Breast cancer, alcohol, and phosphate toxicity.

Alcohol consumption is associated with an increased risk of breast cancer, even at low alcohol intake levels, but public awareness of the breast cancer risk associated with alcohol intake is low. Furthermore, the causative mechanisms underlying alcohol's association with breast cancer are unknown. The present theoretical paper uses a modified grounded theory method to review the research literature and propose that alcohol's association with breast cancer is mediated by phosphate toxicity, the accumulation of excess inorganic phosphate in body tissue. Serum levels of inorganic phosphate are regulated through a network of hormones released from the bone, kidneys, parathyroid glands, and intestines. Alcohol burdens renal function, which may disturb the regulation of inorganic phosphate, impair phosphate excretion, and increase phosphate toxicity. In addition to causing cellular dehydration, alcohol is an etiologic factor in nontraumatic rhabdomyolysis, which ruptures cell membranes and releases inorganic phosphate into the serum, leading to hyperphosphatemia. Phosphate toxicity is also associated with tumorigenesis, as high levels of inorganic phosphate within the tumor microenvironment activate cell signaling pathways and promote cancer cell growth. Furthermore, phosphate toxicity potentially links cancer and kidney disease in onco-nephrology. Insights into the mediating role of phosphate toxicity may lead to future research and interventions that raise public health awareness of breast cancer risk and alcohol consumption.

Phosphate Toxicity and Epithelial to Mesenchymal Transition.

The underlying role of inadequate or excess intake of phosphate is evident in disease states, including metabolic, skeletal, cardiac, kidney and various cancers. Elevated phosphate levels can induce epithelial to mesenchymal transition (EMT) and cell death. EMT and associated lethal, metastatic or fibrinogenic responses are known to be underlying disease processes in fibrotic diseases and various solid tumors. Studies have shown EMT is regulated by induction of different signaling pathways, including TGF-ß, RTK, SRC, Wnt and Notch signal transduction. However, cross-talk amongst these signaling pathways is less understood. We have shown that elevated phosphate levels enhanced EMT partially through activating ERK1/2 pathway, resulting in massive cell death. We thus proposed excess phosphate-mediated lethal EMT as one of the underlying mechanisms of phosphate-induced cytotoxicity, which could explain high phosphate-associated renal fibrosis and cancer metastasis in preclinical and clinical studies. This chapter provides the overview of EMT with the highlights of its regulation by various signaling pathways induced by phosphate toxicity. We further put lately reported lethal EMT in the context of phosphate toxicity with the intent to explain it to excessive phosphate-associated pathologies.

Policosanol attenuates Pi-induced calcification via AMPK-mediated INSIGs expression in rat VSMCs.

Policosanol is a hypocholesterolemic derived from sugar cane and corn that downregulates blood cholesterol levels. It can further lower blood pressure and reduce liver inflammation. Policosanol can also affect vascular calcification, however, its molecular mechanisms are not well understood. This study investigated the effect of policosanol on vascular calcification and its molecular mechanism. Policosanol decreased the expression of inorganic phosphate (Pi)-induced osteogenic genes such as distal-less homeobox 5 (Dlx5) and runt-related transcription factor 2 (Runx2). In addition, following policosanol treatment, adenosine monophosphate-activated protein kinase (AMPK) phosphorylation increased in a time-dependent manner. The constitutively active form of AMPK (CA-AMPK) dramatically suppressed Pi-induced Dlx5 and Runx2 protein levels. Inactivation of AMPK using compound C (Com. C; AMPK inhibitor) recovered policosanol-suppressed Alizarin Red S staining levels. Insulin-induced genes (INSIGs) were induced by CA-AMPK, their overexpression suppressed Pi-induced Dlx5 and Runx2 expression. Taken together, the results demonstrate that policosanol inhibits Pi-induced vascular calcification by regulating AMPK-induced INSIG expression in vascular smooth muscle cells.

A Genome-Wide Screen in Saccharomyces cerevisiae Reveals a Critical Role for Oxidative Phosphorylation in Cellular Tolerance to Lithium Hexafluorophosphate.

Lithium hexafluorophosphate (LiPF6) is one of the leading electrolytes in lithium-ion batteries, and its usage has increased tremendously in the past few years. Little is known, however, about its potential environmental and biological impacts. In order to improve our understanding of the cytotoxicity of LiPF6 and the specific cellular response mechanisms to it, we performed a genome-wide screen using a yeast (Saccharomyces cerevisiae) deletion mutant collection and identified 75 gene deletion mutants that showed LiPF6 sensitivity. Among these, genes associated with mitochondria showed the most enrichment. We also found that LiPF6 is more toxic to yeast than lithium chloride (LiCl) or sodium hexafluorophosphate (NaPF6). Physiological analysis showed that a high concentration of LiPF6 caused mitochondrial damage, reactive oxygen species (ROS) accumulation, and ATP content changes. Compared with the results of previous genome-wide screening for LiCl-sensitive mutants, we found that oxidative phosphorylation-related mutants were specifically hypersensitive to LiPF6. In these deletion mutants, LiPF6 treatment resulted in higher ROS production and reduced ATP levels, suggesting that oxidative phosphorylation-related genes were important for counteracting LiPF6-induced toxicity. Taken together, our results identified genes specifically involved in LiPF6-modulated toxicity, and demonstrated that oxidative stress and ATP imbalance maybe the driving factors in governing LiPF6-induced toxicity.

Limitation of phosphate assimilation maintains cytoplasmic magnesium homeostasis.

Phosphorus (P) is an essential component of core biological molecules. In bacteria, P is acquired mainly as inorganic orthophosphate (Pi) and assimilated into adenosine triphosphate (ATP) in the cytoplasm. Although P is essential, excess cytosolic Pi hinders growth. We now report that bacteria limit Pi uptake to avoid disruption of Mg2+-dependent processes that result, in part, from Mg2+ chelation by ATP. We establish that the MgtC protein inhibits uptake of the ATP precursor Pi when Salmonella enterica serovar Typhimurium experiences cytoplasmic Mg2+ starvation. This response prevents ATP accumulation and overproduction of ribosomal RNA that together ultimately hinder bacterial growth and result in loss of viability. Even when cytoplasmic Mg2+ is not limiting, excessive Pi uptake increases ATP synthesis, depletes free cytoplasmic Mg2+, inhibits protein synthesis, and hinders growth. Our results provide a framework to understand the molecular basis for Pi toxicity. Furthermore, they suggest a regulatory logic that governs P assimilation based on its intimate connection to cytoplasmic Mg2+ homeostasis.

Oxidative stress by Ca2+ overload is critical for phosphate-induced vascular calcification.

Hyperphosphatemia is the primary risk factor for vascular calcification, which is closely associated with cardiovascular morbidity and mortality. Recent evidence showed that oxidative stress by high inorganic phosphate (Pi) mediates calcific changes in vascular smooth muscle cells (VSMCs). However, intracellular signaling responsible for Pi-induced oxidative stress remains unclear. Here, we investigated molecular mechanisms of Pi-induced oxidative stress related with intracellular Ca2+ ([Ca2+]i) disturbance, which is critical for calcification of VSMCs. VSMCs isolated from rat thoracic aorta or A7r5 cells were incubated with high Pi-containing medium. Extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin were activated by high Pi that was required for vascular calcification. High Pi upregulated expressions of type III sodium-phosphate cotransporters PiT-1 and -2 and stimulated their trafficking to the plasma membrane. Interestingly, high Pi increased [Ca2+]i exclusively dependent on extracellular Na+ and Ca2+ as well as PiT-1/2 abundance. Furthermore, high-Pi induced plasma membrane depolarization mediated by PiT-1/2. Pretreatment with verapamil, as a voltage-gated Ca2+ channel (VGCC) blocker, inhibited Pi-induced [Ca2+]i elevation, oxidative stress, ERK activation, and osteogenic differentiation. These protective effects were reiterated by extracellular Ca2+-free condition, intracellular Ca2+ chelation, or suppression of oxidative stress. Mitochondrial superoxide scavenger also effectively abrogated ERK activation and osteogenic differentiation of VSMCs by high Pi. Taking all these together, we suggest that high Pi activates depolarization-triggered Ca2+ influx via VGCC, and subsequent [Ca2+]i increase elicits oxidative stress and osteogenic differentiation. PiT-1/2 mediates Pi-induced [Ca2+]i overload and oxidative stress but in turn, PiT-1/2 is upregulated by consequences of these alterations.NEW & NOTEWORTHY The novel findings of this study are type III sodium-phosphate cotransporters PiT-1 and -2-dependent depolarization by high Pi, leading to Ca2+ entry via voltage-gated Ca2+ channels in vascular smooth muscle cells. Cytosolic Ca2+ increase and subsequent oxidative stress are indispensable for osteogenic differentiation and calcification. In addition, plasmalemmal abundance of PiT-1/2 relies on Ca2+ overload and oxidative stress, establishing a positive feedback loop. Identification of mechanistic components of a vicious cycle could provide novel therapeutic strategies against vascular calcification in hyperphosphatemic patients.

The microRNA miR-134-5p induces calcium deposition by inhibiting histone deacetylase 5 in vascular smooth muscle cells.

Calcium deposition in vascular smooth muscle cells (VSMCs) is a form of ectopic ossification in blood vessels. It can result in rigidity of the vasculature and an increase in cardiac events. Here, we report that the microRNA miR-134-5p potentiates inorganic phosphate (Pi)-induced calcium deposition in VSMCs by inhibiting histone deacetylase 5 (HDAC5). Using miRNA microarray analysis of Pi-treated rat VSMCs, we first selected miR-134-5p for further evaluation. Quantitative RT-PCR confirmed that miR-134-5p was increased in Pi-treated A10 cells, a rat VSMC line. Transfection of miR-134-5p mimic potentiated the Pi-induced increase in calcium contents. miR-134-5p increased the amounts of bone runt-related transcription factor 2 (RUNX2) protein and bone morphogenic protein 2 (BMP2) mRNA in the presence of Pi but decreased the expression of osteoprotegerin (OPG). Bioinformatic analysis showed that the HDAC5 3'untranslated region (3'UTR) was one of the targets of miR-134-5p. The luciferase construct containing the 3'UTR of HDAC5 was down-regulated by miR-134-5p mimic in a dose-dependent manner in VSMCs. Overexpression of HDAC5 mitigated the calcium deposition induced by miR-134-5p. Our results suggest that a Pi-induced increase of miR-134-5p may cause vascular calcification through repression of HDAC5.

Targeting androgen receptor in macrophages inhibits phosphate-induced vascular smooth muscle cell calcification by decreasing IL-6 expression.

Vascular calcification (VC) is a common complication of chronic kidney disease (CKD). However, its mechanisms remain unclear. VC, similar to atherosclerosis, is an inflammatory disease. Vascular smooth muscle cells (VSMCs) play a key role in VC progression. The androgen receptor (AR) in monocytes/macrophages plays an important role in inflammatory diseases. Here, we define the role of macrophage (MФ) AR in inorganic phosphate-induced VSMC calcification. Our results show that the conditioning medium (CM) of silencing AR in macrophages inhibits inorganic phosphate-induced human aortic smooth muscle cell (HASMC) calcification, and alleviates the transdifferentiation of HASMCs into osteoblasts for the protein expression of osteoblasts marker Runt-related transcription factor-2 (Runx2) in HASMCs decreased while that of smooth muscle cell marker SM22α increased. The effect of AR on HASMC calcification might mainly be mediated by the inflammatory cytokine IL-6. Silencing AR in monocytes/macrophages can dramatically decrease IL-6 expression. We also investigated how macrophage AR regulates IL-6. ChIP and luciferase assays indicate that AR directly binds to the ARE sequence in the promoter of the IL-6 gene to accelerate transcription and expression. To our knowledge, this is the first investigation that has established the correlation between AR and VC and identified the contribution of AR in the calcification of VSMCs. In addition, this study describes a novel target for therapeutic intervention in VC.

Quantitative encapsulation and retention of 227Th and decay daughters in core-shell lanthanum phosphate nanoparticles.

Targeted alpha therapy (TAT) offers great promise for treating recalcitrant tumors and micrometastatic cancers. One drawback of TAT is the potential damage to normal tissues and organs due to the relocation of decay daughters from the treatment site. The present study evaluates La(227Th)PO4 core (C) and core +2 shells (C2S) nanoparticles (NPs) as a delivery platform of 227Th to minimize systemic distribution of decay daughters, 223Ra and 211Pb. In vitro retention of decay daughters within La(227Th)PO4 C NPs was influenced by the concentration of reagents used during synthesis, in which the leakage of 223Ra was between 0.4 ± 0.2% and 20.3 ± 1.1% in deionized water. Deposition of two nonradioactive LaPO4 shells onto La(227Th)PO4 C NPs increased the retention of decay daughters to >99.75%. The toxicity of the nonradioactive LaPO4 C and C2S NP delivery platforms was examined in a mammalian breast cancer cell line, BT-474. No significant decrease in cell viability was observed for a monolayer of BT-474 cells for NP concentrations below 233.9 µg mL-1, however cell viability decreased below 60% when BT-474 spheroids were incubated with either LaPO4 C or C2S NPs at concentrations exceeding 29.2 µg mL-1. La(227Th)PO4 C2S NPs exhibit a high encapsulation and in vitro retention of radionuclides with limited contribution to cellular cytotoxicity for TAT applications.

Autophagy protects kidney from phosphate-induced mitochondrial injury.

Hyperphosphatemia is a common complication in patients with advanced chronic kidney disease (CKD) as well as an increased risk of cardiovascular mortality; however, the molecular mechanisms of phosphate-mediated kidney injury are largely unknown. Autophagy is a lysosomal degradation system, which plays protective roles against kidney diseases. Here, we studied the role of autophagy in kidney proximal tubular cells (PTECs) during phosphate overload. Temporal cessation of autophagy in drug-induced PTEC-specific autophagy-deficient mice that were fed high phosphate diet induced mild cytosolic swelling and an accumulation of SQSTM1/p62-and ubiquitin-positive protein aggregates in PTECs, indicating that phosphate overload requires enhanced autophagic activity for the degradation of increasing substrate. Morphological and biochemical analysis demonstrated that high phosphate activates mitophagy in PTECs in response to oxidative stress. PTEC-specific autophagy-deficient mice receiving heminephrectomy and autophagy-deficient cultured PTECs exhibited mitochondrial dysfunction, increased reactive oxygen species production, and reduced ATP production in response to phosphate overload, suggesting that high phosphate-induced autophagy counteracts mitochondrial injury and maintains cellular bioenergetics in PTECs. Thus, potentiating autophagic activity could be a therapeutic option for suppressing CKD progression during phosphate overload.

Diabetes, Diabetic Complications, and Phosphate Toxicity: A Scoping Review.

This article presents a scoping review and synthesis of research findings investigating the toxic cellular accumulation of dysregulated inorganic phosphate-phosphate toxicity-as a pathophysiological determinant of diabetes and diabetic complications. Phosphorus, an essential micronutrient, is closely linked to the cellular metabolism of glucose for energy production, and serum inorganic phosphate is often transported into cells along with glucose during insulin therapy. Mitochondrial dysfunction and apoptosis, endoplasmic reticulum stress, neuronal degeneration, and pancreatic cancer are associated with dysregulated levels of phosphate in diabetes. Ectopic calcification involving deposition of calcium-phosphate crystals is prevalent throughout diabetic complications, including vascular calcification, nephropathy, retinopathy, and bone disorders. A low-glycemic, low-phosphate dietary intervention is proposed for further investigations in the treatment and prevention of diabetes and related diabetic pathologies.

Therapeutic Effect of Iron Citrate in Blocking Calcium Deposition in High Pi-Calcified VSMC: Role of Autophagy and Apoptosis.

In chronic kidney disease (CKD), the first cause of mortality is cardiovascular disease induced mainly by vascular calcification (VC). Recently, iron-based phosphate binders have been proposed in advanced CKD to treat hyperphosphatemia. We studied the effect of iron citrate (iron) on the progression of calcification in high-phosphate (Pi) calcified VSMC. Iron arrested further calcification when added on days 7-15 in the presence of high Pi (1.30 ± 0.03 vs 0.61 ± 0.02; OD/mg protein; day 15; Pi vs Pi + Fe, p < 0.01). We next investigated apoptosis and autophagy. Adding iron to high-Pi-treated VSMC, on days 7-11, decreased apoptotic cell number (17.3 ± 2.6 vs 11.6 ± 1.6; Annexin V; % positive cells; day 11; Pi vs Pi + Fe; p < 0.05). The result was confirmed thorough analysis of apoptotic nuclei both in VSMCs and aortic rings treated on days 7-15 (3.8 ± 0.2 vs 2.3 ± 0.3 and 4.0 ± 0.3 vs 2.2 ± 0.2; apoptotic nuclei; arbitrary score; day 15; Pi vs Pi + Fe; VSMCs and aortic rings; p < 0.05). Studying the prosurvival axis GAS6/AXL, we found that iron treatment on days 9-14 counteracted protein high-Pi-stimulated down-regulation and induced its de novo synthesis. Moreover, iron added on days 9-15 potentiated autophagy, as detected by an increased number of autophagosomes with damaged mitochondria and an increase in autophagic flux. Highlighting the effect of iron on apoptosis, we demonstrated its action in blocking the H2O2-induced increase in calcification added both before high Pi treatment and when the calcification was already exacerbated. In conclusion, we demonstrate that iron arrests further high Pi-induced calcium deposition through an anti-apoptotic action and the induction of autophagy on established calcified VSMC.

Metal-Organocatalyst for Detoxification of Phosphorothioate Pesticides: Demonstration of Acetylcholine Esterase Activity.

In recent years, transition metal complexes have been developed for catalytical degradation of a phosphate ester bond, particularly in RNA and DNA; however, less consideration has been given for development of complexes for the degradation of a phosphorothioate bond, as they are the foremost used pesticides in the environment and are toxic to human beings. In this context, we have developed copper complexes of benzimidazolium based ligands for catalytical degradation of a series of organophosphates (parathion, paraoxon, methyl-parathion) at ambient conditions. The copper complexes (assigned as N1-N3) were characterized using single X-ray crystallography which revealed that all three complexes are mononuclear and distorted square planner in geometry. Further, the solution state studies of the prepared complexes were carried out using UV-visible absorption, fluorescence spectroscopy, and cyclic voltametry. The complexes N1 and N2 have benzimidazolium ionic liquid as base attached with two 2-mercapto-benzimidazole pods, whereas complex N3 contains a nonionic ligand. The synthesized copper complexes were evaluated for their catalytic activity for degradation of organophosphates. It is interesting that the complex containing the ionic ligand efficiently degrades phosphorothioate pesticides, whereas complex N3 was not found to be appropriate for degradation due to a weaker conversion rate. The organophosphate degradation studies were monitored by recording absorbance spectra of parathion in the presence of catalyst, i.e., copper complexes with respect to time. The parathion was hydrolyzed into para-nitrophenol and diethyl thiophosphate. Moreover, to analyze the inhibition activity of the pesticides toward acetylcholine esterase enzyme in the presence of prepared metal complexes, Ellman's assay was performed and revealed that, within 20 min, the inhibition of acetylcholine esterase enzyme decreases by up to 13%.

Prostaglandin E2 receptor EP2 mediates the effect of cyclooxygenase 2 on secondary parathyroid hyperplasia in end-stage renal disease.

BACKGROUND: Secondary hyperparathyroidism (SHPT) in patients with end-stage renal disease (ESRD) is characterized by hyperplasia of the parathyroid glands (PTGs), while the underlying mechanism is not completely understood. Previously we demonstrated a relationship between cyclooxygenase 2 (COX2) overexpression and parathyroid hyperplasia and here we investigate the role of COX2 downstream metabolic product prostaglandin E2 (PGE2) and its receptor EP2 in the pathogenesis of SHPT. METHODS: PTGs isolated from ESRD patients with advanced SHPT were used to test the expression of COX2-microsomal prostaglandin E synthase-1 (mPGES-1)-EP2 pathway. A diffuse proliferative section of the PTGs was used for tissue culture and treated with high phosphate (HPi) medium, COX2-PGE2-EP2 pathway inhibitors or agonists. EP2 short hairpin RNA (shRNA) lentivirus was locally applied to treat an SHPT rat model. RESULTS: In PTGs isolated from ESRD patients, enhanced immunoactivities of COX2, mPGES-1 and EP2 were observed. In primary cultured PTG tissues, HPi induced intact parathyroid hormone (iPTH) secretion, proliferating cell nuclear antigen (PCNA) expression and COX2 activity, while COX2 and EP2 inhibitors attenuated hyperparathyroidism promoted by HPi. Furthermore, PGE2 or EP2 agonist (butaprost) directly stimulated hyperparathyroidism, whereas EP2 receptor antagonist or cyclic adenosine monophosphate inhibitor attenuated the hyperparathyroidism promoted by PGE2 or butaprost. EP2 shRNA treatment significantly reduced excessive expressions of EP2 and PCNA in the PTGs of nephrectomy rats fed an HPi diet, diminished the size of PTGs and downregulated serum iPTH levels. CONCLUSIONS: The COX2 downstream PGE2 and its receptor EP2 may play an important role in HPi-induced parathyroid hyperplasia and may serve as a potential therapeutic target for SHPT in ESRD.

Synthesis and characterization of pyridoxine, nicotine and nicotinamide salts of dithiophosphoric acids as antibacterial agents against resistant wound infection.

The pyridine-derived biomolecules are of considerable interest in developing medicinal compounds with various specific activities. Novel ammonium salts of pyridoxine, (S)-(-)-nicotine and nicotinamide with O,O-diorganyl dithiophosphoric acids (DTPA) were synthesized and characterized. The complexation of chiral monoterpenyl DTPA, including (S)-(-)-menthyl, (R)-(+)-menthyl, (1R)-endo-(+)-fenchyl, (1S,2S,3S,5R)-(+)-isopinocampheolyl derivatives, with pyridoxine and nicotine provided effective antibacterial compounds 3a,b,e,f, and 5a,b,d,f with MIC values against Gram-positive bacteria as low as 10 µM (6 µg/mL). Two selected pyridoxine and nicotine salts based on menthyl DTPA 3a and 5a were similarly active against antibiotic-resistant bacteria from burn wounds including MRSA. The compounds had enhanced amphiphilic and hemolytic properties and effectively altered surface characteristics and matrix-secreting ability of P. aeroginosa and S. aureus. MBC/MIC ratios of 3a and 5a suggested the bactericidal mode of their action. Furthermore, the compounds exhibited moderate cytotoxicity towards human skin fibroblasts (IC50 = 48.6 and 57.6 µM, respectively, 72 h), encouraging their further investigation as potential antimicrobials against skin and wound infections.

Inorganic Polyphosphate Amplifies High Mobility Group Box 1-Mediated Von Willebrand Factor Release and Platelet String Formation on Endothelial Cells.

Objective- Inorganic polyphosphate (polyP) is known to modulate coagulation, inflammation, and metabolic pathways. It also amplifies inflammatory responses of HMGB1 (high mobility group box 1) in endothelial cells. The objective of this study was to evaluate the effect of polyP on von Willebrand factor (VWF) release from endothelial cells with or without HMGB1. Approach and Results- EA.hy926 endothelial cells were treated with different concentrations of polyP70 alone or in combination with different concentrations of HMGB1. VWF release was measured by an ELISA assay in the absence or presence of pharmacological inhibitors of the receptor for advanced glycation end products, P2Y1, and Ca2+. A flow chamber assay was used to monitor polyP70-mediated platelet recruitment and VWF-platelet string formation. PolyP70 and HMGB1 induced VWF release from endothelial cells by a concentration-dependent manner. PolyP70 amplified HMGB1-mediated VWF release from endothelial cells. This was also true if boiled platelet releasate was used as the source of polyP. Gene silencing or pharmacological inhibitors of receptor for advanced glycation end products, P2Y1, and Ca2+ significantly inhibited VWF release. PolyP70 and HMGB1 synergistically promoted VWF-platelet string formation in the flow chamber assay, which was inhibited by the anti-GPIbα (glycoprotein Ib alpha) antibody. VWF release by polyP70-HMGB1 complex required phosphorylation of Src and phospholipase C because inhibitors of Src, phospholipase C, and Ca2+ signaling significantly decreased VWF secretion. The polyP70-HMGB1 complex also increased angiopoietin-2 release, indicating that Weibel-Palade body exocytosis is involved in the VWF release. Conclusions- PolyP70 can promote thrombotic and inflammatory pathways by inducing VWF release and platelet string formation on endothelial cells.

Cisplatin nephrotoxicity as a model of chronic kidney disease.

Cisplatin (CP)-induced nephrotoxicity is widely accepted as a model for acute kidney injury (AKI). Although cisplatin-induced chronic kidney disease (CKD) in rodent has been reported, the role of phosphate in the cisplatin-induced CKD progression is not described. In this study, we gave a single peritoneal injection of CP followed by high (2%) phosphate diet for 20 weeks. High dose CP (20 mg/Kg) led to high mortality; whereas a lower dose (10 mg/Kg) resulted in a full spectrum of AKI with tubular necrosis, azotemia, and 0% mortality 7 days after CP injection. After consuming a high phosphate diet, mice developed CKD characterized by low creatinine clearance, interstitial fibrosis, hyperphosphatemia, high plasma PTH and FGF23, low plasma 1,25(OH)2 Vitamin D3 and αKlotho, and classic uremic cardiovasculopathy. The CP model was robust in demonstrating the effect of aging, sexual dimorphism, and dietary phosphate on AKI and also AKI-to-CKD progression. Finally, we used the CP-high phosphate model to examine previously validated methods of genetically manipulated high αKlotho and therapy using exogenous soluble αKlotho protein supplementation. In this CP CKD model, αKlotho mitigated CKD progression, improved mineral homeostasis, and ameliorated cardiovascular disease. Taken together, CP and high phosphate nephrotoxicity is a reproducible and technically very simple model for the study of AKI, AKI-to-CKD progression, extrarenal complications of CKD, and for evaluation of therapeutic efficacy.

Inhibitory effect of tartrate against phosphate-induced DJ-1 aggregation.

The DJ-1 protein engages in diverse cellular and pathological processes, including tumorigenesis, apoptosis, sperm fertilization, and the progression of Parkinson's disease (PD). The functional dimeric form of DJ-1 transforms into non-functional filamentous aggregates in an inorganic phosphate (Pi)-dependent manner in vitro. Here, we demonstrated that Pi and reactive oxygen species (ROS) induce DJ-1 aggregation in Neuro2A and SH-SY5Y cells. Remarkably, tartrate treatment significantly reduced Pi- and ROS-induced DJ-1 aggregation and restored Pi- and ROS-provoked cell death using quantitative data as mean±standard deviation, and statistics. Mechanistically, tartrate prevented DJ-1 aggregation via occupying the Pi-binding site. These findings revealed an unexpected physiological role of tartrate in the maintenance of DJ-1 function, and thus, a potential use as an inhibitor of DJ-1 aggregation.

Immunotoxicity of organophosphate flame retardants TPHP and TDCIPP on murine dendritic cells in vitro.

Organophosphate flame retardants (PFRs) are commonly used as alternatives for the banned polybrominated diphenyl ethers (PBDEs) and are ubiquitously detected in indoor dust. PFRs can be potentially hazardous to respiratory health via the inhalation of house dust. Dendritic cells (DCs) are crucial in the immunological defense against pathogens in the airways. In respiratory allergy however, an aberrant immune response is induced against innocuous proteins, like house dust mite allergens. In this study, we examined whether exposure to PFRs Triphenylphosphate (TPHP) and Tris(1,3-dichloroisopropyl) phosphate (TDCIPP) affected activation/maturation of DCs at steady state and during exposure to house dust mite allergens (HDM). Bone marrow-derived dendritic cells (BMDCs) were exposed to a concentration range of each PFR (0.1-100 µM) with or without HDM in vitro to analyze the effect on the expression of major histocompatibility complex class II (MHCII), co-stimulatory molecules and cytokine production. Concentrations of TPHP and TDCIPP of ≥50 µM were cytotoxic to BMDCs. At these cytotoxic concentrations, TPHP exposure induced an activated phenotype in steady state DCs, while HDM exposed DCs acquired a tolerogenic phenotype. In contrast, TDCIPP exposure had no effect at steady state DCs but suppressed the expression of MHCII, costimulatory molecules, and the IL-6 production in HDM exposed DCs. The cytotoxic concentrations induced the anti-oxidant enzyme hemeoxigenase-1, which is a marker for oxidative stress. These results demonstrate that PFRs can be immunotoxic for DCs and suggest the necessity to evaluate the effects on the immune system on a cellular level during the risk assessment of these alternative flame retardants.

Potential carcinogenicity predicted by computational toxicity evaluation of thiophosphate pesticides using QSTR/QSCarciAR model.

This study presents in silico prediction of toxic activities and carcinogenicity, represented by the potential carcinogenicity DSSTox/DBS, based on vector regression with a new Kernel activity, and correlating the predicted toxicity values through a QSAR model, namely: QSTR/QSCarciAR (quantitative structure toxicity relationship/quantitative structure carcinogenicity-activity relationship) described by 2D, 3D descriptors and biological descriptors. The results showed a connection between carcinogenicity (compared to the structure of a compound) and toxicity, as a basis for future studies on this subject, but each prediction is based on structurally similar compounds and the reactivation of the substructures of these compounds.