Jose Serrano

CARDIOSELECTIVE AND CUMULATIVE OXIDATION OF MITOCHONDRIAL DNA FOLLOWING SUBCHRONIC DOXORUBICIN ADMINISTRATION

We recently reported the preferential accumulation of 8-hydroxydeoxyguanosine (8OHdG) adducts in cardiac mitochondrial DNA (mtDNA) following acute intoxication of rats with doxorubicin (C.M. Palmeira et al., Biochim. Biophys. Acta, 1321 (1997) 101-106). The concentration of 8OHdG adducts decreased to control values within 2 weeks. Since conventional antineoplastic therapy entails repeated administration of small doses of doxorubicin, it was of interest to characterize the kinetics for the accumulation and repair of 8OHdG adducts in the various DNA fractions. Weekly injections of doxorubicin (2 mg/kg, i.p.) to adult male Sprague-Dawley rats caused a cumulative dose-dependent increase in the concentration of 8OHdG adducts in both mtDNA and nuclear DNA (nDNA) from heart and liver. Following six weekly injections, the concentration of 8OHdG in cardiac mtDNA was 50% higher than liver mtDNA and twice that of cardiac nDNA. In contrast to the rapid repair of 8OHdG observed during the first days following an acute intoxicating dose of doxorubicin, the concentration of 8OHdG adducts remained constant between 1 and 5 weeks following the last injection. This was true for all DNA fractions examined. The cardioselective accumulation and persistence of 8OHdG adducts to mtDNA is consistent with the implication of mitochondrial dysfunction in the cumulative and irreversible cardiotoxicity observed clinically in patients receiving doxorubicin cancer chemotherapy.

Preferential oxidation of cardiac mitochondrial DNA following acute intoxication with doxorubicin

The purpose of this investigation was to determine whether acute doxorubicin intoxication causes a preferential accumulation of 8-hydroxydeoxyguanosine (8OHdG) adducts to mitochondrial DNA (mtDNA) as opposed to nuclear DNA (nDNA), particularly in cardiac tissue. Adult male rats received a single i.p. bolus of doxorubicin (15 mg/kg) and were killed 1-14 days later. Acute intoxication with doxorubicin caused a 2-fold greater increase in 8OHdG adducts to mtDNA compared to nDNA, the concentration of adducts to both nDNA and mtDNA being 20%-40% greater for heart as opposed to liver. For both tissues, the relative abundance of adducts was highest at the earliest time-point examined (24 h) and decreased to control values by 2 weeks. The temporal dilution of 8OHdG adducts was not the result of cell hyperplasia and was only partially due to amplification of the mitochondrial genome, most probably via an increase in DNA copy number rather than a stimulation of mitochondrial biogenesis.

Determination of 8-hydroxydeoxyguanosine in biological tissue by liquid chromatography/electrospray ionization-mass spectrometry/mass spectrometry.

A method based on liquid chromatography (LC) combined with electrospray ionization tandem mass spectrometry for the analysis of the frequency of formation of 8-hydroxydeoxyguanosine (80HdGuo), a biomarker of oxidative damage to DNA, has been developed. This analytical technique has the advantage over gas chromatography combined with mass spectrometry of not requiring analyte derivatization, and over LC combined with ultraviolet and electrochemical detection of being chemospecific. The method was evaluated by determining increased frequency of formation of 80HdGuo in male Sprague-Dawley rats given a single intraperitoneal dose of Adriamycin compared to control rats. Detection of one oxidized deoxyguanosine per 5 x 10(5) deoxyguanosines was readily achieved.

Characterization of dansylated glutathione, glutathione disulfide, cysteine and cystine by narrow bore liquid chromatography/electrospray ionization mass spectrometry

A method using reversed phase high performance liquid chromatography/electrospray ionization-mass spectrometry (RP-LC/ESI-MS) has been developed to confirm the identity of dansylated derivatives of cysteine (C) and glutathione (GSH), and their respective dimers, cystine (CSSC) and glutathione disulfide (GSSG). Cysteine, GSH, CSSC and GSSG are present at low concentrations in rainbow trout (Oncorhynchus mykiss) liver cells. Initially, hepatic cells were sampled from a suspension culture and disrupted upon addition of 10% perchloric acid. The reduced thiols present in the cell extracts were acetylated to prevent dimerization and then the C and GSH species were derivatized with dansyl chloride for fluorescence detection. An LC system using a weak anion exchange column (AE) with fluorescence detection (FLD) was used for sensitive routine analysis; however, it produced peaks of unknown origin in addition to the expected analytes. Analytes were then separated on a C18 RP-LC system using a water/acetonitrile gradient with 0.2% formic acid, and detected using LC/ESI-MS at 3.5 KV which produced an intense ion with a minimum limit of detection of less than 0.5 pmole injected (>10:1 signal-to-noise (S/N). Subsequently, fractions of effluent from the AE-LC/FLD system were analyzed by LC/ESI-MS to confirm the presence of the target analytes in routine cell extracts. Monodansylated GSSG was identified as a product that could possibly affect the quantification of GSH and GSSG.

Identification of potentially mutagenic contaminants in the aquatic environment by liquid chromatographic—thermospray mass spectrometric characterization of in vitro DNA adducts

Abstract Liquid chromatographic—thermospray mass spectrometric (LC—TSP-MS) characterization of chemical adducts of DNA formed during in vitro reactions is proposed as an analytical technique to detect and identify those contaminants in aqueous environmental samples which have the propensity to be genotoxic, i.e. to covalently bond to DNA. The approach for direct-acting chemicals includes the in vitro incubation of DNA with contaminated aqueous samples at 37°C, pH 7.0 for 0.5 to 6 h, followed by enzymatic hydrolysis of the DNA to deoxynucleosides and LC—TSP-MS analysis of the resultant solution. A series of allylic reagents was used as model reactive electrophiles in synthetic aqueous samples to demonstrate that adduct formation was linear with both contaminant concentration and electrophilic reactivity potential. The characterizations can also estimate the proportion of bonding to different sites on a base, for instance, the ratio of O 6 - to 7-alkylguanine (oxygen vs. nitrogen bonding) products, which is an important parameter in assessing the genotoxicology of chemicals.

Thyroid-stimulating hormone (TSH): Measurement of intracellular, secreted, and circulating hormone in Xenopus laevis and Xenopus tropicalis

Thyroid-stimulating hormone (TSH) is an important regulator of the hypothalamic-pituitary-thyroid (HPT) axis in Xenopus laevis. To evaluate the role of this hormone on developing tadpoles, immunologically-based Western blots and sandwich ELISAs were developed for measuring intracellular (within pituitaries), secreted (ex vivo pituitary culture), and circulating (serum) amounts. Despite the small size of the tadpoles, these methods were able to easily measure intracellular and secreted TSH, and circulating TSH was measurable in situations where high levels were induced. The method was validated after obtaining a highly purified and enriched TSH sample using anti-TSH-β antibodies conjugated to magnetic beads. Subsequent mass-spectrometric analysis of the bands from SDS-PAGE and Western procedures identified the presence of amino acid sequences corresponding to TSH subunits. The purified sample was also used to prepare standard curves for quantitative analysis. The Western and ELISA methods had limits of detection in the low nanogram range. While the majority of the developmental work for these methods was done with X. laevis, the methods also detected TSH in Xenopus tropicalis. To our knowledge this is the first report of a specific detection method for TSH in these species, and the first to measure circulating TSH in amphibians. Examples of the utility of the methods include measuring a gradual increase in pituitary TSH at key stages of development, peaking at stages 58-62; the suppression of TSH secretion from cultured pituitaries in the presence of thyroid hormone (T4); and increases in serum TSH following thyroidectomy.

In vivo assessment and potential diagnosis of xenobiotics that perturb the thyroid pathway: Proteomic analysis of Xenopus laevis brain tissue following exposure to model T4 inhibitors.

As part of a multi-endpoint systems approach to develop comprehensive methods for assessing endocrine stressors in vertebrates, differential protein profiling was used to investigate expression patterns in the brain of the amphibian model (Xenopus laevis) following in vivo exposure to a suite of T4 synthesis inhibitors. We specifically address the application of Two Dimensional Polyacrylamide Gel Electrophoresis (2D PAGE), Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) and LC-MS/MS to assess changes in relative protein expression levels. 2D PAGE and iTRAQ proved to be effective complementary techniques for distinguishing protein changes in the developing amphibian brain in response to T4 synthesis inhibition. This information served to evaluate the use of distinctive protein profiles as a potential mechanism to screen chemicals for endocrine activity in anurans. Regulatory pathways associated with proteins expressed as a result of chemical effect are reported. To our knowledge, this is also the first account of the anuran larvae brain proteome characterization using proteomic technologies. Correlation of protein changes to other cellular and organism-level responses will aid in the development of a more rapid and cost-effective, non-mammalian screening assay for thyroid axis-disrupting chemicals.

Analytical procedures and quality assurance criteria for the determination of major and minor deoxynucleosides in fish tissue DNA by liquid chromatography—ultraviolet spectroscopy and liquid chromatography—thermospray mass spectrometry

Analytical procedures and quality assurance criteria have been established for enzymatic hydrolysis of fish tissue DNA to free nucleosides and their subsequent characterization by liquid chromatography-photodiode-array ultraviolet spectroscopy and liquid chromatography-thermospray mass spectrometry. Optimization of enzymatic efficiency to assure minimal loss of modified nucleosides is described. Variability in analyte capacity factors and multiwavelength response have been compared for analyte standards and hydrolysates, and results have been used to derive qualitative and quantitative quality assurance criteria. A comparison of DNA mole percent calculated using single-wavelength quantification and multiwavelength averaging quantification indicates that less variability in data may be expected using the multiwavelength technique. Finally, the composition of DNA from liver of three species of fish widely used in mutagen/carcinogen laboratory and field studies, rainbow trout (Onchorynchus mykiss), medaka (Oryzias latipes), and brown bullhead (Ictalurus nebulosus), has been determined. Identification of deoxyuridine in the DNA hydrolysates of each fish indicates that this analyte should be measured to accurately report DNA deoxynucleoside mole percent, especially when reporting data for the methylation of deoxycytidine.

A comparison of fish pesticide metabolic pathways with those of the rat and goat

ABSTRACT Ecological risk assessments are often limited in their ability to consider metabolic transformations for fish species due to a lack of data. When these types of evaluations are attempted they are often based on parent chemical only, or by assuming similarity to available mammalian metabolic pathways. The metabolism maps for five pesticides (fluazinam, halauxifen‐methyl, kresoxim‐methyl, mandestrobin, and tolclofos‐methyl) were compared across three species. A rapid and transparent process, utilizing a database of systematically collected information for rat, goat, and fish (bluegill or rainbow trout), and using data evaluation tools in the previously described metabolism pathway software system MetaPath, is presented. The approach demonstrates how comparisons of metabolic maps across species are aided by considering the sample matrix in which metabolites were quantified for each species, differences in analytical methods used to identify metabolites in each study, and the relative amounts of metabolites quantified. By incorporating these considerations, more extensive rat and goat metabolism maps were found to be useful predictors of the more limited metabolism of the five pesticides in fish. HighlightsA systematic approach for species comparison of metabolism studies is shown.Major metabolites from fish tissue were represented well in the rat and goat studies.Sample matrix is an important consideration in the fish, rat and goat studies.

Identification of estrogen-responsive vitelline envelope protein fragments from rainbow trout (Oncorhynchus mykiss) plasma using mass spectrometry†

Plasma peptides previously associated with exposure of juvenile male rainbow trout (Oncorhynchus mykiss) to the hormone 17β‐estradiol (E2) were identified using matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF‐MS). Specifically, plasma peptides of interest were fractionated and subsequently identified via spectra obtained by MALDI QqTOF MS/MS and LC‐MALDI TOFTOF MS/MS analysis, de novo sequencing and database matching. The two peptide masses were identified as significant matches for fragments of the C‐terminal propeptides from rainbow trout vitelline envelope protein (VEP)α and VEPγ isoforms. Our findings document the presence of the C‐terminal propeptides from rainbow trout VEPα and VEPγ proteins in the bloodstream of juvenile male rainbow trout exposed to E2 via MALDI‐TOF‐MS detection. We provide three possible explanations for the presence of C‐terminal propeptides in the bloodstream, as well as compare previously obtained hepatic transcriptomic results with the plasma proteomic results obtained in the present study. Mol. Reprod. Dev. 77:963–970, 2010. Published 2010 Wiley‐Liss, Inc.