Intracellular pathways for lignin catabolism in white-rot fungi.

Lignin is a biopolymer found in plant cell walls that accounts for 30% of the organic carbon in the biosphere. White-rot fungi (WRF) are considered the most efficient organisms at degrading lignin in nature. While lignin depolymerization by WRF has been extensively studied, the possibility that WRF are able to utilize lignin as a carbon source is still a matter of controversy. Here, we employ 13C-isotope labeling, systems biology approaches, and in vitro enzyme assays to demonstrate that two WRF, Trametes versicolor and Gelatoporia subvermispora, funnel carbon from lignin-derived aromatic compounds into central carbon metabolism via intracellular catabolic pathways. These results provide insights into global carbon cycling in soil ecosystems and furthermore establish a foundation for employing WRF in simultaneous lignin depolymerization and bioconversion to bioproducts-a key step toward enabling a sustainable bioeconomy.

Physiologically Based Pharmacokinetic Modeling of Salivary Concentrations for Noninvasive Biomonitoring of 2,4-Dichlorophenoxyacetic Acid (2,4-D).

Saliva has become a favorable sample matrix for biomonitoring due to its noninvasive attributes and overall flexibility in collection. To ensure measured salivary concentrations reflect the exposure, a solid understanding of the salivary transport mechanism and relationships between salivary concentrations and other monitored matrices (ie, blood, urine) is needed. Salivary transport of a commonly applied herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), was observed in vitro and in vivo and a physiologically based pharmacokinetic (PBPK) model was developed to translate observations from the cell culture model to those in animal models and further evaluate 2,4-D kinetics in humans. Although apparent differences in experimental in vitro and in vivo saliva:plasma ratios (0.034 and 0.0079) were observed, simulations with the PBPK model demonstrated dynamic time and dose-dependent saliva:plasma ratios, elucidating key mechanisms affecting salivary transport. The model suggested that 2,4-D exhibited diffusion-limited transport to saliva and was additionally impacted by protein binding saturation and permeability across the salivary gland. Consideration of sampling times post-exposure and potential saturation of transport mechanisms are then critical aspects for interpreting salivary 2,4-D biomonitoring observations. This work utilized PBPK modeling in in vitro to in vivo translation to explore benefits and limitations of salivary analysis for occupational biomonitoring.

Evaluation of non-invasive biomonitoring of 2,4-Dichlorophenoxyacetic acid (2,4-D) in saliva.

The objective of this study was to evaluate the potential for non-invasive biomonitoring of 2,4-Dichlorophenoxyacetic acid (2,4-D) in saliva. Using an in vitro rat salivary gland epithelial cell (SGEC) system, a collection of experiments investigating chemical protein binding, temporal and directional transport, as well as competitive transport with para-aminohippuric acid (PAH), a substrate for renal organic anion transporters, was conducted to identify cellular transport parameters required to computationally model salivary transport of 2,4-D. Additionally, a physiological protein gradient was implemented to mimic physiologically relevant concentrations of protein in rat plasma and saliva, and under these conditions the transfer of 2,4-D was markedly slower, driven by increased protein binding (i.e. reduced free 2,4-D species available to cross salivary barrier). The rate of transfer was directly proportional to the amount of unbound 2,4-D and demonstrated no indication of active transport. An in vivo assessment of 2,4-D exposure in rats revealed non-linear protein binding in plasma, indicating saturated protein binding and increased levels of unbound 2,4-D species at higher doses. A strong correlation between 2,4-D concentrations in saliva and unbound 2,4-D in plasma was observed (Pearson correlation coefficient = 0.95). Saliva:plasma 2,4-D ratios measured in vivo (0.0079) were consistent within the linear protein binding range and expected 2,4-D levels from occupational exposures but were significantly different than ratios measured in vitro (physiological conditions) (0.034), possibly due to 2,4-D concentrations in saliva not being at equilibrium with 2,4-D concentrations in blood, as well as physiological features absent in in vitro settings (e.g. blood flow). We demonstrated that 2,4-D is consistently transported into saliva using both in vitro and in vivo models, making 2,4-D a potential candidate for human non-invasive salivary biomonitoring. Further work is needed to understand whether current sensor limits of detection are sufficient to measure occupationally relevant exposures.

Mode of action and pharmacokinetic studies of 2-butoxyethanol in the mouse with an emphasis on forestomach dosimetry.

Chronic inhalation studies with 2-butoxyethanol (BE) conducted by the National Toxicology Program identified the forestomach and liver of B6C3F1 mice as target organs for tumorigenicity (NTP, 2000). Previous studies have shown that the liver tumors likely resulted from chronic hemolysis-induced oxidative stress. For the forestomach lesions seen in mice, chronic contact irritation (cytotoxicity) and regenerative hyperplasia are hypothesized to result in forestomach tumor development. To test this hypothesis, several experiments were conducted to address the sensitivity of the mouse forestomach to BE administered by various routes. Oral administration of undiluted BE was shown to cause irritation and a compensatory proliferative response in the mouse forestomach, confirming that direct contact between the forestomach and BE, which can occur via grooming of BE condensed on the fur during inhalation exposures, can cause irritation. However, only small amounts of BE (<10 mg/kg) were detected on the fur of mice at the end of 6-h, whole-body or nose-only inhalation exposures to the highest concentration used in the NTP chronic inhalation studies (250 ppm). Furthermore, no significant differences were detected in the end-exposure blood concentrations of BE and butoxyacetic acid (BAA) between these types of exposures. In addition, parenteral administration of BE (ip and sc injection) also resulted in forestomach lesions, indicating that there may be sources other than grooming for BE- or BAA-induced forestomach irritation. In the pharmacokinetic study, BE and, to a lesser extent, BAA was eliminated more slowly from the forestomach tissue of mice than from blood or other tissues, following either oral gavage or ip injection. The forestomach was the only tissue with detectable levels of BE at 24 h. BE and BAA were both excreted in the saliva and were present in stomach contents for a prolonged period of time following these routes of exposure, which may further contribute to forestomach tissue dosimetry. Thus, there appear to be multiple mechanisms behind the increased levels of BE and BAA in the forestomach tissue of mice, which together can contribute to a prolonged contact irritation, compensatory hyperplasia, and tumorigenicity in mice. The relevance of these effects in humans, who lack a forestomach, is questionable.

A real-time method to evaluate the nasal deposition and clearance of acetone in the human volunteer.

Nasal dosimetry models have become increasingly quantitative as insights into tissue deposition/clearance and computational fluid dynamics have become available. Validation of these models requires sufficient experimental data. However, investigations into respiratory deposition, particularly in human volunteers, have been historically limited due to methodological limitations. To overcome this, a method for evaluating the nasal wash-in, wash-out phenomena of a highly water-soluble compound in human volunteers was developed and characterized. This methodology was assessed using controlled human inhalation exposures to uniformly labeled [(13)C]acetone at approximately 1 ppm concentration for 30 min under different breathing maneuvers (inhale nose/exhale nose; inhale nose/exhale mouth; inhale mouth/exhale nose). A small-diameter air-sampling probe inserted in the nasopharyngeal cavity of the volunteer was connected directly to an ion-trap mass spectrometer capable of sampling every 0.8 s. A second ion-trap mass spectrometer simultaneously sampled from the volunteer's exhaled breath stream via a breath-inlet device interface. Together, the two mass spectrometers provided real-time appraisal of the [(13)C]acetone concentrations in the nasopharyngeal region and in the exhaled breath stream before, during, and after the different breathing maneuvers. The breathing cycle (depth and frequency) and heart rate were concurrently monitored throughout the exposure using a heart-rate monitor and a human plethysmograph to differentiate inhalation and exhalation. Graphical overlay of the plethysmography results with the mass spectrometer measurements show clear quantifiable differences in [(13)C]acetone levels at the nasal probe as a function of breathing maneuvers. Breath-by-breath analyses of [(13)C]acetone concentrations indicate that between 40 and 75% of the compound is absorbed upon inhalation and nearly all of that absorbed is released back into the breath stream during exhalation.