Use of kinetic and mechanistic data in species extrapolation of bioactivation: Cytochrome P-450 dependent trichloroethylene metabolism at occupationally relevant concentrations

Abstract: Use of Kinetic and Mechanistic Data in S p e c i e s E x t r a p o l a t i o n o f B i o a c t i v a t i o n : Cytochrome P-450 Dependent Trichloroethylene M e t a b o l i s m a t O c c u p a t i o n a l l y R e l e v a n t Concentrations: John C. LIPSCOMB, et al. United States Air Force, Armstrong Laboratory, Toxicology Division-Trichloroethylene (TRI) is an industrial solvent and environmental contaminant; therefore exposure to TRI occurs in diverse human populations. TRI causes hepatocellular carcinoma in B6C3F1 mice, but not rats; this suggests that TRI may be metabolized differently in the two species. We investigated the metabolism of TRI and the effect of TRI on enzymatic activities indicative of specific cytochrome P450 (CYP) forms in hepatic microsomes from mice, rats and humans. Studies in microsomes estimated MichaelisMenten kinetic parameters by saturation analysis. K m values were 35.4, 55.5 and 24.6 µM and V max values were 5,425, 4,826 and 1,440 pmol/min/mg in pooled mouse, rat and human microsomes, respectively. TRI (1,000 ppm) inhibited CYP2E1 dependent activity in all three species and BROD activity in mice and rats; TRI (1,000 ppm) increased CYP1A1/1A2 activity, and had no effect on CYP2A activity. Inhibition studies with mouse hepatic microsomes demonstrated that TRI was a competitive inhibitor of CYP2E1, with K i of 50 ppm. TRI noncompetitively inhibited CYP2B-dependent activities in the rat and mouse. Preincubation of microsomes with TRI and NADPH decreased the absorbence of CO-bound CYP in all three species, but the dose-dependence was most evident in mouse hepatic microsomes. These results have quantified the interspecies difference in CYP-dependent TRI bioactivation and indicate that under both equivalent and occupationally relevant (hepatic) exposure conditions the human is at less risk of forming toxic Trichloroethylene (TRI) is an unsaturated, chlorinated hydrocarbon widely used as an industrial solvent and is now a relatively common ground water pollutant 1) . TRI produces liver and lung toxicity in mice TRI concentrations as low as 10 µM, in vitro, inhibit gap junction mediated intercellular communication in mouse but not rat hepatocytes, and effect which is blocked by CYP inhibitio

Systemic Exposure to PAHs and Benzene in Firefighters Suppressing Controlled Structure Fires

Turnout gear provides protection against dermal exposure to contaminants during firefighting; however, the level of protection is unknown. We explored the dermal contribution to the systemic dose of polycyclic aromatic hydrocarbons (PAHs) and other aromatic hydrocarbons in firefighters during suppression and overhaul of controlled structure burns. The study was organized into two rounds, three controlled burns per round, and five firefighters per burn. The firefighters wore new or laundered turnout gear tested before each burn to ensure lack of PAH contamination. To ensure that any increase in systemic PAH levels after the burn was the result of dermal rather than inhalation exposure, the firefighters did not remove their self-contained breathing apparatus until overhaul was completed and they were >30 m upwind from the burn structure. Specimens were collected before and at intervals after the burn for biomarker analysis. Urine was analyzed for phenanthrene equivalents using enzyme-linked immunosorbent assay and a benzene metabolite (s-phenylmercapturic acid) using liquid chromatography/tandem mass spectrometry; both were adjusted by creatinine. Exhaled breath collected on thermal desorption tubes was analyzed for PAHs and other aromatic hydrocarbons using gas chromatography/mass spectrometry. We collected personal air samples during the burn and skin wipe samples (corn oil medium) on several body sites before and after the burn. The air and wipe samples were analyzed for PAHs using a liquid chromatography with photodiode array detection. We explored possible changes in external exposures or biomarkers over time and the relationships between these variables using non-parametric sign tests and Spearman tests, respectively. We found significantly elevated (P < 0.05) post-exposure breath concentrations of benzene compared with pre-exposure concentrations for both rounds. We also found significantly elevated post-exposure levels of PAHs on the neck compared with pre-exposure levels for round 1. We found statistically significant positive correlations between external exposures (i.e. personal air concentrations of PAHs) and biomarkers (i.e. change in urinary PAH metabolite levels in round 1 and change in breath concentrations of benzene in round 2). The results suggest that firefighters wearing full protective ensembles absorbed combustion products into their bodies. The PAHs most likely entered firefighters’ bodies through their skin, with the neck being the primary site of exposure and absorption due to the lower level of dermal protection afforded by hoods. Aromatic hydrocarbons could have been absorbed dermally during firefighting or inhaled during the doffing of gear that was off-gassing contaminants.National Institute for Occupational Safety and Health (NIOSH) by intramural award under the National Occupational Research AgendaOpe

Muscular contractions in the zebrafish embryo are necessary to reveal thiuram-induced notochord distortions

Author Posting. © The Authors, 2006. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Toxicology and Applied Pharmacology 212 (2006): 24-34, doi:10.1016/j.taap.2005.06.016.Dithiocarbamates form a large group of chemicals that have numerous uses in agriculture and medicine. It has been reported that dithiocarbamates, including thiuram (tetramethylthiuram disulfide), cause wavy distortions of the notochord in zebrafish and other fish embryos. In the present study, we investigated the mechanism underlying the toxicity of thiuram in zebrafish embryos. When embryos were exposed to thiuram (2-1,000 nM: 0.48-240 µg/L) from 3 hours post fertilization (hpf) (30% epiboly) until 24 hpf (Prim-5), all embryos develop wavy notochords, disorganized somites, and have shortened yolk sac extensions. The thiuram response was specific and did not cause growth retardation or mortality at 24 hpf. The thiuram-dependent responses showed the same concentration dependence with a waterborne EC50 values of approximately 7 nM. Morphometric measurements revealed that thiuram does not affect the rate of notochord lengthening. However, the rate of overall body lengthening was significantly reduced in thiuram exposed animals. Other dithiocarbamates, such as ziram, caused similar malformations to thiuram. While expression of genes involved in somitogenesis was not affected, the levels of notochord specific transcripts were altered before and after the onset of malformations. Distortion of the notochord started precisely at 18 hpf, which is concomitant with onset of spontaneous rhythmic trunk contractions. Abolishment of spontaneous contractions using tricaine, α-bungarotoxin, and a paralytic mutant sofa potato, resulted in normal notochord morphology in the presence of thiuram. These results indicate that muscle activity is necessary to reveal the underlying functional deficit and suggest that the developmental target of dithiocarbamates impairs trunk plasticity through an unknown mechanism.This work was supported by grants-in-aid for scientific research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (T.H. and H.T.), and grant-in-aid for JSPS fellows from the Japanese Ministry of Education, Culture, Sports, Science and Technology (W. D.), a grant-in-aid for High Technological Research Center (Rakuno Gakuen University) from the Ministry of Education, Science, Sports and Culture of Japan (H.T.), Technology, cooperative research from active research in Rakuno Gakuen University 2004-7 (H.T.), and NIH/NIEHS grants ES00210 and ES03850 (RT)

Occupational Exposures to Respirable Crystalline Silica During Hydraulic Fracturing

This report describes a previously uncharacterized occupational health hazard: work crew exposures to respirable crystalline silica during hydraulic fracturing. Hydraulic fracturing involves high pressure injection of large volumes of water and sand, and smaller quantities of well treatment chemicals, into a gas or oil well to fracture shale or other rock formations, allowing more efficient recovery of hydrocarbons from a petroleum-bearing reservoir. Crystalline silica (“frac sand”) is commonly used as a proppant to hold open cracks and fissures created by hydraulic pressure. Each stage of the process requires hundreds of thousands of pounds of quartz containing sand; millions of pounds may be needed for all zones of a well. Mechanical handling of frac sand creates respirable crystalline silica dust, a potential exposure hazard for workers. Researchers at the National Institute for Occupational Safety and Health collected 111 personal breathing zone samples at 11 sites in five states to evaluate worker exposures to respirable crystalline silica during hydraulic fracturing. At each of the 11 sites, full-shift samples exceeded occupational health criteria (e.g., the Occupational Safety and Health Administration calculated permissible exposure limit, the NIOSH recommended exposure limit, or the ACGIH threshold limit value), in some cases, by 10 or more times the occupational health criteria. Based on these evaluations, an occupational health hazard was determined to exist for workplace exposures to crystalline silica. Seven points of dust generation were identified, including sand handling machinery and dust generated from the work site itself. Recommendations to control exposures include product substitution (when feasible), engineering controls or modifications to sand handling machinery, administrative controls, and use of personal protective equipment. To our knowledge, this represents the first systematic study of work crew exposures to crystalline silica during hydraulic fracturing. Companies that conduct hydraulic fracturing using silica sand should evaluate their operations to determine the potential for worker exposure to respirable crystalline silica and implement controls as necessary to protect workers. [Supplementary materials are available for this article. Go to the publisher’s online edition of Journal of Occupational and Environmental Hygiene for the following free supplemental resource: a file containing controls and recommendations to limit worker exposures to respirable crystalline silica at hydraulic fracturing work sites.

Correlation between Graphitic Carbon and Elemental Carbon in Diesel Particulate Matter in Workplace Atmospheres

We investigated the suitability of the graphitic carbon (GC) content of diesel particulate matter (DPM), measured using Raman spectroscopy, as a surrogate measure of elemental carbon (EC) determined by thermal optical analysis. The Raman spectra in the range of 800–1800 cm–1 (including the D mode at ∼1322 cm–1 and the G mode at ∼1595 cm–1) were used for GC identification and quantification. Comparison of the Raman spectra for two certified DPM standards (NIST SRM 1650 and SRM 2975), two types of diesel engine exhaust soot, and three types of DPM-enriched workplace aerosols show that the uncertainty of GC quantification based on the D peak height, G peak height, and the total peak area below D and G peaks was about 6.0, 6.7, and 6.9%, respectively. The low uncertainty for different aerosol types suggested possible use of GC as a surrogate measure of EC in workplace atmospheres. A calibration curve was constructed using two laboratory-aerosolized DPM standards to describe the relationship between GC measured by a portable Raman spectrometer and the EC concentration determined by NIOSH Method 5040. The calibration curve was then applied to determine GC-based estimates of the EC contents of diesel engine exhaust samples from two vehicles and seven air samples collected at a hydraulic fracturing worksite. The GC–EC estimates obtained through Raman measurements agreed well with those found by NIOSH Method 5040 for the same samples at EC filter loadings below 2.86 μg/cm2. The study shows that using an appropriate sample collection method that avoids high filter mass loadings, onsite measurement of GC by a portable or hand-held Raman spectrometer can provide a useful indicator of EC in workplace aerosol

Exploring Evaluation Variables for Low-Cost Particulate Matter Monitors to Assess Occupational Exposure

(1) Background: Small, lightweight, low-cost optical particulate matter (PM) monitors are becoming popular in the field of occupational exposure monitoring, because these devices allow for real-time static measurements to be collected at multiple locations throughout a work site as well as being used as wearables providing personal exposure estimates. Prior to deployment, devices should be evaluated to optimize and quantify measurement accuracy. However, this can turn out to be difficult, as no standardized methods are yet available and different deployments may require different evaluation procedures. To gain insight in the relevance of different variables that may affect the monitor readings, six PM monitors were selected based on current availability and evaluated in the laboratory; (2) Methods: Existing strategies that were judged appropriate for the evaluation of PM monitors were reviewed and seven evaluation variables were selected, namely the type of dust, within- and between-device variations, nature of the power supply, temperature, relative humidity, and exposure pattern (peak and constant). Each variable was tested and analyzed individually and, if found to affect the readings significantly, included in a final correction model specific to each monitor. Finally, the accuracy for each monitor after correction was calculated; (3) Results: The reference materials and exposure patterns were found to be main factors needing correction for most monitors. One PM monitor was found to be sufficiently accurate at concentrations up to 2000 &micro;g/m3 PM2.5, with other monitors appropriate at lower concentrations. The average accuracy increased by up to three-fold compared to when the correction model did not include evaluation variables; (4) Conclusions: Laboratory evaluation and readings correction can greatly increase the accuracy of PM monitors and set boundaries for appropriate use. However, this requires identifying the relevant evaluation variables, which are heavily reliant on how the monitors are used in the workplace. This, together with the lack of current consensus on standardized procedures, shows the need for harmonized PM monitor evaluation methods for occupational exposure monitoring

Method for Simultaneous Measurement of Antibodies to 23 Pneumococcal Capsular Polysaccharides

We describe a fluorescent covalent microsphere immunoassay (FCMIA) method for the simultaneous (multiplexed) measurement of immunoglobulin G (IgG) antibodies to 23 pneumococcal capsular polysaccharide (PnPS) serotypes present in the pneumococcal polysaccharide vaccine (PPV23) licensed by the Food and Drug Administration, i.e., PnPSs 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. In addition, the assay incorporates an internal control that allows for contemporaneous evaluation of the effectiveness of pneumococcal cell wall polysaccharide (C-PS) preadsorption and a second control of PnPS 25 (which is not present in any polysaccharide or conjugate vaccine), which can be used to evaluate interassay reproducibility (useful for pre- versus postvaccination studies). The FCMIA was standardized with U.S. reference antipneumococcal serotype standard serum 89S-2. Preadsorption of 89S-2 with each PnPS and C-PS yielded homologous inhibition for serotypes 1, 6B, 9N, 9V, 11A, 12F,14, 15B, 18C, 19A, 19F, 20, 22F, 25, and 33F; heterologous inhibition for serotypes 9V, 10A, 11A, 12F, 15B, 17F, 20, and 23F; and neither homologous nor heterologous inhibition for serotypes 2, 3, 4, and 5. The minimum detectable concentrations for the 24 multiplexed (PnPS and C-PS) FCMIAs ranged from 20 pg/ml for PnPS 3 to 600 pg/ml for PnPS 14. The PnPS FCMIA method has numerous benefits over enzyme-linked immunosorbent assays commonly used to measure anti-PnPS-specific IgG levels, including increased speed, smaller sample volumes, equivalent or better sensitivity, and increased dynamic range