A perspective on In vitro developmental neurotoxicity test assay results: An expert panel review.

For decades, there has been increasing concern about the potential developmental neurotoxicity (DNT) associated with chemicals. Regulatory agencies have historically utilized standardized in vivo testing to evaluate DNT. Owing to considerations including higher-throughput screening for DNT, reduction in animal use, and potential cost efficiencies, the development of alternative new approach methods (NAMs) occurred; specifically, the advent of the DNT in vitro test battery (DNT IVB). SciPinion convened an expert panel to address specific questions related to the interpretation of in vitro DNT test data. The consensus of the expert panel was that the DNT IVB might be used during initial screening, but it is not presently a complete or surrogate approach to determine whether a chemical is a DNT in humans. By itself, the DNT IVB does not have the ability to capture nuances and complexity of the developing nervous system and associated outcomes including behavioral ontogeny, motor activity, sensory function, and learning/memory. Presently, such developmental landmarks cannot be adequately assessed in the DNT IVB or by other NAMs. The expert panel (all who serve as co-authors of this review) recommended that additional data generation and validation is required before the DNT IVB can be considered for application within global regulatory frameworks for decision-making.

Grouping of PFAS for human health risk assessment: Findings from an independent panel of experts.

An expert panel was convened to provide insight and guidance on per- and polyfluoroalkyl substances (PFAS) grouping for the purposes of protecting human health from drinking water exposures, and how risks to PFAS mixtures should be assessed. These questions were addressed through multiple rounds of blind, independent responses to charge questions, and review and comments on co-panelists responses. The experts agreed that the lack of consistent interpretations of human health risk for well-studied PFAS and the lack of information for the vast majority of PFAS present significant challenges for any mixtures risk assessment approach. Most experts agreed that "all PFAS" should not be grouped together, persistence alone is not sufficient for grouping PFAS for the purposes of assessing human health risk, and that the definition of appropriate subgroups can only be defined on a case-by-case manner. Most panelists agreed that it is inappropriate to assume equal toxicity/potency across the diverse class of PFAS. A tiered approach combining multiple lines of evidence was presented as a possible viable means for addressing PFAS that lack analytical and/or toxicological studies. Most PFAS risk assessments will need to employ assumptions that are more likely to overestimate risk than to underestimate risk, given the choice of assumptions regarding dose-response model, uncertainty factors, and exposure information.

Glucose challenge test screening for prediabetes and early diabetes.

AIMS: To test the hypothesis that a 50-g oral glucose challenge test with 1-h glucose measurement would have superior performance compared with other opportunistic screening methods. METHODS: In this prospective study in a Veterans Health Administration primary care clinic, the following test performances, measured by area under receiver-operating characteristic curves, were compared: 50-g oral glucose challenge test; random glucose; and HbA1c level, using a 75-g oral glucose tolerance test as the 'gold standard'. RESULTS: The study population was comprised of 1535 people (mean age 56 years, BMI 30.3 kg/m2 , 94% men, 74% black). By oral glucose tolerance test criteria, diabetes was present in 10% and high-risk prediabetes was present in 22% of participants. The plasma glucose challenge test provided area under receiver-operating characteristic curves of 0.85 (95% CI 0.78-0.91) to detect diabetes and 0.76 (95% CI 0.72-0.80) to detect high-risk dysglycaemia (diabetes or high-risk prediabetes), while area under receiver-operating characteristic curves for the capillary glucose challenge test were 0.82 (95% CI 0.75-0.89) and 0.73 (95% CI 0.69-0.77) for diabetes and high-risk dysglycaemia, respectively. Random glucose performed less well [plasma: 0.76 (95% CI 0.69-0.82) and 0.66 (95% CI 0.62-0.71), respectively; capillary: 0.72 (95% CI 0.65-0.80) and 0.64 (95% CI 0.59-0.68), respectively], and HbA1c performed even less well [0.67 (95% CI 0.57-0.76) and 0.63 (95% CI 0.58-0.68), respectively]. The cost of identifying one case of high-risk dysglycaemia with a plasma glucose challenge test would be $42 from a Veterans Health Administration perspective, and $55 from a US Medicare perspective. CONCLUSIONS: Glucose challenge test screening, followed, if abnormal, by an oral glucose tolerance test, would be convenient and more accurate than other opportunistic tests. Use of glucose challenge test screening could improve management by permitting earlier therapy.

Survival outcomes in patients with early stage, resected pancreatic cancer - a comparison of gemcitabine- and 5-fluorouracil-based chemotherapy and chemoradiation regimens.

PURPOSE: We conducted a comparative survival analysis between patients with resected pancreatic cancer who received adjuvant treatment with either gemcitabine- or 5-fluorouracil-based chemotherapy and chemoradiation regimens. PATIENTS AND METHODS: The Surveillance, Epidemiology and End Results (SEER)-Medicare database was used to identify patients with pancreatic cancer diagnosed from 1998 to 2005 who received curative surgery and adjuvant chemotherapy with either 5-fluorouracil or gemcitabine. These groups were subdivided by treatment with radiotherapy. Patients were followed until death, study end-point or a maximum of 5 years after diagnosis. RESULTS: Three hundred and fifty-nine patients received 5-fluorouracil and 346 received gemcitabine. Compared with chemoradiation with 5-fluorouracil, outcomes for patients who received chemoradiation with gemcitabine did not differ. Patients who received gemcitabine without radiation had increased hazards (poorly differentiated tumours: HR = 1.50, p = 0.01; moderately differentiated tumours, HR = 1.28, p = 0.11). However, outcomes of patients who received 5-fluorouracil without radiation varied with tumour grade. In moderately differentiated tumours, patients had better outcomes with 5-fluorouracil when compared with chemoradiation with 5-fluorouracil (HR = 0.42, p = 0.02). In poorly differentiated tumours, the opposite was true (HR 2.10, p = 0.09). CONCLUSION: Patients with low-grade resected pancreatic cancer may have better outcomes with 5-fluorouracil-based chemotherapy without radiation when compared with 5-fluorouracil with radiation.

An Fe2IVO2 diamond core structure for the key intermediate Q of methane monooxygenase.

A new paradigm for oxygen activation is required for enzymes such as methane monooxygenase (MMO), for which catalysis depends on a nonheme diiron center instead of the more familiar Fe-porphyrin cofactor. On the basis of precedents from synthetic diiron complexes, a high-valent Fe2(micro-O)2 diamond core has been proposed as the key oxidizing species for MMO and other nonheme diiron enzymes such as ribonucleotide reductase and fatty acid desaturase. The presence of a single short Fe-O bond (1.77 angstroms) per Fe atom and an Fe-Fe distance of 2.46 angstroms in MMO reaction intermediate Q, obtained from extended x-ray absorption fine structure and Mössbauer analysis, provides spectroscopic evidence that the diiron center in Q has an Fe2IVO2 diamond core.

Occupational blood exposure among unlicensed home care workers and home care registered nurses: are they protected?

BACKGROUND: Little is known about the risk of blood exposure among personnel providing care to individual patients residing at home. The objective of this study was to document and compare blood exposure risks among unlicensed home care personal care assistants (PCAs) and home care registered nurses (RNs). METHODS: PCAs self-completed surveys regarding blood and body fluid (BBF) contact in group settings (n = 980), while RNs completed mailed surveys (n = 794). RESULTS: PCAs experience BBF contact in the course of providing care for home-based clients at a rate approximately 1/3 the rate experienced by RNs providing home care (8.1 and 26.7 per 100 full time equivalent (FTE), respectively), and the majority of PCA contact episodes did not involve direct sharps handling. However, for PCAs who performed work activities such as handling sharps and changing wound dressings, activities much more frequently performed by RNs, PCAs were at increased risk of injury when compared with RNs (OR = 7.4 vs. 1.4) and (OR = 6.3 vs. 2.5), respectively. CONCLUSION: Both PCAs and RNs reported exposures to sharps, blood, and body fluids in the home setting at rates that warrant additional training, prevention, and protection. PCAs appear to be at increased risk of injury when performing nursing-related activities for which they are inexperienced and/or lack training. Further efforts are needed to protect home care workers from blood exposure, namely by assuring coverage and enforcement of the Occupational Safety and Health Administration (OSHA) Bloodborne Pathogen Standard [Occupational Safety and Health Administration. 1993. Frequently Asked Questions Concerning the Bloodborne Pathogens Standard. Available at: http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=INTERPRETATIONS &p_id=21010#Scope. Accessed May 30, 2008].

Covariation of human microsomal protein per gram of liver with age: absence of influence of operator and sample storage may justify interlaboratory data pooling.

Scaling of metabolic clearance values from liver microsomal data or recombinantly expressed cytochrome P450 enzymes to predict human hepatic clearance requires knowledge of the amount of microsomal protein per gram of liver (MPPGL). Identification of physiological covariates of MPPGL requires analysis of values from large diverse populations, which necessitates pooling of data from numerous sources. To ensure compatibility between results obtained within and between studies, the impact of interoperator differences and sample storage on values of MPPGL was investigated. With use of triplicate samples from one liver (HL86), no statistically significant difference was detected between values of MPPGL prepared from samples stored at -80 degrees C (23.5 +/- 1.2 mg g(-1)) and those determined using fresh tissue (21.9 +/- 0.3 mg g(-1)). Although there was a significant difference in the yield of microsomal protein obtained from another liver sample (HL43) by three different operators (17 +/- 1, 19 +/- 2, and 24 +/- 1 mg g(-1); p = 0.004, analysis of variance), no difference was observed in the estimated MPPGL after application of appropriate correction factors for each operator (28 +/- 1, 30 +/- 5, and 31 +/- 4 mg g(-1)). The result provided justification for pooling reported values of MPPGL for use in covariate analysis. Investigation of the relationship between age and MPPGL provided preliminary evidence that MPPGL values increase from birth to a maximum of 40 mg g(-1) [95% confidence interval for the geometric mean (95% CI mean(geo)): 37-43 mg g(-1) at approximately 28 years followed by a gradual decrease in older age (mean of 29 mg g(-1) at 65 years; 95% CI mean(geo): 27-32 mg g(-1)). Accordingly, appropriate age-adjusted scaling factors should be used in extrapolating in vitro clearance values to clinical studies.

Molecular architecture and conformational flexibility of human RNA polymerase II.

Transcription by RNA polymerase II (RNAPII) is a central process in eukaryotic gene regulation. While atomic details exist for the yeast RNAPII, characterization of the human complex lags behind, mostly due to the inability to obtain large quantities of purified material. Although the complexes have the same protein composition and high sequence similarity, understanding of transcription and of transcription-coupled DNA repair (TCR) in humans will require the use of human proteins in structural studies. We have used cryo-electron microscopy, image reconstruction, and variance analysis to characterize the structure and dynamics of human RNAPII (hRNAPII). Our studies show that hRNAPII in solution parallels the conformational flexibility of the yeast structures crystallized in different states but also illustrate a more extensive conformational range with potential biological significance. This hRNAPII study will serve as a structural platform to build up higher-order transcription and TCR complexes and to gain information that may be unique to the human RNAPII system.

Mechanistic insights into C-H activation from radical clock chemistry: oxidation of substituted methylcyclopropanes catalyzed by soluble methane monooxygenase from Methylosinus trichosporium OB3b.

The soluble methane monooxygenase (MMO) system isolated from Methylosinus trichosporium OB3b catalyzes the adventitious oxidation of alkyl substituted methylcyclopropanes. If the chemical mechanism of C-H activation by MMO involves formation of a radical or carbocation intermediate at the methyl C-H of these 'radical clock' substrates, then cyclopropyl ring opened alcohols may appear in the product mixture due to rearrangement of the intermediate. The lifetime of radical intermediates can be determined from known rearrangement rate constants, k(r). Rearrangement was observed during the oxidation of 1,1,2,2-tetramethylcyclopropane (k(r)=1.7-17. 5x10(8) s(-1), 30 degrees C) but not for cis- or trans-1, 2-dimethylcyclopropane (k(r)=1.2-6.4x10(8) s(-1), 30 degrees C) or the very fast radical clock, trans-2-phenylmethylcyclopropane (k(r)=3.4x10(11) s(-1), 30 degrees C). The results show that the occurrence of rearranged products fails to correlate with either the chemical nature of the C-H bond being broken, which is very similar for all of the methylcyclopropanes studied here, or the magnitude of the radical k(r) value. This study suggests that the steric properties of the substrate play an important role in determining the outcome of the reaction. Substrates with bulky substituents near the C-H bond that is attacked appear to yield intermediates with sufficient lifetimes to rearrange. In contrast, substrates with less steric bulk are postulated to be able to approach the reactive oxygen species in the MMO active site more closely so that intermediates are either rapidly quenched or undergo subsequent interaction with the dinuclear iron cluster of MMO that prevents rearrangement.

Protocatechuate 3,4-dioxygenase. Inhibitor studies and mechanistic implications.

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) from Pseudomonas aeruginosa catalyzes the cleavage of 3,4-dihydroxybenzoate (protocatechuate) into beta-carboxy-cis,cis-muconate. The inhibition constants, Ki, of a series of substrate analogues were measured in order to assess the relative importance of the various functional groups on the substrate. Though important for binding, the carboxylate group is not essential for activity. Compounds with para hydroxy groups are better inhibitors than their meta isomers. Our studies of the enzyme-inhibitor complexes indicate that the 4-OH group of the substrate binds to the active-site iron. Taken together, Mössbauer, EPR, and kinetic data suggest a mechanism where substrate reaction with oxygen is preceded by metal activation of substrate.

The magnetic susceptibility of reduced cytochrome P-450-cam.

The primary electron acceptor of Photosystem II has a midpoint oxidation-reduction potential of +95 mV at pH 7.0 in Photosystem II chloroplast fragments prepared by digitonin treatment. The midpoint potential of the acceptor has a pH dependence of -60 mV/pH unit. At concentrations that inhibit oxygen evolution, o-phenanthroline shifts the midpoint potential of the primary acceptor by +70 mV. The shifted potential retains the same dependence on pH. The effect of o-phenanthroline suggests that it interacts directly with the primary electron acceptor of Photosystem II in a manner similar to that reported previously for the primary electron acceptor in purple photosynthetic bacteria.

Mössbauer and EPR spectroscopy of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) from Pseudomonas aeruginosa has been investigated by EPR and Mössbauer spectroscopy. Low temperature Mössbauer data on the native enzyme (Fe3+, S = 5/2) yields a hyperfine field Hsat=-525 kG at the nucleus. This observation is inconsistent with earlier suggestions, based on EPR data of a rubredoxin-like ligand environment around the iron, i.e. a tetrahedral sulfur coordination. Likewise, the dithionite-reduced enzyme has Mössbauer parameters unlike those of reduced rubredoxin. We conclude that the iron atoms are in a previously unrecognized environment. The ternary complex of the enzyme with 3,4-dihydroxyphenylpropionate and O2 yields EPR signals at g = 6.7 and g = 5.3; these signals result from an excited state Kramers doublet. The kinetics of the disappearance of these signals parallels product formation and the decay of the ternary complex as observed in the optical spectrum. The Mössbauer and EPR data on the ternary complex establish the iron atoms to be a high-spin ferric state characterized by a large and negative zero-field splitting, D = approximately -2 cm-1.

Cytochrome P450cam and its complexes. Mössbauer parameters of the heme iron.

Mössbauer spectroscopy has been used to study the heme iron in various states of cytochrome P450cam from the camphor-hydroxylating system of the bacterium Pseudomonas putida. Native, camphor-free P450cam contains low-spin ferric iron, part of which (approx. 50-70%) is converted to the high-spin ferric state upon addition of camphor. The Mössbauer spectra of the camphor-free enzyme (S equals 1/2) and of the high-spin component (S equals 5/2) of the camphor complex have been successfully simulated using a model based on crystal-field theory and simple convalency considerations. The native low-spin ferric state of P450cam forms a complex with 2-phenylimidazole, with small changes in the g values and Mössbauer spectra. These changes can be accounted for consistently in the crystal-field model referred to above. The addition of putidaredoxin to the camphor-complexed, oxidized P450cam decreases the intensity of the high-spin component and changes its quadrupole splitting. The reduced form of P450cam contrins high-spin ferrous iron, both in the presence and absence of camphor. The complex of reduced P450cam with molecular oxygen is diamagnetic and has a combination of quadrupole splitting and isomer shift that is unusual for a ferrous complex, but strongly resembles that of oxyhemoglobin. These results are compatible with the bound superoxide, Fe3+-O-2, model proposed for oxyhemoglobin (Weiss, J. J. (1964) Nature 202, 83-84). Reduced P450cam and its complexes, oxyP450cam-CO, are all found to be analogous in some respects to the corresponding hemoglobin complexes.

Feasibility and cost-saving potential of outpatient cardiac catheterization.

To determine the feasibility and cost-saving potential of substituting outpatient for inpatient cardiac catheterization, 986 consecutive procedures were studied at a large referral hospital. Patients were classified prospectively as to their eligibility for outpatient cardiac catheterization according to published guidelines. Resource consumption was recorded, and cost savings were then calculated by analyzing the specific supply and personnel costs that could change as a result of inpatient versus outpatient status. Of the total of 986 patients who underwent diagnostic catheterization, 240 (24%) were outpatients, 279 (28%) were inpatients but had no exclusion criteria for outpatient catheterization and 467 (47%) were inpatients who had one or more exclusions for outpatient catheterization. The most common reasons for exclusion from outpatient catheterization were congestive heart failure (22%), unstable angina (15%), noncoronary heart disease (14%), recent myocardial infarction (11%) and severe noncardiac disease (9%). Inpatients with no exclusions for the outpatient procedure tended to be sicker than outpatients because they were older (p = 0.002), had a lower ejection fraction (p = 0.009) and had more triple vessel coronary artery disease (p less than 0.0001). The cost of the catheterization procedure itself was not different between inpatients and outpatients. Laboratory testing was more frequent among inpatients, however, and "room and board" costs were significantly higher. Although the difference in hospital charges for inpatients and outpatients was $580, a rigorous analysis indicated that the potential cost savings was only 38% of this amount, or $218 per eligible patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the quaternary structure of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.

A 2.5 A resolution data set has been collected for crystals of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa. Analysis of the data using the rotation function shows that the alpha 2 beta 2 tetramers associate to form a particle with cubic 23 (T) point group symmetry. Prior to this analysis it was believed that eight tetramers associated to form the holoenzyme. The symmetry of the crystalline holoenzyme also addresses questions concerning its iron content and substrate stoichiometry.

Structure of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa at 2.15 A resolution.

Protocatechuate 3,4-dioxygenase catalyzes the aromatic ring cleavage of 3,4-dihydroxybenzoate by incorporating both atoms of molecular oxygen to yield beta-carboxy-cis,cis-muconate. The structure of this metalloenzyme from Pseudomonas aeruginosa (now reclassified as P. putida) has been refined to an R-factor of 0.172 to 2.15 A resolution. The structure is a highly symmetric (alpha beta Fe3+)12 aggregate with a root-mean-square (r.m.s.) difference of 0.18 A among symmetry-related atoms. The tertiary structure of the two polypeptides (alpha and beta) are highly homologous (r.m.s. difference of 1.05 A over 127 C alpha atoms), suggesting that the ancestral enzyme was originally a homodimer with two active sites. Indeed, a non-functional, vestigial active site retains many of the properties of the functional active site but does not bind iron. The coordination geometry of the non-heme iron catalytic cofactor can best be described as trigonal bipyramidal with Tyr447 (147 beta) and His462 (162 beta) serving as axial ligands, and Tyr408 (108 beta), His460 (160 beta) and Wat837 serving as equitorial ligands. The active site environment has a number of basic residues that may promote binding of the acidic substrate. Within the putative active site cavity which is located between alpha and beta chains, five approximately coplanar solvent molecules suggest a position for the planar substrate Trp449 (149 beta), Ile491 (191 beta), defined by Gly14 (14 alpha) and Pro15 (15 alpha). In this position the guanidino group of Arg457 (157 beta) would be buried by the substrate, suggesting a functional role in catalysis.

Preliminary crystallographic study of protocatechuate 3,4-dioxygenase from Brevibacterium fuscum.

The enzyme protocatechuate 3,4-dioxygenase from the Gram positive organism Brevibacterium fuscum crystallizes in the triclinic space group P1 with unit cell dimensions a = 96.1 A, b = 97.2 A, c = 118.1 A and alpha = 113.9 degrees, beta = 90.7 degrees, gamma = 117.8 degrees. The rod-like crystals diffract to 2.4 A resolution. Rotation function analysis suggests that there are six promoters arranged with local 32 symmetry in the asymmetric unit rather than the previously proposed pentameric complex.

Preliminary crystallographic analysis of methane mono-oxygenase hydroxylase from Methylosinus trichosporium OB3b.

The hydroxylase component of the enzyme methane mono-oxygenase from Methylosinus trichosporium OB3b has been crystallized in the orthorhombic space group C222(1) with unit cell dimensions a = 264.5 A, b = 71.2 A, c = 139.4 A. The crystals grow as square, thick plates and diffract to beyond 2 A resolution. There is one half of the hydroxylase dimer in the asymmetric unit.

Crystal structure of the hydroxylase component of methane monooxygenase from Methylosinus trichosporium OB3b.

Methane monooxygenase (MMO), found in aerobic methanotrophic bacteria, catalyzes the O2-dependent conversion of methane to methanol. The soluble form of the enzyme (sMMO) consists of three components: a reductase, a regulatory "B" component (MMOB), and a hydroxylase component (MMOH), which contains a hydroxo-bridged dinuclear iron cluster. Two genera of methanotrophs, termed Type X and Type II, which differ markedly in cellular and metabolic characteristics, are known to produce the sMMO. The structure of MMOH from the Type X methanotroph Methylococcus capsulatus Bath (MMO Bath) has been reported recently. Two different structures were found for the essential diiron cluster, depending upon the temperature at which the diffraction data were collected. In order to extend the structural studies to the Type II methanotrophs and to determine whether one of the two known MMOH structures is generally applicable to the MMOH family, we have determined the crystal structure of the MMOH from Type II Methylosinus trichosporium OB3b (MMO OB3b) in two crystal forms to 2.0 A resolution, respectively, both determined at 18 degrees C. The crystal forms differ in that MMOB was present during crystallization of the second form. Both crystal forms, however, yielded very similar results for the structure of the MMOH. Most of the major structural features of the MMOH Bath were also maintained with high fidelity. The two irons of the active site cluster of MMOH OB3b are bridged by two OH (or one OH and one H2O), as well as both carboxylate oxygens of Glu alpha 144. This bis-mu-hydroxo-bridged "diamond core" structure, with a short Fe-Fe distance of 2.99 A, is unique for the resting state of proteins containing analogous diiron clusters, and is very similar to the structure reported for the cluster from flash frozen (-160 degrees C) crystals of MMOH Bath, suggesting a common active site structure for the soluble MMOHs. The high-resolution structure of MMOH OB3b indicates 26 consecutive amino acid sequence differences in the beta chain when compared to the previously reported sequence inferred from the cloned gene. Fifteen additional sequence differences distributed randomly over the three chains were also observed, including D alpha 209E, a ligand of one of the irons.

Effects of aging and caloric restriction on hepatic drug metabolizing enzymes in the Fischer 344 rat. II: Effects on conjugating enzymes.

The effects of long-term caloric restriction on the hepatic phase II drug metabolizing enzymes were investigated in the male Fischer 344 rat. Rats that had been restricted to 60% of their pair-fed control consumption from 14 weeks post-partum exhibited altered conjugating enzyme activities at 22 months. Caloric restriction significantly reduced the age-related decrease in glutathione-S-transferase activity towards 1,2-dichloro-4-nitrobenzene, but did not significantly alter the age-related changes in UDP-glucuronyltransferase or sulfotransferase activities towards hydroxysteroids. Caloric restriction appeared to increase hepatic microsomal UDP-glucuronyltransferase activity toward bilirubin and gamma-glutamyltranspeptidase activities. These observations suggest that caloric restriction has multiple effects on the hepatic phase II drug metabolizing enzymes in the rat. Such effects may alter hepatic metabolism and activation or detoxification of drugs and carcinogens.