Compound-specific isotope analysis of benzotriazole and its derivatives

Compound-specific isotope analysis (CSIA) is an important tool for the identification of contaminant sources and transformation pathways, but it is rarely applied to emerging aquatic micropollutants owing to a series of instrumental challenges. Using four different benzotriazole corrosion inhibitors and its derivatives as examples, we obtained evidence that formation of organometallic complexes of benzotriazoles with parts of the instrumentation impedes isotope analysis. Therefore, we propose two strategies for accurate $\delta^{13}$ C and $\delta^{15}$ N measurements of polar organic micropollutants by gas chromatography isotope ratio mass spectrometry (GC/IRMS). Our first approach avoids metallic components and uses a Ni/Pt reactor for benzotriazole combustion while the second is based on the coupling of online methylation to the established GC/IRMS setup. Method detection limits for on-column injection of benzotriazole, as well as its 1-CH $_{3}$ -, 4-CH $_{3}$ -, and 5-CH $_{3}$ -substituted species were 0.1-0.3mM and 0.1-1.0mM for δ13C and δ15N analysis respectively, corresponding to injected masses of 0.7-1.8 nmol C and 0.4-3.0 nmol N, respectively. The Ni/Pt reactor showed good precision and was very long-lived ( $>$ 1000 successful measurements). Coupling isotopic analysis to offline solid-phase extraction enabled benzotriazole-CSIA in tap water, wastewater treatment effluent, activated sludge, and in commercial dishwashing products. A comparison of $\delta ^{13}$ C and $\delta ^{15}$ N values from different benzotriazoles and benzotriazole derivatives, both from commercial standards and in dishwashing detergents, reveals the potential application of the proposed method for source apportionmen

Fate of drugs during wastewater treatment

This is the post-print version of the final paper published in TrAC Trends in Analytical Chemistry. The published article is available from the link below. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. Copyright @ 2013 Elsevier B.V.Recent trends in the determination of pharmaceutical drugs in wastewaters focus on the development of rapid multi-residue methods. This review addresses recent analytical trends in drug determination in environmental matrices used to facilitate fate studies. Analytical requirements for further fate evaluation and tertiary process selection and optimization are also discussed.EPSRC, Northumbrian Water, Anglian Water, Severn Trent Water, Yorkshire Water, and United Utilities

The Fanconi anemia pathway is required for efficient repair of stress-induced DNA damage in haematopoietic stem cells

Within regenerating tissues, aging is characterized by a progressive general deterioration of organ function, thought to be driven by the gradual depletion of functional adult stem cells. Although there are probably multifactorial mechanisms that result in compromized stem cell functionality with advancing age, the accumulation of DNA damage within the stem cell compartment is likely to make a major contribution to this process. However, the physiologic source of DNA damage within the different tissue specific stem cell compartments remains to be determined, as does the fate of stem cells exposed to such damage. Using the haematopoietic system as a model organ, we have recently shown that certain forms of physiologic stress, such as infection-associated inflammation and extensive blood loss, leads to the induction of biologically relevant levels of DNA damage in haematopoietic stem cells (HSCs) by dramatically increasing the proliferative index of this normally quiescent cell population.(1) We were also able to demonstrate that such stress-associated DNA damage was sufficient to completely deplete HSCs and promote severe aplastic anemia (SAA) in the Fanconi anemia (FA) knockout mouse model, which has compromized replication-associated DNA repair. In this “Extra Views” article, we extend this previous work to show that FA mice do not spontaneously develop a haematopoietic phenotype consistent with SAA, even at extreme old age. This suggests that HSC quiescence restricts the acquisition of DNA damage during aging and preserves the functional integrity of the stem cell pool. In line with this hypothesis, we provide an extended time course analysis of the response of FA knockout mice to chronic inflammatory stress and show that enforced HSC proliferation leads to a highly penetrant SAA phenotype, which closely resembles the progression of the disease in FA patients

Compound-specific isotope analysis of benzotriazole and its derivatives.

Compound-specific isotope analysis (CSIA) is an important tool for the identification of contaminant sources and transformation pathways, but it is rarely applied to emerging aquatic micropollutants owing to a series of instrumental challenges. Using four different benzotriazole corrosion inhibitors and its derivatives as examples, we obtained evidence that formation of organometallic complexes of benzotriazoles with parts of the instrumentation impedes isotope analysis. Therefore, we propose two strategies for accurate C and N measurements of polar organic micropollutants by gas chromatography isotope ratio mass spectrometry (GC/IRMS). Our first approach avoids metallic components and uses a Ni/Pt reactor for benzotriazole combustion while the second is based on the coupling of online methylation to the established GC/IRMS setup. Method detection limits for on-column injection of benzotriazole, as well as its 1-CH-, 4-CH-, and 5-CH-substituted species were 0.1-0.3 mM and 0.1-1.0 mM for delta C-13 and delta N-15 analysis respectively, corresponding to injected masses of 0.7-1.8 nmol C and 0.4-3.0 nmol N, respectively. The Ni/Pt reactor showed good precision and was very long-lived (1000 successful measurements). Coupling isotopic analysis to offline solid-phase extraction enabled benzotriazole-CSIA in tap water, wastewater treatment effluent, activated sludge, and in commercial dishwashing products. A comparison of C and N values from different benzotriazoles and benzotriazole derivatives, both from commercial standards and in dishwashing detergents, reveals the potential application of the proposed method for source apportionment

Input Dynamics and Fate in Surface Water of the Herbicide Metolachlor and of its Highly Mobile Transformation Product Metolachlor ESA

A large number of herbicide transformation products has been detected in surface waters and groundwaters of agricultural areas, often even in higher concentrations and more frequently than their parent compounds. However, their input dynamics and fate in surface waters are still rather poorly understood. This study compares the aquatic fate, concentration levels, and dynamics of the transformation product metolachlor ethanesulfonic acid (metolachlor ESA) and its parent compound metolachlor, an often-used corn herbicide. To this end, laboratory photolysis studies were combined with highly temporally resolved concentration measurements and lake mass balance modeling in the study area of Lake Greifensee (Switzerland). It is found that the two compounds show distinctly different concentration dynamics in the lake tributaries. Concentration−discharge relationships for metolachlor ESA in the main tributary showed a high baseflow concentration and increasing discharge dependence during harvest season, whereas baseflow concentrations of metolachlor were negligible and the discharge dependence was restricted to the period immediately following application. From this it was estimated that 70% of the yearly load of metolachlor ESA to the lake was due to groundwater recharge, whereas, for metolachlor, the bigger part of the load, 50−80%, stemmed from event-driven runoff. Lake mass balance modeling showed that the input dynamics of metolachlor and metolachlor ESA are reflected in their concentration dynamics in the lake’s epilimnion and that both compounds show a similar fate in the epilimnion of Lake Greifensee during the summer months with half-lives on the order of 100−200 days, attributable to photolysis and another loss process of similar magnitude, potentially biodegradation. The behavior of metolachlor ESA can likely be generalized to other persistent and highly mobile transformation products. In the future, this distinctly different behavior of mobile pesticide transformation products should find a more appropriate reflection in exposure models used in chemical risk assessment and in pesticide risk management

Loss of TM-Dependent PC-Activation Predisposes to Diabetic Nephropathy: Potential Role of Endothelial Apoptosis.

Abstract Increased levels of soluble thrombomodulin (TM) in patients with diabetes mellitus are considered a marker of endothelial damage. It is unknown whether the loss of endothelial TM-function contributes to the progression of vascular complications in diabetes mellitus. To address whether the loss of TM-dependent protein C (PC) activation contributes to diabetic complications such as diabetic nephropathy two animal models were employed: (1) TMPro mice, which have been previously described and carry a point mutation in the TM-gene (E404P), resulting in a loss of TM-dependent PC-activation, and (2) hPC mice, which carry a transgene resulting in the expression of a mutant “hyperactivatable” PC, which can be activated by thrombin in the absence of TM. The mutant PC could be captured from plasma samples of hPC mice and activated ex vivo by thrombin in the absence of TM. hPC mice had a prolonged bleeding time. Following induction of diabetes by streptozotocin TAT levels were increased in diabetic control (wild type) mice and to a larger extent in TMPro mice, but not in diabetic hPC mice. In comparison to diabetic control mice the kidney weight was increased in diabetic TMPro mice, but not in diabetic hPC mice. Albuminuria was increased in diabetic TMPro mice and reduced in diabetic hPC mice in comparison to diabetic control mice, indicating increased glomerular damage in TMPro mice and partial protection from glomerular damage in hPC mice. Consistently, using a histological score glomerular damage was more severe in diabetic TMPro mice in comparison to diabetic control mice, while diabetic hPC mice were protected. Preliminary data suggest that the observed changes are associated with increased apoptosis in glomeruli of diabetic TMPro mice. Using HUVECs we were able to establish that high glucose concentrations (30 mM) reduce TM-dependent PC activation. The reduced TM-dependent PC activation is associated with increased apoptosis. Glucose induced apoptosis in HUVECs is associated with an increased Bax/Bcl-2 ratio, increased translocation of Bax into mitochondria, and increased caspase-3 activation. Activated PC normalizes the Bax/Bcl-2 ratio, prevents translocation of Bax, and reduces caspase-3 activity. Further studies using TRAPs and inhibitory antibodies established that the antiapoptotic effect of aPC in glucose stressed endothelial cells is mediated through a Par-1 and EPCR-dependent mechanism. The current data strongly suggest that the loss of the endothelial TM-PC system is not just a marker of endothelial damage in diabetic patients, but rather contributes to the progression of diabetic vascular complications.</jats:p