.

UNLABELLED: Glial neoplasms of the human central nervous system have defied treatment, in part because of the limited selectivity of available cytotoxic agents. The thymidine analog 5-iodo-2'-deoxyuridine radiolabeled with the Auger electron emitter 125I (125IUdR) is highly toxic to dividing cells when it is deoxyribonucleic acid incorporated, but it is relatively innocuous when located outside the nucleus. Previous studies have shown that 125IUdR has significant antineoplastic potential against mammalian cells in vitro and direct administration of 125IUdR is effective therapy for ovarian ascites tumors in mice and neoplastic meningitis in rats. Studies using external gamma imaging and autoradiography have also shown that direct intratumoral administration of 123IUdR/125IUdR into intracerebral 9L gliosarcomas in rats results in selective uptake of the radionuclide into tumor cells. Based on these encouraging results, we have evaluated the therapeutic potential of 125IUdR in rats bearing intracerebral 9L gliosarcomas. METHODS: Iodine-125-IUdR was infused intracerebrally over a 2-day period into rats bearing 1-day-old 9L tumors and over a 6-day period into animals with 9-day-old 9L tumors; equimolar concentrations of 127IUdR were infused into control animals. Tumor growth was monitored by contrast-enhanced 1H MRI and animal survival was followed over time. RESULTS: Intracerebral tumors (3-7 mm) were readily detected by MRI. Tumor-bearing rats treated with 127IUdR succumbed within 17-24 days, whereas tumor-bearing animals treated with 125IUdR survived significantly longer, and 10%-20% of the animals were cured of tumors. CONCLUSION: These data substantiate the antineoplastic potential of 5-[125I]iodo-2'-deoxyuridine and indicate that it may be a useful agent for the therapy of solid tumors that are accessible to direct radiopharmaceutical administration.

Novel visible and ultraviolet light photogeneration of hydroxyl radicals by 2-methyl-4-nitro-quinoline-N-oxide (MNO) and 4,4'-dinitro-(2,2')bipyridinyl-N,N'-dioxide (DBD).

Chemicals that upon absorption of light generate hydroxyl radicals (.OH), free of other damaging species under physiological conditions, are useful tools for the study of the biological effects of .OH radical and for its utilization for analytical purposes. We report the novel property of 2-methyl-4-nitro-quinoline-N-oxide (MNO) and 4,4'-dinitro-(2,2')bipyridinyl-N,N'-dioxide (DBD) to act as photogenerators of .OH with UV and visible light. Upon irradiation with 360-400 nm light MNO and DBD generate free radicals that convert coumarin carboxylic acid (CCA) to fluorescent 7-OH-CCA; the .OH radical scavengers dimethylsulfoxide (DMSO) and ethanol eliminate the induction of 7-OH-CCA fluorescence. Upon 400 nm illumination in the presence of MNO, supercoiled plasmid DNA is converted to circular and strand breakage is significantly reduced in the presence of DMSO and completely absent in the absence of MNO. The conversion of CCA to 7-OH-CCA and of supercoiled plasmid to circular DNA are also observed in the absence of oxygen. Taken together, these data indicate that MNO and DBD constitute novel .OH-generating compounds. Because currently known .OH-photogenerating compounds require UV illumination (< 360 nm) that also damages DNA and cells directly, the property of MNO to generate .OH upon 400 nm illumination is advantageous when studies on cells, DNA and other biomolecules are conducted.

Continuous detection of radiation or metal generated hydroxyl radicals within core chromatin particles.

PURPOSE: The aim of this work was to adapt a recently developed fluorometric method for use in the detection of hydroxyl radical (HO.) generated in the immediate vicinity of chromatin core particles reconstituted from pUC19 plasmid DNA and isolated core histones. MATERIALS AND METHODS: The procedure followed involves labelling nucleosomal histones with SECCA, a non-fluorescent coumarin derivative that generates the fluorescent 7-hydroxy-SECCA after reaction with HO.. Core particles are formed using histones and pUC19 DNA in a salt-dialysis procedure. RESULTS: Electron microscopy and micrococcal nuclease digestion are consistent with successful formation of core particles. No significant differences between core particle formation in the unlabelled and SECCA-labelled samples were detected. Exposure to HO. generated by radiation or copper--ascorbic acid--hydrogen peroxide results in a gradual induction of fluorescence. Studies using dimethyl sulphoxide (DMSO) demonstrate that, unlike HO. produced by radiation, the majority of HO. generated by copper--ascorbic acid--hydrogen peroxide occurs primarily in the immediate vicinity of core particles and DNA and cannot be scavenged. CONCLUSIONS: The present procedure demonstrates the feasibility to quantitate HO. generated by several agents in the immediate vicinity of nucleosomes (chromatin-associated HO.) or associated with specific regions within the genome.

Detection of lipid peroxidation on erythrocytes using the excimer-forming property of a lipophilic BODIPY fluorescent dye.

Lipophilic analogues of dipyrrometheneboron (BODIPY-FL) dyes, used for membrane studies, normally fluoresce in the green wavelength region (approximately 516 nm), but at high local concentration, they shift their emission to the red region (approximately 540-600 nm) via excimer formation. A two-wavelength-based method is described that utilizes the excimer-forming property of BODIPY-FL for the sensitive monitoring of membrane lipid peroxidation on erythrocytes (RBCs). Bodipy-FL- C3-EDA, a relatively water-soluble analogue of BODIPY-FL, loses its single fluorescence peak at 516 nm upon reaction with peroxyl radicals generated in aqueous phase by 2,2'-azobis(2-amidinopropane), AAPH, and the loss of fluorescence is prevented by the presence of the peroxyl radical scavenger, Trolox (a water-soluble analogue of vitamin E). Hexadecanoyl-BODIPY-FL (C16-BODIPY, a lipophilic analogue of BODIPY-FL) incorporated into RBC membranes, in addition to the peak at 516 nm, also forms excimer fluorescent peaks at 546 and 590 nm. The relative intensity of the emission peaks depends on the concentration of membrane-incorporated dye. Upon addition of cumene hydroperoxide (CH, 0-10 microM) or benzoyl peroxide (BP, 0-5 microM) to C16-BODIPY-labeled RBC suspensions, gradual changes in the fluorescent peaks occur, the 516 nm peak initially increases, then decreases, while the 546 and 590 nm excimer peaks continuously decrease, as measured by fluorometry or by flow cytometry. The data indicate that lipid peroxidation radicals reacting with C16-BODIPY localized on RBC membranes oxidize the dye and the resulting molecule cannot participate in the excimer formation; this oxidization leads to the observed changes in the fluorescent peaks. The ratio of the fluorescence levels at 590 and 516 nm is a measure of the excimer formation between fluorophores and can be used to monitor the onset of lipid peroxidation in RBCs.

Novel fluorescein-based flow-cytometric method for detection of lipid peroxidation.

The novel property of fluorescein to detect peroxyl radicals is demonstrated. On the basis of this observation, a fluorescein-based, flow-cytometric method to directly and continuously detect free radicals generated in cell membranes during lipid peroxidation has been developed. 5- and 6-Carboxyfluorescein (5-/6-CF) free in solution and fluorescein-labeled polylysine lose their fluorescence gradually upon addition of a peroxyl-radical-generating system (thermal decomposition of 2,2'-azobis(2-amidinopropane) [AAPH]). 5-/6-CF retains its fluorescence when exposed to AAPH in the presence of the peroxyl radical scavenger Trolox. When 5-/6-CF free in solution is incubated with red blood cells exposed to cumene hydroperoxide (CH), a similar loss of fluorescence occurs due to lipid peroxidation on RBC membranes, which is preventable by pretreatment of the cells with Trolox or vitamin E. Undecylamine-fluorescein (C11-fluor), a lipophilic fluorescein conjugate, has been incorporated into the membranes of RBC. Upon addition of CH, a decrease in fluorescence is fluorometrically observed that is proportional to the amount of hydroperoxide added and inhibited by preincubation with Trolox or vitamin E. Flow-cytometric studies are then performed to demonstrate that C11-fluor can monitor free radicals generated during lipid peroxidation on a cell-by-cell basis. When exposed to CH, a time-dependent shift of the flow-cytometric profile toward lower values is observed that is inhibited by Trolox or vitamin E. This approach in conjunction with multiparametric flow cytometry may allow examination of the biologic significance of lipid peroxidation by correlation to other cellular end points on single cells.

Generation of hydroxyl radicals by nucleohistone-bound metal-adriamycin complexes.

A recently developed method has been utilized to demonstrate the generation of hydroxyl radicals (HO.) in the immediate proximity of DNA by copper(II)/iron(III)-adriamycin in the presence of ascorbate and hydrogen peroxide. SECCA, a succinylated derivative of coumarin, generates the fluorescent 7-hydroxy-SECCA following reaction with HO.. SECCA was coupled to polylysine or to histone H1 and then complexed to DNA. When HO. was generated in the proximity of DNA by polylysine-coupled iodine-125, which emits short range Auger electrons, 7-hydroxy-SECCA was produced. DMSO was only moderately efficient in reducing the fluorescence induction, demonstrating the "local" generation of HO. in this system. Copper(II)/iron(III)-adriamycin in the presence of ascorbate and hydrogen peroxide generated the fluorescent 7-hydroxy-SECCA both when SECCA was free in solution and when SECCA was DNA-conjugated. With SECCA free in solution, the fluorescence induction was almost eliminated in the presence of HO. scavengers (ethanol, tert-butanol or DMSO) and the relative efficiency of the scavengers in reducing the fluorescence followed their rate constant with HO.. Furthermore, SECCA incubated with a single oxygen-generating compound demonstrated no fluorescence induction. When SECCA was positioned in close proximity to DNA as a SECCA-histone-H1-DNA complex, the relative efficiency of the scavengers in reducing the fluorescence still followed their rate constant with HO.; overall however the scavengers were much less effective in reducing the fluorescence, due presumably to the formation of HO. radical in the immediate vicinity of DNA. These data suggest that copper(II)/iron(III)-adriamycin produces HO. in the presence of ascorbate and hydrogen peroxide whether unbound or bound to DNA and suggest that in the latter case scavengers would not prevent HO. from attacking chromatin. In addition, the ability of DMSO to trap HO. was shown to decrease as the conformation of the H1-DNA complex becomes more compact indicating the strong dependence of the trapping ability on chromatin conformation.

Measurement of hydroxyl radicals catalyzed in the immediate vicinity of DNA by metal-bleomycin complexes.

A recently developed sensitive fluorimetric assay has been used to examine whether free hydroxyl radicals (HO.) are generated in the immediate vicinity of DNA by Fe(II)-bleomycin. When aqueous solutions of SECCA (the succinimidyl ester of coumarin-3-carboxylic acid) are irradiated with gamma rays or incubated with Fe(II)-bleomycin or Fe (II)-EDTA in the presence of ascorbate and H2O2, 7-hydroxy-SECCA, a fluorescent product of the interaction of HO. with SECCA, is generated. Studies with catalase and several HO. scavengers indicate that the fluorescence induction is mediated by HO. On the contrary, Cu(II)-bleomycin complexes under similar conditions fail to induce 7-hydroxy-SECCA fluorescence. When SECCA is conjugated to DNA via SECCA-polylysine-DNA complexes and incubated in the same iron-containing systems, the relative ability of the scavengers to reduce the fluorescence again demonstrates the generation of 7-hydroxy-SECCA by HO. However, while the fluorescence is practically eliminated by high concentrations of DMSO (100 mumols dm-3) in the systems with Fe(II) or Fe(II)-EDTA, it is not possible to reduce it similarly in the case of Fe(II)- bleomycin. These data demonstrate the generation of HO. by Fe(II)-bleomycin in the immediate vicinity of DNA. Because the experiments simulate the lifetime of HO. expected in cells, these data suggest that, if such DNA-associated HO. radicals are also produced in vivo by bleomycin, these would not be scavengable by intracellular scavengers and they could interact with chromatin.

A fluorimetric method for the detection of copper-mediated hydroxyl free radicals in the immediate proximity of DNA.

An optical method to detect copper-mediated hydroxyl free radicals generated close to DNA and other biomolecules has been developed. Low-molecular-weight polylysines were labeled with SECCA, a derivative of coumarin that generates the fluorescent 7-OH-SECCA following its interaction with hydroxyl free radicals in aqueous solution. These polylysines were then complexed with DNA to place the detector molecule SECCA in the vicinity of the nucleic acid. Following addition of copper sulfate (0-10 mumol dm-3), free radicals were generated by incubation with ascorbic acid (0-1 mmol dm-3) and hydrogen peroxide (0-1 mmol dm-3). A rapid increase in the induced fluorescence was observed corresponding to the formation of the fluorescent 7-OH-SECCA in the polylysine-nucleic acid complex. This fluorescence was not decreased significantly by addition of high concentrations of hydroxyl free-radical scavengers (DMSO, methanol, ethanol and tert-butanol), but was diminished by addition of relatively low concentrations of EDTA (0.1 mmol dm-3), histidine (0.1 mmol dm-3) or catalase (8.3 x 10(-5) mmol dm-3). On the other hand, when such reaction mixtures were incubated with SECCA molecules that were free in solution or SECCA-labeled polylysine in the absence of DNA, the induced fluorescence was diminished by all hydroxyl free-radical scavengers. The efficiency by which the scavengers reduce the fluorescence increases as their hydroxyl rate constant increases. The data indicate that the detector molecule SECCA can be used to detect copper-mediated hydroxyl free radicals generated close to DNA.