Search

Metabolic changes involving the tricarboxylic acid (TCA) cycle have been linked to different non-metabolic cell processes. Among them, apart from cancer and immunity, emerges the DNA damage response (DDR) and specifically DNA damage repair. The oncometabolites succinate, fumarate and 2-hydroxyglutarate (2HG) increase reactive oxygen species levels and create pseudohypoxia conditions that induce DNA damage and/or inhibit DNA repair. Additionally, by influencing DDR modulation, they establish direct relationships with DNA repair on at least four different pathways. The AlkB pathway deals with the removal of N-alkylation DNA and RNA damage that is inhibited by fumarate and 2HG. The MGMT pathway acts in the removal of O-alkylation DNA damage, and it is inhibited by the silencing of the MGMT gene promoter by 2HG and succinate. The other two pathways deal with the repair of double-strand breaks (DSBs) but with opposite effects: the FH pathway, which uses fumarate to help with the repair of this damage, and the chromatin remodeling pathway, in which oncometabolites inhibit its repair by impairing the homologous recombination repair (HRR) system. Since oncometabolites inhibit DNA repair, their removal from tumor cells will not always generate a positive response in cancer therapy. In fact, their presence contributes to longer survival and/or sensitization against tumor therapy in some cancer patients.

Low crude protein (CP) diets can reduce nitrogen (N) excretion and costs by increasing N utilization efficiency. Exogenous proteases may further improve protein digestibility in low CP diets. This study first evaluated in vitro the efficacy of a multiprotease on amino acid (AA) release from feedstuffs and broiler feed. Later, a broiler study evaluated the effect of feeding corn-soybean meal diets containing 3 CP levels (17, 19, and 21% CP) with supplementation on top of 0 or 2,400 U/kg multiprotease on chicken growth performance, total tract CP, and ileal AA digestibilities, and energy utilization. Ross 708 male chickens were placed in 42 cages and assigned to 6 treatments resulting from a 3 × 2 factorial arrangement. Three isocaloric basal diets were formulated to reduce CP, but all diets maintained digestible Lys:CP in 5.47% and the same ideal protein profile. Data were analyzed in a completely randomized design. On average, the multiprotease increased (P < 0.05) in vitro free AA release by 27.81% in most feedstuffs evaluated compared to the control. For broiler feed, 1,200 U/g multiprotease addition improved (P < 0.001) in vitro free AA release by 18.90%. This multiprotease showed interaction effects (P < 0.05) on chicken FCR, energy, and CP digestibility. As expected, BW at 24 d, BW gain, and FCR (8-24 d) worsened (P < 0.001) as dietary CP reduced from 21 to 17%, and multiprotease addition did not improve (P > 0.05) these parameters. BW gain decreased by 12.9% when N intake was reduced from 49.32 to 38.49 g/bird. Multiprotease supplementation improved (P < 0.01) AMEn by 71 kcal/kg, CP digestibility from 59.45 to 63.51%, ileal AA digestibility, and DM digestibility from 67.08 to 73.49%, but only in the 21% CP diet. No differences in ileal AA digestibility due to CP level (P > 0.05) were detected, except for Cys digestibility (P < 0.01). In conclusion, low CP diets reduced growth performance and improved N utilization but negatively affected energy utilization efficiency. Exogenous multiprotease supplementation improved AME, AMEn, protein, ileal AA, and DM digestibility in the 21% CP diet without significantly affecting growth performance., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

High oleic (HO) soybeans may serve as a value-added feed ingredient; providing amino acids and estimating their dietary energy value for broilers is essential. In this study, we determined the apparent metabolizable energy (AME), AME corrected for zero nitrogen retention (AMEn), digestibility, and nitrogen (N) retention of HO full-fat (HO-FF) soybean as compared to solvent-extracted soybean meal (SE-SBM), normal oleic full-fat (NO-FF) and extruded expeller (NO-EE) soybean. A total of 240 Ross-708 male broilers were selected, with 8 replicates per treatment and 6 chicks per cage. The AME and AMEn were estimated using the difference method with a 30% inclusion of test ingredients using a corn-soy reference diet with partial and total excreta collection. The index method with partial excreta collection used titanium dioxide as an inert marker. The same starter diet was provided for all birds for 14 d, followed by the reference and assay diets for the next 6 adaptation days. Total excreta were collected twice a day for 3 d. The AME and AMEn values determined for the HO-FF and NO-FF were higher (P < 0.001) than the NO-EE and SE-SBM. The AME of SE-SBM and NO-EE were similar with both methods, but the AMEn of SE-SBM was lower than the NO-EE only with the partial collection method. The agreement between AME and AMEn values determined by partial and total excreta collection analysis was 98%. Data from the total excreta collection method yielded higher AME and AMEn values (P < 0.001) than those from the partial collection method. In summary, HO-FF and NO-FF soybean meals had similar AME and AMEn values. The HO-FF soybean had 39 and 24% higher AME and AMEn than SE-SBM. Hence, high oleic full-fat soybean meal could serve as a valuable alternative feed ingredient to conventional SE-SBM meals in broiler diets, providing additional energy while providing amino acids and more oleic acid to enrich poultry meat products., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

This consensus of nomenclature and classification for congenital bicuspid aortic valve and its aortopathy is evidence-based and intended for universal use by physicians (both pediatricians and adults), echocardiographers, advanced cardiovascular imaging specialists, interventional cardiologists, cardiovascular surgeons, pathologists, geneticists, and researchers spanning these areas of clinical and basic research. In addition, as long as new key and reference research is available, this international consensus may be subject to change based on evidence-based data1.

This study assessed the impact of a mixture of garlic ( Allium sativum ) and oregano ( Origanum vulgare ) essential oils (EOGOs) on in vitro dry matter digestibility (IVDMD) and in vivo apparent nutrient digestibility. Different EOGO inclusion levels were evaluated to assess the dose response and potential effects of the mixture. Three EOGO inclusion levels (0.5, 0.75, and 1 mL/kg of incubated dry matter) were evaluated in vitro, while four treatments (0.5, 0.75, and 1 mL/day of EOGO and a control group) were tested in vivo on 12 West African sheep. A randomized controlled trial was conducted using a 4 × 4 design. Blood parameters (glucose, blood urea nitrogen, and β-hydroxybutyrate) were measured to observe the effect of EOGO on the metabolism. The results showed that the inclusion of EOGO significantly enhanced IVDMD at low levels ( p < 0.052) compared with the highest levels in treatments containing 0.5 and 0.75 mL/kg of EOGO dry matter. A higher intake of dry matter (DM), crude protein (CP), and neutral detergent fiber (NDF) ( p < 0.05) was observed in the in vivo diets with the inclusion of EOGO. In terms of in vivo apparent digestibility, significant differences were found among treatments in the digestibility coefficients of DM, CP, and NDF. EOGO inclusion increased the digestibility of DM. CP digestibility displayed a cubic effect ( p < 0.038), with the lowest values of digestibility observed at 1 mL EOGO inclusion. Additionally, NDF digestibility showed a cubic effect ( p < 0.012), with the highest value obtained at 0.75 mL of EOGO inclusion. The inclusion levels above 0.75 mL EOGO showed a cubic effect, which indicates that higher concentrations of EOGO may not be beneficial for the digestibility of CP and NDF. Although no significant difference was observed in total digestible nutrients, a linear trend was observed ( p < 0.059). EOGO improved the intake of DM, CP, and NDF. EOGO supplementation improved the digestibility of DM and NDF, with optimal levels observed at 0.5 mL/day. No significant effects were observed in the blood parameters. These results suggest that EOGO has the potential as an additive in ruminal nutrition to improve food digestibility and serve as an alternative to antibiotic additives. The use of EOGO potentially improves fiber digestion and may reduce the use of antibiotics in livestock production. Garlic ( A. sativum ) and oregano ( O. vulgare ) essential oils effectively modulated fiber digestibility at 0.75 mL/day. Garlic ( A. sativum ) and oregano ( O. vulgare ) essential oils have the potential to improve digestibility at low inclusion levels and serve as an alternative to antibiotic additives. The effectiveness of essential oils is greater in a mixture and at lower doses.

High-oleic (HO) soybean may serve as a value-added feed ingredient to enrich poultry meat due to its fatty acid content. However, the amino acid (AA) nutrient digestibility of soybean meal (SBM) made from these soybeans has yet to be determined. The objective of this study was to determine apparent ileal AA digestibility (AID) and standardized ileal AA digestibility (SID) of high-oleic full-fat (HO-FF) SBM compared to normal oleic full-fat (NO-FF), normal oleic extruded expeller (NO-EE), and solvent-extracted SBM (SE-SBM) in broilers. A nitrogen-free basal diet (NFD) was fed to 1 treatment group with 10 chicks/cage to determine basal endogenous losses (BEL). Titanium dioxide was used as an inert marker. The test diets contained 57.5% of the basal NFD and 42.5% of 1 of the 4 soybean sources. A total of 272 Ross-708 male broilers were placed in 40 battery cages with 5 treatments and 8 replicates per treatment. A common starter diet was provided to all the chickens for 14 d. Experimental diets were provided as a mash for 9 d before sample collection. Chickens were euthanized with CO 2 on d 23, and contents of the distal ileum were collected, frozen, and freeze-dried. The BEL were similar to the values found in the literature. At d 23, broilers fed the SE-SBM had the highest body weight gain and best FCR compared to chickens fed the HO-FF and NO-FF treatments (P < 0.001). Broilers fed the SE-SBM and NO-EE experimental diets had (P < 0.001) higher apparent ileal AA digestibility and AA SID than broilers fed the HO-FF and NO-FF treatments. In conclusion, the SID of AA from HO-FF is similar to the digestibilities of other full-fat soybeans found in the literature and is lower than that of NO-EE and SE-SBM., (Copyright © 2023. Published by Elsevier Inc.)

The comet assay in Drosophila has been used in the last few years to study DNA damage responses (DDR) in different repair-mutant strains and to compare them to analyze DNA repair. We have used this approach to study interactions between DNA repair pathways in vivo. Additionally, we have implemented an ex vivo comet assay, in which nucleoids from treated and untreated cells were incubated ex vivo with cell-free protein extracts from individuals with distinct repair capacities. Four strains were used: wild-type OregonK ( OK ), nucleotide excision repair mutant mus201 , dmPolQ protein mutant mus308 , and the double mutant mus201;mus308 . Methyl methanesulfonate (MMS) was used as a genotoxic agent. Both approaches were performed with neuroblasts from third-instar larvae; they detected the effects of the NER and dmPolQ pathways on the DDR to MMS and that they act additively in this response. Additionally, the ex vivo approach quantified that mus201 , mus308 , and the double mutant mus201;mus308 strains presented, respectively, 21.5%, 52.9%, and 14.8% of OK strain activity over MMS-induced damage. Considering the homology between mammals and Drosophila in repair pathways, the detected additive effect might be extrapolated even to humans, demonstrating that Drosophila might be an excellent model to study interactions between repair pathways., Competing Interests: The authors declare no conflicts of interest.

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., (© 2023. The Author(s).)

A systematic investigation on the cellular uptake, intracellular dissolution, and in vitro biological effects of ultra-small (<10 nm) iron hydroxide adipate/tartrate coated nanoparticles (FeAT-NPs) was carried out in intestinal Caco-2, hepatic HepG2 and ovarian A2780 cells, and the nucleotide excision repair (NER) deficient GM04312 fibroblasts. Quantitative evaluation of the nanoparticles uptake, as well as their transformation within the cell cytosol, was performed by inductively coupled plasma mass spectrometry (ICP-MS), alone or in combination with high performance liquid chromatography (HPLC). The obtained results revealed that FeAT-NPs are effectively taken up in a cell type-dependent manner with a minimum dissolution after 3 h. These results correlated with no effects on cell proliferation and minor effects on cell viability and reactive oxygen species (ROS) production for all the cell lines under study. Moreover, the comet assay results revealed significant DNA damage only in GM04312 cells. In vivo genotoxicity was further studied in larvae from Drosophila melanogaster, using the eye-SMART test. The obtained results showed that FeAT-NPs were genotoxic only with the two highest tested concentrations (2 and 5 mmol·L−1 of Fe) in surface treatments. These data altogether show that these nanoparticles represent a safe alternative for anemia management, with high uptake level and controlled iron release.

Hypoxia-inducible factors (HIF) 2α and 1α are the major oxygen-sensing molecules in eukaryotic cells. HIF2α has been pathogenically linked to paraganglioma and pheochromocytoma (PPGL) arising in sympathetic paraganglia or the adrenal medulla (AM), respectively. However, its involvement in the pathogenesis of paraganglioma arising in the carotid body (CB) or other parasympathetic ganglia in the head and neck (HNPGL) remains to be defined. Here, we retrospectively analyzed HIF2α by immunohistochemistry in 62 PPGL/HNPGL and human CB and AM, and comprehensively evaluated the HIF-related transcriptome of 202 published PPGL/HNPGL. We report that HIF2α is barely detected in the AM, but accumulates at high levels in PPGL, mostly (but not exclusively) in those with loss-of-function mutations in VHL and genes encoding components of the succinate dehydrogenase (SDH) complex. This is associated with upregulation of EPAS1 and the HIF2α-regulated genes COX4I2 and ADORA2A . In contrast, HIF2α and HIF2α-regulated genes are highly expressed in CB and HNPGL, irrespective of VHL and SDH dysfunctions. We also found that HIF2α and HIF1α protein expressions are not correlated in PPGL nor HNPGL. In addition, HIF1α-target genes are almost exclusively overexpressed in VHL -mutated HNPGL/PPGL. Collectively, the data suggest that involvement of HIF2α in the physiology and tumor pathology of human paraganglia is organ-of-origin-dependent and HIF1α-independent.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background and Aim: The use of antibiotics as growth promoters in the feed of poultry, has contributed to an increase in the antimicrobial resistance of foodborne pathogens worldwide. Hence, the development of new effective alternatives to antibiotics that do not hinder productivity is imperative. For this, the aim of the present study was to determine whether oregano essential oil (OEO) extracted from Lippia origanoides is a suitable alternative to growth-promoting antibiotics (GPAs) for improving the performance, egg quality, and intestinal morphometry of ISA Brown laying hens., Materials and Methods: A total of ninety-six 70-week-old ISA Brown laying hens were randomly assigned to four treatment groups with four replicates per treatment and six hens per replicate. The treatments consisted of four different diets that were formulated according to the nutritional requirements of the genetic line and the production phase with and without the addition of GPA and OEO: NC, which did not contain OEO or GPA; GPA, which included 50 ppm zinc bacitracin as a GPA; 80OEO, which included 80 ppm OEO and no GPA; and 150OEO, which included 150 ppm OEO and no GPA., Results: All of the OEO and GPA treatment groups had a better feed conversion ratio than the NC group. However, the addition of 150 ppm OEO to the diet improved the percentage egg production and egg mass, as well as the external and internal quality of the egg compared with the other treatments. In addition, both the 80OEO and 150OEO treatments improved the yolk color, shell thickness, and shell color, as well as parameters related to the intestinal morphometry compared with the NC group., Conclusion: The findings of this study indicate that 150 ppm OEO can be used as a substitute for GPA to improve the performance, egg quality, and parameters related to the intestinal morphometry of ISA Brown laying hens., (Copyright: © Ramirez, et al.)

Besides their crucial role in cell electrogenesis and maintenance of basal membrane potential, the voltage-dependent K + channel Kv11.1/hERG1 shows an essential impact in cell proliferation and other processes linked to the maintenance of tumour phenotype. To check the possible influence of channel expression on DNA damage responses, HEK293 cells, treated with the genotoxic agent methyl methanesulfonate (MMS), were compared with those of a HEK-derived cell line (H36), permanently transfected with the Kv11.1-encoding gene, and with a third cell line (T2) obtained under identical conditions as H36, by permanent transfection of another unrelated plasma membrane protein encoding gene. In addition, to gain some insights about the canonical/conduction-dependent channel mechanisms that might be involved, the specific erg channel inhibitor E4031 was used as a tool. Our results indicate that the expression of Kv11.1 does not influence MMS-induced changes in cell cycle progression, because no differences were found between H36 and T2 cells. However, the canonical ion conduction function of the channel appeared to be associated with decreased cell viability at low/medium MMS concentrations. Moreover, direct DNA damage measurements, using the comet assay, demonstrated for the first time that Kv11.1 conduction activity was able to modify MMS-induced DNA damage, decreasing it particularly at high MMS concentration, in a way related to PARP1 gene expression. Finally, our data suggest that the canonical Kv11.1 effects may be relevant for tumour cell responses to anti-tumour therapies.

EAG ( ether-à-go-go or KCNH ) are a subfamily of the voltage-gated potassium (Kv) channels. Like for all potassium channels, opening of EAG channels drives the membrane potential toward its equilibrium value for potassium, thus setting the resting potential and repolarizing action potentials. As voltage-dependent channels, they switch between open and closed conformations (gating) when changes in membrane potential are sensed by a voltage sensing domain (VSD) which is functionally coupled to a pore domain (PD) containing the permeation pathway, the potassium selectivity filter, and the channel gate. All Kv channels are tetrameric, with four VSDs formed by the S1-S4 transmembrane segments of each subunit, surrounding a central PD with the four S5-S6 sections arranged in a square-shaped structure. Structural information, mutagenesis, and functional experiments, indicated that in "classical/ Shaker -type" Kv channels voltage-triggered VSD reorganizations are transmitted to PD gating via the α-helical S4-S5 sequence that links both modules. Importantly, these Shaker -type channels share a domain-swapped VSD/PD organization, with each VSD contacting the PD of the adjacent subunit. In this case, the S4-S5 linker, acting as a rigid mechanical lever (electromechanical lever coupling), would lead to channel gate opening at the cytoplasmic S6 helices bundle. However, new functional data with EAG channels split between the VSD and PD modules indicate that, in some Kv channels, alternative VSD/PD coupling mechanisms do exist. Noticeably, recent elucidation of the architecture of some EAG channels, and other relatives, showed that their VSDs are non-domain swapped. Despite similarities in primary sequence and predicted structural organization for all EAG channels, they show marked kinetic differences whose molecular basis is not completely understood. Thus, while a common general architecture may establish the gating system used by the EAG channels and the physicochemical coupling of voltage sensing to gating, subtle changes in that common structure, and/or allosteric influences of protein domains relatively distant from the central gating machinery, can crucially influence the gating process. We consider here the latest advances on these issues provided by the elucidation of eag1 and erg1 three-dimensional structures, and by both classical and more recent functional studies with different members of the EAG subfamily., (Copyright © 2020 Barros, de la Peña, Domínguez, Sierra and Pardo.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Voltage-dependent potassium channels (Kv channels) are crucial regulators of cell excitability that participate in a range of physiological and pathophysiological processes. These channels are molecular machines that display a mechanism (known as gating) for opening and closing a gate located in a pore domain (PD). In Kv channels, this mechanism is triggered and controlled by changes in the magnitude of the transmembrane voltage sensed by a voltage-sensing domain (VSD). In this review, we consider several aspects of the VSD⁻PD coupling in Kv channels, and in some relatives, that share a common general structure characterized by a single square-shaped ion conduction pore in the center, surrounded by four VSDs located at the periphery. We compile some recent advances in the knowledge of their architecture, based in cryo-electron microscopy (cryo-EM) data for high-resolution determination of their structure, plus some new functional data obtained with channel variants in which the covalent continuity between the VSD and PD modules has been interrupted. These advances and new data bring about some reconsiderations about the use of exclusively a classical electromechanical lever model of VSD⁻PD coupling by some Kv channels, and open a view of the Kv-type channels as allosteric machines in which gating may be dynamically influenced by some long-range interactional/allosteric mechanisms.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cisplatin, one of the most extensively used metallodrugs in cancer treatment, presents the important drawback of patient resistance. This resistance is the consequence of different processes including those preventing the formation of DNA adducts and/or their quick removal. Thus, a tool for the accurate detection and quantitation of cisplatin-induced adducts might be valuable for predicting patient resistance. To prove the validity of such an assumption, highly sensitive plasma mass spectrometry (ICP-MS) strategies were applied to determine DNA adduct levels and intracellular Pt concentrations. These two metal-relative parameters were combined with an evaluation of biological responses in terms of genomic stability (with the Comet assay) and cell cycle progression (by flow cytometry) in four human cell lines of different origins and cisplatin sensitivities (A549, GM04312, A2780 and A2780cis), treated with low cisplatin doses (5, 10 and 20 μM for 3 hours). Cell viability and apoptosis were determined as resistance indicators. Univariate linear regression analyses indicated that quantitation of cisplatin-induced G-G intra-strand adducts, measured 1 h after treatment, was the best predictor for viability and apoptosis in all of the cell lines. Multivariate linear regression analyses revealed that the prediction improved when the intracellular Pt content or the Comet data were included in the analysis, for all sensitive cell lines and for the A2780 and A2780cis cell lines, respectively. Thus, a reliable cisplatin resistance predictive model, which combines the quantitation of adducts by HPLC-ICP-MS, and their repair, with the intracellular Pt content and induced genomic instability, might be essential to identify early therapy failure.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Epigenetic alterations are increasingly implicated in the initiation and progression of cancer. Genome-wide (global) hypomethylation seems to occur in early neoplasia and is a feature of genomic DNA derived from solid tumour tissues like ovarian cancer. Thus, analytical methods that provide sensitive and quantitative information about cytosine methylation in DNA are currently required. In this work, we compare two different anion-exchange columns for the separation of methylated cytosine from the other DNA nucleotides: a silica-based (Tracer Extrasil SAX) column and a polystyrene/divinyl benzene-based (Mono-Q™) column. Under the optimised conditions, linearity range, precision and detection limits of the developed high-performance liquid chromatography (HPLC) method were evaluated and compared using conventional ultraviolet (UV) absorbance detection at 270 nm. Good separation of the five target nucleotides, including 5-methyl-2'-deoxycytidine monophosphate (5mdCMP) and 2'-deoxycytidine monophosphate (dCMP) was achieved on the Mono-Q™ column with a gradient elution of ammonium acetate buffer (1 M, pH 6.9) at a flow rate of 1 mL min(-1). The coupling of this column to inductively coupled plasma mass spectrometry (ICP-MS) permitted also phosphorous ((31)P) specific detection of the nucleotides. Both detection systems offered adequate analytical performance characteristics, with detection limits of 30 and 40 μg L(-1) for 5mdCMP by HPLC-UV and HPLC-ICP-MS, respectively. However, the latter method allowed the determination of the global DNA methylation level (%) without the need for external calibration. Different genomic DNA samples were analysed including calf thymus DNA and DNA from two human cancer cell lines (adenocarcinoma epithelial A549 and ovarian carcinoma A2780) using the proposed strategy. In the line A2780, the cisplatin-sensitive and cisplatin-resistant variants were analysed, finding no significant differences in the methylation percentage after treatment with cisplatin.

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.

The comet assay, a very useful tool in genotoxicity and DNA repair testing, is being applied to Drosophila melanogaster since around 15 years ago, by several research groups. This organism is a valuable model for all kind of processes related to human health, including DNA damage response. The assay has been performed mainly in vivo using different larvae cell types (from brain, midgut, hemolymph, and imaginal disk), but also in vitro with the S2 cell line. Since its first application, it has been used to analyze the genotoxicity and action mechanisms of different chemicals, demonstrating good sensitivity and proving its usefulness. Moreover, it is the only assay that can be used to analyze DNA repair in somatic cells in vivo, comparing the effects of chemicals in different repair strains, and to quantitate repair activities in vitro. Additionally, the comet assay in Drosophila, in vivo and in vitro, has been applied to study the influence of protein overexpression on genome integrity and degradation. Although the assay is well established, it could benefit from some research to determine optimal experimental design to standardize it, and then to allow comparisons among laboratories independently of the chosen cell type.

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.

Cisplatin is a chemotherapeutic drug widely used in the treatment of several tumours, but this chemotherapy presents problems in terms of side-effects and patient resistance. The detection and determination of cisplatin-induced adducts and the relationship with the physiological or clinical effects of this drug under different repair conditions could be a good measure to assess patient's response to such chemotherapy. A new methodological approach to detect and quantify cisplatin adducts by use of high-performance liquid chromatography with inductively coupled plasma mass-spectrometric detection (HPLC-ICP-MS) and isotope-dilution analysis (IDA), is evaluated for its application in vivo, under different repair conditions. This analysis is combined with the use of the Comet assay, which detects DNA strand-breaks, and the w/w(+) SMART assay, which monitors induction of somatic mutation and recombination in Drosophila melanogaster in vivo under different conditions of nucleotide-excision repair. Results show that (i) cisplatin induces in Drosophila several adducts not detected in mammals. The two most abundant cisplatin-induced adducts, identified by electrospray-mass spectrometry as G monoadduct and G-G intrastrand cross-links, were quantified individually; (ii) cisplatin induces higher levels of G monoadducts and G-G cross-links in NER-proficient than in NER-deficient cells; (iii) the level of adducts correlates with their biological consequences, both in terms of DNA strand-breaks (tail-moment values), and of somatic mutation and recombination (frequency of mosaic eyes and clones in 10(4) cells), when the repair status is considered. This work demonstrates the validity and potential of the adduct detection and quantification methodology in vivo, and its use to correlate adducts with their genetic consequences., (© 2011 Elsevier B.V. All rights reserved.)

The D. melanogaster mus308 gene, highly conserved among higher eukaryotes, is implicated in the repair of cross-links and of O-ethylpyrimidine DNA damage, working in a DNA damage tolerance mechanism. However, despite its relevance, its possible role on the processing of different DNA ethylation damages is not clear. To obtain data on mutation frequency and on mutation spectra in mus308 deficient (mus308(-)) conditions, the ethylating agent diethyl sulfate (DES) was analysed in postmeiotic male germ cells. These data were compared with those corresponding to mus308 efficient conditions. Our results indicate that Mus308 is necessary for the processing of oxygen and N-ethylation damage, for the survival of fertilized eggs depending on the level of induced DNA damage, and for an influence of the DNA damage neighbouring sequence. These results support the role of mus308 in a tolerance mechanism linked to a translesion synthesis pathway and also to the alternative end-joinig system.

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.

The antitumoral effect of cisplatin [cis-diamminodichloroplatinum(II)] in mammals is related to its binding to DNA components. However, there is a lack of specific chemical methods to selectively detect those adducts formed in vivo at low concentrations. In this work, a new sensitive and selective method of determining cisplatin-DNA adducts based on the use of element-selective mass spectrometry is proposed, and the method is then applied to detect cisplatin adducts induced in vivo in somatic cells of Drosophila melanogaster. The bioanalytical strategy proposed here allows the determination of the most important DNA adduct formed between adjacent guanine units of the same DNA strand with cisplatin, and it is based on the coupling of capillary liquid chromatography (cap-LC) to inductively coupled plasma mass spectrometry (ICP-MS). This set-up allows the simultaneous monitoring of the Pt (from the drug) and P (from the DNA components) present in these adducts, once they have been cleaved by enzymatic hydrolysis of the DNA samples. Using this instrumental set-up, the adducts of cisplatin formed in vivo when D. melanogaster flies are exposed to different cisplatin concentrations can be detected and their concentration determined. The results obtained show a direct correlation between the concentration of cisplatin adducts, the induced genotoxic damage (measured as DNA strand breaks using the Comet assay) and the cisplatin concentration. [figure: see text] The work illustrates the complementary use of bioanalytical and biological information to study cisplatin interactions with DNA is vivo at biologically relevant concentrations of the drug.

Objective: The goal of this study was to show the ability of 3D MDCT angiography to display the arterial and venous vascular anatomy of pulmonary sequestration in children., Conclusion: MDCT angiography with 3D rendering shows the anomalous feeding artery and the draining veins that allow a diagnosis of pulmonary sequestration. These features may prove useful in distinguishing intra- and extralobar sequestration and in surgical planning.

In spite of differences between female and male germ cells, and although both of them contribute to the gene pool of future generations, most germ cell mutagenicity studies in higher eukaryotes have been carried out on males. To study the response of female germ cells to mutagen/carcinogen exposure, the mutagenicity of two model chemicals like diethyl sulfate (DES) and hexamethylphosphoramide (HMPA), and the monofunctional methylating chemotherapeutic drug streptozotocin (STZ), has been analysed on repair efficient females of Drosophila melanogaster. Results previously obtained with N-ethyl-N-nitrosourea (ENU), another model chemical, have also been included in the analysis. The activity of bypass tolerance mechanism (BTM; represented by the mus308 locus) and nucleotide excision repair (NER) on the removal of oxygen and nitrogen ethylations was studied by determining DES mutagenicity in NER deficient females, comparing it with existing results for ENU, and by analysing both chemicals on BTM deficient females. Results indicate that (1) all chemicals are mutagenic on repair efficient females; (2) a measure of mutagenic activity ranked from the lowest DES to STZ, HMPA, and ENU as the highest. This order correlates with the repair of the respectively induced DNA damages, and with the mutagenic and carcinogenic potency of these compounds, considering the toxicity of cross-linking agents; (3) NER efficiently repairs nitrogen ethylation damage and seems to contribute to the processing of oxygen damage in female germ cells; and (4) BTM is involved on the processing of oxygen ethylation damage, whereas the results on nitrogen ethylation are not clear. Finally, these results indicate that differences between male and female germ cells affect the response to chemical exposure, and therefore demonstrate the necessity of analysing also female cells in germinal mutagenicity studies. In addition, these studies can provide important mechanistic information about germ cell chemical mutagenesis, and even when the analysis of oogonia is not possible, since all female germ cells are pre-meiotic, studies of oocytes could be a model for pre-meiotic cells.

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., (Copyright 2003 Wiley-Liss, Inc.)

To check the possibilities of the recently developed comet assay, to be used in mechanistic studies in Drosophila melanogaster, neuroblast cells of third instar larvae are used to analyse in vivo, the effect of two repair deficient mutations: mus201, deficient on nucleotide excision repair, and mus308, deficient in a mechanism of damage bypass, on the genotoxicity of methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENU). The obtained results reveal: (1) MMS-induced breaks are most probably consequence of N-alkylation damage mediated abasic (AP) site breakage; (2) MMS and at least part of the EMS induced damage leading to DNA strand breaks are efficiently repaired by the nucleotide excision repair mechanism; (3) ENU and part of EMS induced damage need a functional Mus308 protein to be processed, otherwise they can lead to DNA strand breaks. In addition, the results of this work confirm the validity of neuroblast cells to conduct the comet assay, and the usefulness of this assay in in vivo mechanistic studies related to DNA repair in D. 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., (Copyright 2002 Wiley-Liss, Inc.)

Although N-nitrosodiethylamine (NDEA) is a potent carcinogen in rodents and a probable human carcinogen, little attempts were made to characterize its mutation spectrum in higher eukaryotes. We have compared forward mutation frequencies at multiple (700) loci with the mutational spectrum induced at the vermilion gene of Drosophila, after exposure of post- and pre-meiotic male germ cells to NDEA. Among 30 vermilion mutants collected from post-meiotic stages were 12 G:C-->A:T transitions (40%), 8 A:T-->T:A transversions (27%), and 4 structural rearrangements (13%). The remainder were three A:T-->G:C transitions, two G:C-->C:G transversions and one G:C-->T:A transversion. The results show that although NDEA induces predominantly transitions (40% G:C-->A:T and 10% A:T-->G:C), the frequencies of transversions (37%, of which 27% of A:T-->T:A transversions) and especially of rearrangements (13%) are remarkably high. This mutation spectrum differs significantly from that produced by the direct-ethylating agent N-ethylnitrosourea (ENU), although the relative distribution of ethylated DNA adducts is similar for both carcinogens. These differences, in particular the occurrence of rearrangements, are most likely the result of the requirement of NDEA for bioactivation. Since all four rearrangements were collected from non-metabolizing spermatozoa (or late spermatids), it is hypothesized that they derived from acetaldehyde, a stable metabolite of NDEA. Due to its cytotoxicity, attempts to isolate vermilion mutants from NDEA-exposed pre-meiotic cells were largely unsuccessful, because only two mutants (one A:T-->G:C transition and one 1bp insertion) were collected from those stages. Our results show that NDEA is capable of generating carcinogenic lesions other than base pair substitutions.

DNA damage caused by oxygen alkylation of bases (mainly at O6-G, O4-T and O2-T positions in DNA) has been correlated with the mutagenic and carcinogenic potency of monofunctional alkylating agents. In all kinds of organisms, repair of O6-alkylG is carried out mainly by the enzyme O6-methyl guanine-DNA methyltransferase (MGMT). However, little is known about the repair of the O-alkylT adducts or about the contribution of nucleotide excision repair (NER) to this process, especially in higher eukaryotes. To study the influence of the NER system on the repair of O-alkylation damage, the molecular mutation spectrum induced by N-ethyl-N-nitrosourea (ENU) in an NER-deficient Drosophila strain, carrying a mutation at the mus201 locus, was obtained and compared with a previously published spectrum for NER-proficient conditions. This comparison reveals a clear increase in the frequency of base pair changes, including GC --> AT and AT --> GC transitions and AT --> TA transversions. In addition, one deletion and two frameshift mutations, not found under NER-proficient conditions, were isolated in the NER-deficient mutant. The results demonstrate that: (1) N-alkylation damage contributes considerably (more than 20%) to the mutagenic activity of ENU under NER-deficient conditions, confirming that the NER system repairs this kind of damage; and (2) that in germ cells of Drosophila in vivo, NER seems to repair O6-ethylguanine and/or O2-ethylcytosine, O4-ethylthymine, and possibly also O2-ethylthymine.

The mus308 locus of D. melanogaster was originally characterized by virtue of a mutant phenotype that resulted in specific hypersensitivity to cross-linking agents. However, the gene product has also been implicated in the repair of lesions other than cross-links. The gene was recently sequenced, and it encodes a protein with motifs characteristic of both DNA polymerases and helicases. We present mutability studies, using the recessive lethal (RL) test, which show that N-ethyl-N-nitrosourea (ENU) induces hypermutability in mus308-deficient conditions, although only in early broods. Further studies elucidated the role of MUS308 in repair processes by characterizing the spectrum of molecular mutations induced by in vivo ENU in postmeiotic germ cells, in mus308 conditions. These revealed that, in comparison to repair-proficient conditions, there is an increase in the frequency of GC --> AT and AT --> GC transitions, and AT --> TA transversions. Moreover, frameshift mutations, which have not previously been reported to form part of the ENU spectrum, were also found. These results indicate that MUS308 is needed to process ENU-induced lesions, and support the hypothesis that the mus308 gene plays a role in post-replication bypass of O-alkylpyrimidines, probably mediated by recombination, which serves to increase the time available for error-free repair of these persistent and highly mutagenic lesions.

The vermilion gene in Drosophila has extensively been used for the molecular analysis of mutations induced by chemicals in germ cells in vivo. The gene is located on the X-chromosome and is a useful target for the study of mutagenesis since all types of mutations are generated. We have critically evaluated this system with respect to sensitivity for mutation induction and selectivity for different types of mutations, using a database of more than 600 vermilion mutants induced in postmeiotic male germ cells by 18 mutagens. From most of these mutants the mutation has been analysed. These data showed 336 base substitutions, 96 intra-locus DNA rearrangements and 78 multi-locus deletions (MLD). Mutants containing a MLD were either heterozygous sterile or homozygous and hemizygous lethal. The distribution of both basepair (bp) changes and intra-locus rearrangements over the coding region of the vermilion gene was uniform with no preferences concerning 5' or 3' regions, certain exons, splice sites, specific amino acid changes or nonsense mutations. Possible hotspots for base substitutions seem to be related to the type of DNA damage rather than to the vermilion system. Gene mutations other than bp changes were examined on sequence characteristics flanking the deletion breakpoints. Induction frequencies of vermilion mosaic mutants were, in general, higher than those of vermilion complete mutants, suggesting that persistent lesions are the main contributors to the molecular spectra. Comparison of induction frequencies of vermilion mutants and sex-linked recessive lethal (SLRL) mutants for the 18 mutagens showed that the sensitivity of the vermilion gene against a mutagenic insult is representative for genes located on the X-chromosome. The effect of nucleotide excision repair (NER) on the formation of SLRL mutants correlated with an increase of transversions in the vermilion spectra under NER deficient conditions. Furthermore, the clastogenic potency of the mutagens, i.e., the efficiency to induce chromosomal-losses vs. SLRL forward mutations, shows a positive correlation with the percentage of DNA deletions in the molecular spectra of vermilion mutants.

The role of a defect for nucleotide excision repair (NER) in oocytes on the repair of DNA ethyl adducts induced by diethyl sulfate (DES) in male germ cells of Drosophila was analysed. Frequencies of mutations at multiple loci (recessive lethal mutations) and at the vermilion gene induced in NER+ conditions (cross NER+ x NER+) were compared with those fixed in a NER- background (NER- x NER+). The M(NER-)/M(NER+) mutability ratios for two DES concentrations, 10 mM and 15 mM, were 2.21 and 1.49, respectively, indicating that NER repairs part of the DES-induced damage. The majority of 28 fertile vermilion mutations produced by DES in NER- are transitions, both GC-AT (46.4%) and AT-GC (21.4%) transitions are found, the consequences of O6-ethylguanine and O4-ethylthymine, respectively. Transversions (21.5%), one +1 frameshift mutation (3.6%) and two deletions (7.1%) are most likely the result of N-alkylation damage. Furthermore, the DES-induced mutation spectra show interesting differences in relation to the exposure dose. All 10 mutants isolated in this and a previous [L.M. Sierra, A. Pastink, M.J.M. Nivard, E.W. Vogel, DNA base sequence changes induced by DES in postmeiotic male germ cells of Drosophila melanogaster, Mol. Gen. Genet. 237 (1993) 370-374] study from experiments with low DES-effectiveness are exclusively transitions, independent whether the females were of the NER+ or NER-genotype. This indicates that at lower DES effectiveness only O-alkylation damage is relevant, and that N-alkylation damage is repaired. In experiments revealing high DES-effectiveness, vermilion mutations representing N-alkylation damage reached 43% (9/21) with NER- and 26% (7/27) with NER+ females, suggesting (i) that NER becomes involved at high adduct levels because then the base excision repair (BER) may be saturated, and (ii) that this involvement of NER causes the relative decrease from 43% to 26% N-alkylation mediated sequence changes.

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., (Copyright 1999 Elsevier Science B.V.)

Molecular mutation spectra induced by N-ethyl-N-nitrosourea have been obtained in several organisms and test systems, frequently showing different results. In Drosophila melanogaster this spectrum has been analyzed in postmeiotic stages, resulting in good agreement between the adduct spectrum and mutational events, the majority being GC-->AT transitions (61%). However, when collecting data about in vivo ENU-induced mutations in mouse germ cell stages mostly damage at A:T sites (89%) was observed. In this work we analyze the molecular spectrum induced with ENU in pre-meiotic repair-active male germ cells of D.melanogaster, using the specific locus test (SLT) with the vermilion locus as target. Results show that the most mutagenic sites in spermatogonial stem cells of Drosophila are A:T pairs (85%), with AT-->TA transversions (50%) and AT-->GC transitions (35%) as the most frequent mutations. Differences from the post-meiotic spectrum may be explained by the active repair of some adducts, such as O6-ethylguanine and N-alkyl-induced abasic sites. In addition, these results show the relevance of the minor lesions O4-ethylthymine and O2-ethylthymine in the production of mutations, as a consequence of their poor repair. Finally, since there is a striking similarity to the ENU-induced mutation spectrum in mouse, these results reveal that Drosophila continues to be an excellent model system.

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 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.

Previous studies on structure-activity relationships (SARs) between types of DNA modifications and tumour incidence revealed linear positive relationships between the log TD50 estimates and s-values for a series of mostly monofunctional alkylating agents. The overall objective of this STEP project was to further elucidate the mechanistic principles underlying these correlations, because detailed knowledge on mechanisms underlying the formation of genotoxic damage is an absolute necessity for establishing guidance values for exposures to genotoxic agents. The analysis included: (1) the re-calculation and further extension of TD50 values in mmol/kg body weight for chemicals carcinogenic in rodents. This part further included the checking up data for Swain-Scott s-values and the use of the covalent binding index (CBI); (2) the elaboration of genetic toxicity including an analysis of induced mutation spectra in specific genes at the DNA level, i.e., the vermilion gene of Drosophila, a plasmid system (pX2 assay) and the HPRT gene in cultured mammalian cells (CHO-9); and (3) the measurement of specific DNA alkylation adducts in animal models (mouse, rat, hamster) and mammalian cells in culture. The analysis of mechanisms controlling the expression of mammalian DNA repair genes (alkyltransferases, glycosylases) as a function of the cell type, differentiation stage, and cellular microenvironment in mammalian cells. The 3 classes of genotoxic carcinogens selected for the project were: (1) chemicals forming monoalkyl adducts upon interaction with DNA; (2) genotoxins capable of forming DNA etheno-adducts; and (3) N-substituted aryl compounds forming covalent adducts at the C8 position of guanine in DNA. In general, clear SARs and AARs (activity-activity relationships) between physiochemical parameters (s-values, O6/N7-alkylguanine ratios, CBI), carcinogenic potency in rodents and several descriptors of genotoxic activity in germ cells (mouse, Drosophila) became apparent when the following descriptors were used: TD50 estimates (lifetime doses expressed in mg/kg b.wt. or mmol/kg b.wt.) from cancer bioassays in rodents; the degree of germ-cell specificity, i.e., the ability of a genotoxic agent to induce mutations in practically all cell stages of the male germ-cell cycle of Drosophila (this project) and the mouse (literature search), as opposed to a more specific response in postmeiotic stages of both species; the Mexr-/Mexr+ hypermutability ratio, determined in a repair assay utilizing Drosophila germ cells; mutation spectra induced at single loci (the 7 loci used in the specific-locus test of the mouse (published data), and the vermilion gene of Drosophila); and doubling doses (DD) in mg/kg (mmol/kg) for specific locus test results on mice. By and large, the TD50 values, the inverse of which can be considered as measures of carcinogenic potency, were shown to be predictable from knowledge of the in vivo doses associated with the absorbed amounts of the investigated alkylators and with the second-order constant, kc, reaction at a critical nucleophilic strength, nc. For alkylating agents kc can be expressed as the second-order rate constant for hydrolysis, kH2O, and the substrate constant s:kH2OTD50 is a function of a certain accumulated degree of alkylation, here given as the (average) daily increment, ac, for 2 years exposure of the rodents. The TD*50 in mmol/kg x day) could then be written: [formula: see text] This expression would be valid for monofunctional alkylators provided the reactive species are uncharged. This is the case for most SN2 reagents. Although it appears possible to predict carcinogenic potency from measured in vivo doses and from detailed knowledge of reaction-kinetic parameter values, it is at present not possible to quantify the uncertainty of such predictions. One main reason for this is the complication due to uneven distribution in the body, with effects on the dose in target tissues. The estimation can be impro