Effect of exogenus protease on performance, nutrient digestibility, intestinal histomorphometric, meat quality characteristics, carcass yield in broilers fed low protein diets.

The objective of the present study was to evaluate the effects of increasing doses of protease on broilers from 1 to 42 days of age. A total of 1290 Ross AP broilers were used, distributed among five treatments: positive control diet, negative control diet (NC), NC + 50 ppm of protease, NC + 100 ppm of protease, and NC + 200 ppm of protease. Each treatment contained six replicates of 43 animals each. The inclusion of proteases in the diet had effects (P < 0.05) on body weight, feed intake, weight gain, and feed conversion in the 12 to 21 day period; body weight, weight gain, and feed intake in the 29 to 42 day period; nutrient digestibility (energy metabolizability coefficient and crude protein at 28 days); and intestinal parameters (crypt and muscle width of jejunum and ileum at 28 days and villus length, crypt length, and jejunum thickness muscle layer at 42 days). These results indicate that the inclusion of protease in broiler feed can improve production parameters when the amount of crude protein in the diet is reduced.

Enhanced detection of DNA sequences using end-point PCR amplification and online gel electrophoresis (GE)-ICP-MS: determination of gene copy number variations.

The design and evaluation of analytical methods that permit quantitative analysis of specific DNA sequences is exponentially increasing. For this purpose, highly sensitive methodologies usually based on labeling protocols with fluorescent dyes or nanoparticles are often explored. Here, the possibility of label-free signal amplification using end-point polymerase chain reaction (PCR) are exploited using on-column agarose gel electrophoresis as separation and inductively coupled plasma-mass spectrometry (ICP-MS) for the detection of phosphorus in amplified DNA sequences. The calibration of the separation system with a DNA ladder permits direct estimation of the size of the amplified gene fragment after PCR. With this knowledge, and considering the compound-independent quantification capabilities exhibited by ICP-MS for phosphorus (it is only dependent on the number of P atoms per molecule), the correlation of the P-peak area of the amplified gene fragment, with respect to the gene copy numbers (in the starting DNA), is then established. Such a relationship would permit the determination of copy number variations (CNVs) in genomic DNA using ICP-MS measurements. The method detection limit, in terms of the required amount of starting DNA, is ∼6 ng (or 1000 cells if 100% extraction efficiency is expected). The suitability of the proposed label-free amplification strategy is applied to CNVs monitoring in cells exposed to a chemical agent capable of deletion induction, such as cisplatin.

Long-term biomonitoring of breast cancer patients under adjuvant chemotherapy: the comet assay as a possible predictive factor.

Most chemotherapy treatments induce DNA damage in the exposed patients. Using the comet assay and peripheral blood mononuclear cells (PBMC), we have quantified this induced DNA damage and studied its relationship with GSTM1 and GSTT1 polymorphisms, and clinical parameters. For this purpose, 29 Caucasian women, breast cancer patients under CMF or CEF adjuvant chemotherapy were included in the study. The clinical parameters considered were (i) therapies side effects, like haematological and biochemical toxicities, (ii) prognostic and predictive factors, like hormonal receptor expression, tumour differentiation degree, sickness stage, and nodal status, and (iii) the effectiveness of the chemotherapy measured as five years relapse probability. The results were also related to the confounding factor age. Comet assay results indicate that 13 patients were characterised by absence of induced DNA strand breaks, and 16 patients presented induced DNA strand breaks along the treatment. Relationships between comet variables and clinical parameters, found with principal component analysis, correlations, one-way ANOVA and multivariate logistic regression analyses revealed that: (1) baseline levels of DNA damage are related to GSTM1 genotype and to hormonal receptor expression; (2) GSTM1 genotype also influences comet results after chemotherapy, as it does the AST level; (3) the tail moment values of the cycle 6.1 and the sickness stage might predict cancer relapse at five years: for the Stage, OR = 13.8 (IIB versus I+IIA), 95% CI 0.80-238.97, and for 6.1 cycle TM, OR = 1.3, 95%, CI 0.97-1.79, with a potential model (10* Stage (I-IIA = 0, IIB = 1) + 6.1 cycle), that has a good predictive capacity, with an area under ROC curve of 0.872 (CI 0.62-1.00). To our knowledge, this is the first time such a predictive value is found for the comet assay. Nevertheless, before the comet assay could be used as a tool for oncologists, this relationship should be confirmed in more patients, and problems of standardisation and data interpretation should be solved.

Chromatographic methods coupled to mass spectrometry for the determination of oncometabolites in biological samples-A review.

It is now well-established that dysregulation of the tricarboxylic acid (TCA) cycle enzymes succinate dehydrogenase, fumarate hydratase, and isocitrate dehydrogenase leads to the abnormal cellular accumulation of succinate, fumarate, and 2-hydroxyglutarate, respectively, which contribute to the formation and malignant progression of numerous types of cancers. Thus, these metabolites, called oncometabolites, could potentially be useful as tumour-specific biomarkers and as therapeutic targets. For this reason, the development of analytical methodologies for the accurate identification and determination of their levels in biological matrices is an important task in the field of cancer research. Currently, hyphenated gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) techniques are the most powerful analytical tools in what concerns high sensitivity and selectivity to achieve such difficult task. In this review, we first provide a brief description of the biological formation of oncometabolites and their oncogenic properties, and then we present an overview and critical assessment of the GC-MS and LC-MS based analytical approaches that are reported in the literature for the determination of oncometabolites in biological samples, such as biofluids, cells, and tissues. Advantages and drawbacks of these approaches will be comparatively discussed. We believe that the present review represents the first attempt to summarize the applications of these hyphenated techniques in the context of oncometabolite analysis, which may be useful to new and existing researchers in this field.

Targeting HER2 protein in individual cells using ICP-MS detection and its potential as prognostic and predictive breast cancer biomarker.

The human epidermal growth factor receptor 2 (HER2) is a transmembrane protein that has become one of the most specific prognostic and predictive biomarker of breast cancer. Its early detection is key for optimizing the patient clinical outcome. This work is focused on the detection of HER2 in individual cells using an antibody containing lutetium (Lu) as reporter group that is monitored by introducing the individual cells into the inductively coupled plasma mass spectrometer (ICP-MS). This Lu-containing antibody probe is used to label different breast cancer cell lines considered HER2 negative (MDA-MB-231) and positive (SKBR-3 and BT-474). Optimizations regarding the amount of the probe necessary to ensure complete labelling reactions are conducted in the different cell models. Concentrations in the range of 0.006 fg Lu/cell and 0.030 fg Lu/cell could be found in the HER2 negative and HER2 positive cells, respectively. In addition, the selectivity of the labelling reaction is tested by using two different metal-containing antibody probes for HER2 (containing Lu) and for transferrin receptor 1 (containing Nd), respectively, within the same cell population. Finally, the methodology is applied to the targeting of HER2 positive cells in complex cell mixtures containing variable amounts of BT-474 and MDA-MB-231 cells. The obtained results showed the excellent capabilities of the proposed strategy to discriminate among cell populations. This finding could help for scoring HER2 positive tumors improving existing technologies.

Quantitative profiling of in vivo generated cisplatin-DNA adducts using different isotope dilution strategies.

Platinum compounds are the major group of metal-based chemotherapeutic drug used in current practice and still a topic of intense investigation. The relative contribution of structurally defined cisplatin adducts with DNA to induce apoptosis and the cellular processing of these lesions is still poorly understood mostly due to the lack of sensitive and accurate analytical tools for in vivo studies. In this regard, two novel sensitive and selective strategies are proposed here to quantify cisplatin-DNA adducts generated in Drosophila melanogaster larvae and in head and neck squamous cell carcinoma cultures. The methods involve the isolation and enzymatic digestion of the DNA in the samples exposed to cisplatin and further quantification by high-performance liquid chromatography with inductively coupled plasma mass spectrometric detection (HPLC-ICPMS). Two different strategies, based on isotope dilution analysis (IDA), have been attempted and evaluated for quantification: species-unspecific (the postcolumn addition of a 194Pt-enriched solution) and the species-specific (by means of a synthesized isotopically enriched cisplatin (194Pt) adduct). For the second approach, the synthesis and characterization of the cisplatin adduct in a custom oligonucleotide containing the sequence (5'-TCCGGTCC-3') was necessary. The adducted oligo was then added to the DNA samples either before or after enzymatic hydrolysis. The results obtained using these two strategies (mixing before and after enzymatic treatment) permit to address, quantitatively, the column recoveries as well as the efficiency of the enzymatic hydrolysis. Species-specific spiking before enzymatic digestion provided accurate and precise analytical results to clearly differentiate between Drosophila samples and carcinoma cell cultures exposed to different cisplatin concentrations.

Sensitive determination of the human epidermal growth factor receptor 2 (HER2) by immuno-polymerase chain reaction with inductively coupled plasma-mass spectrometry detection.

Sensitive and selective analytical methods are necessary for the determination of clinical biomarkers of breast cancer. The human epidermal growth factor receptor 2 (HER2) is an important breast cancer biomarker since tumors with HER2 protein overexpression (HER2-positive tumors) turn out to be more aggressive and likely to recur. Therefore, accurate determination of serum HER2 values is critical to optimize clinical outcomes in patients with breast cancer. To gain sensitivity and selectivity in the determination of HER2, a sandwich immune assay (highly selective) has been implemented using a detection antibody labelled with a DNA marker. Further amplification of the label using the polymerase chain reaction (PCR), followed by phosphorous quantification of the PCR product (amplicon) using inductively coupled plasma mass spectrometry (ICP-MS), completes this novel assay. Considering that the concentration of the amplicon is proportional to the amount of antigen (HER2) that is recognized by the labelled detection antibody, the concentration of HER2 can be directly obtained by P-analysis. For this aim, a DNA marker of 123 base pairs has been connected to the detection antibody of a sandwich immune assay conducted in pre-coated plates containing the capture antibody of HER2. After the recognition occurred, the PCR amplification was conducted and the PCR product analysed by ICP-MS. Detection limits of 2.5 pg mL-1 of HER2 could be achieved using 35 PCR cycles (7-fold lower than the commercial ELISA method). The developed methodology has been applied to the determination of HER2 in biological samples (human serum and cell culture supernatant of breast cancer cells, MDA-MB-231) obtaining mean method recoveries of about 87% and 81%, respectively.

Multiplex polymerase chain reaction in combination with gel electrophoresis-inductively coupled plasma mass spectrometry: A powerful tool for the determination of gene copy number variations and gene expression changes.

During the last few years multiplex real-time or quantitative polymerase chain reaction PCR (qPCR) has become the method of choice for multiplex gene expression changes and gene copy number variations (CNVs) analysis. However, such determinations require the use of different fluorescent labels for the different amplified sequences, which increases significantly the costs of the analysis and limits the applicability of the technique for simultaneous amplification of many targets of interest in a single reaction. In this regard, the use of the coupling between gel electrophoresis (GE) separation with inductively coupled plasma mass spectrometry (ICP-MS) detection allows the label-free determination of multiplex PCR-amplified sequences (amplicons) by monitoring the P present in the DNA backbone. The quantitative dimension is obtained since under optimal and controlled multiplex PCR conditions the peak areas of the separated amplicons are directly proportional to the amount of DNA template in the original sample. Moreover, the calibration of the GE-ICP-MS system with a DNA ladder permits direct estimation of the size (bp) of the PCR products. The suitability of the proposed multiplex strategy has been evaluated addressing two different situations: determination of CNVs and gene expression changes in human ovarian cancer cells. In the first case, the results obtained for the simultaneous quantitation of CNVs of four genes (HER2, CCNE1, GSTM1, ACTB) on DNA obtained from OVCAR-3 cells were in accordance with the literature data, and also with the results obtained by conventional simplex qPCR. In the second case, multiplex gene expression changes of BAX, ERCC1 and CTR1 genes, using ACTB as constitutive gene, on A2780cis respect to A2780 cells, resistant and sensitive to cisplatin, respectively, provided the same information as single reaction reverse transcription (RT)-qPCR.

Quantitative evaluation of cellular uptake, DNA incorporation and adduct formation in cisplatin sensitive and resistant cell lines: Comparison of different Pt-containing drugs.

The use of Pt-containing compounds as chemotherapeutic agents facilitates drug monitoring by using highly sensitive elemental techniques like inductively coupled plasma mass spectrometry (ICP-MS). However, methodological problems arise when trying to compare different experiments due to the high variability of biological parameters. In this work we have attempted to identify and correct such variations in order to compare the biological behavior of cisplatin, oxaliplatin and pyrodach-2 (a novel platinum-containing agent). A detailed study to address differential cellular uptake has been conducted in three different cell lines: lung adenocarcinoma (A549); cisplatin-sensitive ovarian carcinoma (A2780); and cisplatin-resistant ovarian carcinoma (A2780cis). The normalization of Pt results to cell mass, after freeze-drying, has been used to minimize the errors associated with cell counting. Similarly, Pt accumulation in DNA has been evaluated by referencing the Pt results to the DNA concentration, as measured by (31)P monitoring using flow-injection and ICP-MS detection. These strategies have permitted to address significantly lower Pt levels in the resistant cells when treated with cisplatin or oxaliplatin as well as an independent behaviour from the cell type (sensitive or resistant) for pyrodach-2. Similarly, different levels of incorporation in DNA have been found for the three drugs depending on the cell model revealing a different behavior regarding cell cisplatin resistance. Further speciation experiments (by using complementary HPLC-ICP-MS and HPLC-ESI-Q-TOF MS) have shown that the main target in DNA is still the N7 of the guanine but with different kinetics of the ligand exchange mechanism for each of the compounds under evaluation.

O-ethylthymidine adducts are the most relevant damages for mutation induced by N-ethyl-N-nitrosourea in female germ cells of Drosophila melanogaster.

Responses to genotoxic agents vary not only among organisms, test systems, and cellular stages, but also between sexes; little, however, is known about the mutagenic consequences of chemical exposures to female germ cells. In this study, the mutagenicity of N-ethyl-N-nitrosourea (ENU) was analyzed in female germ cells of Drosophila melanogaster using the recessive-lethal test and the vermilion system, which simultaneously generates information on induced mutation frequency and mutation spectrum. ENU was mutagenic in all stages of oogenesis, although there were differences among the stages. In mature and immature oocytes, ENU-induced mutations in the vermilion locus were 43.5% A:T-->G:C transitions, 39.1% A:T-->T:A transversions, 8.7% G:C-->A:T transitions, and 8.7% A:T-->C:G transversions, indicating that the most important premutagenic lesions induced by this chemical are O(4)-ethylthymine and O(2)-ethylthymine. The low frequency of mutation involving O(6)-ethylguanine (i.e., G:C-->A:T transitions) could be a consequence of the repair of these lesions by O(6)-methylguanine DNA methyltransferase. Comparison of these results with those previously obtained in male germ cells stresses the importance of the repair activity of the analyzed cells, because the mutation spectrum in female germ cells was similar to the spectrum obtained with repair-proficient spermatogonial cells and different from repair-deficient postmeiotic cells. The results also indicate that studies with female germ cells could be an alternative to the use of premeiotic male germ cells, especially when the analysis of these cells is difficult or almost impossible and when studies of in vivo DNA repair in premeiotic germ cells are performed.

Effect of nucleotide excision repair on ENU-induced mutation in female germ cells of Drosophila melanogaster.

The role of nucleotide excision repair (NER) in the repair of alkylation damage in the germ cells of higher eukaryotes has been studied mainly by treating postmeiotic male germ cells. Little is known about repair in actively repairing female germ cells. In this study, we treated NER-deficient (ner(-)) mus201(D1) Drosophila females with N-ethyl-N-nitrosourea (ENU) and determined both the mutant frequencies in the multiple locus recessive lethal (RL) test and in the single locus vermilion gene and determined the ENU mutation spectrum in the vermilion gene. The results show that ENU is mutagenic in all cell stages and that the induced frequencies increase with cell maturation, from oogonia to mature oocytes. In addition, the induced spectrum consists mainly of A:T-->T:A transversions (43.8%), A:T-->G:C transitions (21.9%), and A:T-->C:G transversions (15.6%). G:C-->A:T (3.1%) transitions, other transversions (9.4%), frameshifts (3.1%), and deletions (3.1%) were also found. Comparison of these results with those previously obtained for repair-proficient (ner(+)) female germ cells reveal: 1) Differences in the RL and vermilion mutation frequencies for ner(+) and ner(-) germ cells, indicating that NER is involved in the repair of ENU-induced damage to these cells. 2) At least 15.6% of mutations in ner(-) cells may be the consequence of N-ethylation damage and mutations of this type were not detected in ner(+) cells. 3) Although differences were found in transition frequencies between ENU-treated ner(+) and ner(-) germ cells (52.2% vs. 25%), suggesting that a functional NER is involved in processing O-ethylated damage, the role of NER in repairing O-ethylated adducts is uncertain.

Is the white-ivory assay of Drosophila melanogaster a useful tool in genetic toxicology?

The white-ivory assay of Drosophila is based on the detection of reversions to wild-type phenotype of ommatidia with the white-ivory mutation. A tandem quadruplication of this gene is used in order to increase the reversion probability. Although the exact mechanism implicated in reversion is not known, revertant spots are believed to arise as a consequence of intrachromosmal recombination or related phenomena. Since the white-ivory assay has not been broadly used, the number of chemicals tested until now is still limited. In this work, we have assayed 25 chemicals belonging to several chemical groups, i.e., crosslinking agents, DNA-topoisomerase inhibitors, antimetabolites/nucleotide pool inhibitors, cyclic-adduct inducers, halogenated hydrocarbons, bulky-adduct inducers, intercalating agents, oxidative damage inducers, and a multiple damage inducer, to validate this test. Cross-linking agents, halogenated hydrocarbons, and the multiple damage inducer, dounomycin, were positive. On the contrary, the three antimetabolites/nucleotide pool inhibitors tested were negative. The other chemical groups showed disparate results, since some chemicals were positive, whereas others were negative in each group. A comparison with the results obtained in the w/ w+ and mwh/flr3 assays shows that the wi assay detects a more restricted spectrum of damages than those, although, with respect to carcinogenicity, its sensitivity (0.76, with the 62 chemicals tested until now) is similar to that estimated for the mentioned somatic assays. The conclusion of this work, then, is that the wi assay is not recommended as a general screening test, because the background reversion frequencies show a high variability among solvents, the range of lesion-recognition is lower than in the w/ w+ and mwh/flr3 SMARTs, and the mechanism implicated in the white-ivory reversion is poorly understood.

White-ivory assay of Drosophila melanogaster under deficient repair conditions.

The prediction ability of a test to detect genotoxic activity may be increased, at least from a theoretical point of view, by carrying it out under deficient repair conditions. The white-ivory (w[i]) assay of Drosophila melanogaster is a somatic mutation and recombination test (SMART) that essentially differs from other SMARTs by the endpoints that can be detected. In this article, we study the consequences, with the w(i) assay, of the introduction of two mutations, mus201 and mei-41, which produce deficiency in two different repair mechanisms: the nucleotide excision repair system and in a G2/M cell-cycle checkpoint, respectively. Ten chemicals, previously classified as positive in the w(i) assay, have been assayed in both deficient repair conditions. As in the w/w+ and mwh/flr3 SMARTs, the results obtained with the w(i) assay show that the use of deficient repair strains does not improve the detection of genotoxic effects. However, the utilization of these deficient repair strains has been shown to be a useful tool in mechanistic studies. In fact, it seems that the nucleotide excision repair system mainly eliminates some spontaneous and chemically-induced damages involved in the reversion of w(i), whereas the repair system deficient in mei-41 flies is partly necessary to recover revertant w(i) spots.

The hypermutability conferred by the mus308 mutation of Drosophila is not specific for cross-linking agents.

The hypersensitivity of the mus308 mutant of D. melanogaster to cross-linking agents has been suggested to be the consequence of a possible defect of this mutant in DNA cross-link repair. Moreover, the mus308 mutation has been proposed as an animal model for the study of Fanconi's anemia. In order to obtain more information about the function controlled by this locus, we have measured the mutability of the mus308 mutant to several mutagens with different modes of action using the sex-linked recessive lethal test. We show that this mutation confers hypermutability not only to the cross-linking agents tested, hexamethylphosphoramide and hexamethylmelamine, but to the point mutagen N-ethyl-N-nitrosourea as well, whereas the response to methyl methanesulfonate was normal. The results suggest that the mus308 locus is not defective in a repair pathway specific for cross-links but is rather involved in a step of a more general post-replication repair process responsible for the removal of non-excised adducts.

The w/w+ SMART assay of Drosophila melanogaster detects the genotoxic effects of reactive oxygen species inducing compounds.

The somatic mutation and recombination w/w+ eye assay has been used for genotoxic evaluation of a broad number of chemicals with different action mechanisms yielding high values of sensitivity, specificity and accuracy. The aim of this work was to determine the utility of this assay in the evaluation of reactive oxygen species inducers. For this, we have tested eight compounds: diquat, paraquat, menadione, juglone, plumbagin, streptonigrin, tert-butyl hydroperoxide and 4-nitroquinoline 1-oxide, using the Drosophila Oregon K strain which had previously shown advantageous conditions to test this type of compounds. Diquat was the only chemical for which the results were clearly negative, probably because its high toxicity, whereas indications of a marginal genotoxicity raised for menadione. The remaining compounds were evaluated as positives. The conclusion of these experiments is that the w/w+ assay is capable to detect genotoxic effects induced by compounds that generate reactive oxygen species through different action mechanisms.

'Non-genotoxic' carcinogens evaluated using the white-ivory assay of Drosophila melanogaster.

Seven carcinogenic compounds (urethane, ethionine, auramine O, safrole, amitrole, acetamide and thioacetamide) were tested using the white-ivory (Wi) assay of Drosophila melanogaster. These compounds were chosen because they were considered as Ames-test negative but produced positive results in the yeast DEL assay, which estimates the introduction of intrachromosomal recombination. Only one compound, urethane, produced positive results in the Wi assay, while the remaining were classified as negative. These results indicate that, in contrast with which has been postulated in yeast, these carcinogens do not induce any event associated to intrachromosomal recombination in D. melanogaster.

Methodological aspects of the white-ivory assay of Drosophila melanogaster.

The white-ivory somatic assay of Drosophila melanogaster was developed to detect genotoxic agents which induce loss of a tandem duplication. Although the mechanism of this loss is not known, some suggestions point to intrachromosomal recombination as the main reversion mechanism. Since the few papers published to date on this assay present controversial methodologies, prior to a larger study of chemicals with different mechanisms of action, we have carried out an analysis to optimize some conditions of this assay. For this purpose, we have used three different strains and four well characterized mutagenic chemicals: N-ethyl-N-nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and hexamethyl phosphoramide (HMPA). The results obtained allow us to conclude that: (i) the best strain for this assay is C(1)DX,y,f/Dp(1:1:1:1)wi,y2, although the use of strain FM6,l(1)66a/Dp(1:1:1:1)wi,y2;st/st could be considered for some mechanistical studies; (ii) developmental reasons make it necessary to use as estimate of reversion frequency the proportion of eyes showing at least one spot; (iii) reversion frequency cannot be used as estimate of mutation efficiency, neither can spot size evaluate time of spot induction; (iv) the four chemicals clearly induce loss of the wi duplication; according to their activities they rank ENU > HMPA > MMS approximately EMS.

Acrolein genotoxicity in Drosophila melanogaster. II. Influence of mus201 and mus308 mutations.

The influence of mus201 and mus308 mutants on acrolein mutagenicity was analyzed with the Drosophila melanogaster sex-linked recessive lethal test (SLRL), using the maternal approach, to further study the mechanisms of action of this chemical. The hypermutability indices obtained were 2.59 for mus201 and 0.52 for mus308 conditions. Statistical analysis indicates that whereas part of the acrolein-induced lesions are repaired by excision mechanism, as expected for a cyclic agent, there is no demonstrable influence of the mus308 locus on the mutagenicity of this chemical.

Acrolein genotoxicity in Drosophila melanogaster. III. Effects of metabolism modification.

In order to investigate the role of metabolism in acrolein genotoxicity in D. melanogaster, the action of several metabolism modifiers, namely phenobarbital, an inducer of xenobiotic metabolism, phenylimidazole and iproniazid, inhibitors of oxidative activities of cytochrome P450, and diethyl maleate, a glutathione-depleting agent, have been assayed using the sex-linked recessive lethal (SLRL) test, with two different administration routes (feeding and injection). The results support the hypothesis that acrolein is not only a direct mutagen but is also transformed, by oxidative activities of cytochrome P450 after glutathione conjugation, into an active metabolite, possibly glycidaldehyde. Moreover, acrolein is deactivated by an enzymatic activity induced by phenobarbital.