Translating dosimetry of Dibenzo[def,p]chrysene (DBC) and metabolites across dose and species using physiologically based pharmacokinetic (PBPK) modeling.

Dibenzo[def,p]chrysene (DBC) is an environmental polycyclic aromatic hydrocarbon (PAH) that causes tumors in mice and has been classified as a probable human carcinogen by the International Agency for Research on Cancer. Animal toxicity studies often utilize higher doses than are found in relevant human exposures. Additionally, like many PAHs, DBC requires metabolic bioactivation to form the ultimate toxicant, and species differences in DBC and DBC metabolite metabolism have been observed. To understand the implications of dose and species differences, a physiologically based pharmacokinetic model (PBPK) for DBC and major metabolites was developed in mice and humans. Metabolism parameters used in the model were obtained from experimental in vitro metabolism assays using mice and human hepatic microsomes. PBPK model simulations were evaluated against mice dosed with 15 mg/kg DBC by oral gavage and human volunteers orally microdosed with 29 ng of DBC. DBC and its primary metabolite DBC-11,12-diol were measured in blood of mice and humans, while in urine, the majority of DBC metabolites were obeserved as conjugated DBC-11,12-diol, conjugated DBC tetrols, and unconjugated DBC tetrols. The PBPK model was able to predict the time course concentrations of DBC, DBC-11,12-diol, and other DBC metabolites in blood and urine of human volunteers and mice with reasonable accuracy. Agreement between model simulations and measured pharmacokinetic data in mice and human studies demonstrate the success and versatility of our model for interspecies extrapolation and applicability for different doses. Furthermore, our simulations show that internal dose metrics used for risk assessment do not necessarily scale allometrically, and that PBPK modeling provides a reliable approach to appropriately account for interspecies differences in metabolism and physiology.

Human Microdosing with Carcinogenic Polycyclic Aromatic Hydrocarbons: In Vivo Pharmacokinetics of Dibenzo[def,p]chrysene and Metabolites by UPLC Accelerator Mass Spectrometry.

Metabolism is a key health risk factor following exposures to pro-carcinogenic polycyclic aromatic hydrocarbons (PAHs) such as dibenzo[def,p]chrysene (DBC), an IARC classified 2A probable human carcinogen. Human exposure to PAHs occurs primarily from the diet in nonsmokers. However, little data is available on the metabolism and pharmacokinetics in humans of high molecular weight PAHs (≥4 aromatic rings), including DBC. We previously determined the pharmacokinetics of DBC in human volunteers orally administered a microdose (29 ng; 5 nCi) of [14C]-DBC by accelerator mass spectrometry (AMS) analysis of total [14C] in plasma and urine. In the current study, we utilized a novel "moving wire" interface between ultraperformance liquid chromatography (UPLC) and AMS to detect and quantify parent DBC and its major metabolites. The major [14C] product identified in plasma was unmetabolized [14C]-DBC itself (Cmax = 18.5 ±15.9 fg/mL, Tmax= 2.1 ± 1.0 h), whereas the major metabolite was identified as [14C]-(+/-)-DBC-11,12-diol (Cmax= 2.5 ±1.3 fg/mL, Tmax= 1.8 h). Several minor species of [14C]-DBC metabolites were also detected for which no reference standards were available. Free and conjugated metabolites were detected in urine with [14C]-(+/-)-DBC-11,12,13,14-tetraol isomers identified as the major metabolites, 56.3% of which were conjugated (Cmax= 35.8 ± 23.0 pg/pool, Tmax = 6-12 h pool). [14C]-DBC-11,12-diol, of which 97.5% was conjugated, was also identified in urine (Cmax = 29.4 ± 11.6 pg/pool, Tmax = 6-12 h pool). Parent [14C]-DBC was not detected in urine. This is the first data set to assess metabolite profiles and associated pharmacokinetics of a carcinogenic PAH in human volunteers at an environmentally relevant dose, providing the data necessary for translation of high dose animal models to humans for translation of environmental health risk assessment.

Furan carcinogenicity: DNA binding and genotoxicity of furan in rats in vivo.

SCOPE: Furan is a potent hepatotoxicant and liver carcinogen in rodents. However, short-term tests for genotoxicity of furan are inconclusive. The aim of this study was to assess the potential of furan to covalently bind to DNA, and to assess furan genotoxicity in rats in vivo. MATERIALS AND METHODS: Accelerator mass spectrometry was used to determine the (14) C-content in DNA following administration of [3,4-(14) C]-furan (0.1 and 2.0 mg/kg bw) to F344 rats. DNA damage, micronuclei, chromosomal aberrations, and sister chromatid exchanges were analyzed in F344 rats treated with furan for up to 28 days. CONCLUSION: The (14) C-content in liver DNA was significantly increased in a dose-dependent manner, with mean concentrations of 7.9 ± 3.5 amol (14) C/µg DNA and 153.3 ± 100.2 amol (14) C/µg DNA, corresponding to 16.5 ± 7.4 and 325.2 ± 212.7 adducts/10(9) nucleotides at 0.1 and 2.0 mg/kg bw, respectively. There was no evidence for genotoxicity of furan in peripheral blood and bone marrow cells. However, a dose-related increase in the incidence of chromosomal aberrations in rat splenocytes and some indication of DNA damage in liver were observed. Collectively, results from this study indicate that furan may operate-at least in part-by a genotoxic mode of action.

Ultrahigh efficiency moving wire combustion interface for online coupling of high-performance liquid chromatography (HPLC).

We describe a 100%-efficient moving-wire interface (MWI) for online coupling of high-performance liquid chromatography (HPLC) that transmits 100% of the carbon in nonvolatile analytes to a CO(2)-gas-accepting ion source. This interface accepts a flow of analyte in solvent, evaporates the solvent, combusts the remaining analyte, and directs the combustion products to the instrument of choice. Effluent is transferred to a periodically indented wire by a coherent jet to increase efficiency and maintain peak resolution. The combustion oven is plumbed such that gaseous combustion products are completely directed to an exit capillary, avoiding the loss of combustion products to the atmosphere. This system achieves almost-complete transfer of the analyte at HPLC flow rates up to 125 µL/min at a wire speed of 6 cm/s. This represents a 30× increase in efficiency and an 8× increase in maximum wire loading, compared to the spray transfer technique used in earlier MWIs.

Quantitative metabolism using AMS: Choosing a labeled precursor.

Biological radioisotope studies suffer from a lack of sensitive measurement techniques and therefore traditionally require large amounts of labeled material to produce a measurable signal. Such quantities of materials are often significantly higher than naturally-occurring levels preventing these studies from replicating physiological conditions. AMS affords the sensitivity necessary to perform biological radioisotope studies with low levels of labeled material that preserve physiological conditions. The choice of labeled material can substantially affect the ease of interpretation and comprehensiveness of these studies. Here, the benefits and limitations of whole-cell labeling with (14)C-glucose and targeted pathway labeling with (14)C-nicotinic acid are discussed and compared.

Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae.

Nicotinamide adenine dinucleotide (NAD+) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD+ decomposition products. NAD+ biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD+ biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD+ and NADH (the reduced form of NAD+) analyses on BY4742 wild-type, NAD+ salvage pathway knockout (npt1Delta) and NAD+ de novo pathway knockout (qpt1Delta) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized 14C labelled nicotinic acid in the culture media combined with HPLC speciation and both UV and 14C detection to quantitate the total amounts of NAD+ and NADH and the amounts derived from the salvage pathway. We observed that wild-type and qpt1Delta yeast exclusively utilized extracellular nicotinic acid for NAD+ and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions, suggesting that the de novo pathway plays little role if a functional salvage pathway is present. We also observed that NAD+ concentrations decreased in all three strains under CR. However, unlike the wild-type strain, NADH concentrations did not decrease and NAD+: NADH ratios did not increase under CR for either knockout strain. Lifespan analyses revealed that CR resulted in a lifespan increase of approximately 25% for the wild-type and qpt1Delta strains, while no increase in lifespan was observed for the npt1Delta strain. In combination, these data suggest that having a functional salvage pathway is required for lifespan extension under CR.

Limited dynamic range of immune response gene expression observed in healthy blood donors using RT-PCR.

The use of quantitative gene expression analysis for the diagnosis, prognosis, and monitoring of disease requires the ability to distinguish pathophysiological changes from natural variations. To characterize these variations in apparently healthy subjects, quantitative real-time PCR was used to measure various immune response genes in whole blood collected from blood bank donors. In a single-time-point study of 131 donors, of 48 target genes, 43 were consistently expressed and 34 followed approximately log-normal distribution. Most transcripts showed a limited dynamic range of expression across subjects. Specifically, 36 genes had standard deviations (SDs) of 0.44 to 0.79 cycle threshold (C(T)) units, corresponding to less than a 3-fold variation in expression. Separately, a longitudinal study of 8 healthy individuals demonstrated a total dynamic range (> 2 standard error units) of 2- to 4-fold in most genes. In contrast, a study of whole blood gene expression in 6 volunteers injected with LPS showed 15 genes changing in expression 10- to 90-fold within 2 to 5 h and returning to within normal range within 21 hours. This work demonstrates that (1) the dynamic range of expression of many immune response genes is limited among healthy subjects; (2) expression levels for most genes analyzed are approximately log-normally distributed; and (3) individuals exposed to an infusion of bacterial endotoxin (lipopolysaccharide), show gene expression profiles that can be readily distinguished from those of a healthy population. These results suggest that normal reference ranges can be established for gene expression assays, providing critical standards for the diagnosis and management of disease.