Grouping of Petroleum Substances as Example UVCBs by Ion Mobility-Mass Spectrometry to Enable Chemical Composition-Based Read-Across.

Substances of Unknown or Variable composition, Complex reaction products, and Biological materials (UVCBs), including many refined petroleum products, present a major challenge in regulatory submissions under the EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) and US High Production Volume regulatory regimes. The inherent complexity of these substances, as well as variability in composition obfuscates detailed chemical characterization of each individual substance and their grouping for human and environmental health evaluation through read-across. In this study, we applied ion mobility mass spectrometry in conjunction with cheminformatics-based data integration and visualization to derive substance-specific signatures based on the distribution and abundance of various heteroatom classes. We used petroleum substances from four petroleum substance manufacturing streams and evaluated their chemical composition similarity based on high-dimensional substance-specific quantitative parameters including m/z distribution, drift time, carbon number range, and associated double bond equivalents and hydrogen-to-carbon ratios. Data integration and visualization revealed group-specific similarities for petroleum substances. Observed differences within a product group were indicative of batch- or manufacturer-dependent variation. We demonstrate how high-resolution analytical chemistry approaches can be used effectively to support categorization of UVCBs based on their heteroatom composition and how such data can be used in regulatory decision-making.

A chemical-biological similarity-based grouping of complex substances as a prototype approach for evaluating chemical alternatives.

Comparative assessment of potential human health impacts is a critical step in evaluating both chemical alternatives and existing products on the market. Most alternatives assessments are conducted on a chemical-by-chemical basis and it is seldom acknowledged that humans are exposed to complex products, not individual substances. Indeed, substances of Unknown or Variable composition, Complex reaction products, and Biological materials (UVCBs) are ubiquitous in commerce yet they present a major challenge for registration and health assessments. Here, we present a comprehensive experimental and computational approach to categorize UVCBs according to global similarities in their bioactivity using a suite of in vitro models. We used petroleum substances, an important group of UVCBs which are grouped for regulatory approval and read-across primarily on physico-chemical properties and the manufacturing process, and only partially based on toxicity data, as a case study. We exposed induced pluripotent stem cell-derived cardiomyocytes and hepatocytes to DMSO-soluble extracts of 21 petroleum substances from five product groups. Concentration-response data from high-content imaging in cardiomyocytes and hepatocytes, as well as targeted high-throughput transcriptomic analysis of the hepatocytes, revealed distinct groups of petroleum substances. Data integration showed that bioactivity profiling affords clustering of petroleum substances in a manner similar to the manufacturing process-based categories. Moreover, we observed a high degree of correlation between bioactivity profiles and physico-chemical properties, as well as improved groupings when chemical and biological data were combined. Altogether, we demonstrate how novel in vitro screening approaches can be effectively utilized in combination with physico-chemical characteristics to group complex substances and enable read-across. This approach allows for rapid and scientifically-informed evaluation of health impacts of both existing substances and their chemical alternatives.

Large-conductance Ca2+-dependent K+ channels regulate basal uteroplacental blood flow in ovine pregnancy.

OBJECTIVES: The mechanisms regulating basal uteroplacental blood flow (UBF) and the greater than 30-fold increase observed in normal pregnancy remain unclear. Although vascular growth contributes in early gestation, vasodilation accounts for the exponential rise seen in the last third of pregnancy. Large conductance potassium channels (BK(Ca)) are expressed in uterine vascular smooth muscle (VSM), but the extent of their role in regulating UBF in pregnancy is unclear. Therefore, we determined if BK(Ca) regulate basal UBF during ovine pregnancy. METHODS: Studies were performed at 113 to 127 days and 135 to 150 days of gestation in eight pregnant ewes instrumented with uterine artery flow probes and uterine arterial and venous catheters. Tetraethylammonium chloride (TEA), a BK(Ca)-specific inhibitor at less than 1.0 mM, was infused intra-arterially into the pregnant uterine horn over 60 minutes to achieve levels of 0.001-0.35 mM while continuously monitoring UBF, arterial pressure (MAP), and heart rate (HR). Uterine arterial and venous blood was collected simultaneously to measure uterine cyclic guanosine monophosphate (cGMP) synthesis. RESULTS: Intra-arterial TEA dose-dependently decreased basal UBF in the early (R = 0.81, n = 36, P <.001) and late (R = 0.72, n = 31, P <.001) study periods without altering contralateral UBF, MAP, and HR. The IC(50) was 0.2 mM and basal UBF decreased >or=80% at 0.35 mM in both periods. Although UBF fell greater than 40% at estimated plasma TEA levels of 0.3 mM, uterine arterial cGMP was unchanged, uterine venous cGMP rose, and uterine cGMP synthesis was unchanged; therefore, upstream events associated with BK(Ca) activation were unaffected by blockade. CONCLUSIONS: These are the first data demonstrating that BK(Ca) are essential in the maintenance of basal UBF in the last third of ovine pregnancy.

Estrogen selectively up-regulates eNOS and nNOS in reproductive arteries by transcriptional mechanisms.

OBJECTIVE: To determine the mechanism(s) whereby daily and acute estradiol-17beta (E(2)beta) exposure modifies endothelium-derived nitric oxide synthase (eNOS) and vascular smooth muscle (VSM) neuronal nitric oxide synthase (nNOS) in reproductive and nonreproductive arteries and to localize NOS isoform expression within the vessel wall. METHODS: Oophorectomized nonpregnant ewes received E(2)beta (1 microg/kg per day) or no E(2)beta for 5-6 days or acute E(2)beta (1 microg/kg) on day 6-7 with or without daily E(2)beta. Uterine, mammary, mesenteric, and femoral arteries were collected at completion of each study, adventitia were removed, and samples were frozen and stored at -80C. After separating endothelium and VSM, NOS isoform mRNA was measured using reverse transcription-polymerase chain reaction. VSM nNOS protein was determined by Western analysis. RESULTS: Basal eNOS and nNOS mRNA was greatest (P <.02) in reproductive artery endothelium and VSM, respectively. Daily E(2)beta was required for maximum uterine vascular responses to acute E(2)beta and was associated with increased reproductive artery endothelial eNOS mRNA (>1.5-fold, P <.02) and uterine VSM nNOS mRNA (>2.5-fold, P <.003) and protein (21%, P <.05). Acute E(2)beta in the presence and absence of daily E(2)beta also increased uterine eNOS 68% and 28% (P =.01), respectively, within 90 minutes but did not affect VSM nNOS. Mammary eNOS increased 71% only after E(2)beta withdrawal; VSM nNOS was unchanged. Neither NOS isoform was altered in nonreproductive arteries by daily or acute E(2)beta. CONCLUSIONS: Basal eNOS and nNOS isoform expression is greatest in arteries from reproductive tissues, and isoform responses to E(2)beta are cell specific and transcriptionally regulated. Furthermore, optimal uterine vascular responses to acute E(2)beta exposure require daily E(2)beta exposure that enhances basal NOS expression and abundance.