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Methionine is one of the most frequently supplemented amino acids in raising of poultry. However, an overdose of methionine can cause hyperhomocysteinemia. Folic acid, taking part in the process of homocysteine remethylation, is a factor affecting the reduction of the concentration of this amino acid. The study was carried out in 2 stages. The experiment of step I was to investigate the effect of methionine and/or folic acid administration in ovo in the early stage of embryogenesis (E4), and the experiment of the second stage - in the late stage of embryogenesis (E17) on the following biochemical parameters of chicken blood: glucose concentration in whole blood and concentration of homocysteine and uric acid in plasma of domestic chickens (Gallus gallus domesticus). Our results confirm that methionine supplementation may increase the concentration of uric acid and homocysteine. Moreover, we demonstrated that folic acid administered during embryogenesis decreased homocysteine concentration, also in groups simultaneously supplemented with methionine, especially in the initial stage of postnatal life of the bird., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

We present a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of sodium lauryl sulfate and sodium laureth sulfate homologues in the range of alkyl chain length C 12 -C 16 with 0-5 ethoxy groups. The method is based on scanning the precursor ions fragmenting to m/z 80 and 97 (Precursor Ion Scanning mode), which makes it specific for species with easily cleavable sulfate groups. By monitoring fragmentation of thus discovered quasi-molecular ions we were able to unequivocally identify all sulfate species present in complex mixtures of alkyl and alkyl-ether sulfates with molecular weight ranging from 200 to 600 m/z. Because of the intrinsic sulfate-sensitivity, the presented method can be also applied to non-sodium salts of alkyl- and alkyl-ether sulfates (e.g. ammonium, mono- or triethanolamine, etc.), which are often used by cosmetic manufacturers to justify the misleading SLS- and SLES-free claims (where SLS and SLES refer to sodium lauryl sulfate and sodium laureth sulfate, respectively). The use of reversed phase liquid chromatography (RPLC) column with C4 instead of C18 shortened significantly the overall analysis time and allowed us to use a semiquantitative method (based on single standard for Quantitative Analysis of Multi-component System, QAMS) to determine several SLS and SLES homologues in one run with the limit of quantification (LOQ) = 0.4 µg/mL and of detection (LOD) in the range 0.12-0.97 µg/mL. The method was successfully applied to 17 commercially available cosmetic/household products allowing verification of their manufacturers' declarations., 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. Published by Elsevier B.V.)

Vitamin D has a potential role in protecting against cardiovascular disease (CVD). Serum 25-hydroxyvitamin D (25D) is the most widely used indicator of vitamin D status in the human body. 25D is estimated as total of 25-hydroxyvitamin D2 (25D2) and 25-hydroxyvitamin D3 (25D3). However, the presence of 3-epi-25-hydroxyvitamin D3 (3epi25D3) can affect 25D measurement. In this research a novel validated UPLC-MS/MS technique was developed to measure three vitamin D metabolites, 25D2, 25D3 and 3epi25D3 in human plasma. A liquid-liquid extraction using hexane was applied for isolation of the analytes from the samples. A chromatographic separation was achieved in a Kinetex F5 analytical column with isocratic elution (water and methanol with 0.1% methanoic acid, 20:80 v/v). Mass spectrometry detection of the metabolites was performed in a triple-quadruple tandem mass spectrometer under positive ion mode. Concentrations of the analytes were estimated in plasma samples of 54 patients. Validation parameters of the UPLC-MS/MS method, including linearity, precision, accuracy, and stability, fulfilled the requirements for bioanalytical assays. The deficient concentration of 25D (<20 ng/mL) was stated in over 60% of patients. 3epi25D3 was present in 78% of samples and its relative amount ranged from 0 to 54.1% of 25D concentration. The analysis of 25D2, 25D3 and 3epi25D3 by the validated UPLC-MS/MS method in plasma of patients with CVD permitted the classification of the patients with insufficient levels of 25D. 3epi25D3 might be relevant in the classification of vitamin D status., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The spatial network structure of Dergall is based on substances nontoxic to humans and the environment which, when applied on solid surfaces, creates a coating that reduces bacterial cell adhesion. The bacteriostatic properties of siloxanes are based on a purely physical action mechanism which excludes development of drug-resistant microorganisms. The aims of the present study were to 1) evaluate a Dergall layer formed on the eggshell surface regarding the potential harmful effects on the chick embryo; 2) evaluate antimicrobial activity and estimate the prolongation time of Dergall's potential antimicrobial activity. Dergall at a concentration of 0.6% formed a layer on the eggshell surface. In vitro testing of the potential harmful effects of Dergall by means of a hen embryo test of the chorioallantoic membrane showed no irritation reaction at a concentration of 3% and lower. The hatchability of the groups sprayed with a Dergall water solution with a concentration of 0 to 5% was 89.1 to 93.8% for fertilized eggs (P > 0.05) but decreased to 63.7% (P < 0.05) in the group sprayed with a 6% concentration of the solution. This phenomenon was caused by embryo mortality in the first week of incubation. At the concentration of 0.6%, Dergall exhibited strong antibacterial properties against bacteria such as Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, Shigella flexneri, and Salmonella typhimurium. For Streptococcus pyogenes, the highest antibacterial activity of Dergall was reported in the concentrations of 100 and 50%. For Pseudomonas aeruginosa, no antibacterial activity of Dergall was generally observed, but in vivo testing showed a strong decrease of all gram-negative bacteria growth. Moreover, a prolonged antimicrobial effect lasting until 3 D after disinfection was observed, which makes Dergall a safe and efficient disinfectant., (Copyright © 2019. Published by Elsevier Inc.)

Chronic kidney disease (CKD) is a pathological condition associated with renal osteodystrophy for which there are limited treatment options. Gut-derived serotonin (GDS) is one of the key signaling factors controlling the osteoblast proliferation. Previously, we shown that inhibition of GDS synthesis by LP533401 improved bone mineral status of rats with 5/6 nephrectomy-induced CKD model. Here, we investigated whether the use of LP533401 can modify GDS-dependent molecular pathway involved in osteoblast formation and bone mineralization in CKD rats. The 8-weeks of pharmacological manipulation after a complete CKD development reduced GDS and lead to the advantage of endogenous vitamin D [25(OH)D] over serotonin and parathyroid hormone (PTH) in rats treated with LP533401. The imbalance between GDS - 25(OH)D - PTH resulted in the intensified expression of cAMP- responsive element-binding protein (Creb), whereas the expression of myelocytomatosis oncogene (c-Myc) was simultaneously reduced. This lead to disruption of Foxo1- activating transcription factor 4 (Atf4) complex, and decrease in the expression of the major osteogenic markers. The weakening of excessive osteoblastogenesis was associated with better bone mineral status in all rats with CKD, and especially in LP533401-treated animals. In conclusion, the inhibition of GDS synthesis resulted in the mitigation of osteoblastogenesis observed in CKD, which translated into improvement of bone mineral status. This study provides key mechanistic insights into how modification of GDS-dependent molecular pathway affects bone mineral status in CKD and lays the groundwork for translating the role of functional serotonin signaling in the origin of impaired bone mineral status in patients with CKD., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

A micro-electrochemical reaction cell was coupled to an electrospray mass spectrometer in order to track redox transformations for two representative medicinal gold compounds - i.e. [(2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranosato-S)(triethylphosphine)gold(I)] and [Au(bipy dmb -H)(OH)][PF 6 ] (where bipy dmb -H is deprotonated 6-(1,1-dimethylbenzyl)-2,2'-bipyridine), known as Auranofin and Aubipy c respectively - in parallel to square wave voltammetry (SWV) measurements. Irreversible oxidation of thio-glucose tetraacetate was the dominant reaction for the gold(I) compound Auranofin; oxidation was accompanied by hydrolysis leading to progressive deacetylation. Two main active forms were identified for this prodrug: the triethylphosphinegold(I) cation and a gold(I) thioglucose species, with a variable number of acetyl groups. For the gold(III) complex Aubipy c irreversible reduction of the gold(III) center was highlighted, accompanied by a ligand exchange process. The free gold(I) ion is proposed to be the final species that subsequently binds transport proteins in the bloodstream. Molecule specific mass spectrometry determinations provide complementary data to square wave voltammetry helping to understand the nature of the electrochemical conversions of complex or unstable compounds. Finally, it was possible to establish that oxidizing conditions during drug preparation and administration should be avoided in the case of Auranofin; conversely, reduction conditions typical for the blood or the cytosol environment are suitable to obtain the active gold(I) species from the gold(III) complex Aubipy c ., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Platinum (Pt) occurs at very low levels in parent rock and soils in unpolluted areas, however concentrations of this element in urban areas is steadily increasing. At the levels recorded in urban environments, Pt is not yet phytotoxic, but it already poses a threat to human health, particularly when present in airborne particulate matter. In this study an attempt was made to evaluate Pt(II) uptake, distribution and toxicity in Arabidopsis thaliana L. plants. Arabidopsis thaliana plants were hydroponically grown with increasing Pt(II) concentrations in the range of 0.025-100µM. Pt(II) was taken up by the roots and translocated to the rosette. At lower Pt(II) concentrations (≤ 2.5μM) hormesis was recorded, plant growth was stimulated, the efficiency of the photosynthetic apparatus improved and biomass accumulation increased. Higher Pt(II) concentrations were phytotoxic, causing growth inhibition, impairment of the photosynthetic apparatus, membrane injuries and a reduction in biomass accumulation. Exposure of A. thaliana to Pt(II) also resulted in an increased content of phytochelatins throughout the plant and glutathione in the rosette. Uptake and translocation of Pt(II) to harvestable organs of A. thaliana suggests that species of higher biomass accumulation from the Brassicaceae family can probably be used for the phytoextraction of Pt-polluted sites., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The routine use of postoperative pleural cavity drainage after the Nuss procedure is not widely accepted, and its limited use depends on experience. This study analyzed the influence of pleural drainage in the surgical treatment of patients with pectus excavatum on the prevention of pneumothorax and the efficacy of using drainage after a corrective operation., Methods: From November 2013 to May 2015, 103 consecutive patients with pectus excavatum, aged 11 to 39 years, underwent surgical treatment by the Nuss procedure. Patients were prospectively randomized into two groups. In 58 patients, a 28F chest tube was routinely introduced into the right pleural cavity during procedure for 2 consecutive days (group I). In the remaining 45 patients, the drain was not inserted (group II)., Results: No statistically significant differences were found between the study groups, including sex, age, body mass index, or clinical subjective and objective factors in the preoperative evaluation. Group II manifested more complications in the early postoperative period; however, this was not statistically significant (group I vs group II; p = 0.0725). Pneumothorax requiring additional chest tube placement was statistically significant (group I vs group II; p = 0.0230). Other complications were also more frequent among patients from group II, although this did not reach statistical significance. Follow up was 22.9 ± 6.4 months., Conclusions: Routine drainage of the pleural cavity during the Nuss procedure significantly reduces the incidence of postoperative pneumothorax and should be considered as a routine procedure., (Copyright © 2017 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Vascular calcification (VC) is highly prevalent in patients with chronic kidney disease (CKD) and is strongly associated with cardiovascular mortality and morbidity. Accumulating evidence over the past decade has challenged the hypothesis of close interaction between bone and VC what raises the possibility of a common underlying pathophysiological mechanism. Lately, bone regulatory proteins such as: osteoprotegerin (OPG) and Receptor Activator for Nuclear Factor κB Ligand (RANKL) has attracted attention of researchers as a possible key mediators of bone-vascular calcification imbalance. The literature search was carried out using the MEDLINE/PubMed database and a combination of keywords and MeSH terms, and only papers published since January 2005 to July 2016 were selected. The search resulted in 562 potential articles. After selection according to the eligibility criteria, 107 studies fulfilled were included (102 full texts and 5 was case reports). OPG and RANKL plays essential role in the regulation of bone metabolism and may be regarded as a possible link between VC, bone and mineral metabolism in CKD patients. Further studies are required to determine the diagnostic significance of these proteins in evaluation of progression and severity of VC process in CKD patients. Finally, the efficacy and safety, especially in regard to VC, of anti-RANKL therapy in CKD patients requires well-designed prospective, randomized trials., (Copyright © 2016 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.)

Background: The aim of this study was a comparison of early and late results in surgical treatment of funnel chest using the Nuss method in patients in various age groups to find the optimal age to perform the corrective procedure., Methods: Six hundred eighty patients operated on from June 2002 to October 2012 were included in the retrospective analysis. Patients were divided into 3 different age groups: group A = 156 patients from 7 to 14 years, group B = 328 patients aged 15 to 20 years, and group C = 196 patients older than 20 years of age. The mean follow-up was 33 months., Results: Early non-life-threatening complications developed in 238 (35.0%) patients and frequency increased with age (group A, 24.3%; group B, 37.8%; group C, 38.8%; p = 0.0063). Good and very good corrective effects were achieved in 97.7 % of the entire patient population. Recurrence of the deformity was observed more often in younger patients (group A, 3.2 %) than in the other patients (group B, 1.2%; group C, 1.5%), although the difference between the studied groups was not significant (p = 0.3251)., Conclusions: Good cosmetic results obtained with the use of the Nuss operation were not related to the age of the patients. The high incidence of minor complications in older patients seems to be an acceptable cost of a good cosmetic outcome and stable correction. Surgical morbidity is lowest in younger patients; however, the frequency of the recurrence of deformation is higher than in other groups., (Copyright © 2016 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Atomic Force Microscope (AFM) is a widely used tool in force spectroscopy studies. Presently, this instrument is accessible from numerous vendors, albeit commercial solutions are expensive and almost always hardware and software closed. Approaches for open setups were published, as with modern low cost and readily available piezoelectric actuators, data acquisition interfaces and optoelectronic components building such force spectroscopy AFM is relatively easy. However, suitable software to control such laboratory made instrument was not released. Developing it in the lab requires significant time and effort. Our Nanopuller software described in this paper is intended to eliminate this obstacle. With only minimum adjustments this program can be used to control and acquire data with any suitable National Instruments universal digital/analog interface and piezoelectric actuator analog controller, giving significant freedom and flexibility in designing force spectroscopy experiment. Since the full code, written in a graphical LabVIEW environment is available, our Nanopuller can be easily customized. In this paper we describe the program and test its performance in controlling different setups. Successful and accurate force curve acquisition for standard samples (single molecules of I27O reference titin polyprotein and DNA as well as red blood cells) is shown., (Copyright © 2016 Elsevier B.V. All rights reserved.)

An Escherichia coli hemH mutant accumulates protoporphyrin IX, causing photosensitivity of cells to visible light. Here, we have shown that intracellular free iron in hemH mutants is double that observed in hemH(+) strain. The aim of this study was to recognize the influence of this increased free iron concentration on AlkB-directed repair of alkylated DNA by analyzing survival and argE3 → Arg(+) reversion induction after λ>320 nm light irradiation and MMS-treatment in E. coli AB1157 hemH and alkB mutants. E.coli AlkB dioxygenase constitutes a direct single-protein repair system using non-hem Fe(II) and cofactors 2-oxoglutarate (2OG) and oxygen (O2) to initiate oxidative dealkylation of DNA/RNA bases. We have established that the frequency of MMS-induced Arg(+) revertants in AB1157 alkB(+)hemH(-)/pMW1 strain was 40 and 26% reduced comparing to the alkB(+)hemH(-) and alkB(+)hemH(+)/pMW1, respectively. It is noteworthy that the effect was observed only when bacteria were irradiated with λ>320 nm light prior MMS-treatment. This finding indicates efficient repair of alkylated DNA in photosensibilized cells in the presence of higher free iron pool and AlkB concentrations. Interestingly, a 31% decrease in the level of Arg(+) reversion was observed in irradiated and MMS-treated hemH(-)alkB(-) cells comparing to the hemH(+)alkB(-) strain. Also, the level of Arg(+) revertants in the irradiated and MMS treated hemH(-) alkB(-) mutant was significantly lower (by 34%) in comparison to the same strain but MMS-treated only. These indicate AlkB-independent repair involving Fe ions and reactive oxygen species. According to our hypothesis it may be caused by non-enzymatic dealkylation of alkylated dNTPs in E. coli cells. In in vitro studies, the absence of AlkB protein in the presence of iron ions allowed etheno(ϵ) dATP and ϵdCTP to spontaneously convert to dAMP and dCMP, respectively. Thus, hemH(-) intra-cellular conditions may favor Fe-dependent dealkylation of modified dNTPs., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Electrospray ionization mass spectrometry (ESI-MS) techniques have been used to characterize the speciation of a Ru(III) anticancer drug, indazolium trans-[tetrachloridobis(1H-indazole) ruthenate(III)], upon its binding to transferrin and the impact of cellular reducing components on drug-transferrin adducts. Using time-of-flight ESI-MS, the polymorphism of apo- (iron-free) and holo-form (iron-saturated) of the protein was confirmed. While the ruthenium moieties bound to each of five isoforms under simulated extracellular conditions are essentially identical in numbers for apo- and holo-transferrin, distinct differences were found in the composition of Ru(III) species attached to either of the protein forms, which are dominated by differently coordinated aquated complexes. On the other hand, at least one of the RuN bonds in metal-organic framework remains intact even after prolonged interaction with the protein. Triple quadrupole tandem ESI-MS measurements demonstrated that the ruthenium species released from drug adducts with holo-transferrin in simulated cancer cytosol are underwent strong ligand exchange (as compared to the protein-bound forms) but most strikingly, they contain the metal center in the reduced Ru(II) state. In vitro probing the extra- and intracellular interactions of promising Ru(III) drug candidate performed by ESI-MS is thought to shed light on the transportation to tumor cells by transferrin and on the activation to more reactive species by the reducing environment of solid tumors., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Mutation of the heat shock gene, htpG, causes severe defects of several cellular functions in Escherichia coli. A null htpG mutant constructed by gene replacement was impaired in the biosynthesis and secretion of several enzymes, and in biofilm formation and proteolysis. A significant decrease in the activity of β-lactamase in the ΔhtpG mutant was observed at 42°C. The alkaline phosphatase activity in sonicates of cells propagated at this raised temperature was lower in the ΔhtpG mutant than in the wild-type strain. The ability of the ΔhtpG mutant to degrade abnormal proteins was also impaired compared with the wild-type, but was increased at 42°C. Assays based on bioluminescence and crystal violet staining demonstrated that biofilm formation was diminished in the ΔhtpG mutant at the elevated temperature. All these defects can be complemented upon introducing htpG wild allele., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Aims: Dyslipidemia and oxidative stress are commonly present in patients during maintenance dialysis treatment. However, the significance of oxidized LDL (oxLDL) as a marker of oxidative stress in uremia is still unresolved. The aim of this study was to establish the role of oxLDL and oxLDL/LDL ratio as markers of lipoprotein abnormalities and oxidative stress in the dialyzed patients., Main Methods: Plasma oxLDL level was measured by ELISA, and oxLDL/LDL ratio was calculated in 106 dialyzed patients and 20 controls. The linkages between oxLDL, oxLDL/LDL ratio and lipid profile and oxidative stress markers malondialdehyde (MDA) and Cu/Zn superoxide dismutase (Cu/Zn SOD) levels were also analyzed., Key Findings: OxLDL levels and oxLDL/LDL ratio were similar in hemodialyzed patients and controls, whereas these parameters were lower in peritoneally dialyzed patients when compared to healthy individuals. In contrast, both MDA and Cu/Zn SOD levels were significantly higher in uremics than in controls. oxLDL and oxLDL/LDL ratio positively correlated with lipid profile (except of HDL), whereas there were no positive associations between these parameters and both MDA and Cu/Zn SOD. Multiple regression analysis confirmed that increased oxLDL/HDL and TC/HDL ratios and total cholesterol levels are the parameters which independently predicted oxLDL in dialyzed patients. In the case of oxLDL/LDL ratio, the independent variables were oxLDL/HDL ratio, total cholesterol and HDL levels., Significance: oxLDL levels and oxLDL/LDL ratio seem to be the markers of lipoprotein abnormalities rather than the markers of oxidative stress in the population of dialyzed patients., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The mode of action of metal-based anticancer drugs, including their accumulation in blood, transport, delivery to cancer cell, and cell processing (together with release of an active form and possibly targeting) is largely dependent on protein binding. Among analytical methods capable of providing a better understanding of metallodrug-protein interactions, capillary electrophoresis (CE) with inductively coupled plasma mass spectrometry (ICP-MS) detection is arguably a premier technique. Since its advent to the area of metallodrug proteomics in 2004, the benefits of CE-ICP-MS became evident, stimulating further research and methodological developments. This hyphenated technique's merits comprise an ability to separate rapidly and efficiently the parent drug and protein-bound drug form(s), with no alteration of original speciation in the sample, to identify the metal-containing species due to specific ICP-MS response, to measure the binding parameters (e.g. rate and equilibrium constants), and finally to quantify the metal-protein adducts in real-world samples. This review is aimed on offering the reader a summary of applications of CE-ICP-MS to various metallodrug-protein systems, with a focus on experimental strategies in use for the assessment of binding reactivity and affinity, monitoring in vitro cellular transformations and serum binding profiles, and ex vivo metallodrug-proteomic analysis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Disturbances in hemostasis and abnormal angiogenesis are components in the plaque growth and destabilization. The role of the vascular endothelial growth factor (VEGF) in the perturbation of hemostasis in chronic kidney disease (CKD) is still unknown. In this preliminary study, we investigate the relation between VEGF and the parameters of coagulation: tissue factor (TF), its inhibitor (TFPI), and fibrinolytic system: urokinase-type plasminogen activator (uPA), its soluble receptor (suPAR), tissue-type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), plasmin/antiplasmin complexes (PAP) in the patients with mild-to-moderate, and severe CKD and healthy controls. All indices (except TFPI) were raised in CKD patients, particularly in those with severe CKD, compared with controls. The strong positive correlations were between VEGF and some parameters, both coagulation (TF, TFPI, TF/TFPI ratio) and fibrinolytic system (uPA, suPAR, PAP). The relationships were also between the individual hemostatic parameters. In multiple regression analysis, VEGF and kidney dysfunction markers (urea and creatinine levels) were independently associated with uPA, and VEGF was independently associated with suPAR levels. Moreover, PAP was independently associated with age and suPAR. This study represents the first to investigate the relation between VEGF and the activation both coagulation and fibrinolysis in CKD patients. VEGF and the parameters of hemostatic system activation were higher in the CKD group than in the controls with a significant correlation between them. VEGF was independently associated with uPA/suPAR system, whereas suPAR was independently related to PAP levels, suggesting a new link between abnormal angiogenesis and hyperfibrinolysis in this population., (Copyright © 2012 Mosby, Inc. All rights reserved.)

Objective: To investigate the role of autoantibodies against oxidized LDL (oxLDL-Ab) in clearing of oxLDL from circulation in dialyzed uremic patients, as well as the significance of this process in the carotid atherosclerosis and cardiovascular complications., Methods: oxLDL, oxLDL-Ab and intima-media thickness (IMT) were measured in 132 uremic patients on hemodialysis (HD), peritoneal dialysis (PD) or conservative treatment both with and without cardiovascular disease (CVD) and in 29 healthy controls. The ratio of oxLDL to oxLDL-Ab (oxLDL/oxLDL-Ab), reflecting the balance between in vivo oxidative LDL modification and the clearance of oxLDL from circulation was also calculated., Results: oxLDL were lower, whereas oxLDL-Ab were higher in patients without CVD compared to those with CVD. oxLDL/oxLDL-Ab ratio was also significantly lower in dialyzed subjects without CVD compared to their counterparts with CVD and healthy controls. IMT values were significantly higher in uremic patients compared with controls, and patients with CVD had particularly elevated IMT compared with those without CVD. The inverse correlation was between IMT and oxLDL-Ab, whereas the IMT was strongly and positively associated with oxLDL/oxLDL-Ab ratio. There was the weak inverse relationship between oxLDL and oxLDL-Ab levels in dialyzed patients. Multiple stepwise regression analysis identified oxLDL/oxLDL-Ab ratio as one of the predictors of IMT in the whole dialyzed group., Conclusions: oxLDL/oxLDL-Ab ratio could be a new biomarker significantly and independently associated with carotid atherosclerosis in dialyzed patients. A titer of oxLDL-Ab and oxLDL/oxLDL-Ab ratio seem to play an important role for distinguishing patients with and without cardiovascular complications., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

This report presents an unusual case of late, aortic hemorrhage that occurred during the removal of the stabilizing bar 3 years after a Nuss operation. The primary reason for this complication was a rotation of the sternum bar, which caused chronic damage to the aorta and development of an aortomediastinal fistula. Cardiopulmonary bypass and implantation of an aortic prosthesis were required for successful treatment of this complication., (Copyright © 2011 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: Disturbances of the fibrinolytic system and kynurenine (KYN) pathway of tryptophan (TRP) metabolism have been postulated as important factors in the pathogenesis of cardiovascular disease (CVD). However, no data are yet available on the associations between these 2 systems in relation to CVD prevalence in patients with chronic renal failure (CRF)., Methods: We evaluated TRP and its metabolites, KYN, 3-hydroxykynurenine, and quinolinic acid (QA), and their relations with the parameters of the fibrinolytic system, such as tissue-type plasminogen activators and urokinase-type (uPA) plasminogen activators, plasmin-antiplasmin (PAP) complexes, plasminogen activator inhibitor-1, and Cu/Zn superoxide dismutase (Cu/Zn SOD) levels as the marker of oxidative stress in a population of 50 patients with CRF and 20 healthy controls. In addition, the relations of these parameters to CVD prevalence in patients with CRF were also determined., Results: Compared with controls, the CRF group had significantly increased KYNs, PAP, uPA, soluble urokinase PA receptor (suPAR), plasminogen activator inhibitor-1 (all P < 0.0001), and tissue-type plasminogen activators (P < 0.01) concentrations. TRP levels were significantly lower (P < 0.0001), whereas Cu/Zn SOD levels did not differ significantly between patients with CRF and controls. An accelerated PAP formation was positively correlated with age, inflammatory state, creatinine, uPA/suPAR system, 3-hydroxykynurenine, QA, Cu/Zn SOD, and CVD prevalence, whereas it was inversely associated with TRP levels and estimated glomerular filtration rate. Multivariable analysis confirmed that QA, TRP, suPAR, and Cu/Zn SOD levels were the independent variables significantly associated with the hyperfibrinolytic state in patients with CRF., Conclusions: This cross-sectional study has demonstrated that hyperfibrinolysis was associated with uPA/suPAR system, KYNs, oxidative status, and CVD prevalence in patients with CRF.

Purpose: The kynurenine pathway (KP) is a major route of tryptophan metabolism. Several metabolites of this pathway are proposed to be involved in the pathogenesis of Alzheimer's disease. The aim of this study was to evaluate peripheral KP in patients with Alzheimer type dementia and a detailed analysis of correlation between kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), anthranilic acid (AA), quinolinic acid (QUIN) and degree of neuropsychological changes in AD., Material/methods: The plasma concentration of tryptophan and its products degradation by kynurenine pathway were analyzed in 34 patients suffering from Alzheimer type dementia and 18 controls in similar age using high-performance liquid chromatography technique., Results: In demented patients we found lower tryptophan and KYNA concentrations. There was a non-significant increase of KYN, 3-HK and AA levels, and a Marked increase of QUIN in Alzheimer's disease group. We observed positive correlations between cognitive function tests and plasma KYNA levels, and inversely correlations between these tests and QUIN levels in Alzheimer type dementia., Conclusions: Increased TRP degradation and simultaneous altered kynurenines levels were found in plasma of AD patients. It proves activation of peripheral kynurenine pathway in this type of dementia. The alterations of two main KYN metabolites: KYNA and QUIN seem to be associated with the impairment of the cognitive function in AD patients. This appears to offer Novel therapeutic opportunities, with the development of new compounds as a promising perspective for brain neuroprotection.

Purpose: The endothelium dysfunction is an important component of atherosclertic cardiovascular disease. It has been also suggested that kynurenine pathway activation may be involved in the pathogenesis of this disease., Material/methods: This is a cross-sectional study in chronic kidney disease (CKD) patients (n=106; 60 Males). The plasma markers of endothelial dysfunction and kynurenine (KYN), 3-hydroxykynurenine (3-HKYN), kynurenic acid (KYNA), anthranilic acid (AA) and quinolinic acid (QA) were measured in relation to an early indicator of the systemic atherosclerosis - intima-media thickness (IMT)., Results: Kynurenines, von Willebrand factor (vWF), thrombomodulin (TM), soluble adhesion molecules (sICAM-1, sVCAM-1) and IMT in each uraemic group were significantly higher than in healthy people. In contrast, no significant differences in sE-selectin and sP-selectin concentrations were observed between CKD patients and controls. Kynurenines were positively associated with vWF, TM, sICAM-1 and sVCAM-1, whereas sP-selectin was inversely associated with the most of kynurenines. IMT was positively correlated both with kynurenines: KYN, 3-HKYN, QA as well as with endothelial markers: TM, vWF, sICAM-1 and sVCAM-1 (all p<0.01). Finally, multiple regression analysis identified age, vWF, sVCAM-1 and QA levels as the independent variables significantly associated with increased IMT in this population (adjusted r² = 0.51)., Conclusions: This study suggests a relationship between kynurenine pathway activation, endothelial dysfunction and the progression of atherosclerosis in CKD patients. It opens a new idea that the inhibition of kynurenine pathway may provide an effective strategy to slow down endothelial dysfunction and thereby the prevalence of atherosclerosis in this population.

Background: Cellular adhesion molecules and oxidative stress play a role in the pathogenesis of atherosclerosis in patients with chronic kidney disease (CKD). Recently, it has been postulated that the kynurenine (KYN) pathway could be involved in the pathogenesis of atherosclerosis., Methods: We evaluated the KYN, kynurenic acid (KYNA), anthranilic acid (AA), and their relations with cellular adhesion molecules: soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular adhesion molecule-1 (sVCAM-1), sE-selectin, sP-selectin, and Cu/Zn superoxide dismutase (Cu/Zn SOD) levels as the markers of oxidative stress in the population of 132 patients with CKD and 28 healthy controls., Results: Compared with the controls, 2 groups of dialyzed patients had significantly increased KYN (both P < 0.01), KYNA, AA, sICAM-1, sVCAM-1, and Cu/Zn SOD levels (all P < 0.001, respectively). KYN, AA, sICAM-1, and sVCAM-1 concentrations were significantly higher in undialyzed patients with CKD compared with healthy subjects (P < 0.001, P < 0.01, and both P < 0.05, respectively). sICAM-1 and sVCAM-1 were positively associated with KYN (P < 0.0001 and P < 0.01), KYNA (P < 0.05 and P < 0.0001), AA (P < 0.01 and P < 0.0001), and with Cu/Zn SOD (both P < 0.0001, respectively) in the whole CKD group. The positive relationship existed between sICAM-1, sVCAM-1 and age, high-sensitivity C-reactive protein, creatinine, and the duration of dialysis therapy. Multivariable analysis showed that KYN was a strong independent correlate of sICAM-1, whereas Cu/Zn SOD and platelets independently and significantly predicted sVCAM-1 in patients with CKD., Conclusions: This study demonstrated that KYN is independently and significantly associated with elevated sICAM-1, whereas oxidative status and platelets independently and significantly predicted increased sVCAM-1 levels in patients with CKD.

Increased oxidative stress (SOX), inflammation and accelerated atherosclerosis have been reported in end-stage renal disease (ESRD), but their associations with kynurenine pathway activation remain unknown. We determined the plasma concentrations of kynurenine (KYN), kynurenic acid (KYNA) and quinolinic acid (QA); three distinct SOX markers: Cu/Zn superoxide dismutase (Cu/Zn SOD), total peroxide and malondialdehyde (MDA), high sensitivity C-reactive protein (hs CRP) as a indicator of inflammation, and intima-media thickness (IMT)--an early reflection of the systemic atherosclerosis in the population of 124 patients with ESRD. In uraemia, the concentrations of KYN, KYNA and QA were increased by 37-105%, by 84-428%, and by 394-1018% of the control values; respectively. These changes were accompanied by significant increase in kyna/kyn and qa/kyn ratios, reflecting increased activity of kynurenine pathway enzymes. KYN, QA and qa/kyn ratio were positively associated with inflammation, SOX markers, and IMT values in uraemics. Moreover, multiple stepwise regression analysis identified age, presence of diabetes mellitus, QA and qa/kyn ratio as the independent variables significantly associated with increased IMT in this population. In conclusion, the results of the present study suggest a relationship between kynurenine pathway activation and increased oxidative stress, inflammation and the progression of atherosclerosis in end-stage renal disease patients.

Increased oxidative stress (SOX), inflammation and prevalence of cardiovascular disease (CVD) have been reported in end-stage renal disease (ESRD), but their associations with kynurenine (KYN) pathway activation remain unknown. We determined the plasma concentrations of tryptophan (TRP), KYN, 3-hydroxykynurenine (3-HKYN); two distinct SOX markers: Cu/Zn superoxide dismutase (Cu/Zn SOD) and total peroxide; and high sensitivity C-reactive protein (hs CRP) as a indicator of inflammation in 146 ESRD patients and healthy controls. Analysis of TRP degradation through the KYN pathway demonstrated that in uremia the concentrations of this aminoacid were decreased by 40-60% in comparison with controls. In contrast, the plasma levels of KYN and 3-HKYN in ESRD patients were increased by 32-96% and 184-306%, respectively. These changes were accompanied by significant increase in the kyn/trp ratios by 140-240%, and 3-hkyn/kyn ratios by 40-154% in uremics compared to controls. ESRD patients showed a significant increase in Cu/Zn SOD, total peroxide and hs CRP levels between controls and all patients group. KYN and 3-HKYN were positively associated with inflammation and SOX markers in uremics. Logistic regression analysis showed that age, gender, presence of DM (all p<0.001), elevated hs CRP (p<0.01) and 3-HKYN levels (p<0.05) were independently associated with the presence of CVD in this population. These results suggest a relationship between KYN pathway activation and increased SOX, inflammation and CVD prevalence in ESRD patients.

Purpose: Anticoagulant tissue factor pathway inhibitor (TFPI) is released from its endothelial stores by heparin, which may lead to its untoward depletion. We investigated the effects of sulodexide--a commercially available mixture of heparan and dermatan sulfate, on plasma TFPI release and depletion., Material and Methods: An open-label pilot trial of intravenous and/or oral sulodexide effects on plasma immunoreactive total TFPI antigen level was performed in 11 healthy men. The drug was initially administered i.v. at a single dose of 120 mg, thenorally for 12 days (50 mg b.i.d), and again by i.v route after 2 weeks., Results: Sulodexide injections induced marked increases in plasma TFPI; they were more pronounced on day 14 than on study initiation (3-fold vs. 2-fold after 10 min) and still evident after 120 min. TFPI levels did not change when measured at 120 min after oral sulodexide administration. The percentage increment in plasma TFPI after 10 min from initial sulodexide injection inversely correlated with baseline TFPI levels (r = - 0.664, P = 0.026). On day 14, the association became strong (r = - 0.970, P < 0.0001) and evident also after 120 min (r = - 0.810, P < 0.002). Baseline TFPI levels decreased over the trial; on day 14 they were lower by 34% than on study initiation (P = 0.001)., Conclusions: TFPI release by i.v. sulodexide and its depletion during oral administration of this heparinoid compound constitute novel and likely important hemostatic effects of the drug.

Varying the counter-ion is a highly supportive practice in tackling the problem of poor water-solubility of metal complexes of pharmaceutical importance. As a matter of fact, the relevant structural modification may alter the metabolic pathways and possibly the mode of action of a drug. To prove that this does not take place for one of the lead anticancer metal-based developmental compounds, indazolium trans-[RuCl(4)(1H-indazole)(2)] (KP1019), its reactivity toward human serum proteins was assessed under simulated physiological conditions and compared to that of a much more soluble analogue, sodium trans-[RuCl(4)(1H-indazole)(2)] (KP1339). For such kinetic assaying, capillary electrophoresis (CE) interfaced online with inductively coupled plasma mass spectrometry (ICP-MS) to specifically monitor changes in the metal speciation following the formation of ruthenium-protein adducts was applied. The rate constants of interaction with albumin and transferrin were determined at pharmacologically fitting drug-to-protein ratios as on average 0.0319+/-0.0021 min(-1) and 0.0931+/-0.0019 min(-1) (KP1019) and 0.0316+/-0.0018 min(-1) and 0.0935+/-0.0053 min(-1) (KP1339), respectively. The results of this brief study showed that changing from organic to inorganic counter-ion at the stage of formulation could commonly be recommended for improving ruthenium-based drug solubility and bioavailability.

Purpose: Visfatin and apelin are novel adipocytokines that have recently generated much interest. The aim of the study was to assess visfatin and apelin in correlation with markers of endothelial cell injury and inflammation in 22 patients with chronic kidney disease-CKD and 22 age- and sex-matched healthy volunteers., Methods: We assessed visfatin, apelin, markers of coagulation: TAT (thrombin-antithrombin complexes), prothrombin fragments 1+2; fibrinolysis: tPA (tissue plasminogen activator), PAI-1 (plasminogen activator inhibitor), PAP (plasmin-antiplasmin complexes); endothelial function/injury: vWF (von Willebrand factor), thrombomodulin, ICAM (intracellular adhesion molecule), VCAM (vascular cell adhesion molecule), CD146, CD40L, CD44, E-selectin, inflammation: hsCRP., Results: Triglycerides, hsCRP, creatinine, vWF, prothrombin fragments 1+2, TAT, thrombomodulin, ICAM, VCAM, CD146, CD44, CD40L, PAI-1, PAP, visfatin and E-selectin were elevated in chronic kidney disease patients when compared with the control group. Visfatin correlated significantly in patients with chronic kidney disease, in univariate analysis, with CD40L (r=-0.27, p<0.05), apelin (r=0.27, p<0.05), ICAM (r=0.26, p<0.05), VCAM (r=0.31, p<0.05) and tended to correlate with CD146 (r=0.21, p=0.10). Apelin correlated significantly with E-selectin (r=0.31, p<0.05) and VCAM (r=0.31, p<0.05). In the healthy volunteers visfatin correlated significantly with ICAM (r=-0.37, p<0.05) and serum creatinine (0.38, p<0.05)., Conclusions: Elevated visfatin in CKD patients may be due to renal failure and/or inflammation. Adipocytokines related to adhesion molecules might support the importance of inflammation/endothelial cell injury in the pathogenesis of atherosclerosis and its consequences in CKD.

The elucidation of structures of glutathione (GSH) complexes play an important role in the fundamental understanding of biochemical pathways of metal ion deactivation in plants. This article attempts to feature key studies for stoichiometry of metal complexes with glutathione and its constituent amino acids to obtain a better understanding of the different metal affinities of the complexation sites of glutathione. The SEC-ICP-MS experiments have indicated that oxidation process of glutathione was accelerated by metal ion presence in following order Cu(+), Pb(2+) and Cd(2+). The redox activity of metal ions was confirmed by ESI-MS experiments, which allowed to observe formation of glutathione disulphide (GSSG) in time. The stoichiometry of Cd(2+), Cu(+) and Pb(2+) complexes with GSH was defined by observing the isotope pattern of investigated metals and hydrogen loss or transfer during binding. The complexes with metal bound to sulphur of 1:1 and 1:2 stoichiometry were found in case of cadmium and lead. The number of hydrogen atoms lost during metal binding and the SEC-ESI-MS results allowed to elucidate that copper is bound by GSSG in ratio 1:1 and 1:2. Additionally, size exclusion chromatography coupled to electrospray MS allowed to differentiate more stable complexes from weak ones that could be created in the gas phase.

Matrix metalloproteinases (MMPs)/their inhibitors (TIMPs) system and elevated oxidative stress (SOX) have been implicated as important factors in atherosclerosis and vascular remodeling. The aim of the present study was to investigate whether MMPs/TIMPs system is associated with SOX in hemodialyzed (HD) patients. We compared the serum levels of metalloproteinases and their inhibitors, markers of SOX, inflammation and atherosclerosis between HD patients (n=40) with and without cardiovascular disease (CVD) and controls (n=20). Cu/Zn superoxide dismutase (Cu/Zn SOD) were elevated (all p<0.001), whereas total lipid peroxide levels were similar in HD patients and controls. The autoantibodies against oxidized LDL (OxLDL-Ab) levels were increased only in patients with CVD (p<0.05). Intima media thickness (IMT) in both CVD and in patients without CVD significantly exceeded those in the control (p<0.001 and p<0.01, respectively). Serum pre-HD values of MMP-2, TIMP-1 and TIMP-2 were significantly increased in HD patients, especially in the CVD group (all p<0.001), and they were associated with those of Cu/Zn SOD, IMT and CVD prevalence. Multiple regression analysis showed that MMP-2, Cu/Zn SOD levels and age independently and significantly predicted elevated IMT in HD patients. Our results suggest the association between SOX and the MMPs/TIMPs system in HD patients, which could represent one of the mechanisms involved in the progression of atherosclerosis in this population.

Increased oxidative stress (SOX) has been reported in continuous ambulatory peritoneal dialysis (CAPD) patients, but its influence on beta-chemokine levels and progression of atherosclerosis remains unknown. We determined three distinct SOX markers: Cu/Zn superoxide dismutase (Cu/Zn SOD), total peroxide and autoantibodies against oxidized LDL (OxLDL-Ab); high sensitivity C-reactive protein (hs CRP); beta-chemokines: monocyte chemoattractant protein-1 (CCL2), macrophage inflammatory proteins (CCL3 and CCL4) and regulated upon activation, normal T cell expressed and secreted (CCL5) and the intima-media thickness (IMT) values in CAPD patients both with and without cardiovascular disease (CVD) and healthy controls. CAPD patients both with and without CVD had significantly increased IMT (p<0.001 and <0.01), Cu/Zn SOD (both p<0.001) and CCL2 levels (p<0.001 and <0.01, respectively) as compared to controls. CCL4 (p<0.01) and hs CRP (p<0.05) were increased only in patients with CVD, whereas there were no differences in the total peroxide, OxLDL-Ab and CCL3 levels between patients and controls. CCL5 concentrations were significantly decreased in both patients subgroup (both p<0.001) versus controls. Multivariate analysis showed that age (p<0.001), male sex (p<0.01), CCL4 and CCL2 levels (both p<0.05) were the independent variables linked to IMT values. Our data suggest a possible role of enhanced beta-chemokine levels in the carotid atherosclerosis in patients treated with CAPD, in addition to age and male sex.

Purpose: Renal osteodystrophy is a common complication of chronic renal failure and renal replacement therapy. Successful kidney transplantation reverses many of these abnormalities, however, the improvement is often incomplete. The osteoclast specific 5b isoform of tartrate-resistant acid phosphatase (TRAP) 5b has recently been proposed a specific and sensitive marker of bone resorption. The aim of the study was to assess correlations of TRAP 5b with markers of bone resorption and formation in kidney transplant recipients, hemodialyzed and peritoneally dialyzed patients and healthy volunteers., Material and Methods: We assessed PTH, markers of bone formation-alkaline phosphatase and its bone isoform, osteocalcin, markers of bone resorption--procollagen type I carboxy-terminal extension peptide, procollagen type I cross-linked carboxy-terminal telopeptide, serum CrossLaps-Ctx, beta2-microglobulin and urinary deoxypyridynoline (DPD), expressed as DPD/creatinine ratio. (BMD) bone mineral density measurements were determined for femoral neck and lumbar spine (L2-L4) using DEXA., Results: In dialyzed patients markers of bone formation and resorption were significantly higher than in healthy volunteers, whereas in kidney transplant recipients these disturbances were less pronounced. TRAP 5b correlated positively with age and mainly with markers of bone resorption in kidney transplant recipients, dialyzed patients and healthy volunteers. TRAP 5b did not correlate with BMD in any groups studied., Conclusion: Since TRAP 5b correlated mainly with markers of bone resorption, it may serve as a new additional marker of bone resorption in the assessment of renal osteodystrophy.

A solution of a Zn-complex of recombinant mouse MT-1 isoform (Zn(7)-MT-1) was prepared and titrated with Cd(2+) ions. A method based on the coupling of capillary zone electrophoresis (CZE) with electrospray MS detection was developed for the analysis of the stoichiometry of the species formed during the titration. The method offered the possibility of the on-line removal of up to 100 mM Tris or phosphate buffer solutions that would otherwise suppress the electrospray signal. By allowing the determination of the metal stoichiometry of the complex species present in solution the method was shown to be complementary to circular dichroism and UV-VIS spectrophotometry conventionally used for similar studies. The titration of the Zn(7)-MT complex with Cd(II) showed the sequential displacement of the Zn by Cd. The unusually high stability of the Cd(6)Zn(1)-MT species was observed which suggests a structural role of the remaining Zn(II) ion.

Earlier evidence, in Part I of this paper, has shown that cytotoxic and antitumor 1-nitroacridines did not primarily exert their potent inhibitory effects on cultured mammalian cells by physicochemical binding with DNA, although it undoubtedly occurred (Chem.-Biol. Interact., 43 (1983) 131). As a result it was investigated (i) whether 9-14C- or 1'-14C-labeled derivatives of their representative, 1-nitro-9-/3'-dimethylamino-n-propylamino/acridine (Ledakrin or Nitracrine), were capable of covalent binding with nucleic acids and other suitable macromolecules in target cells in vivo and/or (ii) whether activation of the agent in the cell was a necessary prerequisite for such binding. Using the criteria of resistance to exhaustive extractions with trichloroacetic acid and/or organic solvents, [14C]Ledakrin was found to bind covalently, with relatively little discrimination, with: (i) intracellular macromolecules, including DNA, of cultured tumor HeLa cells (370-2500 DNA base pairs/one Ledakrin molecule; (ii) experimental animal tumor Ehrlich ascites (Eat) cells in vivo (650-5880 DNA base pairs/one Ledakrin molecule); (iii) bacterial Bacillus subtilis SB 1058 cells (7000-33 000 Ledakrin links/one cell genome); (iv) NADPH-fortified rat liver homogenates in vitro (25.6 nmol/mg microsomal protein under air). These results far exceed the common levels reported for alkylating agents or chemical carcinogens. Unlike [ethyl-14C]quinacrine, compared in vitro, covalent macromolecules binding with Ledakrin in vitro, and most probably in vivo, can be equated to NADPH-dependent activation(s) by oxidoreductase systems and the presence of DNA alone was not satisfactory in itself to attain Ledakrin binding. Fractionation of the enzymatic digest of 14C-associated DNA, isolated from Eat cells exposed in vivo to [9-14C]Ledakrin, by Sephadex LH-20 column chromatography followed by mass spectrometry analyses of modified nucleosides, indicated that both mono- and dinucleosidical Ledakrin metabolites were the products of an in vivo reaction. This implied that the lethal reaction of the drug could be its cross-linking of the target macromolecules and/or its monofunctional attack on vitally important cellular components.

To define a critical lesion in presumable target DNA cause in vivo by the antitumor and cytotoxic 1-nitroacridines, Ehrlich ascites tumor (Eat) cells implanted into mice, HeLa cells grown in monolayer culture or Bacillus subtilis SB 1058 strain cells were exposed to Ledakrin [Nitracrine; 1-nitro-9-(3'-dimethylamino-n-propylamino)acridine], its non-antitumor congeners, or mitomycin C tested for comparison; total intracellular DNA was isolated from control or treated cells and denatured by heat, alkali or formamide, after which the chemically-induced fraction of interstrand cross-linked DNA molecules was assessed by thermal denaturation-renaturation curve analysis, hydroxylapatite column chromatography, or partitioning in a Dextran T500-polyethylene glycol 6000 biphasic system. Ledakrin, as compared to mitomycin C, was a very effective cross-linking agent, inducing one covalent cross-link per approx. 20 X 10(3) (B. subtilis), 56 X 10(3) (HeLa) or 80 X 10(3) (Eat) DNA base pairs. The first cross-links were introduced in B. subtilis cell genomes at minimal bactericidal concentrations of Ledakrin of mitomycin C. Ledakrin failed to induce discernible cross-linking of bihelical DNA when complexed with in cell-free system. Unlike the other two 1-nitroacridines which cross-linked DNA of cultured HeLa or B. subtilis cells, the non-antitumor 2-, 3- or 4-nitroacridines did not cause such effect and diminished cross-linking by Ledakrin or mitomycin C. Hence, upon metabolic activation in mammalian or bacterial cell Ledakrin and, most probably other 1-nitroacridines, become very effective DNA cross-linking agents and such effects appear to be responsible for the antitumor and potent cytotoxic activities of 1-nitroacridines.

The mode of action of cytotoxic and antitumor 1-nitroacridines and their isomeric derivatives was studied by comparing their effects in cell-free systems and towards cultured tumor HeLa cells, assuming that the nitroacridines considered exert cytotoxic effects by physicochemical binding with the DNA. All the nitroacridines impaired biosyntheses of DNA, RNA and protein in cultured HeLa cells and a causal relationship between nitroacridine inhibition of macromolecular biosyntheses and lethal effects of the agents appears likely. In cell-free systems, the nitroacridines bound with two independent sites on the DNA, forming complexes with enhanced resistance to DNA strand separation upon melting and inhibited the DNA polymerase reaction by altering activity of template and/or of enzyme. The 1-nitroacridines were poorly effective in cell-free systems and were the most potent inhibitors toward the growth of HeLa cells among the derivatives studied. It is concluded that the primary events responsible for cytotoxic effects of antitumor 1-nitroacridines and of their isomeric derivatives are different. The metabolic activation of 1-nitroacridines to more reactive intermediates which will attach to and alter the structure and/or function of DNA of sensitive cells is suggested.