Serum 5'-Nucleotidase as a Novel Predictor of Adverse Clinical Outcomes after Percutaneous Coronary Intervention in Patients with Coronary Artery Disease.

Background: The correlation between 5 ' -Nucleotidase ( 5 ' -NT) and the clinical outcomes in coronary artery disease (CAD) patients following percutaneous coronary intervention (PCI) is not clear. This study aims to clarify this relationship. Methods: The PRACTICE study enrolled 15,250 patients between December 2016 and October 2021. After filtering out those without 5 ' -NT data, a total of 6555 patients were analyzed with a median follow-up of 24 months. Based on the receiver operating characteristic (ROC) curve analysis, a 5 ' -NT level of 5.57 U/L was selected as the optimal cutoff value. All research samples were divided into high-value ( ≥ 5.57 U/L, n = 2346) and low-value groups ( < 5.57 U/L, n = 4209). Key clinical outcomes included all-cause death (ACD), cardiovascular death (CD), major adverse cardiovascular events (MACE), and major adverse cardiovascular and cerebrovascular events (MACCE). After separating patients into high and low value groups, multivariate Cox regression analysis was used to correct for potential confounding variables. Finally, risk ratios and their 95% confidence intervals (CIs) were calculated. Results: During the follow-up period, 129 instances of ACD were recorded-49 cases (1.2%) in the low-value group and 80 cases (3.4%) in the high-value group. Similarly, 102 CDs occurred, including 42 low-value group cases (1.0%) and 60 high-value group cases (2.6%). A total of 363 MACE occurred, including 198 low-value group cases (4.7%) and 165 high-value group cases (7%). A total of 397 cases of MACCE occurred, including 227 low-value group cases (5.4%) and 170 high-value group cases (7.2%). As serum 5 ' -NT increased, the incidence of ACD, CD, MACE and MACCE increased. After multivariate Cox regression, high 5 ' -NT levels were linked with a 1.63-fold increase in ACD risk (hazard ratio [HR] = 2.630, 95% CI: [1.770-3.908], p < 0.001) when compared to low 5 ' -NT patients. Similarly, the risk of CD, MACE, and MACCE increased by 1.298-fold (HR = 2.298, 95% CI: [1.477-3.573], p < 0.001), 41% (HR = 1.410, 95% CI: [1.124-1.768], p = 0.003) and 30.5% (HR = 1.305, 95% CI: [1.049-1.623], p = 0.017), respectively. Conclusions: high serum 5 ' -NT levels were independently correlated with adverse clinical outcomes in CAD patients following PCI, affirming its potential as a prognostic indicator.

Cell wall-anchored 5'-nucleotidases in Gram-positive cocci.

5'-nucleotidases (5'-NTs) are enzymes that catalyze the hydrolysis of nucleoside monophosphates to produce nucleosides and phosphate. Since the identification of adenosine synthase A (AdsA) in Staphylococcus aureus in 2009, several other 5'-NTs have been discovered in Gram-positive cocci, mainly in streptococci. Despite some differences in substrate specificity, pH range and metal ion requirements, all characterized 5'-NTs use AMP and ADP, and in some cases ATP, to produce the immunosuppressive adenosine, which dampens pro-inflammatory immune responses. Several 5'-NTs are also able to use dAMP as substrate to generate deoxy-adenosine which is cytotoxic for macrophages. A synergy between 5'-NTs and exonucleases which are commonly expressed in Gram-positive cocci has been described, where the nucleases provide dAMP as a cleavage product from DNA. Some of these nucleases produce dAMP by degrading the DNA backbone of neutrophil extracellular traps (NETs) resulting in a "double hit" strategy of immune evasion. This Micro Review provides an overview of the biochemical properties of Gram-positive cell wall-anchored 5'-NTs and their role as virulence factors. A potential use of 5'-NTs for vaccine development is also briefly discussed.

Serotonergic Drugs Inhibit Chikungunya Virus Infection at Different Stages of the Cell Entry Pathway.

Chikungunya virus (CHIKV) is an important reemerging human pathogen transmitted by mosquitoes. The virus causes an acute febrile illness, chikungunya fever, which is characterized by headache, rash, and debilitating (poly)arthralgia that can reside for months to years after infection. Currently, effective antiviral therapies and vaccines are lacking. Due to the high morbidity and economic burden in the countries affected by CHIKV, there is a strong need for new strategies to inhibit CHIKV replication. The serotonergic drug 5-nonyloxytryptamine (5-NT) was previously identified as a potential host-directed inhibitor for CHIKV infection. In this study, we determined the mechanism of action by which the serotonin receptor agonist 5-NT controls CHIKV infection. Using time-of-addition and entry bypass assays, we found that 5-NT predominantly inhibits CHIKV in the early phases of the replication cycle, at a step prior to RNA translation and genome replication. Intriguingly, however, no effect was seen during virus-cell binding, internalization, membrane fusion and genomic RNA (gRNA) release into the cell cytosol. In addition, we show that the serotonin receptor antagonist methiothepin mesylate (MM) also has antiviral properties toward CHIKV and specifically interferes with the cell entry process and/or membrane fusion. Taken together, pharmacological targeting of 5-HT receptors may represent a potent way to limit viral spread and disease severity.IMPORTANCE The rapid spread of mosquito-borne viral diseases in humans puts a huge economic burden on developing countries. For many of these infections, including those caused by chikungunya virus (CHIKV), there are no specific treatment possibilities to alleviate disease symptoms. Understanding the virus-host interactions that are involved in the viral replication cycle is imperative for the rational design of therapeutic strategies. In this study, we discovered an antiviral compound, elucidated its mechanism of action, and propose serotonergic drugs as potential host-directed antivirals for CHIKV.

Substrate binding modes of purine and pyrimidine nucleotides to human ecto-5'-nucleotidase (CD73) and inhibition by their bisphosphonic acid derivatives.

Human ecto-5-nucleotidase (CD73) is involved in purinergic signalling, which influences a diverse range of biological processes. CD73 hydrolyses AMP and is the major control point for the levels of extracellular adenosine. Inhibitors of CD73 thus block the immunosuppressive action of adenosine, a promising approach for cancer immunotherapy. Interestingly, ADP and ATP are competitive inhibitors of CD73, with the most potent small-molecule inhibitors to date being non-hydrolysable ADP analogues. While AMP is the major substrate of the enzyme, CD73 has been reported to hydrolyse other 5'-nucleoside monophosphates. Based on a fragment screening campaign at the BESSY II synchrotron, we present the binding modes of various deoxyribo- and ribonucleoside monophosphates and of four additional fragments binding to the nucleoside binding site of the open form of the enzyme. Kinetic analysis of monophosphate hydrolysis shows that ribonucleotide substrates are favoured over their deoxyribose equivalents with AMP being the best substrate. We characterised the initial step of AMP hydrolysis, the binding mode of AMP to the open conformation of CD73 and compared that to other monophosphate substrates. In addition, the inhibitory activity of various bisphosphonic acid derivatives of nucleoside diphosphates was determined. Although AMPCP remains the most potent inhibitor, replacement of the adenine base with other purines or with pyrimidines increases the Ki value only between twofold and sixfold. On the other hand, these nucleobases offer new opportunities to attach substituents for improved pharmacological properties.

4-Aminopyridine based amide derivatives as dual inhibitors of tissue non-specific alkaline phosphatase and ecto-5'-nucleotidase with potential anticancer activity.

Ecto-nucleotidase members i.e., ecto-5'-nucleotidase and alkaline phosphatase, hydrolyze extracellular nucleotides and play an important role in purinergic signaling. Their overexpression are implicated in a variety of pathological states, including immunological diseases, bone mineralization, vascular calcification and cancer, and thus they represent an emerging drug targets. In order to design potent and selective inhibitors, new derivatives of 4-aminopyridine have been synthesized (10a-10m) and their structures were established on the basis of spectral data. The effect of nature and position of substituent was interestingly observed and justified on the basis of their detailed structure activity relationships (SARs) against both families of ecto-nucleotidase. Compound 10a displayed significant inhibition (IC50 ±â€¯SEM = 0.25 ±â€¯0.05 µM) that was found ≈168 fold more potent as compared to previously reported inhibitor suramin (IC50 ±â€¯SEM = 42.1 ±â€¯7.8 µM). This compound exhibited 6 times more selectivity towards h-TNAP over h-e5'NT. The anticancer potential and mechanism were also established using cell viability assay, flow cytometric analysis and nuclear staining. Molecular docking studies were also carried out to gain insight into the binding interaction of potent compounds within the respective enzyme pockets and herring-sperm DNA.

Involvement of purinergic system in inflammation and toxicity induced by copper in zebrafish larvae.

The use of zebrafish (Danio rerio) is increasing as an intermediate preclinical model, to prioritize drug candidates for mammalian testing. As the immune system of the zebrafish is quite similar to that of mammals, models of inflammation are being developed for the screening of new drugs. The characterization of these models is crucial for studies that seek for mechanisms of action and specific pharmacological targets. It is well known that copper is a metal that induces damage and cell migration to hair cells of lateral line of zebrafish. Extracellular nucleotides/nucleosides, as ATP and adenosine (ADO), act as endogenous signaling molecules during tissue damage by exerting effects on inflammatory and immune responses. The present study aimed to characterize the inflammatory status, and to investigate the involvement of the purinergic system in copper-induced inflammation in zebrafish larvae. Fishes of 7 days post-fertilization were exposed to 10 µM of copper for a period of 24 h. The grade of oxidative stress, inflammatory status, copper uptake, the activity and the gene expression of the enzymes responsible for controlling the levels of nucleotides and adenosine were evaluated. Due to the copper accumulation in zebrafish larvae tissues, the damage and oxidative stress were exacerbated over time, resulting in an inflammatory process involving IL-1ß, TNF-α, COX-2 and PGE2. Within the purinergic system, the mechanisms that control the ADO levels were the most involved, mainly the reactions performed by the isoenzyme ADA 2. In conclusion, our data shed new lights on the mechanisms related to copper-induced inflammation in zebrafish larvae.

Extracellular ATP and its derivatives provide spatiotemporal guidance for bone adaptation to wide spectrum of physical forces.

ATP is a ubiquitous intracellular molecule critical for cellular bioenergetics. ATP is released in response to mechanical stimulation through vesicular release, small tears in cellular plasma membranes, or when cells are destroyed by traumatic forces. Extracellular ATP is degraded by ecto-ATPases to form ADP and eventually adenosine. ATP, ADP, and adenosine signal through purinergic receptors, including seven P2X ATP-gated cation channels, seven G-protein coupled P2Y receptors responsive to ATP and ADP, and four P1 receptors stimulated by adenosine. The goal of this review is to build a conceptual model of the role of different components of this complex system in coordinating cellular responses that are appropriate to the degree of mechanical stimulation, cell proximity to the location of mechanical injury, and time from the event. We propose that route and amount of ATP release depend on the scale of mechanical forces, ranging from vesicular release of small ATP boluses upon membrane deformation, to leakage of ATP through resealable plasma membrane tears, to spillage of cellular content due to destructive forces. Correspondingly, different P2 receptors responsive to ATP will be activated according to their affinity at the site of mechanical stimulation. ATP is a small molecule that readily diffuses through the environment, bringing the signal to the surrounding cells. ATP is also degraded to ADP which can stimulate a distinct set of P2 receptors. We propose that depending on the magnitude of mechanical forces and distance from the site of their application, ATP/ADP profiles will be different, allowing the relay of information about tissue level injury and proximity. Lastly, ADP is degraded to adenosine acting via its P1 receptors. The presence of large amounts of adenosine without ATP, indicates that an active source of ATP release is no longer present, initiating the transition to the recovery phase. This model consolidates the knowledge regarding the individual components of the purinergic system into a conceptual framework of choreographed responses to physical forces.

Biochemistry and toxicology of proteins and peptides purified from the venom of Vipera berus berus.

The isolation and characterization of individual snake venom components is important for a deeper understanding of the pathophysiology of envenomation and for improving the therapeutic procedures of patients. It also opens possibilities for the discovery of novel toxins that might be useful as tools for understanding cellular and molecular processes. The variable venom composition, toxicological and immunological properties of the common vipers (Vipera berus berus) have been reviewed. The combination of venom gland transcriptomics, bottom-up and top-down proteomics enabled comparison of common viper venom proteomes from multiple individuals. V. b. berus venom contains proteins and peptides belonging to 10-15 toxin families: snake venom metalloproteinase, phospholipases A2 (PLA2), snake venom serine proteinase, aspartic protease, L-amino acid oxidase (LAAO), hyaluronidase, 5'-nucleotidase, glutaminyl-peptide cyclotransferase, disintegrin, C-type lectin (snaclec), nerve growth factor, Kunitz type serine protease inhibitor, snake venom vascular endothelial growth factor, cysteine-rich secretory protein, bradykinin potentiating peptide, natriuretic peptides. PLA2 and LAAO from V. b. berus venom produce more pronounced cytotoxic effects in cancer cells than normal cells, via induction of apoptosis, cell cycle arrest and suppression of proliferation. Proteomic data of V. b. berus venoms from different parts of Russia and Slovakian Republic have been compared with analogous data for Vipera nikolskii venom. Proteomic studies demonstrated quantitative differences in the composition of V. b. berus venom from different geographical regions. Differences in the venom composition of V. berus were mainly driven by the age, sex, habitat and diet of the snakes. The venom variability of V. berus results in a loss of antivenom efficacy against snakebites. The effectiveness of antibodies is discussed. This review presents an overview with a special focus on different toxins that have been isolated and characterized from the venoms of V. b. berus. Their main biochemical properties and toxic actions are described.

Identification and Expression Analysis of CD73 Inhibitors in Cervical Cancer.

AIMS: The present study was conducted to examine the inhibitory effects of synthesized sulfonylhydrazones on the expression of CD73 (ecto-5'-NT). BACKGROUND: CD73 (ecto-5'-NT) represents the most significant class of ecto-nucleotidases, which are mainly responsible for the dephosphorylation of adenosine monophosphate to adenosine. Inhibition of CD73 played an important role in the treatment of cancer, autoimmune disorders, precancerous syndromes, and some other diseases associated with CD73 activity. OBJECTIVE: Keeping in view the significance of CD73 inhibitor in the treatment of cervical cancer, a series of sulfonylhydrazones (3a-3i) derivatives synthesized from 3-formylchromones were evaluated. METHODS: All sulfonylhydrazones (3a-3i) were evaluated for their inhibitory activity towards CD73 (ecto-5'-NT) by the malachite green assay and their cytotoxic effect was investigated on the HeLa cell line using MTT assay. Secondly, the most potent compound was selected for cell apoptosis, immunofluorescence staining, and cell cycle analysis. After that, CD73 mRNA and protein expression were analyzed by real-time PCR and Western blot. RESULTS: Among all compounds, 3h, 3e, 3b, and 3c were found to be the most active against rat-ecto- 5'-NT (CD73) enzyme with IC50 (µM) values of 0.70 ± 0.06 µM, 0.87 ± 0.05 µM, 0.39 ± 0.02 µM, and 0.33 ± 0.03 µM, respectively. These derivatives were further evaluated for their cytotoxic potential against cancer cell line (HeLa). Compounds 3h and 3c showed the cytotoxicity at IC50 value of 30.20 ± 3.11 µM and 86.02 ± 7.11 µM, respectively. Furthermore, compound 3h was selected for cell apoptosis, immunofluorescence staining, and cell cycle analysis, which showed a promising apoptotic effect in HeLa cells. Additionally, compound 3h was further investigated for its effect on the expression of CD73 using qRT-PCR and western blot. CONCLUSION: Among all synthesized compounds (3a-3i), Compound 3h (E)-N'-((6-ethyl-4-oxo-4Hchromen- 3-yl) methylene)-4-methylbenzenesulfonohydrazide was identified as the most potent compound. Additional expression studies conducted on the HeLa cell line proved that this compound successfully decreased the expression level of CD73 and thus, inhibited the growth and proliferation of cancer cells.

Activities of purine converting enzymes in heart, liver and kidney mice LDLR-/- and Apo E-/.

Nucleotide metabolism plays a major role in a number of vital cellular processes such as energetics. This, in turn, is important in pathologies such as atherosclerosis. Three month old atherosclerotic mice with knock outs for LDLR and apolipoprotein E (ApoE) were used for the experiments. Activities of AMP-deaminase (AMPD), ecto5'-nucleotidase (e5NT), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) were measured in heart, liver and kidney cortex and medulla by analysing conversion of substrates into products using HPLC. The activity of ecto5'-nucleotidase differ in hearts of LDLR-/- and ApoE-/- mice with no differences in ADA and AMPD activity. We noticed highest activity of e5NT in kidney medulla of the models. This model of atherosclerosis characterize with an inhibition of enzyme responsible for production of protective adenosine in heart but not in other organs and different metabolism of nucleotides in kidney medulla.

Alpha-bisabolol Promotes Glioma Cell Death by Modulating the Adenosinergic System.

BACKGROUND/AIM: Glioblastoma multiforme is the most malignant type of glioma. Alpha-bisabolol is an essential oil reported as a potent cell death agent. In the present work, we evaluated the effect of alpha-bisabolol on ecto-5'-nucleotidase/CD73, the most well-characterized enzymatic source of adenosine, present in lipid rafts. MATERIALS AND METHODS: Glioma cells were treated with alpha-bisabolol and, in some experiments, pre-treated with an A3 antagonist. MTT assay (viability), malachite green method (ecto-5'-nucleotidase/CD73 activity) and quantitative polymerase chain reaction (qPCR) (A3 mRNA) were carried out. RESULTS: Alpha-bisabolol led to a decrease in C6 and U138-MG glioma cells viability, accompanied by an increase in ecto-5'-NT/CD73 activity. Pre-treatment with an A3 antagonist reverted the effect of α-bisabolol with an increase of mRNA expression of this receptor. CONCLUSION: Our data indicated the participation of ecto-5'-nucleotidase/CD73 and A3 receptor in the anti-proliferative effect of α-bisabolol on glioma cells.

Evaluation of serum adenosine deaminase and 5'-nucleotidase activities as probable markers in ovarian cancer.

Adenosine deaminase (ADA) and 5'-nucleotidase (5'-NT) activities were measured in sera of patients with ovarian cancer and patients with benign ovarian tumour. The results were compared with that of a control group consisting of healthy women. ADA levels were significantly increased (P<0.001) in the ovarian cancer group (n=50) but not in the benign group (n=28) when compared to the controls (n=20). The results indicate that ADA and 5'-NT levels may help to differentiate malignant conditions from benign tumours of the ovary in addition to the existing tests such as serum CA-125 levels and histopathological study.

Serum adenosine deaminase, 5' nucleotidase and malondialdehyde in acute infective hepatitis.

Serum adenosine deaminase (ADA), 5' nucleotidase (5'NT) and malondialdehyde (MDA) were estimated in patients with acute infective hepatitis (AIH) along with the routine parameters of liver disease. Present study is done to evaluate these special parameters in patients with clinical history of AIH and to assess the utility of these parameters as diagnostic/ prognostic indices of liver function and to correlate special parameters with routine live function tests (LFT). ADA, 5'NT and MDA along with routine LFT was estimated in 25 patients with AIH and 25 samples from healthy voluntary blood donars served as the control group. Routine LFT was estimated by standard clinical chemistry procedures on dade behring analyser and ADA, 5'NT and MDA were estimated by berthlot reaction, fiske and subbarao method and thiobarbituric acid method respectively.Statistical analysis showed that serum ADA, 5'NT and MDA were significantly higher in patients as compared with the controls. There was a significant positive correlation between ADA and total bilirubin and MDA and total bilirubin. Hence we can conclude that these tests would be more sensitive to diagnose the patients with AIH and that the raised bilirubin levels could be looked upon, as a protective mechanism which the liver has evolved in order to combat oxidative stress.

Immunohistochemical and functional analysis of ectonucleoside triphosphate diphosphohydrolase 1 (CD39) and ecto-5'-nucleotidase (CD73) in pig aortic valves.

Extracellular nucleotides control mechanisms such as thrombosis or inflammation that are important in several pathologies, including heart valve disease and calcification. Ectonucleoside triphosphate diphosphohydrolase 1 (eNTPD1, CD39) and ecto-5'-nucleotidase (e5NT, CD73) are ectoenzymes that convert adenosine triphosphate to adenosine diphosphate, adenosine monophosphate and finally to adenosine. Changes in activities of these enzymes influence extracellular nucleotide concentrations and therefore could be involved in valve pathology. This study aimed to analyze type of cells, specific area, level of expression and biochemical function of CD39 and CD73 in pig aortic valves. Samples were collected from aortic valves of domestic pigs. Histological sections were cut from paraffin embedded tissue blocks. Following incubation with primary antibody against CD39 or CD73, washing and secondary goat anti-rabbit secondary antibodies, slides were viewed with NanoZoomer scanner. Substantial expression CD39 and CD73 was observed in two main types of valve cells: endothelial and valve interstitial cells. Subsequently, biochemical function of CD39 and CD73 was evaluated in cells cultured from pig aortic valve. Breakdown of extracellular nucleotides added to cell medium was analyzed with high performance liquid chromatography. In the interstitial cells, the CD73 products formation was much faster than in endothelium, while for the CD39 activity this relation was opposite. Expression and high concentration of CD39 and CD73 products in endothelium are expected, but presence of CD73 in valve interstitial cells is a surprise. We conclude that CD39 and CD73 and their enzymatic activities that convert extracellular nucleotides are highly expressed and could have special function in the valve.

In vitro and cellular effects of 4-pyridone-3-carboxamide riboside on enzymes of nucleotide metabolism.

4-Pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR) is an endogenously produced nucleoside that has recently been identified as a substrate for intracellular phosphorylation to form nucleotide derivatives. Low level of 4PYR is normally present in human plasma, but 4PYR massively accumulates in patients with renal failure. This study aimed to evaluate effects of 4PYR and its monophosphate derivative (4PYMP) on several enzymes of nucleotide metabolism in homogenates and intact cells. Activities of adenosine monophosphate deaminase (AMPD), adenosine deaminase, ecto-5'-nucleotidase (e5NT), adenine phosphoribosyltransferase (APRT), hypoxanthine/guanine phosphoribosyltransferase, purine nucleoside phosphorylase, and S-adenosylhomocysteine hydrolase (SAHH) were evaluated in erythrocyte lysates, rat heart homogenates, and in the intact rat cardiomyocytes by high performance liquid chromatography-based assays. 4PYMP caused significant inhibition of AMPD in both erythrocyte lysate and heart homogenate with 50% inhibitory concentration (IC50) of 74 and 55 µM, respectively. Inhibition of e5NT in heart homogenates was also noted with IC50 of 63 µM. 4PYMP slightly inhibited APRT and 4PYR caused moderate activation of SAHH. No effects on other enzymes studied were noted. Inhibition of AMPD by 4PYMP in homogenates was confirmed in the intact cell experiments with isolated cardiomyocytes that were allowed to accumulate 4PYMP by incubation with 4PYR. We conclude that among pathways studied, most important is the effect of 4PYMP on AMPD and that such effect could be one of the consequences of elevated plasma 4PYR concentration.

Identification of novel chromone based sulfonamides as highly potent and selective inhibitors of alkaline phosphatases.

A new series of structurally diverse chromone containing sulfonamides has been developed. Crystal structures of three representative compounds (2a, 3a and 4a) in the series are reported. All compounds were screened for their inhibitory potential against alkaline phosphatases (ALPs). Two main classes of ALP isozymes were selected for this study, the tissue non-specific alkaline phosphatase (TNALP) from bovine and porcine source and the tissue-specific intestinal alkaline phosphatases (IALPs) from bovine source. All sulfonamide compounds had a marked preference for IALP (K(i), up to 0.01 ± 0.001 µM) over TNALPs. Kinetics studies of the compounds showed competitive mode of inhibition. Molecular docking studies were carried out in order to characterize the selective inhibition of the compounds. An additional interesting aspect of these chromone sulfonamides is their inhibitory activity against ecto-5'-nucleotidase enzyme.

The adverse outcome pathway concept: a pragmatic tool in toxicology.

Adverse outcome pathways (AOPs) are novel tools in toxicology and human risk assessment with broad potential. AOPs are designed to provide a clear-cut mechanistic representation of critical toxicological effects that span over different layers of biological organization. AOPs share a common structure consisting of a molecular initiating event, a series of intermediate steps and key events, and an adverse outcome. Development of AOPs ideally complies with OECD guidelines. This also holds true for AOP evaluation, which includes consideration of the Bradford Hill criteria for weight-of-evidence assessment and meeting a set of key questions defined by the OECD. Elaborate AOP frameworks have yet been proposed for chemical-induced skin sensitization, cholestasis, liver fibrosis and liver steatosis. These newly postulated AOPs can serve a number of ubiquitous purposes, including the establishment of (quantitative) structure-activity relationships, the development of novel in vitro toxicity screening tests and the elaboration of prioritization strategies.