Towards a qAOP Framework for Predictive Toxicology - Linking Data to Decisions

The adverse outcome pathway (AOP) is a conceptual construct that facilitates organisation and interpretation of mechanistic data representing multiple biological levels and deriving from a range of methodological approaches including in silico, in vitro and in vivo assays. AOPs are playing an increasingly important role in the chemical safety assessment paradigm and quantification of AOPs is an important step towards a more reliable prediction of chemically induced adverse effects. Modelling methodologies require the identification, extraction and use of reliable data and information to support the inclusion of quantitative considerations in AOP development. An extensive and growing range of digital resources are available to support the modelling of quantitative AOPs, providing a wide range of information, but also requiring guidance for their practical application. A framework for qAOP development is proposed based on feedback from a group of experts and three qAOP case studies. The proposed framework provides a harmonised approach for both regulators and scientists working in this area

Nanoparticle-and liposome-carried drugs: New strategies for active targeting and drug delivery across blood-brain barrier

The blood-brain barrier (BBB), the unusual microvascular endothelial interface between the central nervous system (CNS) and the circulatory system, is a major hindrance to drug delivery in the brain parenchyma. Besides the absence of fenestrations and the abundance of tight junctions, ATP-binding cassette (ABC) transporters critically reduce drug entry within the CNS, as they carry many drugs back into the bloodstream. Nanoparticle-and liposome-carried drugs, because of their increased cellular uptake and reduced efflux through ABC transporters, have been developed in recent times to circumvent the low drug permeability of the BBB. This review discusses the role of ABC transporters in controlling drug penetration into the brain parenchyma, the rationale for using nanoparticle-and liposome-based strategies to increase drug delivery across the BBB and new therapeutic strategies for using nanoparticle-and liposome-carried drugs in different conditions, ranging from CNS tumors and neurodegenerative diseases to viral infections and epilepsy. © 2013 Bentham Science Publishers

Omega 3 fatty acids chemosensitize drug resistant colon cancer cells by downregulating cholesterol synthesis and altering lipid rafts environment

Background. High levels of cholesterol in plasmamembrane and in particular in lipid rafts are critical for the activity of P-glycoprotein (Pgp) and multidrug resistance related protein 1 (MRP1), two drug efflux transporters that determine a multidrug resistant phenotype. It has been reported that omega 3 fatty acids prevent colon cancers and modulate cholesterol homeostasis in gut. The mechanisms by which they affect cholesterol homeostasis, and the effects on membrane environment and transporters activity have not been investigated yet. Aim. We aim to clarify how omega 3 fatty acids modulate the cholesterol metabolism, the membrane properties and the activity of drug efflux transporters in colon cancer. Methods. Human chemosensitive colon cancer HT29 cells and their chemoresistant counterpart HT29-dx were treated with omega 3 docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Results. HT29-dx cells had higher plasmamembrane cholesterol and cholesterol synthesis than HT29 cells: the phenotype of HT29-dx cells was due to the lower expression of the oncosuppressor Trc8, a E3 ligase that ubiquitinates 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoAR). DHA and EPA lowered membrane- and lipid rafts-associated cholesterol and decreased cholesterol synthesis in HT29-dx cells, by down-regulating HMGCoAR protein in a SREBP2- and LXR-independent way. Of note, they activated Trc8 and restored HMGCoAR ubiquitination. DHA and EPA were also incorporated in membrane and lipid rafts, where they decreased the saturated/unsaturated fatty acids ratio. In consequence of the altered membrane environment, DHA and EPA disassociated Pgp and MRP1 from lipid rafts, decreased transporters activity and restored the antitumor effects of several chemotherapeutic drugs in resistant cells. Conclusions. We propose omega 3 fatty acids as potential chemosensitizer agents in colon cancer, thanks to their effects on cholesterol homeostasis and plasmamembrane microenvironment

Omega 3 fatty acids chemosensitize multidrug resistant colon cancer cells by down-regulating cholesterol synthesis and altering detergent resistant membranes composition* Equal Contribution °Last CoAuhors

BACKGROUND: The activity of P-glycoprotein (Pgp) and multidrug resistance related protein 1 (MRP1), two membrane transporters involved in multidrug resistance of colon cancer, is increased by high amounts of cholesterol in plasma membrane and detergent resistant membranes (DRMs). It has never been investigated whether omega 3 polyunsatured fatty acids (PUFAs), which modulate cholesterol homeostasis in dyslipidemic syndromes and have chemopreventive effects in colon cancer, may affect the response to chemotherapy in multidrug resistant (MDR) tumors. METHODS: We studied the effect of omega 3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in human chemosensitive colon cancer HT29 cells and in their MDR counterpart, HT29-dx cells. RESULTS: MDR cells, which overexpressed Pgp and MRP1, had a dysregulated cholesterol metabolism, due to the lower expression of ubiquitin E3 ligase Trc8: this produced lower ubiquitination rate of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoAR), higher cholesterol synthesis, higher cholesterol content in MDR cells. We found that DHA and EPA re-activated Trc8 E3 ligase in MDR cells, restored the ubiquitination rate of HMGCoAR to levels comparable with chemosensitive cells, reduced the cholesterol synthesis and incorporation in DRMs. Omega 3 PUFAs were incorporated in whole lipids as well as in DRMs of MDR cells, and altered the lipid composition of these compartments. They reduced the amount of Pgp and MRP1 contained in DRMs, decreased the transporters activity, restored the antitumor effects of different chemotherapeutic drugs, restored a proper tumor-immune system recognition in response to chemotherapy in MDR cells. CONCLUSIONS: Our work describes a new biochemical effect of omega 3 PUFAs, which can be useful to overcome chemoresistance in MDR colon cancer cells

Digoxin and ouabain induce the efflux of cholesterol via liver X receptor signalling and the synthesis of ATP in cardiomyocytes

Cardioactive glycosides exert positive inotropic effects on cardiomyocytes through the inhibition of Na+/K+-ATPase. We showed previously that in human hepatoma cells, digoxin and ouabain increase the rate of the mevalonate cascade and therefore have Na+/K+-ATPase-independent effects. In the present study we found that they increase the expression and activity of 3-hydroxy-3 methylglutaryl-CoA reductase and the synthesis of cholesterol in cardiomyocytes, their main target cells. Surprisingly this did not promote intracellular cholesterol accumulation. The glycosides activated the liver X receptor transcription factor and increased the expression of ABCA1 (ATP-binding cassette protein A1) transporter, which mediates the efflux of cholesterol and its delivery to apolipoprotein A-I. By increasing the synthesis of ubiquinone, another derivative of the mevalonate cascade, digoxin and ouabain simultaneously enhanced the rate of electron transport in the mitochondrial respiratory chain and the synthesis of ATP. Mice treated with digoxin showed lower cholesterol and higher ubiquinone content in their hearts, and a small increase in their serum HDL (high-density lipoprotein) cholesterol. The results of the present study suggest that cardioactive glycosides may have a role in the reverse transport of cholesterol and in the energy metabolism of cardiomyocyte

Probabilistic modelling of developmental neurotoxicity based on a simplified adverse outcome pathway network.

In a century where toxicology and chemical risk assessment are embracing alternative methods to animal testing, there is an opportunity to understand the causal factors of neurodevelopmental disorders such as learning and memory disabilities in children, as a foundation to predict adverse effects. New testing paradigms, along with the advances in probabilistic modelling, can help with the formulation of mechanistically-driven hypotheses on how exposure to environmental chemicals could potentially lead to developmental neurotoxicity (DNT). This investigation aimed to develop a Bayesian hierarchical model of a simplified AOP network for DNT. The model predicted the probability that a compound induces each of three selected common key events (CKEs) of the simplified AOP network and the adverse outcome (AO) of DNT, taking into account correlations and causal relations informed by the key event relationships (KERs). A dataset of 88 compounds representing pharmaceuticals, industrial chemicals and pesticides was compiled including physicochemical properties as well as in silico and in vitro information. The Bayesian model was able to predict DNT potential with an accuracy of 76%, classifying the compounds into low, medium or high probability classes. The modelling workflow achieved three further goals: it dealt with missing values; accommodated unbalanced and correlated data; and followed the structure of a directed acyclic graph (DAG) to simulate the simplified AOP network. Overall, the model demonstrated the utility of Bayesian hierarchical modelling for the development of quantitative AOP (qAOP) models and for informing the use of new approach methodologies (NAMs) in chemical risk assessment

Low cholesterol efflux capacity and abnormal lipoprotein particles in youth with type 1 diabetes: a case control study

Abstract Background Patients with type 1 diabetes (T1DM) have increased mortality from cardiovascular disease (CVD). Risk factors for CVD include an elevation of LDL (LDLp) and small HDL (sHDLp) particles, and a decrease in reverse cholesterol transport i.e. HDL-cholesterol efflux capacity (CEC). Our objective was to compare lipoprotein particles and CEC between T1DM and healthy controls (HC) and to explore the associations between NMR lipid particles and cholesterol efflux. Methods 78 patients with T1DM and 59 HC underwent fasting lipoprotein profile testing by NMR and measurements of CEC by cell-based method. The associations between NMR lipid particles with CEC were analyzed using multivariable linear regression models. Results Youth with T1DM had higher total LDLp 724 [(563–985) vs 622 (476–794) nmol/L (P = 0.011)] (Maahs et al. in Circulation 130(17):1532–58, 2014; Shah et al. in Pediatr Diabetes 16(5):367–74, 2015), sHDLp [11.20 (5.7–15.3) vs 7.0 (3.2–13.1) μmol/L (P = 0.021)], and lower medium HDLp [11.20 (8.5–14.5) vs 12.3 (9–19.4), (P = 0.049)] and lower CEC (0.98 ± 0.11% vs 1.05 ± 0.15%, P = 0.003) compared to HC. Moreover, CEC correlated with sHDLp (β = − 0.28, P = 0.045) and large HDLp (β = 0.46, P < 0.001) independent of age, sex, ethnicity, BMIz, HbA1c, hsCRP and total HDLp in the diabetic cohort. Conclusions Youth with T1DM demonstrated a more atherogenic profile including higher sHDL and LDLp and lower CEC. Future efforts should focus on considering adding lipoprotein particles and CEC in CVD risk stratification of youth with T1DM. Trial registration Clinical Trials Registration Number NCT0227509