Metals on the Menu-Analyzing the Presence, Importance, and Consequences.

Metals are integral components of the natural environment, and their presence in the food supply is inevitable and complex. While essential metals such as sodium, potassium, magnesium, calcium, iron, zinc, and copper are crucial for various physiological functions and must be consumed through the diet, others, like lead, mercury, and cadmium, are toxic even at low concentrations and pose serious health risks. This study comprehensively analyzes the presence, importance, and consequences of metals in the food chain. We explore the pathways through which metals enter the food supply, their distribution across different food types, and the associated health implications. By examining current regulatory standards for maximum allowable levels of various metals, we highlight the importance of ensuring food safety and protecting public health. Furthermore, this research underscores the need for continuous monitoring and management of metal content in food, especially as global agricultural and food production practices evolve. Our findings aim to inform dietary recommendations, food fortification strategies, and regulatory policies, ultimately contributing to safer and more nutritionally balanced diets.

Pesticide Use and Degradation Strategies: Food Safety, Challenges and Perspectives.

While recognizing the gaps in pesticide regulations that impact consumer safety, public health concerns associated with pesticide contamination of foods are pointed out. The strategies and research directions proposed to prevent and/or reduce pesticide adverse effects on human health and the environment are discussed. Special attention is paid to organophosphate pesticides, as widely applied insecticides in agriculture, veterinary practices, and urban areas. Biotic and abiotic strategies for organophosphate pesticide degradation are discussed from a food safety perspective, indicating associated challenges and potential for further improvements. As food systems are endangered globally by unprecedented challenges, there is an urgent need to globally harmonize pesticide regulations and improve methodologies in the area of food safety to protect human health.

A new acetylcholinesterase allosteric site responsible for binding voluminous negatively charged molecules - the role in the mechanism of AChE inhibition.

Acetylcholinesterase (AChE) inhibitors are important in the treatment of neurodegenerative diseases. Two inhibitors, 12-tungstosilicic acid (WSiA) and 12-tungstophosphoric acid (WPA), which have polyoxometalate (POM) type structure, have been shown to inhibit AChE activity in nM concentration. Circular dichroism and tryptophan fluorescence spectroscopy demonstrated that the AChE inhibition was not accompanied by significant changes in the secondary structure of the enzyme. The molecular docking approach has revealed a new allosteric binding site, termed ß-allosteric site (ß-AS), which is considered responsible for the inhibition of AChE by POMs. To the best of our knowledge, this is the first study reporting a new allosteric site that is considered responsible for AChE inhibition by voluminous and negatively charged molecules such as POMs. The selected POMs were further subjected to genotoxicity testing using human peripheral blood cells as a model system. It was shown that WSiA and WPA induced a mild cytostatic but not genotoxic effects in human lymphocytes, which indicates their potential to be used as medicinal drugs. The identification of non-toxic compounds capable of binding to an allosteric site that so far has not been considered responsible for enzyme inhibition could be fundamental for the development of new drug design strategies and the discovery of more efficient AChE modulators.

Conjugates of Gold Nanoparticles and Antitumor Gold(III) Complexes as a Tool for Their AFM and SERS Detection in Biological Tissue.

Citrate-capped gold nanoparticles (AuNPs) were functionalized with three distinct antitumor gold(III) complexes, e.g., [Au(N,N)(OH)2][PF6], where (N,N)=2,2'-bipyridine; [Au(C,N)(AcO)2], where (C,N)=deprotonated 6-(1,1-dimethylbenzyl)-pyridine; [Au(C,N,N)(OH)][PF6], where (C,N,N)=deprotonated 6-(1,1-dimethylbenzyl)-2,2'-bipyridine, to assess the chance of tracking their subcellular distribution by atomic force microscopy (AFM), and surface enhanced Raman spectroscopy (SERS) techniques. An extensive physicochemical characterization of the formed conjugates was, thus, carried out by applying a variety of methods (density functional theory-DFT, UV/Vis spectrophotometry, AFM, Raman spectroscopy, and SERS). The resulting gold(III) complexes/AuNPs conjugates turned out to be pretty stable. Interestingly, they exhibited a dramatically increased resonance intensity in the Raman spectra induced by AuNPs. For testing the use of the functionalized AuNPs for biosensing, their distribution in the nuclear, cytosolic, and membrane cell fractions obtained from human lymphocytes was investigated by AFM and SERS. The conjugates were detected in the membrane and nuclear cell fractions but not in the cytosol. The AFM method confirmed that conjugates induced changes in the morphology and nanostructure of the membrane and nuclear fractions. The obtained results point out that the conjugates formed between AuNPs and gold(III) complexes may be used as a tool for tracking metallodrug distribution in the different cell fractions.

UV-C light irradiation enhances toxic effects of chlorpyrifos and its formulations.

UV-C irradiation is widely used in the food industry. However, the health effects from dietary exposure to the irradiated pesticide residues retained in foodstuffs are underestimated. In this study, technical chlorpyrifos (TCPF) and its oil in water (EW) and emulsifiable concentrate (EC) formulations were irradiated by UV-C, and their photodegradation products were subjected to toxicity assessment, including determination of acetylcholinesterase (AChE) activity, genotoxicity and oxidative stress using human blood cells as a model system. Toxicity studies were performed using the chlorpyrifos concentrations in the range of those proposed as the maximum residue levels in plant commodities. TCPF, EW and EC photodegradation products induced DNA damage and oxidative stress, and their genotoxicity did not decrease as a function of irradiation time. Irradiated TCPF and EC are more potent AChE inhibitors than irradiated EW. Accordingly, the application of UV-C irradiation must be considered when processing the plants previously treated with chlorpyrifos formulations.

Copper-polyaniline nanocomposite: Role of physicochemical properties on the antimicrobial activity and genotoxicity evaluation.

Copper nanoparticles (Cu NPs) have proven to own excellent antimicrobial efficacy, but the problems of easy oxidation and aggregation limit their practical application. Here, nanocomposite based on polyaniline (PANI) and Cu NPs solved this problem and brought additional physicochemical properties that are markedly advantageous for antimicrobial applications. Current work exploits this potential, to examine its time- and concentration-dependent antimicrobial activity, employing E. coli, S. aureus, and C. albicans as a model microbial species. Regarding the presence of polaronic charge carriers in the fibrous polyaniline network, effects of Cu NPs' size and their partially oxidized surfaces (the data were confirmed by HRTEM, FESEM, XRD, Raman and XPS analysis), as well as rapid copper ions release, Cu-PANI nanocomposite showed efficient bactericidal and fungicidal activities at the concentrations ≤1 ppm, within the incubation time of 2 h. Beside the quantitative analysis, the high levels of cellular disruption for all tested microbes were evidenced by atomic force microscopy. Moreover, the minimum inhibitory and bactericidal concentrations of the Cu-PANI nanocomposite were lower than those reported for other nanocomposites. Using such low concentrations is recognized as a good way to avoid its toxicity toward the environment. For this purpose, Cu-PANI nanocomposite is tested for its genotoxicity and influence on the oxidative status of the human cells in vitro.

Ruthenium(II)-N-alkyl phenothiazine complexes as potential anticancer agents.

In recent years, the search for effective anticancer compounds based on transition metal complexes has been the focus of medical investigations. The synergy between the ruthenium(II) and N-alkylphenothiazine counter-ions (chlorpromazine hydrochloride, thioridazine hydrochloride and trifluoperazine dihydrochloride, respectively) through the formation of three different complexes (1-3) was investigated. We explored whether the selected counter-ions and complexes might affect redox homeostasis and genome integrity of normal human blood cells, and induce an inhibition of Na+/K+-ATPase and AChE at pharmacologically relevant doses. Our results have shown that counter-ions and complexes did not affect the activity of Na+/K+-ATPase, while AChE activity was inhibited in a dose-dependent manner. All investigated compounds disturbed the viability and redox homeostasis of lymphocytes. Complexes 1 and 2 displayed potent cytotoxic and prooxidant action while complex 3 behaved as a weaker genotoxic inducer. Still, the tested complexes appeared to be less genotoxic and more cytostatic than the corresponding counter-ions. The effects of selected complexes were also tested in PC12 and U2OS cancer cells with special attention being given to the ability of phenothiazines to affect dopamine D2 receptors. Using the confocal laser scanning microscopy, we observed that all the complexes reduced cell viability. Although all investigated complexes have been bound to the dopamine receptor D2-eGFP, only complex 3 reduced its surface density and increased its lateral mobility in investigated cell lines. Albeit the role of alternative targets for complex 3 cannot be ruled out, its effects should be further examined as potential treatment strategy against cancer cells that overexpress D2.

Modulators of Acetylcholinesterase Activity: From Alzheimer's Disease to Anti-Cancer Drugs.

BACKGROUND: Acetylcholinesterase (AChE) is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in numerous cholinergic pathways in the central and peripheral nervous systems. The enzyme inactivation leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission. Hence, acetylcholinesterase inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs for different neurodegenerative diseases (such as Alzheimer's and Parkinson's) as well as toxins. At the same time, there are increasing evidence that in non-neuronal context, AChE is involved in the regulation of cell proliferation, differentiation, apoptosis and cell-cell interaction. An irregular expression of AChE has been found in different types of tumors, suggesting the involvement of AChE in the regulation of tumor development. Having all this in mind, there is a possibility that some AChE inhibitors could be used as anti-cancer agents. OBJECTIVE: This contribution will discuss a broad range of possible application of different AChE inhibitors as drugs, from well-known anti-Alzheimer's disease drugs to their use in cancer treatment in future. Emphasis will be put on various known AChE inhibitors classes, whose application as drugs could be controversy, as well as on newly investigated natural products, which can also modulate AChE activity. CONCLUSION: It is not clear a patient treated for neurodegenerative condition prone to increased risk for some types of cancer and vice versa. This is necessary to keep in mind during rational drug design process for all therapies, which are based on AChE as a target molecule.

The influence of oxo-bridged binuclear gold(III) complexes on Na/K-ATPase activity: a joint experimental and theoretical approach.

The in vitro effects of oxo-bridged binuclear gold(III) complexes, i.e., [(bipy2Me)2Au2(µ-O)2][PF6]2 (Auoxo6), Au2[(bipydmb-H)2(µ-O)][PF6] (Au2bipyC) and [Au2(phen2Me)2(µ-O)2](PF6)2 (Au2phen) on Na/K-ATPase, purified from the porcine cerebral cortex, were investigated. All three studied gold complexes inhibited the enzyme activity in a concentration-dependent manner achieving IC50 values in the low micromolar range. Kinetic analysis suggested an uncompetitive mode of inhibition for Auoxo6 and Au2bipyC, and a mixed type one for Au2phen. Docking studies indicated that the inhibitory actions of all tested complexes are related to E2-P enzyme conformation binding to ion channel and intracellular part between N and P sub-domain. In addition, Au2phen was able to inhibit the enzyme by interacting with its extracellular part as well. Toxic effects of the gold(III) complexes were evaluated in vitro by following lactate dehydrogenase activity in rat brain synaptosomes and incidence of micronuclei and cytokinesis-block proliferation index in cultivated human lymphocytes. All investigated complexes turned out to induce cytogenetic damage consisting of a significant decrease in cell proliferation and an increase in micronuclei in a dose-dependent manner. On the other hand, lactate dehydrogenase activity, an indicator of membrane integrity/viability, was not affected by Auoxo6 and Au2bipyC, while Au2phen slightly modified its activity.

The impact of concentration and administration time on the radiomodulating properties of undecylprodigiosin in vitro.

Undecylprodigiosin pigment (UPP) is reported to display cytotoxic activity towards various types of tumours. Nevertheless, its efficacy in modifying the cellular response to ionising radiation is still unknown. In this study, the radiomodulating effects of UPP were investigated. The effects of UPP were assessed in vitro by treating cultures of human peripheral blood with UPP and ionising radiation using two treatment regimens, the UPP pre-irradiation treatment and UPP post-irradiation treatment. The activity of UPP was investigated evaluating its effects on the radiation-induced micronuclei formation, cell proliferation, and induction of apoptosis. The redox modulating effects of UPP were examined measuring the catalase activity and the level of malondialdehyde, as a measure of oxidative stress. The results showed that UPP effects on cellular response to ionising radiation depend on its concentration and the timing of its administration. At low concentration, the UPP displayed radioprotective effects in γ-irradiated human lymphocytes while at higher concentrations, it acted as a radiosensitiser enhancing either mitotic catastrophe or apoptosis depending on the treatment regimen. The UPP modified redox processes in cells, particularly when it was employed prior to γ-irradiation. Our data highlight the importance of further research of the potential of UPP to sensitize tumour cells to radiation therapy by inhibiting pathways that lead to treatment resistance.

Biological effects of bacterial pigment undecylprodigiosin on human blood cells treated with atmospheric gas plasma in vitro.

It is known that some bacterial species are more resilient to different kinds of irradiation due to the naturally developed protective mechanisms and compounds such as pigments. On the other hand, reasoned tissue engineering using plasma remains a critical task and requires very precise control of plasma parameters in order to mitigate its potential detrimental effects. Here we isolated a natural protective agent, microbially produced undecylprodigiosin ((5'Z)-4'-methoxy-5'-[(5-undecyl-1H-pyrrol-2-yl)methylene]-1H,5'H-2,2'-bipyrrole), and investigated its effects on human blood cells independently and in combination with plasma. Two approaches were applied; the first, undecylprodigiosin (UP pigment) was added to the blood cultures, which then were exposed to plasma (pre-treatment); and the second- the blood cultures were exposed to plasma and then treated with pigment (post-treatment). The interactions of plasma and UP pigment with blood cells were investigated by conducting a series of biological tests providing the information regarding their genotoxicity, cytotoxicity and redox modulating activities. The exposure of cells to plasma induced oxidative stress as well as certain genotoxic and cytotoxic effects seen as elevated micronuclei incidence, decreased cell proliferation and enhanced apoptosis. In blood cultures treated with UP pigment alone, we found that both cytotoxic and protective effects could be induced depending on the concentration used. The highest UP pigment concentration increased lipid peroxidation and the incidence of micronuclei by more than 70% with maximal suppression of cell proliferation. On the contrary, we found that the lowest UP pigment concentration displayed protective effects. In combined treatments with plasma and UP pigment, we found that UP pigment could provide spatial shielding to plasma exposure. In the pre-treatment approach, the incidence of micronuclei was reduced by 35.52% compared to control while malondialdehyde level decreased by 36% indicating a significant mitigation of membrane damage induced by plasma. These results open perspectives for utilizing UP pigment for protection against overexposures in the field of plasma medicine.

Current experience in testing mitochondrial nutrients in disorders featuring oxidative stress and mitochondrial dysfunction: rational design of chemoprevention trials.

An extensive number of pathologies are associated with mitochondrial dysfunction (MDF) and oxidative stress (OS). Thus, mitochondrial cofactors termed "mitochondrial nutrients" (MN), such as α-lipoic acid (ALA), Coenzyme Q10 (CoQ10), and l-carnitine (CARN) (or its derivatives) have been tested in a number of clinical trials, and this review is focused on the use of MN-based clinical trials. The papers reporting on MN-based clinical trials were retrieved in MedLine up to July 2014, and evaluated for the following endpoints: (a) treated diseases; (b) dosages, number of enrolled patients and duration of treatment; (c) trial success for each MN or MN combinations as reported by authors. The reports satisfying the above endpoints included total numbers of trials and frequencies of randomized, controlled studies, i.e., 81 trials testing ALA, 107 reports testing CoQ10, and 74 reports testing CARN, while only 7 reports were retrieved testing double MN associations, while no report was found testing a triple MN combination. A total of 28 reports tested MN associations with "classical" antioxidants, such as antioxidant nutrients or drugs. Combinations of MN showed better outcomes than individual MN, suggesting forthcoming clinical studies. The criteria in study design and monitoring MN-based clinical trials are discussed.

Inhibition of Na(+)/K(+)-ATPase and cytotoxicity of a few selected gold(III) complexes.

Na(+)/K(+)-ATPase is in charge of maintaining the ionic and osmotic intracellular balance by using ATP as an energy source to drive excess Na(+) ions out of the cell in exchange for K(+) ions. We explored whether three representative cytotoxic gold(III) compounds might interfere with Na(+)/K(+)-ATPase and cause its inhibition at pharmacologically relevant concentrations. The tested complexes were [Au(bipy)(OH)2][PF6] (bipy=2,2'-bipyridine), [Au(py(dmb)-H)(CH3COO)2] (py(dmb)-H=deprotonated 6-(1,1-dimethylbenzyl)-pyridine), and [Au(bipy(dmb)-H)(OH)][PF6] (bipy(dmb)-H=deprotonated 6-(1,1-dimethylbenzyl)-2,2'-bipyridine). We found that all of them caused a pronounced and similar inhibition of Na(+)/K(+)-ATPase activity. Inhibition was found to be non-competitive and reversible. Remarkably, treatment with cysteine resulted in reversal or prevention of Na(+)/K(+)-ATPase inhibition. It is very likely that the described effects may contribute to the overall cytotoxic profile of these gold complexes.

Oxidative stress and mitochondrial dysfunction across broad-ranging pathologies: toward mitochondria-targeted clinical strategies.

Beyond the disorders recognized as mitochondrial diseases, abnormalities in function and/or ultrastructure of mitochondria have been reported in several unrelated pathologies. These encompass ageing, malformations, and a number of genetic or acquired diseases, as diabetes and cardiologic, haematologic, organ-specific (e.g., eye or liver), neurologic and psychiatric, autoimmune, and dermatologic disorders. The mechanistic grounds for mitochondrial dysfunction (MDF) along with the occurrence of oxidative stress (OS) have been investigated within the pathogenesis of individual disorders or in groups of interrelated disorders. We attempt to review broad-ranging pathologies that involve mitochondrial-specific deficiencies or rely on cytosol-derived prooxidant states or on autoimmune-induced mitochondrial damage. The established knowledge in these subjects warrants studies aimed at elucidating several open questions that are highlighted in the present review. The relevance of OS and MDF in different pathologies may establish the grounds for chemoprevention trials aimed at compensating OS/MDF by means of antioxidants and mitochondrial nutrients.

Radiation-induced mitotic catastrophe in FANCD2 primary fibroblasts.

PURPOSE: As the Fanconi anemia (FA) pathway is required for appropriate cell cycle progression through mitosis and the completion of cell division, the aim of the present study was to determine the destiny of FA cells after irradiation in vitro and to elucidate any difference in radiosensitivity between FA and control cells. MATERIALS AND METHODS: Analyses of phosphorylated histone H2AX (γ-H2AX) foci, micronuclei formation and cell cycle analysis were performed in unirradiated (0 min) and irradiated primary FA fibroblasts and in a control group at different post-irradiation times (30 min, 2 h, 5 h and 24 h). RESULTS: The accumulation of γ-H2AX foci in irradiated FA fibroblasts was observed. At 24 h post-irradiation, 57% of FA cells were γ-H2AX foci-positive, significantly higher than in the control (p < 0.01). The cell cycle analysis has shown the transient G2/M arrest in irradiated FA fibroblasts. The portion of cells in the G2/M phase showed initial increase at 30 min post-irradiation and afterwards decreased over time reaching the pretreatment level 24 h after irradiation. Irradiated FA fibroblasts progressed to abnormal mitosis, as is shown by the production of cells with different nuclear morphologies from binucleated to multinucleated surrounded with micronuclei, and also by a high percentage of foci-positive micronuclei. The majority of radiation-induced micronuclei were γ-H2AX foci-positive, indicating that radiation-induced micronuclei contain fragments of damaged chromosomes. In contrast, in the control group, most of the micronuclei were classified as γ-H2AX foci-negative, which indicates that cells with unrepaired damage were blocked before entering mitosis. CONCLUSION: The results clearly indicate that mitotic catastrophe might be an important cell-death mechanism involved in the response of FA fibroblasts to ionizing radiation.

Bone marrow cell transcripts from Fanconi anaemia patients reveal in vivo alterations in mitochondrial, redox and DNA repair pathways.

Fanconi anaemia (FA) is a genetic cancer predisposition disorder associated with cytogenetic instability, bone marrow failure and a pleiotropic cellular phenotype, including low thresholds of responses to oxidative stress, cross-linking agents and selected cytokines. This study was aimed at defining the scope of abnormalities in gene expression using the publicly available FA Transcriptome Consortium (FTC) database (Gene Expression Omnibus, 2009 and publicly available as GSE16334). We evaluated the data set that included transcriptomal analyses on RNA obtained from low-density bone marrow cells (BMC) from 20 patients with FA and 11 healthy volunteers, by seeking to identify changes in expression of over 22,000 genes, including a set of genes involved in: (i) bioenergetic pathways; (ii) antioxidant activities; (iii) response to stress and metal-chelating proteins; (iv) inflammation-related cytokines and (v) DNA repair. Ontological analysis of genes expressed at magnitudes of 1.5-fold or greater demonstrated significant suppression of genes in the categories of (i) energy metabolism; (ii) antioxidant activities; and (iii) stress and chelating proteins. Enhanced expression was found for 16 of 26 genes encoding inflammatory cytokines. A set of 20 of 21 transcripts for DNA repair activities were down-regulated; four of these transcripts related to type II topoisomerase. The data provide evidence for alterations in gene regulation of bioenergetic activities, redox-related activities, stress and metal-chelating proteins, and of some selected DNA repair activities in patients with FA.

Genotoxic assessment of carbon nanotubes.

Carbon nanotubes are unique one-dimensional macromolecules with promising application in biology and medicine. Since their toxicity is still under debate, here we describe an investigation of genotoxic properties of purified single-walled carbon nanotubes (SWCNT), multiwall carbon nanotubes (MWCNT), and amide-functionalized purified SWCNT. We used two different cell systems: cultured human lymphocytes where we employed cytokinesis-block micronucleus test and human fibroblasts where we investigate the induction of DNA double-strand breaks (DSBs) employing H2AX phosphorylation assay.

In vitro effects of some gold complexes on Na(+)/K(+) ATPase activity and cell proliferation.

The in vitro influence of gold(III) complexes, H[AuCl4], [Au(DMSO)2Cl2]Cl and [Au(bipy)Cl2]Cl (bipy = 2,2'-bipyridine), upon commercially available Na(+)/K(+) ATPase activity, purified from porcine brain cortex, was investigated. Additionally, the complexes were tested on human lymphocytes, and incidence of micronuclei and cell proliferation index was determined. Concentration-dependent inhibition of the enzyme for all three compounds was obtained, but with differing potencies. Calculated IC50 from Hill analysis were (in M): 5.75×10(-7), 5.50×10(-6) and 3.98×10(-5), for H[AuCl4], [Au(DMSO)2Cl2]Cl and [Au(bipy)Cl2]Cl, respectively, while Hill coefficient values, n, were above 1 in all cases. This inhibition can be prevented using -SH donating ligands such as L-Cys and glutathione, and these ligands can also cause a recovery of the enzyme activity after the induced inhibition. Kinetic analysis demonstrated that each of the studied gold(III) complexes affects Na(+)/K(+) ATPase reducing maximum enzymatic velocity, Vmax, but not significantly changing the affinity for the substrate (KM value), implying a noncompetitive mode of the interaction. Furthermore, among investigated gold(III) complexes, the [Au(bipy)Cl2]Cl complex exhibits a strong cytotoxic effect on human lymphocytes, which suggests its potential for use in antitumor therapy.

From clinical description, to in vitro and animal studies, and backward to patients: oxidative stress and mitochondrial dysfunction in Fanconi anemia.

Fanconi anemia (FA) is a rare genetic disease associated with deficiencies in DNA repair pathways. A body of literature points to a pro-oxidant state in FA patients, along with evidence for oxidative stress (OS) in the FA phenotype reported by in vitro, molecular, and animal studies. A highlight arises from the detection of mitochondrial dysfunction (MDF) in FA cell lines of complementation groups A, C, D2, and G. As yet lacking, in vivo studies should focus on FA-associated MDF, which may help in the understanding of the mitochondrial basis of OS detected in cells and body fluids from FA patients. Beyond the in vitro and animal databases, the available analytical devices may prompt the direct observation of metabolic and mitochondrial alterations in FA patients. These studies should evaluate a set of MDF-related endpoints, to be related to OS endpoints. The working hypothesis is raised that, parallel to OS, nitrosative stress might be another, so far unexplored, hallmark of the FA phenotype. The expected results may shed light on the FA pathogenesis and might provide grounds for pilot chemoprevention trials using mitochondrial nutrients.

Dysfunctional telomeres in primary cells from Fanconi anemia FANCD2 patients.

BACKGROUND: Fanconi anemia (FA) is characterized by sensitivity to DNA cross-linking agents, mild cellular, and marked clinical radio sensitivity. In this study we investigated telomeric abnormalities of non-immortalized primary cells (lymphocytes and fibroblasts) derived from FA patients of the FA-D2 complementation group, which provides a more accurate physiological assessment than is possible with transformed cells or animal models. RESULTS: We analyzed telomere length, telomere dysfunction-induced foci (TIFs), sister chromatid exchanges (SCE), telomere sister chromatid exchanges (T-SCE), apoptosis and expression of shelterin components TRF1 and TRF2. FANCD2 lymphocytes exhibited multiple types of telomeric abnormalities, including premature telomere shortening, increase in telomeric recombination and aberrant telomeric structures ranging from fragile to long-string extended telomeres. The baseline incidence of SCE in FANCD2 lymphocytes was reduced when compared to control, but in response to diepoxybutane (DEB) the 2-fold higher rate of SCE was observed. In contrast, control lymphocytes showed decreased SCE incidence in response to DEB treatment. FANCD2 fibroblasts revealed a high percentage of TIFs, decreased expression of TRF1 and invariable expression of TRF2. The percentage of TIFs inversely correlated with telomere length, emphasizing that telomere shortening is the major reason for the loss of telomere capping function. Upon irradiation, a significant decrease of TIFs was observed at all recovery times. Surprisingly, a considerable percentage of TIF positive cells disappeared at the same time when incidence of γ-H2AX foci was maximal. Both FANCD2 leucocytes and fibroblasts appeared to die spontaneously at higher rate than control. This trend was more evident upon irradiation; the percentage of leucocytes underwent apoptosis was 2.59- fold higher than that in control, while fibroblasts exhibited a 2- h delay before entering apoptosis. CONCLUSION: The results of our study showed that primary cells originating from FA-D2 patients display shorten telomeres, elevated incidence of T-SCEs and high frequency of TIFs. Disappearance of TIFs in early response to irradiation represent distinctive feature of FANCD2 cells that should be examined further.