A microplate assay for DNA damage determination (fast micromethod).

A rapid and convenient procedure for DNA damage determination in cell suspensions and solid tissues on single microplates was developed. The procedure is based on the ability of commercially available fluorochromes to interact preferentially with dsDNA in the presence of ssDNA, RNA, and proteins at high pH (>12.0), thus allowing direct measurements of DNA denaturation without sample handling or stepwise DNA separations. The method includes a simple and rapid 40-min sample lysis in the presence of EDTA, SDS, and high urea concentration at pH 10, followed by time-dependent DNA denaturation at pH 12.4 after NaOH addition. The time course and the extent of DNA denaturation is followed in a microplate fluorescence reader at room temperature for less than 1 h. The method requires only 30 ng DNA per single well and could conveniently be used whenever fast analysis of DNA integrity in small samples has to be done, e.g., in patients' lymphocytes after irradiation or chemotherapy (about 3000 cells per sample), in solid tissues or biopsies after homogenization (about 25 microg tissue per well), or in environmental samples for genotoxicity assessment.     

Identification of oxidized mitochondrial proteins in alcohol-exposed human hepatoma cells and mouse liver

Heavy alcohol consumption can damage various cells and organs partly through production of reactive oxygen species (ROS) and mitochondrial dysfunction. Treatment with antioxidants can significantly reduce the degree of damage. Despite well established roles of ROS in alcohol-induced cell injury, the proteins that are selectively oxidized by ROS are poorly characterized. We hypothesized that certain cysteinyl residues of target proteins are oxidized by ROS upon alcohol exposure, and these modified proteins may play roles in mitochondrial dysfunction. A targeted proteomics approach utilizing biotin-N-maleimide (biotin-NM) as a specific probe to label oxidized cysteinyl residues was employed to investigate which mitochondrial proteins are modified during and after alcohol exposure. Human hepatoma HepG2 cells with transduced CYP2E1 (E47 cells) were used as a model to generate ROS through CYP2E1-mediated ethanol metabolism. Following exposure to 100 mM ethanol for 4 and 8 h, the biotin-NM-labeled oxidized proteins were purified with agarose coupled to either streptavidin or monoclonal antibody against biotin. The purified proteins were resolved by two-dimensional gel electrophoresis and protein spots that displayed differential abundances were excised from the gel, in-gel digested with trypsin and analyzed for identity utilizing either matrix-assisted laser desorption-time of flight mass spectrometry or microcapillary reversed-phase liquid chromatography-tandem mass spectrometry. The results demonstrate that heat shock protein 60, protein disulfide isomerase, mitochondrial aldehyde dehydrogenases, prohibitin, and other proteins were oxidized after alcohol exposure. The identity of some of the proteins purified with streptavidin-agarose was also confirmed by immunoblot analyses using the specific antibody to each target protein. This method was also used to identify oxidized mitochondrial proteins in the alcohol-fed mouse liver. These results suggest that exposure to ethanol causes oxidation of various mitochondrial proteins that may negatively affect their function and contribute to alcohol-induced mitochondrial dysfunction and cellular injury.     

Comparison of ornithine decarboxylase from rat liver, rat hepatoma and mouse kidney.

Comparisons were made of ornithine decarboxylase isolated from Morris hepatoma 7777, thioacetamide-treated rat liver and androgen-stimulated mouse kidney. The enzymes from each source were purified in parallel and their size, isoelectric point, interaction with a monoclonal antibody or a monospecific rabbit antiserum to ornithine decarboxylase, and rates of inactivation in vitro, were studied. Mouse kidney, which is a particularly rich source of ornithine decarboxylase after androgen induction, contained two distinct forms of the enzyme which differed slightly in isoelectric point, but not in Mr. Both forms had a rapid rate of turnover, and virtually all immunoreactive ornithine decarboxylase protein was lost within 4h after protein synthesis was inhibited. Only one form of ornithine decarboxylase was found in thioacetamide-treated rat liver and Morris hepatoma 7777. No differences between the rat liver and hepatoma ornithine decarboxylase protein were found, but the rat ornithine decarboxylase could be separated from the mouse kidney ornithine decarboxylase by two-dimensional gel electrophoresis. The rat protein was slightly smaller and had a slightly more acid isoelectric point. Studies of the inactivation of ornithine decarboxylase in vitro in a microsomal system [Zuretti & Gravela (1983) Biochim. Biophys. Acta 742, 269-277] showed that the enzymes from rat liver and hepatoma 7777 and mouse kidney were inactivated at the same rate. This inactivation was not due to degradation of the enzyme protein, but was probably related to the formation of inactive forms owing to the absence of thiol-reducing agents. Treatment with 1,3-diaminopropane, which is known to cause an increase in the rate of degradation of ornithine decarboxylase in vivo [Seely & Pegg (1983) Biochem. J. 216, 701-717] did not stimulate inactivation by microsomal extracts, indicating that this system does not correspond to the rate-limiting step of enzyme breakdown in vivo.     

A fast and sensitive micromethod for the manual sequencing of peptides using O-phthalaldehyde as derivatizing reagent

A general procedure for the manual sequencing of peptides using the fluorogenic reagent O-phthalaldehyde (OPA) is described. The method can be applied in two different ways. One of them involves back hydrolysis of the anilinothiazolinones resulting from the Edman degradation of the peptide and subsequent detection of the free amino acids as OPA derivatives. The other is a subtractive analysis in which the amino acid composition of the remaining peptide is determined after each degradation cycle. The direct procedure can be coupled to the subtractive one in order to assure the accuracy of the sequence analysis. The method is fast and simple, and allows determination of 10 pmol of amino acid per cycle using standard reagents and instrumentation. Sensitivity can be greatly enhanced provided that ultrapure chemicals are employed. Small peptides (8-10 residues) were sequenced from 200 pmol sample, using a high-performance liquid chromatography assembly coupled to a fluorescence detector.     

Regulation of expression of tyrosine aminotransferase in somatic cell hybrids between rat hepatoma cells and mouse fibroblasts

Somatic cell hybrids between rat hepatoma cells and mouse 3T3 fibroblasts fail to produce the liver-specific enzyme tyrosine aminotransferase. A novel approach using gamma-irradiation to induce chromosome loss from the non-expressing parent cell was applied to dissect genetically the factors in 3T3 cells that interact with the regulation of expression of tyrosine aminotransferase in these hybrids. Suppression of basal and steroid-inducible tyrosine aminotransferase activities was progressively relieved with increasing dose of radiation. The wide range in degree of reexpression suggests a complex of regulatory mechanisms. Suppression of steroid-inducibility was not linked to the mouse X-chromosome. Nor did the mouse genome affect the modulation of enzyme activity induced by insulin and by serum.     

A fast and sensitive algorithm for aligning ESTs to the human genome

There is a pressing need to align the growing set of expressed sequence tags (ESTs) with the newly sequenced human genome. However, the problem is complicated by the exon/intron structure of eukaryotic genes misread nucleotides in ESTs, and the millions of repetitive sequences in genomic sequences. To solve this problem, algorithms that use dynamic programming have been proposed. In reality, however, these algorithms require an enormous amount of processing time. In an effort to improve the computational efficiency of these classical DP algorithms, we developed software that fully utilizes lookup-tables to detect the start- and endpoints of an EST within a given DNA sequence efficiently, and subsequently promptly identify exons and introns. In addition, the locations of all splice sites must be calculated correctly with high sensitivity and accuracy, while retaining high computational efficiency. This goal is hard to accomplish in practice, due to misread nucleotides in ESTs and repetitive sequences in the genome. Nevertheless, we present two heuristics that effectively settle this issue. Experimental results confirm that our technique improves the overall computation time by orders of magnitude compared with common tools, such as SIM4 and BLAT, and simultaneously attains high sensitivity and accuracy against a clean dataset of documented genes.     

Localisation of centromeric proteins to a fraction of mouse minor satellite DNA on a mini-chromosome in human,mouse and chicken cells

Centromeres are required for faithful segregation of chromosomes in cell division. It is not clear how centromere sites are specified on chromosomes in vertebrates. We have previously introduced a mini-chromosome, named ST1, into a variety of cell lines including human HT1080, mouse LA9 and chicken DT40. This mini-chromosome, segregating faithfully in these cells, contains mouse minor and major, and human Y -satellite DNA repeats. In this study, after determining the organisation of the satellite repeats, we investigated the location of the centromere on the mini-chromosome by combined immunocytochemistry and fluorescence in situ hybridisation analysis. Centromeric proteins were consistently co-localised with the minor satellite repeats in all three cell lines. When chromatin fibres were highly stretched, centromeric proteins were only seen on a small portion of the minor satellite repeats. These results indicate that a fraction of the minor satellite repeats is competent in centromere function not only in mouse but also in human and chicken cells.Kang Zeng and Jose I. de las Heras contributed equally to this work     

Studies of poly(A+-RNA in mouse hepatoma and cortisone-stimulated rat liver

The content of poly(A)-containing RNA in subcellar fractions has been investigated both in cortisone-treated rat liver and experimental hepatoma cells. The fractions included nuclei, cytoplasm, mitochondria, free and membrane-bound polyribosomes. 1) In both cases of genome activation (cortisone induction and hepatoma cells) an increase in poly(A) content of all subcellular fractions except free polyribosomes was observed. 2) Cortisone was found to induce elongation of poly(A) segments detected in both nuclei and cytoplasm. 3) An increase in the poly(A) block size also was found to be stimulated in nuclei and cytoplasm of hepatoma cells. 4) The observed elongation in poly(A) length occurred against the background of an increase of the population of of poly(A)-RNA's.     

Nature of DNA lesions induced in human hepatoma cells, human colonic cells and human embryonic lung fibroblasts by the antiretroviral drug 3'-azido-3'-deoxythymidine

This study tried to clarify the question if nuclear genotoxicity played a role in 3'-azido-3'-deoxythymidine (AZT) toxicity. We investigated cytotoxic and DNA-damaging effects of AZT on human hepatoma HepG2 and human colonic CaCo-2 cells as well as on human diploid lung fibroblasts HEL. The amount of induced DNA damage was measured by standard alkaline single cell gel electrophoresis (SCGE). The nature of induced DNA lesions was evaluated (1) by modified SCGE, which includes treatment of lysed cells with DNA repair enzymes Endo III and Fpg and enables to recognize oxidized bases of DNA, and (2) by SCGE processed in parallel at pH 13.0 (standard technique) and pH 12.1, which enables to recognize alkali labile DNA lesions and direct DNA strand breaks. Cytotoxicity of AZT was evaluated by the trypan blue exclusion technique. Our findings showed that 3-h treatment of cells with AZT decreased the viability of all cell lines studied. SCGE performed in the presence of DNA repair enzymes proved that AZT induced oxidative lesions to DNA in all cell types. In hepatoma HepG2 cells and embryonic lung fibroblasts HEL the majority of AZT-induced DNA strand breaks were pH-independent, i.e. they were identified at both pH values (12.1 and 13.0). These DNA lesions represented direct DNA breaks. In colonic Caco-2 cells DNA lesions were converted to DNA strand breaks particularly under strong alkaline conditions (pH>13.0), which is characteristic for alkali-labile sites of DNA. DNA strand break rejoining was investigated by the standard comet assay technique during 48 h of post-AZT-treatment in HepG2 and Caco-2 cells. The kinetics of DNA rejoining, considered an indicator of DNA repair, revealed that AZT-induced DNA breaks were repaired in both cell types slowly, though HepG2 cells seemed to be more repair proficient with respect to AZT-induced DNA lesions.     

Phenolic ring deiodination in cultured rat hepatoma cells, and subcellular localization of deiodinases in cultured rat hepatoma, monkey hepatocarcinoma cells and normal rat liver homogenates

The cultured rat hepatoma cell (R117-21B) homogenates metabolized 3,[3′,5′-¹²⁵I]triiodothyronine by phenolic ring deiodination and produced radioactive iodide and 3,3′-diiodothyronine. Thyroxine (T₄) was converted to 3,3′,5-triidothyronine (T₃). The production of ¹²⁵I⁻ presented the deiodinase activity. The optimal pH for phenolic ring deiodination was observed to be pH 6.0–7.0. This enzyme reaction was accelerated by dithiothreitol. Propylthiouracil strongly inhibited the phenolic ring deiodination at 0.1 mM, whereas an effect of 20 mM methylmercaptoimidazol on the deiodination was very weak or absent.Excess unlabeled iodothyronines (T₄, T₃ and 3,5-diiodo-l-thyronine inhibited the phenolic ring deiodination of labeled 3,3′,5′-triiodothyronine, althought their inhibitory effect was slightly different. Triiodothyroacetic acid was a better inhibitor than T₃. Diiodotyrosine did not affect phenolic ring deiodination in cultured rat hepatoma cell homogenates.Phenolic and nonphenolic ring deiodinase activities of cultured monkey hepatocarcinoma cell and rat liver homogenates were also studied by the use of 3,[3′,5′-¹²⁵I]triiodothyronine and [3,5-¹²⁵I]thyroxine, respectively. Both deiodinase activities were observed in particulate fractions (mitochondrial and microsomal) of cultured cell and rat liver homogenates.     

Rapid and sensitive determination of protein-nitrotyrosine by ELISA: Application to human plasma

3-Nitrotyrosine (3NT) is known as an important indicator of nitrosative stress and has been linked to various diseases. Our aim was to develop an indirect ELISA (enzyme-linked immunosorbent assay) method suitable for the detection of protein-bound 3NT in clinical plasma and serum samples. Nitrated protein standards and reduced protein standards were prepared. Limit of detection was determined for standards; recovery and reproducibility were determined for human plasma samples. The limit of detection for this method is 1.82±0.56 pmol/mg protein. Mean recovery of standards was 95%. 3NT concentration in plasma samples of obese and normal weight subjects was determined to be between 2 pmol/mg and 19 pmol/mg. No time-consuming sample preparation or expensive laboratory equipment is required, and applied antibodies are commercially available. Sensitivity, rapid analysis time, possibilities of high throughput applications and small sample volumes make this ELISA attractive for use in clinical laboratories.     

Lipid peroxidation inhibitory factors in liver and muscle of rat, mouse, and chicken

Glutathione- or sulfhydryl-dependent antioxidant factors that act to prevent lipid peroxidation have been reported in both microsomes and cytoplasm from rat liver. The cytoplasmic factor has been identified in several other tissues and species, but the distribution of the microsomal factor has not been reported. Chicken and mouse livers had much lower activities of the glutathione-dependent membrane-associated and cytoplasmic antioxidant factors than rat liver. Peroxidative damage to membranes has been hypothesized as a mechanism of tissue damage in muscular dystrophy. However, neither the chicken, mouse, nor rat had significant activities of the antioxidant factors in muscle. There was also no significant difference between normal and dystrophic chicken livers in the activity of the antioxidant factors associated with the microsomes or the cytoplasm, nor of the liver microsomal factor in normal and dystrophic mice. The results do not support an important role for the antioxidant factors in the pathogenesis of muscular dystrophy, and raise questions as to whether such factors are physiologically important in species other than rat or in tissues other than liver.     

High-performance liquid chromatography method for determination of N-glucuronidation of 4-aminobiphenyl by mouse, rat, and human liver microsomes

A simple and sensitive method for determination of the N-glucuronidation activity of mouse, rat, and human liver microsomes toward the carcinogenic arylamine 4-aminobiphenyl (4-ABP) using high-performance liquid chromatography with ultraviolet detection has been developed. The method uses chemically synthesized 4-ABP-N-glucuronide (4-ABP-G) as a standard for method validation. Validation was done with respect to specificity, linearity, precision, accuracy, and lower limits of detection. The method was specific since there were no interference peaks from the reaction matrix. The calibration curve for 4-ABP-G was linear from 50 to 5000 pmol/200 microl with R2=0.999. The newly developed method has good precision and accuracy. The intra- and interday precisions were less than 5 and 10%, respectively, and the highest values for intra- and interday accuracies were -4.6 and -12%, respectively. The lower limit of detection was 10 pmol/200 microl. The developed method was used to determine the glucuronidation activity of mouse, rat, and human liver microsomes. Human liver microsomes were the most active in 4-ABP glucuronidation (344.1 pmol/min/mg) followed by rats (30.6 pmol/min/mg) and then mice (12.3 pmol/min/mg). Human UGT1A4 supersomes were much more active than UGT1A9 (184.4 mol/min/mg versus 25.2 mol/min/mg). These results are consistent with those of earlier studies that used the radioactive [C14]UDPGA.