Unraveling the parahormetic mechanism underlying the health-protecting effects of grapeseed procyanidins.

Proanthocyanidins (PACs), the predominant constituents within Grape Seed Extract (GSE), are intricate compounds composed of interconnected flavan-3-ol units. Renowned for their health-affirming properties, PACs offer a shield against a spectrum of inflammation associated diseases, such as diabetes, obesity, degenerations and possibly cancer. While monomeric and dimeric PACs undergo some absorption within the gastrointestinal tract, their larger oligomeric and polymeric counterparts are not bioavailable. However, higher molecular weight PACs engage with the colonic microbiota, fostering the production of bioavailable metabolites that undergo metabolic processes, culminating in the emergence of bioactive agents capable of modulating physiological processes. Within this investigation, a GSE enriched with polymeric PACs was employed to explore in detail their impact. Through comprehensive analysis, the present study unequivocally verified the gastrointestinal-mediated transformation of medium to high molecular weight polymeric PACs, thereby establishing the bioaccessibility of a principal catabolite termed 5-(3',4'-dihydroxyphenyl)-γ-valerolactone (VL). Notably, our findings, encompassing cell biology, chemistry and proteomics, converge to the proposal of the notion of the capacity of VL to activate, upon oxidation to the corresponding quinone, the nuclear factor E2-related factor 2 (Nrf2) pathway-an intricate process that incites cellular defenses and mitigates stress-induced responses, such as a challenge brought by TNFα. This mechanistic paradigm seamlessly aligns with the concept of para-hormesis, ultimately orchestrating the resilience to stress and the preservation of cellular redox equilibrium and homeostasis as benchmarks of health.

Salivary proteomic profile of young healthy subjects.

Background: The incidence of noncommunicable diseases (NCDs) has been rapidly ramped up worldwide. Hence, there is an urgent need to non-invasively detect NCDs possibly by exploiting saliva as a 'liquid biopsy' to identify biomarkers of the health status. Since, the absence of standardized procedures of collection/analysis and the lack of normal ranges makes the use of saliva still tricky, our purpose was to outline a salivary proteomic profile which features healthy individuals. Methods: We collected saliva samples from 19 young blood donors as reference population and the proteomic profile was investigated through mass-spectrometry. Results: We identified 1,004 proteins of whose 243 proteins were shared by all subjects. By applying a data clustering approach, we found a set of six most representative proteins across all subjects including Coronin-1A, F-actin-capping protein subunit alpha, Immunoglobulin J chain, Prosaposin, 78 kDa glucose-regulated protein and Heat shock 70 kDa protein 1A and 1B. Conclusion: All of these proteins are involved in immune system activation, cellular stress responses, proliferation, and invasion thus suggesting their use as biomarkers in patients with NCDs.

Co-administration of H-ferritin-doxorubicin and Trastuzumab in neoadjuvant setting improves efficacy and prevents cardiotoxicity in HER2 + murine breast cancer model.

Neoadjuvant chemotherapy has been established as the standard of care for HER2-positive breast cancer since it allows cancer down-staging, up to pathological complete response. The standard of care in the neoadjuvant setting for HER2-positive breast cancer is a combination of highly cytotoxic drugs such as anthracyclines and the anti-HER2 monoclonal antibody. Despite this cocktail allows a pathological complete response in up to 50%, their co-administration is strongly limited by intrinsic cardiotoxicity. Therefore, only a sequential administration of anthracyclines and the anti-HER2 treatment is allowed. Here, we propose the anthracycline formulation in H-Ferritin nanocages as promising candidate to solve this unmet clinical need, thanks to its capability to increase anthracyclines efficacy while reducing their cardiotoxicity. Treating a murine model of HER2-positive breast cancer with co-administration of Trastuzumab and H-Ferritin anthracycline nanoformulation, we demonstrate an improved tumor penetration of drugs, leading to increased anticancer efficacy and reduced of cardiotoxicity.

Complete (1)H and (13)C NMR spectral assignment of alpha- and beta-adenosine, 2'-deoxyadenosine and their acetate derivatives.

(1)H and (13)C NMR chemical shifts of alpha- and beta-anomers of adenosine, 2'-deoxyadenosine and their acetate derivatives were completely and definitely assigned using the concerted application of one- and two-dimensional experiments (gCOSY, gNOESY, gHSQC and gHMBC). The influence of the stereochemistry of the purine base on the NMR data of the hydrogen and carbon atoms of the furanose moiety was estimated.

Mitochondrial function, apoptosis and cell cycle delay in the WEHI-3B leukaemia cell line and its variant Ciprofloxacin-resistant WEHI-3B/CPX.

OBJECTIVE: The susceptibility of two cell lines, WEHI-3B myelomonocytic leukaemia and its variant Ciprofloxacin-resistant WEHI-3B/CPX to undergo apoptosis induced by Ciprofloxacin was studied and compared. MATERIALS AND METHODS: Apoptosis was checked by measuring the DNA fragmentation and determining the ratio of apoptotic/necrotic cells. The relationship between the induction of apoptosis and G(1), S or G(2) block in the cell cycle has also been investigated and cytogenetical evaluation of chromosomal aberrations in both cell lines has been carried out. The regulation of expression of Bax and Bcl-2 was also checked by western blotting after Ciprofloxacin treatment. RESULTS: We observed that the resistance of the subline was caused by a small percentage of cells that underwent apoptosis during continuous exposure to Ciprofloxacin in comparison with the parental cell line, whereas the percentage of necrotic cells remained unchanged. The WEHI-3B cells showed a G(2) block and a higher degree of cytogenetic damage after drug exposure. The two cell lines expressed the same level of Bax and Bcl-2 following stimulation by Ciprofloxacin. Only in the resistant subclone, the ratio Bcl-2/Bax reversed in the anti-apoptotic gene expression. CONCLUSION: The resistance to ciprofloxacin observed is not related to mitochondrial function and although Bcl-2/Bax ratio behaviour does not fully explain the resistance of the WEHI3B/CPX subclone it is consistent with phenotypic character of resistance to CPX. The toxic effect on sensitive cells could be mediated by the cell cycle arrest whereas in the resistant clone, the prolonged G(2) phase could play a key role to favour cell cycle progression and proliferation.

Inhibition of CFU-E/BFU-E and CFU-GM colony growth by cyclophosphamide, 5-fluorouracil and taxol: development of a high-throughput in vitro method.

The major side effect of anticancer drugs is damage to the hematopoietic system. These compounds may interfere with cell proliferation and differentiation in different blood lineages causing many diseases such as neutropenia, aplastic anaemia or trombocytopenia. The clonogenic assays are useful in vitro tools for evaluating and predicting acute xenobiotics myelotoxicity. A miniaturisation of these assays, in order to reduce costs and increase the number of compounds that could be tested, is under investigation. The in vitro sensitivity of human burst-forming unit erythroid (BFU-E) and colony-forming unit granulocyte-macrophage (CFU-GM) to three anticancer drugs: cyclophosphamide (CTX), 5-fluorouracil (5-FU) and taxol (TAX) was evaluated both in 35 mm plate and 96-well plate systems and the dose-response curves, IC50 values and IC90 values were compared. The correlation between in vitro data and clinical plasma levels confirms that severe hematotoxicity is the primary adverse effect of these drugs with an evident selectivity on erythroid progenitors for cyclophosphamide. IC50 and IC90 values, calculated on the basis of results obtained with the traditional assay, correlate with those obtained in microplate, as well as the dose-response curves, indicating that the 96 well plate assay could be a useful and reliable tool for high-throughput screening in early stages of drug development.

Complete 1H and 13C NMR spectral assignment of 17-hydroxy epimeric sterols with planar A or A and B rings.

Complete 1H and 13C spectral assignments of 17beta- and 17alpha-hydroxy epimers of three biologically active sterols (boldenone, 3-methoxyestradiol and 3-methoxydihydroequilenin) were achieved making use of one- and two-dimensional NMR techniques (1D-HOHAHA, DEPT, COSY, NOESY, TOCSY, HSQC and COLOC).

Application of the CFU-GM assay to predict acute drug-induced neutropenia: an international blind trial to validate a prediction model for the maximum tolerated dose (MTD) of myelosuppressive xenobiotics.

In a previous study of prevalidation, a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed, to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up, by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper, we describe an international blind trial designed to apply this model to the clinical neutropenia, by testing 20 drugs, including 14 antineoplastics (Cytosar-U, 5-Fluorouracil, Myleran, Thioguanine, Fludarabine, Bleomycin, Methotrexate, Gemcitabine, Carmustine, Etoposide, Teniposide, Cytoxan, Taxol, Adriamycin); two antivirals (Retrovir, Zovirax,); three drugs for other therapeutic indications (Cyclosporin, Thorazine, Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin, Bleomycin, Etoposide, Fludarabine, 5-Fluorouracil, Myleran, Taxol, Teniposide, Thioguanine, and Thorazine), IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine, Cyclosporin, Cytosar-U, Cytoxan, Gemcitabine, Indocin, Lindane, Methotrexate, Retrovir, and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had "actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD, instead of the 4-fold range required by the model, whereas the prediction with Cytosar-U was approximately 10-fold in error. A possible explanation for the failure in the prediction of these three drugs, which are pyrimidine analogs, is discussed. We concluded that our model correctly predicted the human MTD for 20 drugs out of 23, since the other three drugs (Topotecan, PZA, and Flavopiridol) were tested in the prevalidation study. The high percentage of predicitivity (87%), as well as the reproducibility of the SOP testing, confirm that the model can be considered scientifically validated in this study, suggesting promising applications to other areas of research in developing validated hematotoxicological in vitro methods.

Refinement of the colony-forming unit-megakaryocyte (CFU-MK) assay for its application to pharmaco-toxicological testing.

In vitro colony-forming unit (CFU) assays can be used in the evaluation of potentially haematotoxic compounds during preclinical testing. The use of undifferentiated haematopoietic cells, able to proliferate and commit towards a specific blood cell lineage, enable selective toxicity to be detected. We optimized the colony-forming unit-megakaryocyte (CFU-MK) assay for toxicological applications. We used a collagen-based colony-forming assay to examine the sensitivity of four cell types: mononuclear human cord blood cells (CBC), mononuclear human bone marrow cells (BMC), cord blood enriched CD34+CD38- cells, and bone marrow enriched CD34+CD38- cells, to the toxic effects of five drugs known to cause thrombocytopenia in humans. The enrichment of CD34+CD38- cells was achieved by using a negative cell separation technique. Our results showed that a comparable toxicity was detected both with CBC, BMC and CD34+CD38- cells enriched from cord blood, whereas CD34+CD38- cells from bone marrow were more resistant to some drugs. The assay showed a high reproducibility of the endpoint measured (IC(50)), independently of the cell type used and donor source. The present study demonstrates that the refined CFU-MK assay is reproducible and can be used for in vitro toxicology studies with CBC as well as BMC.

Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFU-GM) assay.

This report describes an international prevalidation study conducted to optimise the Standard Operating Procedure (SOP) for detecting myelosuppressive agents by CFU-GM assay and to study a model for predicting (by means of this in vitro hematopoietic assay) the acute xenobiotic exposure levels that cause maximum tolerated decreases in absolute neutrophil counts (ANC). In the first phase of the study (Protocol Refinement), two SOPs were assessed, by using two cell culture media (Test A, containing GM-CSF; and Test B, containing G-CSF, GM-CSF, IL-3, IL-6 and SCF), and the two tests were applied to cells from both human (bone marrow and umbilical cord blood) and mouse (bone marrow) CFU-GM. In the second phase (Protocol Transfer), the SOPs were transferred to four laboratories to verify the linearity of the assay response and its interlaboratory reproducibility. After a further phase (Protocol Performance), dedicated to a training set of six anticancer drugs (adriamycin, flavopindol, morpholino-doxorubicin, pyrazoloacridine, taxol and topotecan), a model for predicting neutropenia was verified. Results showed that the assay is linear under SOP conditions, and that the in vitro endpoints used by the clinical prediction model of neutropenia are highly reproducible within and between laboratories. Valid tests represented 95% of all tests attempted. The 90% inhibitory concentration values (IC(90)) from Test A and Test B accurately predicted the human maximum tolerated dose (MTD) for five of six and for four of six myelosuppressive anticancer drugs, respectively, that were selected as prototype xenobiotics. As expected, both tests failed to accurately predict the human MTD of a drug that is a likely protoxicant. It is concluded that Test A offers significant cost advantages compared to Test B, without any loss of performance or predictive accuracy. On the basis of these results, we proposed a formal Phase II validation study using the Test A SOP for 16-18 additional xenobiotics that represent the spectrum of haematotoxic potential.

Inhibition of CFU-E/BFU-E by 3'-azido-3'-deoxythymidine, chlorpropamide, and protoporphirin IX zinc (II): a comparison between direct exposure of progenitor cells and long-term exposure of bone marrow cultures.

Erythropoiesis occurs in two stages: proliferation amplifies cell number, and differentiation stimulates the acquisition of the functional properties of red blood cells. The erythroid colony-forming unit (CFU-E) amplifies the differentiation process in response to erythropoietic stress in vitro, whereas the burst-forming unit (BFU-E), which is not particularly sensitive to erythropoietin stimulation, gives rise to the CFU-E and, when stimulated, produces morphologically-identifiable erythroid colonies. The aim of this work was to evaluate the toxic effects of the antiviral agent, 3'-azido-3'-deoxythymidine (AZT), the antidiabetic drug, chlorpropamide (CLP), and the heme-analogous compound, protophorphirin IX zinc (II) (ZnPP), on the proliferation of erythroblastic progenitors by using human umbilical-cord blood cells and murine progenitors from long-term bone marrow cultures. All these agents may interfere with the hemopoietic process, causing myelotoxicity as an adverse effect via different mechanisms. Our results showed selective toxicity of the three drugs on the erythroid progenitors (IC(50): AZT 0.35 +/- 0.13 microM, ZnPP 23.34 +/- 1.16 microM, CLP 1.07 +/- 0.27 mM), with respect to the myeloid progenitors (IC(50): AZT 0.8 microM, ZnPP 103.9 +/- 3.9 microM and CLP > 2800 microM). The IC(50) values were well correlated with peak plasma levels reached in vivo by the drugs. There was a marked similarity between the drug sensitivities of the human and murine progenitors but differences in toxicity exerted by the drugs on the basis of the time of exposure. Drug treatment of long-term cultures, followed by the clonogenic assay of progenitors collected from them in the absence of the drugs, generally resulted in a lower hematotoxicity.

Comparison of in vitro drug-sensitivity of human granulocyte-macrophage progenitors from two different origins: umbilical cord blood and bone marrow.

Predictive in vitro hematotoxicity assays using human cells will provide estimation of tolerable level and aid considerably the development of agents with greater therapeutic activity and less toxicity. Human hematopoietic cells can be derived from three sources: human bone marrow by sternal or femoral aspiration, mobilized peripheral blood, or umbilical cord blood samples collected from placentas after deliveries. Because of the difficulties to have a continuous supply of bone marrow cells from normal human donors and the related ethical problems, we performed a study to compare the sensitivity of human bone marrow cells (h-BMC) and human cord blood cells (h-CBC) to chemicals in order to confirm if h-CBC can readily replace bone marrow cells in checking the sensitivity of GM-CFU progenitors to drugs as preliminarily reported in literature. Our results showed that the prediction of IC50 values in human model is quite similar by using h-BMC or h-CBC. On the contrary, the type of medium influenced in a significant way the ICs determination of some drugs.

Modulation of proto-oncogene expression by polychlorinated biphenyls in 3T3-L1 cell line.

The effects of two substituted polychlorinated biphenyls, the 3,4,5,3',4,5' (PCB-169) and the 2,3,4,2',4',5' (PCB-138) forms, were examined on the expression of c-myc, c-jun, c-ras, and jun-b in 3T3-L1 cells. Northern blot analysis demonstrated that the two PCBs, which exhibit a coplanar and di-ortho-substituted configuration, activated these oncogenes differently. PCB-138 markedly induced overexpression of ras, jun, and myc, whereas PCB-169 led to the overexpression of jun-b. High-performance liquid chromatography analysis of the cell samples treated in medium without serum revealed a higher intracellular concentration of the 2,3,4,2',4',5'-hexachlorobiphenyl (hexaCB), whereas the 3,4,5,3',4'5'-hexaCB reached the same concentration in the sonicated samples of cells with or without serum. These results indicated that there was a relationship between PCB structure, bioavailability, and the capacity to stimulate oncogene expression.

13th meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): alternative testing methodologies and conceptual issues.

Substantial world-wide resources are being committed to develop improved toxicological testing methods that will contribute to better protection of human health and the environment. The development of new methods is intrinsically driven by new knowledge emanating from fundamental research in toxicology, carcinogenesis, molecular biology, biochemistry, computer sciences, and a host of other disciplines. Critical evaluations and strong scientific consensus are essential to facilitate adoption of alternative methods for use in the safety assessment of drugs, chemicals, and other environmental factors. Recommendations to hasten the development of new alternative methods included increasing emphasis on the development of mechanism-based methods, increasing fundamental toxicological research, increasing training on the use of alternative methods, integrating accepted alternative methods into toxicity assessment, internationally harmonizating chemical toxicity classification schemes, and increasing international cooperation to develop, validate, and gain acceptance of alternative methods.

Death from cardiovascular disease in Italy, 1972-1981: decline in mortality rates and possible causes.

Mortality rates for cardiovascular disease vary widely between countries, and epidemiological patterns (trends in incidence rates, prevalence of risk factors, availability of medical care) are heterogeneous even among industrialized nations. We studied mortality from cardiovascular disease in Italy from 1972 to 1981 and compared mortality to trends in risk factors during the same period. Age-adjusted mortality rates for acute ischaemic heart disease (IHD) have increased in Italy from 1972 to reach a peak in 1978 (180.53/100,000 in males, 51.55/100,000 in females), then declined between 1978 and 1981, by 7% in males and 5% in females. The decline was more evident in males and in the younger age groups. Deaths from chronic IHD reached a peak in 1973 in females and in 1975 in males, then decreased, respectively by 24.8% and 35.7% until 1981. Mortality for cerebrovascular disease declined from 1972 to 1981 by 16.2% in males and 21.5% in females. Data from national statistics and sample surveys in different areas of Italy show an increase in total calorie intake, in animal proteins, fats and dairy products and raised average serum cholesterol levels plus an increase in smoking prevalence but a possible decline in blood pressure levels. The roles of hypertension treatment and of access to specialized medical care are discussed as possible contributors to the new declining trend of IHD, and the need is stressed for preventive strategies in health promotion.