Mild pentachlorophenol-mediated uncoupling of mitochondria depletes ATP but does not cause an oxidized redox state or dopaminergic neurodegeneration in Caenorhabditis elegans.

Aims: Mitochondrial dysfunction is implicated in several diseases, including neurological disorders such as Parkinson's disease. However, there is uncertainty about which of the many mechanisms by which mitochondrial function can be disrupted may lead to neurodegeneration. Pentachlorophenol (PCP) is an organic pollutant reported to cause mitochondrial dysfunction including oxidative stress and mitochondrial uncoupling. We investigated the effects of PCP exposure in Caenorhabditis elegans, including effects on mitochondria and dopaminergic neurons. We hypothesized that mild mitochondrial uncoupling by PCP would impair bioenergetics while decreasing oxidative stress, and therefore would not cause dopaminergic neurodegeneration. Results: A 48-hour developmental exposure to PCP causing mild growth delay (∼10 % decrease in growth during 48 h, covering all larval stages) reduced whole-organism ATP content > 50 %, and spare respiratory capacity âˆ¼ 30 %. Proton leak was also markedly increased. These findings suggest a main toxic mechanism of mitochondrial uncoupling rather than oxidative stress, which was further supported by a concomitant shift toward a more reduced cellular redox state measured at the whole organism level. However, exposure to PCP did not cause dopaminergic neurodegeneration, nor did it sensitize animals to a neurotoxic challenge with 6-hydroxydopamine. Whole-organism uptake and PCP metabolism measurements revealed low overall uptake of PCP in our experimental conditions (50 µM PCP in the liquid exposure medium resulted in organismal concentrations of < 0.25 µM), and no measurable production of the oxidative metabolites tetra-1,4-benzoquinone and tetrachloro-p-hydroquinone. Innovation: This study provides new insights into the mechanistic interplay between mitochondrial uncoupling, oxidative stress, and neurodegeneration in C. elegans. These findings support the premise of mild uncoupling-mediated neuroprotection, but are inconsistent with proposed broad "mitochondrial dysfunction"-mediated neurodegeneration models, and highlight the utility of the C. elegans model for studying mitochondrial and neurotoxicity. Conclusions: Developmental exposure to pentachlorophenol causes gross toxicological effects (growth delay and arrest) at high levels. At a lower level of exposure, still causing mild growth delay, we observed mitochondrial dysfunction including uncoupling and decreased ATP levels. However, this was associated with a more-reduced cellular redox tone and did not exacerbate dopaminergic neurotoxicity of 6-hydroxydopamine, instead trending toward protection. These findings may be informative of efforts to define nuanced mitochondrial dysfunction-related adverse outcome pathways that will differ depending on the form of initial mitochondrial toxicity.

The Affinity of Brominated Phenolic Compounds for Human and Zebrafish Thyroid Receptor ß: Influence of Chemical Structure.

Brominated phenolic compounds (BPCs) are found in the environment, and in human and wildlife tissues, and some are considered to have endocrine disrupting activities. The goal of this study was to determine how structural differences of 3 BPC classes impact binding affinities for the thyroid receptor beta (TRß) in humans and zebrafish. BPC classes included halogenated bisphenol A derivatives, halogenated oxidative transformation products of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), and brominated phenols. Affinities were assessed using recombinant TRß protein in competitive binding assays with 125I-triiodothyronine (125I-T3) as the radioligand. Zebrafish and human TRß displayed similar binding affinities for T3 (Ki = 0.40 and 0.49 nM) and thyroxine (T4, Ki = 6.7 and 6.8 nM). TRß affinity increased with increasing halogen mass and atomic radius for both species, with the iodinated compounds having the highest affinity within their compound classes. Increasing halogen mass and radius increases the molecular weight, volume, and hydrophobicity of a compound, which are all highly correlated with increasing affinity. TRß affinity also increased with the degree of halogenation for both species. Human TRß displayed higher binding affinities for the halogenate bisphenol A compounds, whereas zebrafish TRß displayed higher affinities for 2,4,6-trichlorophenol and 2,4,6-trifluorophenol. Observed species differences may be related to amino acid differences within the ligand binding domains. Overall, structural variations impact TRß affinities in a similar manner, supporting the use of zebrafish as a model for TRß disruption. Further studies are necessary to investigate how the identified structural modifications impact downstream receptor activities and potential in vivo effects.

Nonionic Ethoxylated Surfactants Induce Adipogenesis in 3T3-L1 Cells.

Recent studies have demonstrated that a number of environmental contaminants can act as metabolic disruptors and modulate metabolic function both in vitro and in vivo. 3T3-L1 mouse preadipocytes are commonly utilized to assess perturbations to adipogenesis, providing insight into environmental contaminants that may impact in vivo metabolic health. This study sought to assess whether various alkylphenol ethoxylates and alcohol ethoxylates (APEOs and AEOs, respectively), ubiquitous contaminants used in common household products, could disrupt metabolic health. 3T3-L1 cells were exposed to increasing concentrations of individual ethoxylated surfactants and base hydrophobes, and assessed for triglyceride accumulation (relative to a rosiglitazone-induced maximum response) and preadipocyte proliferation (relative to a differentiated vehicle control). We report herein that nonionic APEOs and AEOs promoted triglyceride accumulation and/or preadipocyte proliferation in 3T3-L1 cells at concentrations from 0.1 to 10 µM. Activity appeared to be an effect of the polyethoxylate chain length, as the alkylphenol/alcohol hydrophobes exhibited minimal or no adipogenic activity. In addition, nonylphenol ethoxylates (NPEO) of various ethoxylate chain lengths exhibited biphasic adipogenic activity, with increasing triglyceride accumulation and preadipocyte proliferation from NPEO (0, average ethoxylate number) through NPEO (4), and then decreasing activities from NPEO (4) through NPEO (20). Our results suggest potential metabolic impacts of these compounds at environmentally relevant concentrations, demonstrating a need to further assess molecular mechanisms and better characterize environmental concentrations of the specific AEOs and APEOs that are inducing the greatest degree of adipogenic activity herein.

The Association Between Geographic Density of Infectious Disease Physicians and Limb Preservation in Patients With Diabetic Foot Ulcers.

BACKGROUND: Avoiding major (above-ankle) amputation in patients with diabetic foot ulcers is best accomplished by multidisciplinary care teams with access to infectious disease specialists. However, access to infectious disease physicians is partially influenced by geography. We assessed the effect of living in a hospital referral region with a high geographic density of infectious disease physicians on major amputation for patients with diabetic foot ulcers. We studied geographic density, rather than infectious disease consultation, to capture both the direct and indirect (eg, informal consultation) effects of access to these providers on major amputation. METHODS: We used a national retrospective cohort of 56440 Medicare enrollees with incident diabetic foot ulcers. Cox proportional hazard models were used to assess the relationship between infectious disease physician density and major amputation, while controlling for patient demographics, comorbidities, and ulcer severity. RESULTS: Living in hospital referral regions with high geographic density of infectious disease physicians was associated with a reduced risk of major amputation after controlling for demographics, comorbidities, and ulcer severity (hazard ratio, .83; 95% confidence interval, .75-.91; P < .001). The relationship between the geographic density of infectious disease physicians and major amputation was not different based on ulcer severity and was maintained when adjusting for socioeconomic factors and modeling amputation-free survival. CONCLUSIONS: Infectious disease physicians may play an important role in limb salvage. Future studies should explore whether improved access to infectious disease physicians results in fewer major amputations.

Overcoming the Challenges of Unstructured Data in Multisite, Electronic Medical Record-based Abstraction.

BACKGROUND: Unstructured data encountered during retrospective electronic medical record (EMR) abstraction has routinely been identified as challenging to reliably abstract, as these data are often recorded as free text, without limitations to format or structure. There is increased interest in reliably abstracting this type of data given its prominent role in care coordination and communication, yet limited methodological guidance exists. OBJECTIVES: As standard abstraction approaches resulted in substandard data reliability for unstructured data elements collected as part of a multisite, retrospective EMR study of hospital discharge communication quality, our goal was to develop, apply and examine the utility of a phase-based approach to reliably abstract unstructured data. This approach is examined using the specific example of discharge communication for warfarin management. RESEARCH DESIGN: We adopted a "fit-for-use" framework to guide the development and evaluation of abstraction methods using a 4-step, phase-based approach including (1) team building; (2) identification of challenges; (3) adaptation of abstraction methods; and (4) systematic data quality monitoring. MEASURES: Unstructured data elements were the focus of this study, including elements communicating steps in warfarin management (eg, warfarin initiation) and medical follow-up (eg, timeframe for follow-up). RESULTS: After implementation of the phase-based approach, interrater reliability for all unstructured data elements demonstrated κ's of ≥0.89-an average increase of +0.25 for each unstructured data element. CONCLUSIONS: As compared with standard abstraction methodologies, this phase-based approach was more time intensive, but did markedly increase abstraction reliability for unstructured data elements within multisite EMR documentation.

Detection of pharmaceuticals and personal care products (PPCPs) in near-shore habitats of southern Lake Michigan.

Pharmaceuticals and personal care products (PPCPs) have been documented throughout the United States freshwaters but research has focused largely on lotic systems. Because PPCPs are designed to have a physiological effect, it is likely that they may also influence aquatic organisms. Thus, PPCPs may negatively impact aquatic ecosystems. The objectives of this research were to quantify PPCP abundance in near-shore habitats of southern Lake Michigan and identify factors related to PPCP abundance. Stratified sampling was conducted seasonally at four southern Lake Michigan sites. All sites and depths had measurable PPCP concentrations, with mean individual compound concentrations of acetaminophen (5.36 ng/L), caffeine (31.0 ng/L), carbamazepine (2.23 ng/L), cotinine (4.03 ng/L), gemfibrozil (7.03 ng/L), ibuprofen (7.88 ng/L), lincomycin (4.28 ng/L), naproxen (6.32 ng/L), paraxanthine (1,7-dimethylxanthine; 46.2 ng/L), sulfadimethoxine (0.94 ng/L), sulfamerazine (0.92 ng/L), sulfamethazine (0.92 ng/L), sulfamethoxazole (26.0 ng/L), sulfathiazole (0.92 ng/L), triclocarban (5.72 ng/L), trimethoprim (5.15 ng/L), and tylosin (3.75 ng/L). Concentrations of PPCPs varied significantly among sampling times and locations (river mouth vs offshore), with statistical interactions between the main effects of site and time as well as time and location. Concentrations of PPCPs did not differ with site or depth. Temperature, total carbon, total dissolved solids, dissolved oxygen, and ammonium concentrations were related to total pharmaceutical concentrations. These data indicate that PPCPs are ubiquitous and persistent in southern Lake Michigan, potentially posing harmful effects to aquatic organisms.

Using a triple-quadrupole mass spectrometer in accurate mass mode and an ion correlation program to identify compounds.

Atomic masses and isotopic abundances are independent and complementary properties for discriminating among ion compositions. The number of possible ion compositions is greatly reduced by accurately measuring exact masses of monoisotopic ions and the relative isotopic abundances (RIAs) of the ions greater in mass by +1 Da and +2 Da. When both properties are measured, a mass error limit of 6-10 mDa (< 31 ppm at 320 Da) and an RIA error limit of 10% are generally adequate for determining unique ion compositions for precursor and fragment ions produced from small molecules (less than 320 Da in this study). 'Inherent interferences', i.e., mass peaks seen in the product ion mass spectrum of the monoisotopic [M+H]+ ion of an analyte that are -2, -1, +1, or +2 Da different in mass from monoisotopic fragment ion masses, distort measured RIAs. This problem is overcome using an ion correlation program to compare the numbers of atoms of each element in a precursor ion to the sum of those in each fragment ion and its corresponding neutral loss. Synergy occurs when accurate measurement of only one pair of +1 Da and +2 Da RIAs for the precursor ion or a fragment ion rejects all but one possible ion composition for that ion, thereby indirectly rejecting all but one fragment ion-neutral loss combination for other exact masses. A triple-quadrupole mass spectrometer with accurate mass capability, using atmospheric pressure chemical ionization (APCI), was used to measure masses and RIAs of precursor and fragment ions. Nine chemicals were investigated as simulated unknowns. Mass accuracy and RIA accuracy were sufficient to determine unique compositions for all precursor ions and all but two of 40 fragment ions, and the two corresponding neutral losses. Interrogation of the chemical literature provided between one and three possible compounds for each of the nine analytes. This approach for identifying compounds compensates for the lack of commercial ESI and APCI mass spectral libraries, which precludes making tentative identifications based on spectral matches.

Use of artificial neural networks for the accurate prediction of peptide liquid chromatography elution times in proteome analyses.

The use of artificial neural networks (ANNs) is described for predicting the reversed-phase liquid chromatography retention times of peptides enzymatically digested from proteome-wide proteins. To enable the accurate comparison of the numerous LC/MS data sets, a genetic algorithm was developed to normalize the peptide retention data into a range (from 0 to 1), improving the peptide elution time reproducibility to approximately 1%. The network developed in this study was based on amino acid residue composition and consists of 20 input nodes, 2 hidden nodes, and 1 output node. A data set of approximately 7000 confidently identified peptides from the microorganism Deinococcus radiodurans was used for the training of the ANN. The ANN was then used to predict the elution times for another set of 5200 peptides tentatively identified by MS/MS from a different microorganism (Shewanella oneidensis). The model was found to predict the elution times of peptides with up to 54 amino acid residues (the longest peptide identified after tryptic digestion of S. oneidensis) with an average accuracy of approximately 3%. This predictive capability was then used to distinguish with high confidence isobar peptides otherwise indistinguishable by accurate mass measurements as well as to uncover peptide misidentifications. Thus, integration of ANN peptide elution time prediction in the proteomic research will increase both the number of protein identifications and their confidence.