RESUMEN
Organophosphate flame retardants (OPFRs) pose the significant risks to the environment and human health and have become a serious public health issue. Tricresyl phosphates (TCPs), a group of aryl OPFRs, exhibit neurotoxicity and endocrine disrupting toxicity. However, the binding mechanisms between TCPs and human serum albumin (HSA) remain unknown. In this study, through fluorescence and ultraviolet-visible (UV-vis) absorption spectroscopy, molecular docking and molecular dynamics (MD), tri-para-cresyl phosphate (TpCP) was selected to explore potential interactions between HSA and TCPs. The results of the fluorescence spectroscopy demonstrated that a decrease in the fluorescence intensity of HSA and a blue shift were observed with the increasing concentrations of TpCP. The binding constant (Ka) was 2.575 × 104 L/mol, 4.701 × 104 L/mol, 5.684 × 104 L/mol and 9.482 × 104 L/mol at 293 K, 298 K, 303 K, and 310 K, respectively. The fluorescence process between HSA and TpCP involved a mix of static and dynamic quenching mechanism. The gibbs free energy (ΔG0) of HSA-TpCP system was -24.452 kJ/mol, -25.907 kJ/mol, -27.363 kJ/mol, and - 29.401 kJ/mol at 293 K, 298 K, 303 K, and 310 K, respectively, suggesting that the HSA-TpCP reaction was spontaneous. The enthalpy change (ΔH0) and thermodynamic entropy change (ΔS0) of the HSA-TpCP system were 60.83 kJ/mol and 291.08 J/(mol·>k), respectively, indicating that hydrophobic force was the major driving force in the HSA-TpCP complex. Furthermore, multispectral analysis also revealed that TpCP could alter the microenvironment of tryptophan residue and the secondary structure of HSA and bind with the active site I of HSA. Molecular docking and MD simulations confirmed that TpCP could spontaneously form a stable complex with HSA, which was consistent with the fluorescence experimental results. This study provides novel insights into the mechanisms of underlying the transportation and distribution of OPFRs in humans.
Asunto(s)
Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Espectrometría de Fluorescencia , Termodinámica , Humanos , Albúmina Sérica Humana/química , Albúmina Sérica Humana/metabolismo , Retardadores de Llama/metabolismo , Espectrofotometría Ultravioleta , Sitios de Unión , Tritolilfosfatos/química , Tritolilfosfatos/metabolismo , Albúmina Sérica/química , Albúmina Sérica/metabolismo , Enlace de HidrógenoRESUMEN
Organophosphate triesters (OPEs) have caused great concern as a class of emerging environmental contaminants due to their widespread use and their toxicity to organisms. However, the phototransformation behavior of OPE is still not fully understood, which is important for understanding their environmental fate. In the present study, the photodegradation of tricresyl phosphate (TCP), one of the most widely detected OPEs in aqueous environments, was investigated including the direct photolysis and in the presence of several natural water factors, NO2-, Fe3+ and humic acid. The degradation process followed the pseudo-first-order kinetics, with rate constant increasing slightly with increasing initial TCP concentration. The presence of NO2- and Fe3+ was observed to promote the photochemical loss of TCP, while humic acid played a negative role on TCP transformation. Electron spin resonance (EPR) analysis showed that carbon-centered radical was produced in the photolysis process of TCP, and hydroxyl radical contributed to the promotion of rate constant for Fe3+ and NO2-. Four photolysis products were tentatively identified by HPLC-LTQ-Orbitrap MS analysis, and the possible degradation pathways of TCP were proposed. These findings provide a meaningful reference for the fate and transformation of OPEs in natural water.
Asunto(s)
Fotólisis , Tritolilfosfatos/química , Contaminantes Químicos del Agua/química , Cloruros/química , Compuestos Férricos/química , Sustancias Húmicas/análisis , Radical Hidroxilo/análisis , Cinética , Nitrito de Sodio/química , Tritolilfosfatos/análisis , Tritolilfosfatos/efectos de la radiación , Agua/química , Contaminantes Químicos del Agua/análisis , Contaminantes Químicos del Agua/efectos de la radiaciónRESUMEN
The quality of aircraft cabin air has been an area of concern for several decades. Many investigations have linked the presence of organophosphates in air to Aerotoxic Syndrome with adverse symptoms reported by thousands of aircraft crew across the globe. Currently the source of organophosphates has been under debate, with studies pointing towards tricresylphosphates (TCP) in aircraft oil as the main source due to leaks in engine seals resulting in fumes entering the cabin. However, comparisons of oil and cabin samples have shown that the cabin samples contain a much higher proportion of ortho-substituted TCP than is commonly detected in oil. The aim of this experiment was to investigate the potential for palladium catalysts (present in aircraft air conditioning systems) to convert meta- and para-substituted TCP to produce ortho-substituted TCP through transisomerisation. This experiment was performed in a controlled laboratory setting aimed to represent the conditions likely to be experienced in aircraft. Samples were introduced to a stainless steel micro reactor tube containing the pelletized palladium catalyst using a HPLC pump with a 0.2â¯ml/min feed flow rate. The temperature maintained at 400⯰C over a period of 1â¯h and samples collected using a condensing vesicle. These were then diluted and transferred to a 2â¯mL vial for analysis by gas chromatography mass spectrometry. No evidence supporting the transisomerisation of tricresylphosphate was obtained. This indicates that more emphasis should be placed on identifying other potential sources of ortho substituted TCP.
Asunto(s)
Contaminación del Aire Interior , Aeronaves/normas , Paladio/química , Tritolilfosfatos/química , Cromatografía de Gases y Espectrometría de Masas , Gases/análisis , Gases/química , Humanos , Isomerismo , Organofosfatos/análisis , Organofosfatos/química , Tritolilfosfatos/análisisRESUMEN
Understanding the atmospheric fate of organophosphorus plasticizers is important for their environmental risk assessment. However, limited information is available at present. In this study, density functional theory (DFT) calculations were performed to investigate the transformation mechanism and kinetics of tri-p-cresyl phosphate (TpCP) initiated by OH. Results show that the initial reactions are dominated by H-abstraction and OH addition to form TpCP-radical, TpCP-OH adducts and aryl phosphodiester. The H-abstraction pathways are more favorable than the OH addition pathways. The TpCP-radical and TpCP-OH adducts can further react with O2 in the atmosphere to finally form benzaldehyde phosphate, hydroxylated TpCP and bicyclic radicals. Based on the transition state theory, the calculated rate constant (kOH) of TpCP with OH at Tâ¯=â¯298â¯K is 1.9â¯×â¯10-12 cm3molecule-1s-1 with an atmospheric lifetime of 4.2 days, which demonstrates that gaseous TpCP is atmospherically persistent. This study provides a comprehensive investigation of the OH-initiated oxidation of TpCP, which is useful for understanding its mechanism of transformation and evaluating the risk in atmospheric environment.
Asunto(s)
Contaminantes Atmosféricos/análisis , Atmósfera/química , Radical Hidroxilo/química , Plastificantes/análisis , Tritolilfosfatos/análisis , Contaminantes Atmosféricos/química , Cinética , Oxidación-Reducción , Plastificantes/química , Medición de Riesgo , Tritolilfosfatos/químicaRESUMEN
Phosphorus flame retardant tricresyl phosphate (TCP) adsorption on graphene nanomaterials from aqueous solutions was explored using batch and column modes. Comparative studies were performed regarding the kinetics and equilibrium of TCP adsorption on graphene oxide (GO) and graphene (G) in batch mode. The adsorption kinetics exhibited a rapid TCP uptake, and experimental data were well described by the pseudo-second-order kinetic model. Adsorption isotherm data of TCP on the two adsorbents displayed an improved TCP removal performance with increasing temperature at pH 5, while experimental data were well described by the Langmuir isotherm model with a maximum adsorption capacity of 87.7 mg·g-1 for G, and 30.7 mg·g-1 for GO) at 303 K. The thermodynamic parameters show that the adsorption reaction is a spontaneous and endothermic process. In addition, dynamic adsorption of TCP in a fixed G column confirmed a faster approach to breakthrough at high flow rate, high influent TCP concentration, and low filling height of adsorbent. Breakthrough data were successfully described by the Thomas and Yoon-Nelson models.
Asunto(s)
Grafito/química , Nanoestructuras , Tritolilfosfatos/química , Contaminantes Químicos del Agua/química , Purificación del Agua/métodos , Adsorción , Concentración de Iones de Hidrógeno , Cinética , Óxidos , Temperatura , Termodinámica , Agua/químicaRESUMEN
Tri-ortho-cresyl phosphate (ToCP) is an anti-wear, flame retardant additive used in industrial lubricants, hydraulic fluids and gasoline. The neurotoxic effects of ToCP arise from the liver-activated metabolite 2-(o-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (cresyl saligenin phosphate or CBDP), which inhibits esterase enzymes including butyrylcholinesterase (BChE). Following BChE adduction, CBDP undergoes hydrolysis to form the aged adduct ortho-cresyl phosphoserine (oCP-BChE), thus providing a biomarker of CBDP exposure. Previous studies have identified ToCP in aircraft cabin and cockpit air, but assessing human exposure has been hampered by the lack of a laboratory assay to confirm exposure. This work presents the development of an immunomagnetic-UHPLC-MS/MS method for the quantitation of unadducted BChE and the long-term CBDP biomarker, oCP-BChE, in human serum. The method has a reportable range from 2.0 ng/ml to 150 ng/ml, which is consistent with the sensitivity of methods used to detect organophosphorus nerve agent protein adducts. The assay demonstrated high intraday and interday accuracy (≥85%) and precision (RSD ≤ 15%) across the calibration range. The method was developed for future analyses of potential human exposure to CBDP. Analysis of human serum inhibited in vitro with CBDP demonstrated that the oCP-BChE adduct was stable for at least 72 h at 4, 22 and 37 °C. Compared to a previously reported assay, this method requires 75% less sample volume, reduces analysis time by a factor of 20 and demonstrates a threefold improvement in sensitivity. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.
Asunto(s)
Butirilcolinesterasa/sangre , Separación Inmunomagnética/métodos , Espectrometría de Masas en Tándem/métodos , Tritolilfosfatos/sangre , Butirilcolinesterasa/química , Cromatografía Líquida de Alta Presión/métodos , Humanos , Límite de Detección , Reproducibilidad de los Resultados , Tritolilfosfatos/químicaRESUMEN
Flight crews complain of illness following a fume event in aircraft. A chemical in jet engine oil, the neurotoxicant tri-o-cresyl phosphate, after metabolic activation to cresyl saligenin phosphate makes a covalent adduct on butyrylcholinesterase (BChE). We developed a mass spectrometry method for detection of the cresyl phosphate adduct on human BChE as an indicator of exposure. Monoclonal mAb2, whose amino acid sequence is provided, was crosslinked to cyanogen bromide-activated Sepharose 4B and used to immunopurify plasma BChE treated with cresyl saligenin phosphate. BChE was released with acetic acid, digested with pepsin, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MSMS) on the Triple TOF 5600 mass spectrometer. Peptide FGES198AGAAS with an added mass of 170 Da from cresyl phosphate on serine 198 (Ser198) was detected as parent ion 966.4 Da. When characteristic daughter ions were monitored in the MSMS spectrum, the limit of detection was 0.1% cresyl saligenin phosphate inhibited plasma BChE. This corresponds to 2×10(-9) g in 0.5 ml or 23×10(-15) moles of inhibited BChE in 0.5 ml of plasma. In conclusion, a sensitive assay for exposure to tri-o-cresyl phosphate was developed. Laboratories that plan to use this method are cautioned that a positive result gives no proof that tri-o-cresyl phosphate is toxic at low levels.
Asunto(s)
Contaminantes Atmosféricos/química , Aeronaves , Butirilcolinesterasa/sangre , Butirilcolinesterasa/química , Exposición a Riesgos Ambientales/análisis , Inmunoensayo/métodos , Tritolilfosfatos/química , Contaminantes Atmosféricos/toxicidad , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Butirilcolinesterasa/inmunología , Humanos , Límite de Detección , Espectrometría de Masas , Datos de Secuencia Molecular , Tritolilfosfatos/toxicidadRESUMEN
About 18% of fighter pilots complain of ill symptoms that begin during flight and persist for days. A possible source of toxicity is the air supplied through the on board oxygen generating system. The air passes through the jet engine before it is enriched for oxygen and breathed through an oxygen mask. While in the jet engine, the air can become contaminated with jet engine lubricating oil. A potentially toxic component in jet engine oil is tri-ortho-cresyl phosphate (TOCP), which is metabolically activated to the highly reactive cresyl saligenin phosphate. The cresyl saligenin phosphate reacts with butyrylcholinesterase (BChE) to make a covalent adduct on serine 198. The purpose of this work was to determine whether the blood of healthy, active-duty F-16 pilots has measurable levels of the cresyl phosphate adduct. BChE was immunopurified from 0.5ml plasma by binding to immobilized monoclonal mAb2. BChE protein was released with acetic acid, digested with pepsin and analyzed by LC-MS/MS on the 5600 Triple TOF mass spectrometer. Positive controls for quantifying the limit of detection were plasma samples containing known amounts of cresyl saligenin phosphate treated plasma. The cresyl phosphate adduct eluted at 31.3min with an observed parent ion mass of 966.4m/z and characteristic daughter ions 778.3, 673.3, and 602.3m/z. Control experiments demonstrated that as little as 0.1% of the 1-2µg BChE recovered from 0.5ml plasma could be detected as the cresyl phosphate adduct on peptide FGES198AGAAS. Mass spectrometry analysis of plasma from fifteen healthy F-16 pilots showed that none had evidence of exposure to TOCP. It was concluded that the on-board oxygen generating system, when operating properly, did not deliver tri-ortho-cresyl phosphate in the oxygen supply.
Asunto(s)
Aeronaves , Salud , Exposición Profesional/análisis , Oxígeno/química , Tritolilfosfatos/química , Tritolilfosfatos/toxicidad , Contaminantes Atmosféricos/química , Contaminantes Atmosféricos/toxicidad , Butirilcolinesterasa/sangre , Butirilcolinesterasa/metabolismo , Humanos , Isomerismo , Dispositivos de Protección RespiratoriaRESUMEN
Tri-o-cresyl-phosphate (TOCP) is a common additive in jet engine lubricants and hydraulic fluids suspected to have a role in aerotoxic syndrome in humans. TOCP is metabolized to cresyl saligenin phosphate (CBDP), a potent irreversible inhibitor of butyrylcholinesterase (BChE), a natural bioscavenger present in the bloodstream, and acetylcholinesterase (AChE), the off-switch at cholinergic synapses. Mechanistic details of cholinesterase (ChE) inhibition have, however, remained elusive. Also, the inhibition of AChE by CBDP is unexpected, from a structural standpoint, i.e., considering the narrowness of AChE active site and the bulkiness of CBDP. In the following, we report on kinetic X-ray crystallography experiments that provided 2.7-3.3 Å snapshots of the reaction of CBDP with mouse AChE and human BChE. The series of crystallographic snapshots reveals that AChE and BChE react with the opposite enantiomers and that an induced-fit rearrangement of Phe297 enlarges the active site of AChE upon CBDP binding. Mass spectrometry analysis of aging in either H(2)(16)O or H(2)(18)O furthermore allowed us to identify the inhibition steps, in which water molecules are involved, thus providing insights into the mechanistic details of inhibition. X-ray crystallography and mass spectrometry show the formation of an aged end product formed in both AChE and BChE that cannot be reactivated by current oxime-based therapeutics. Our study thus shows that only prophylactic and symptomatic treatments are viable to counter the inhibition of AChE and BChE by CBDP.
Asunto(s)
Acetilcolinesterasa/metabolismo , Butirilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/metabolismo , Compuestos Organofosforados/metabolismo , Tritolilfosfatos/metabolismo , Acetilcolinesterasa/química , Animales , Butirilcolinesterasa/química , Células CHO , Dominio Catalítico , Inhibidores de la Colinesterasa/química , Cricetinae , Cristalografía por Rayos X , Humanos , Espectrometría de Masas , Ratones , Simulación de Dinámica Molecular , Compuestos Organofosforados/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tritolilfosfatos/químicaRESUMEN
This paper summarises the results of voltammetric studies on the herbicide 3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea (Linuron), using a carbon paste electrode containing tricresyl phosphate (TCP-CPE) as liquid binder. The principal experimental conditions, such as the pH effect, investigated in Britton-Robinson buffer solutions (pH 2.0-7.0), the peak characteristics for the analyte of interest, or instrumental parameters for the differential pulse voltammetric mode were optimized for the method. As found out, the best electroanalytical performance of the TCP-CPE was achieved at pH 2.0, whereby the oxidation peak of Linuron appeared at ca. +1.3 V vs. SCE. The analytical procedure developed offers good linearity in the concentration range of 1.25-44.20 µg mL(-1) (1.77 × 10(-4)-5.05 × 10(-6) mol L(-1)), showing-for the first time-the applicability of the TCP-CPE for anodic oxidations in direct voltammetry (without accumulation). The method was then verified by determining Linuron in a spiked river water sample and a commercial formulation and the results obtained agreed well with those obtained by the reference HPLC/UV determination.
Asunto(s)
Carbono/química , Técnicas Electroquímicas/métodos , Herbicidas/análisis , Linurona/análisis , Tritolilfosfatos/química , Electrodos , Herbicidas/química , Concentración de Iones de Hidrógeno , Linurona/química , Reproducibilidad de los Resultados , Ríos/químicaRESUMEN
Over 1 billion pounds of organophosphorus (OP) chemicals are manufactured worldwide each year, including 70 million pounds of pesticides sprayed in the US. Current methods to monitor environmental and occupational exposures to OPs such as chlorpyrifos (CPS) have limitations, including low specificity and sensitivity, and short time windows for detection. Biomarkers for the OP tricresyl phosphate (TCP), which can contaminate bleed air from jet engines and cause an occupational exposure of commercial airline pilots, crewmembers and passengers, have not been identified. The aim of our work has been to identify, purify, and characterize new biomarkers of OP exposure. Butyrylcholinesterase (BChE) inhibition has been a standard for monitoring OP exposure. By identifying and characterizing molecular biomarkers with longer half-lives, we should be able to clinically detect TCP and OP insecticide exposure after longer durations of time than are currently possible. Acylpeptide hydrolase (APH) is a red blood cell (RBC) cytosolic serine proteinase that removes N-acetylated amino acids from peptides and cleaves oxidized proteins. Due to its properties, it is an excellent candidate for a biomarker of exposure. We have been able to purify APH and detect inhibition by both CPS and metabolites of TCP. The 120-day lifetime of the RBC offers a much longer window for detecting exposure. The OP-modified serine conjugate in the active site tryptic peptide has been characterized by mass spectrometry. This research uses functional proteomics and enzyme activities to identify and characterize useful biomarkers of neurotoxic environmental and occupational OP exposures.
Asunto(s)
Biomarcadores/metabolismo , Compuestos Organofosforados/toxicidad , Plaguicidas/toxicidad , Secuencia de Aminoácidos , Animales , Butirilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacología , Eritrocitos/metabolismo , Humanos , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Péptido Hidrolasas/análisis , Péptido Hidrolasas/metabolismo , Proteómica/métodos , Tritolilfosfatos/químicaRESUMEN
Tricresyl phosphate (TCP) is used as an anti-wear additive in aircraft turbine engine oil. Concerns about its toxicity are largely based on the tri-o-cresyl phosphate isomer content. However, the presence of other and more toxic isomers has been previously suggested. In this work, the structural isomers of TCP have been determined by two methods (experimental and semi-theoretical). First, the TCP isomers were separated by gas chromatography (GC) and identified by mass spectrometry (MS). Second, after base cleavage of TCP, GC was used to quantify the cresol precursors. These results were used to calculate the TCP isomer distribution based on the assumption of a statistical distribution of the TCP isomers. The results from the two determinations showed reasonable agreement for three of the four oils studied. The o-cresyl isomers were found to be present almost exclusively as the more toxic mono-o-cresyl isomers in the concentration range 13-150 mg/L. The ability to analyse for the mono-o-cresyl isomers allows the toxicity of TCP to be based on the latter isomers rather than on the less toxic tri-o-cresyl phosphate isomer.
Asunto(s)
Aeronaves , Cromatografía de Gases/métodos , Espectrometría de Masas/métodos , Petróleo/análisis , Tritolilfosfatos/análisis , Isomerismo , Estructura Molecular , Reproducibilidad de los Resultados , Tritolilfosfatos/químicaRESUMEN
Neurotoxicity of tricresyl phosphates (TCPs) and jet engine oil (JEO) containing TCPs were evaluated in studies conducted in both rat and hen. Results for currently produced samples ("conventional" and "low-toxicity") were compared with published findings on older samples to identify compositional changes and relate those changes to neurotoxic potential. Finally, a human risk assessment for exposure by oral ingestion of currently produced TCPs in JEO at 3% (JEO + 3%) was conducted. TCPs and certain other triaryl phosphates administered as single doses inhibited brain neuropathy target esterase (B-NTE; neurotoxic esterase) in the rat and the hen (hen 3.25 times as sensitive), and both species were deemed acceptable for initial screening purposes. Neither rat nor hen was sensitive enough to detect statistically significant inhibition of B-NTE after single doses of IEO + 3% "conventional" TCP. Subacute administration of 2 g/kg/d of JEO + 3% "conventional" TCP to the hen produced B-NTE inhibition (32%), which did not result in organophosphorus-induced delayed neurotoxicity (OPIDN). Subchronic administration of JEO + 3% TCP but not JEO + 1% TCP at 2 g/kg/d produced OPIDN. Thus, the threshold for OPIDN was between 20 and 60 mg "conventional" TCP/kg/d in JEO for 10 wk. The current "conventional" TCPs used in JEO and new "low-toxicity" TCPs now used in some JEO are synthesized from phenolic mixtures having reduced levels of ortho-cresol and ortho-xylenols resulting in TCPs of very high content of meta- and para-substituted phenyl moieties; this change in composition results in lower toxicity. The "conventional" TCPs still retain enough inhibitory activity to produce OPIDN, largely because of the presence of ortho-xylyl moieties; the "low-toxicity" TCPs are largely devoid of ortho substituents and have extremely low potential to cause OPIDN. The TCPs produced in the 1940s and 1950s were more than 400 times as toxic as the "low-toxicity" TCPs produced today. Analysis of the doses required to produce OPIDN in a subchronic hen study suggests that the minimum toxic dose of "conventional" TCP for producing OPIDN in a 70-kg person would be 280 mg/d, and for JEO containing 3% TCP, 9.4 g/d. Food products could be inadvertently contaminated with neat "conventional" TCP but it is unlikely that food such as cooking oil would be contaminated with enough JEO + 3% TCP to cause toxicity. Further, at the dosage required for neurotoxicity, it would be virtually impossible for a person to receive enough JEO + 3% TCP in the normal workplace (or in an aircraft) to cause such toxicity. There is no record of a JEO formulated with the modern "conventional" TCP causing human neurotoxicity.
Asunto(s)
Encéfalo/efectos de los fármacos , Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Colinesterasas/efectos de los fármacos , Aceites Combustibles/toxicidad , Neurotoxinas/toxicidad , Tritolilfosfatos/toxicidad , Administración Oral , Animales , Ataxia/inducido químicamente , Encéfalo/enzimología , Hidrolasas de Éster Carboxílico/efectos de los fármacos , Pollos , Colinesterasas/sangre , Femenino , Aceites Combustibles/análisis , Cromatografía de Gases y Espectrometría de Masas , Humanos , Masculino , Neurotoxinas/administración & dosificación , Neurotoxinas/química , Exposición Profesional/efectos adversos , Ratas , Ratas Long-Evans , Tritolilfosfatos/administración & dosificación , Tritolilfosfatos/químicaRESUMEN
Under extreme conditions of thermal degradation, a potent neurotoxin, trimethylolpropane phosphate (TMPP), may be formed by reaction of tricresyl phosphate (TCP, an anti-wear additive) and trimethylolpropane esters (TMPE) of carboxylic acids that may be contained in turbine engine lubricant formulations. The neurotoxin is only formed at extreme temperatures (350-700 degrees C), which has minimized concern for use of the synthetic lubricants under intended applications; however, since researchers are often not privy to the proprietary composition of such lubricants, extreme caution should be exercised in studies of or applications of synthetic lubricants degraded under extreme conditions.