Expression of a novel hydrolase MhpC in Brevibacillus parabrevis BCP-09 and its characteristics for degrading synthetic pyrethroids.

Synthetic pyrethroids are widely used insecticides which may cause chronic diseases in non-target organisms upon long-term exposure. Microbial degradation offers a reliable method to remove them from the environment. This study focused on Brevibacillus parabrevis BCP-09 and its enzymes for degrading pyrethroids. The predicted deltamethrin-degrading genes phnA and mhpC were used to construct recombinant plasmids. These plasmids, introduced into Escherichia coli BL21(DE3) cells and induced with L-arabinose. The results indicated that the intracellular crude enzyme efficiently degraded deltamethrin by 98.8 %, ß-cypermethrin by 94.84 %, and cyfluthrin by 73.52 % within 24 h. The hydrolytic enzyme MhpC possesses a catalytic triad Ser/His/Asp and a typical "Gly-X-Ser-X-Gly" conservative sequence of the esterase family. Co-cultivation of induced E. coli PhnA and E. coli MhpC resulted in degradation rates of 41.44 ± 3.55 % and 60.30 ± 4.55 %, respectively, for deltamethrin after 7 d. This study states that the degrading enzymes from B. parabrevis BCP-09 are an effective method for the degradation of pyrethroids, providing available enzyme resources for food safety and environmental protection.

Isonitrile biosynthesis by non-heme iron(II)-dependent oxidases/decarboxylases.

The isonitrile group is a compact, electron-rich moiety coveted for its commonplace as a building block and bioorthogonal functionality in synthetic chemistry and chemical biology. Hundreds of natural products containing an isonitrile group with intriguing bioactive properties have been isolated from diverse organisms. Our recent discovery of a conserved biosynthetic gene cluster in some Actinobacteria species highlighted a novel enzymatic pathway to isonitrile formation involving a non-heme iron(II) and α-ketoglutarate-dependent dioxygenase. Here, we focus this chapter on recent advances in understanding and probing the biosynthetic machinery for isonitrile synthesis by non-heme iron(II) and α-ketoglutarate-dependent dioxygenases. We will begin by describing how to harness isonitrile enzymatic machinery through heterologous expression, purification, synthetic strategies, and in vitro biochemical/kinetic characterization. We will then describe a generalizable strategy to probe the mechanism for isonitrile formation by combining various spectroscopic methods. The chapter will also cover strategies to study other enzyme homologs by implementing coupled assays using biosynthetic pathway enzymes. We will conclude this chapter by addressing current challenges and future directions in understanding and engineering isonitrile synthesis.

Association mechanism of bicalutamide and human serum albumin for potential clinical implications.

The binding mechanism of molecular interaction between bicalutamide and human serum albumin (HSA) in a pH 7.4 phosphate buffer was studied using various spectroscopic techniques in combination with molecular modeling. Fluorescence data revealed that the fluorescence quenching of HSA by bicalutamide was a static quenching procedure. The binding constants and number of binding sites were evaluated at different temperatures. The thermodynamic parameters, ΔH and ΔS, were calculated to be 4.30 × 104 J·mol-1 and 245 J·mol-1·K-1, respectively, suggesting that the binding of bicalutamide to HSA was driven mainly by hydrophobic interactions and hydrogen bonds. The displacement studies indicated neither Sudlow's site I nor II but subdomain IB as the main binding site for bicalutamide on HSA. The binding distance between bicalutamide and HSA was determined to be 3.54 nm based on the Förster theory. Analysis of circular dichroism, synchronous, and 3D fluorescence spectra demonstrated that HSA conformation was slightly altered in the presence of bicalutamide.

Functional Validation of Endogenous Redox Partner Cytochrome P450 Reductase Reveals the Key P450s CYP6P9a/-b as Broad Substrate Metabolizers Conferring Cross-Resistance to Different Insecticide Classes in Anopheles funestus.

The versatility of cytochrome P450 reductase (CPR) in transferring electrons to P450s from other closely related species has been extensively exploited, e.g., by using An. gambiae CPR (AgCPR), as a homologous surrogate, to validate the role of An. funestus P450s in insecticide resistance. However, genomic variation between the AgCPR and An. funestus CPR (AfCPR) suggests that the full metabolism spectrum of An. funestus P450s might be missed when using AgCPR. To test this hypothesis, we expressed AgCPR and AfCPR side-by-side with CYP6P9a and CYP6P9b and functionally validated their role in the detoxification of insecticides from five different classes. Major variations were observed within the FAD- and NADP-binding domains of AgCPR and AfCPR, e.g., the coordinates of the second FAD stacking residue AfCPR-Y456 differ from that of AgCPR-His456. While no significant differences were observed in the cytochrome c reductase activities, when co-expressed with their endogenous AfCPR, the P450s significantly metabolized higher amounts of permethrin and deltamethrin, with CYP6P9b-AfCPR membrane metabolizing α-cypermethrin as well. Only the CYP6P9a-AfCPR membrane significantly metabolized DDT (producing dicofol), bendiocarb, clothianidin, and chlorfenapyr (bioactivation into tralopyril). This demonstrates the broad substrate specificity of An. funestus CYP6P9a/-b, capturing their role in conferring cross-resistance towards unrelated insecticide classes, which can complicate resistance management.

Biocatalytic Synthesis of Ruxolitinib Intermediate via Engineered Imine Reductase.

An enzyme catalyzed strategy for the synthesis of a chiral hydrazine from 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 is presented. An imine reductase (IRED) from Streptosporangium roseum was identified to catalyze the reaction between 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 to produce trace amounts of (R)-3-cyclopentyl-3-hydrazineylpropanenitrile 4. We employed a 2-fold approach to optimize the catalytic performance of this enzyme. First, a transition state analogue (TSA) model was constructed to illuminate the enzyme-substrate interactions. Subsequently, the Enzyme_design and Funclib methods were utilized to predict mutants for experimental evaluation. Through three rounds of site-directed mutagenesis, site saturation mutagenesis, and combinatorial mutagenesis, we obtained mutant M6 with a yield of 98% and an enantiomeric excess (ee) of 99%. This study presents an effective method for constructing a hydrazine derivative via IRED-catalyzed reductive amination of ketone and hydrazine. Furthermore, it provides a general approach for constructing suitable enzymes, starting from nonreactive enzymes and gradually enhancing their catalytic activity through active site modifications.

Expression Pattern of RpCSP6 from Rhopalosiphum padi and Its Binding Mechanism with Deltamethrin: Insights into Chemosensory Protein-Mediated Insecticide Resistance.

The mechanisms of insecticide resistance are complex. Recent studies have revealed a novel mechanism involving the chemosensory system in insecticide resistance. However, the specific binding mechanism between olfactory-related genes and insecticides needs to be clarified. In this study, the binding mechanism between pyrethroid insecticide deltamethrin and RpCSP6 from Rhopalosiphum padi was investigated by using computational and multiple experimental methods. RpCSP6 was expressed in different tissues and developmental stages of R. padi and can be induced by deltamethrin. Knockdown of RpCSP6 significantly increased the susceptibility of R. padi to deltamethrin. The binding affinity of RpCSP6 to 24 commonly used insecticides was measured. Seven key residues were found to steadily interact with deltamethrin, indicating their significance in the binding affinity to the insecticide. Our research provided insights for effectively analyzing the binding mechanism of insect CSPs with insecticides, facilitating the development of new and effective insecticides that target insect CSPs.

Substrate access tunnel engineering of a Fe-type nitrile hydratase from Pseudomonas fluorescens ZJUT001 for substrate preference adjustment and catalytic performance enhancement.

Substrate access tunnel engineering is a useful strategy for enzyme modification. In this study, we improved the catalytic performance of Fe-type Nitrile hydratase (Fe-type NHase) from Pseudomonas fluorescens ZJUT001 (PfNHase) by mutating residue Q86 at the entrance of the substrate access tunnel. The catalytic activity of the mutant PfNHase-αQ86W towards benzonitrile, 2-cyanopyridine, 3-cyanopyridine, and 4-hydroxybenzonitrile was enhanced by 9.35-, 3.30-, 6.55-, and 2.71-fold, respectively, compared to that of the wild-type PfNHase (PfNHase-WT). In addition, the mutant PfNHase-αQ86W showed a catalytic efficiency (kcat/Km) towards benzonitrile 17.32-fold higher than the PfNHase-WT. Interestingly, the substrate preference of PfNHase-αQ86W shifted from aliphatic nitriles to aromatic nitrile substrates. Our analysis delved into the structural changes that led to this altered substrate preference, highlighting an expanded entrance tunnel region, theenlarged substrate-binding pocket, and the increased hydrophobic interactions between the substrate and enzyme. Molecular dynamic simulations and dynamic cross-correlation Matrix (DCCM) further supported these findings, providing a comprehensive explanation for the enhanced catalytic activity towards aromatic nitrile substrates.

The case for sporadic cyanogenic glycoside evolution in plants.

Cyanogenic glycosides are α-hydroxynitrile glucosides present in approximately 3000 different plant species. Upon tissue disruption, cyanogenic glycosides are hydrolyzed to release toxic hydrogen cyanide as a means of chemical defense. Over 100 different cyanogenic glycosides have been reported, with structural diversity dependent on the precursor amino acid, and subsequent modifications. Cyanogenic glycosides represent a prime example of sporadic metabolite evolution, with the metabolic trait arising multiple times throughout the plant lineage as evidenced by recruitment of different enzyme families for biosynthesis. Here, we review the latest developments within cyanogenic glycoside biosynthesis, and argue possible factors driving sporadic evolution including shared intermediates and crossovers with other metabolic pathways crossovers, and metabolite multifunctionality beyond chemical defense.

Investigation of the molecular structure of CHBP, biological activities and SARS-CoV-2 protein binding interaction by molecular and biomolecular spectroscopy approaches.

The objective of this investigation is to learn more about the structural, electrical, spectroscopic, and physiochemical characteristics of biologically active cyano-4'-hydroxybiphenyl (CHBP). The title molecule's optimized conformational analysis was computed using the DFT/B3LYP/6-311++G (d, p) level of theory. The observed wavenumbers were compared with theoretical FT-IR and FT-Raman spectra. 1H and 13C NMR experimental spectra in CDCl3 solution (solvent phase) were recorded and the chemical shift was calculated. NBO analysis was used to examine the transfer of charge as well as the intermolecular and intramolecular bonding of orbitals. The TD-DFT (time-dependent DFT) approach was used to estimate theoretical values for both the gas and solvent (ethanol) in the corresponding transitional research, which was conducted using UV-Vis's spectra. Energy gap (Eg = 0.26764 eV) implies that the strong potential for charge transfer, and the stability of the CHBP compound. CHBP compound's has bioactive nature, its drug-likeness and biological properties were evaluated. The predicted topological polar surface area of 44.02 \AA2 for the molecule falls within the permissible range of < 140 \AA2. Based on the docking results, the most stable docking score value is -6.84 kcal/mol. In that interaction, MET 165 affects both phenyl rings in a pi-sulphur fashion and a single bond hydrogen with protein moieties GLN 192. This suggests that the pi-alkyl in PRO 168 is a hydroxyl substitutional ring. Our findings demonstrate the CHBP compound is a good inhibitor against the SAR COVID-19 viral protein.

Biosynthesis of the Unusual Epoxy Isonitrile-Containing Antibiotics Aerocyanidin and Amycomicin.

Aerocyanidin and amycomicin are two antibiotics derived from long-chain acids with a rare epoxy isonitrile moiety, the complexity of which renders the total synthesis of these two natural products rather challenging. How this functionality is biosynthesized has also remained obscure. While the biosynthetic gene clusters for these compounds have been identified, both appear to be deficient in genes encoding enzymes seemingly necessary for the oxidative modifications observed in these antibiotics. Herein, the biosynthetic pathways of aerocyanidin and amycomicin are fully elucidated. They share a conserved pathway to isonitrile intermediates that involves a bifunctional thioesterase and a nonheme iron α-ketoglutarate-dependent enzyme. In both cases, the isonitrile intermediates are then loaded onto an acyl carrier protein (ACP) catalyzed by a ligase. The isonitrile-tethered ACP is subsequently processed by polyketide synthase(s) to undergo chain extension, thereby assembling a long-chain γ-hydroxy isonitrile acid skeleton. The epoxide is installed by the cupin domain-containing protein AecF to conclude the biosynthesis of aerocyanidin. In contrast, three P450 enzymes AmcB, AmcC, and AmcQ are involved in epoxidation and keto formation to finalize the biosynthesis of amycomicin. These results thus explain the sequence of oxidation events that result in the final structures of aerocyanidin and amycomicin as well as the biosynthesis of the key γ-hydroxy epoxy isonitrile functional group.

Investigation of etravirine uptake and distribution in single aortic endothelial cells in vitro using Raman imaging.

Etravirine (ETV) is an antiretroviral agent that belongs to the class of non-nucleoside reverse transcriptase inhibitors. This study explores the uptake and distribution of ETV in human aortic endothelial cells (HAECs) using Raman spectroscopy combined with chemometrics. The distinctive chemical structure of ETV facilitates tracking of its uptake by observing the Raman band at 2225 cm-1 in the Raman-silent region. The perinuclear distribution pattern in HAECs depends on drug concentration and incubation time. The uptake of ETV is observed within 5 minutes at a concentration of 10 µM, as evidenced by Raman images. Lower ETV concentrations, reflective of those found in human plasma, are detectable in HAECs by applying chemometric methods to Raman spectra from the perinuclear region. The ETV accumulation process is crucial in advancing our understanding of the drug's impact on biochemical alterations within endothelial cells. Additionally, ETV emerges as a promising Raman reporter for marking subcellular compartments, leveraging the 2225 cm-1 band in the cellular Raman silent region. This research contributes valuable insights into the behavior of ETV at the subcellular level, shedding light on its potential applications and impact on subcellular dynamics.

Turning glucosinolate into allelopathic fate: investigating allyl isothiocyanate variability and nitrile formation in eco-friendly Brassica juncea from South Korea.

Leaf mustard (Brassica juncea L.) is explored for its biofumigant properties, derived from its secondary metabolites, particularly allyl isothiocyanate (AITC), produced during the enzymatic breakdown of glucosinolates like sinigrin. The research examines eight leaf mustard cultivars developed in Yeosu city, South Korea, focusing on their genetic characteristics, AITC concentration and nitriles formation rates from glucosinolates. Results indicate that the allelopathic effects, largely dependent on AITC concentration and enzymatic activity, vary across cultivar. Sinigrin and AITC constitute 79% and 36%, respectively, of glucosinolate and its hydrolysis products. The cultivar 'Nuttongii' demonstrates significant potential for inhibiting weeds, exhibiting the highest AITC concentration at 27.47 ± 6.46 µmole g-1 These outcomes highlight the importance of selecting mustard cultivars for biofumigation based on their glucosinolate profiles and hydrolysis product yields. The study also identifies a significant genetic influence on AITC and nitrile formation, suggesting that epithiospecifier protein modulation could enhance both allelopathic and other beneficial effects. Collectively, the research underscores the promise of mustard as a sustainable, environmentally friendly alternative to traditional herbicides.

Molecular Modification Strategies of Nitrilase for Its Potential Application in Agriculture.

Some feed source plants will produce secondary metabolites such as cyanogenic glycosides during metabolism, which will produce some poisonous nitrile compounds after hydrolysis and remain in plant tissues. The consumption of feed-source plants without proper treatment affect the health of the animals' bodies. Nitrilases can convert nitriles and have been used in industry as green biocatalysts. However, due to their bottleneck problems, their application in agriculture is still facing challenges. Acid-resistant nitrilase preparations, high-temperature resistance, antiprotease activity, strong activity, and strict reaction specificity urgently need to be developed. In this paper, the application potential of nitrilase in agriculture, especially in feed processing industry was explored, the source properties and catalytic mechanism of nitrilase were reviewed, and modification strategies for nitrilase application in agriculture were proposed to provide references for future research and application of nitrilase in agricultural and especially in the biological feed scene.

BioLindlar Catalyst: Ene-Reductase-Promoted Selective Bioreduction of Cyanoalkynes to Give (Z)-Cyanoalkenes.

The direct synthesis of alkenes from alkynes usually requires the use of transition-metal catalysts. Unfortunately, efficient biocatalytic alternatives for this transformation have yet to be discovered. Herein, the selective bioreduction of electron-deficient alkynes to alkenes catalysed by ene-reductases (EREDs) is described. Alkynes bearing ketone, aldehyde, ester, and nitrile moieties have been effectively reduced with excellent conversions and stereoselectivities, observing clear trends for the E/Z ratios depending on the nature of the electron-withdrawing group. In the case of cyanoalkynes, (Z)-alkenes were obtained as the major product, and the reaction scope was expanded to a wide variety of aromatic substrates (up to >99 % conversion, and Z/E stereoselectivities of up to >99/1). Other alkynes containing aldehyde, ketone, or ester functionalities also proved to be excellent substrates, and interestingly gave the corresponding (E)-alkenes. Preparative biotransformations were performed on a 0.4 mmol scale, producing the desired (Z)-cyanoalkenes with good to excellent isolated yields (63-97 %). This novel reactivity has been rationalised through molecular docking by predicting the binding poses of key molecules in the ERED-pu-0006 active site.

In silico studies on the molecular interactions of steroid hormones and steroid hormone mimicking drugs in the androgen receptor binding cleft - Implications for prostate cancer treatment.

Occupancy of prostate cancer (PCa) cell androgen receptors (AR) signals proliferation, therefore testosterone biosynthesis inhibitors and AR antagonists are important PCa treatments. Conversely, androgen mimics (e.g., prednisone) used in management of PCa might cause proliferation. The balance between PCa proliferation and inhibition predicts treatment success. We used in silico molecular modelling to explore interactions between ARs, androgens (testosterone, dihydrotestosterone (DHT)) and drugs used to treat (bicalutamide) and manage (dexamethasone, prednisone, hydrocortisone) PCa. We found that hydrogen (H-) bonds between testosterone, DHT and Arg752, Asn705 and Thr877 followed by ligand binding cleft hydrophobic interactions signal proliferation, whereas bicalutamide antagonism is via Phe764 interactions. Hydrocortisone, dexamethasone and prednisone H-bond Asn705 and Thr877, but not Arg752 in the absence of a water molecule. Studies with a bicalutamide agonist AR mutation showed different amino acid interactions, indicating testosterone and DHT would not promote proliferation as effectively as via the native receptor. However, hydrocortisone and bicalutamide form Arg752 and Asn705 H-bonds indicating agonism. Our results suggest that as PCa progresses the resulting mutations will change the proliferative response to androgens and their drug mimics, which have implications for the treatment of prostate cancer.

Heterogeneous g-C3N4/polyaniline composites enhanced the conversion of organics into methane during anaerobic wastewater treatment.

In this study, g-C3N4/PANI was prepared by in situ oxidative polymerization. Graphite-phase carbon nitride (g-C3N4) with surface defects was deposited onto the surface of conductive polyaniline (PANI) to form a p-n heterojunction. This construction aimed to create an efficient heterogeneous catalyst, increasing the surface defect level and active sites of the composite, and augmenting its capability to capture and transfer extracellular electrons under anaerobic conditions. This addresses the challenge of low efficiency in direct interspecies electron transfer between bacteria and archaea during anaerobic digestion for methane production. The results showed that the prepared g-C3N4/PANI increased the CH4 yield and CH4 production rate by 82% and 96%, respectively. Notably, the conductivity and XPS test results showed that the ratio of g-C3N4 to PANI was 0.15, and the composite exhibited favorable conductivity, with a uniform distribution of pyrrolic nitrogen, pyridinic nitrogen, and graphitic nitrogen, each accounting for approximately 30%. Furthermore, g-C3N4/PANI effectively enhanced the metabolic efficiency of intermediate products such as acetate and butyrate. Analysis of the microbial community structure revealed that g-C3N4/PANI led to a significant increase in the abundance of hydrogenotrophic methanogen Methanolinea (from 48% to 64%) and enriched Clostridium (a rise of 1%) with direct interspecies electron transfer capability. Microbial community function analysis demonstrated that the addition of g-C3N4/PANI boosted the activities of key enzymes involved in anaerobic digestion, including phosphate transacetylase (PTA), phospho-butyryl transferase (PTB), and NAD-independent lactate dehydrogenase (NNLD), by 47%, 135%, and 153%, respectively. This acceleration in enzymatic activity promoted the metabolism of acetyl-CoA, butyryl-CoA, and pyruvate. Additionally, the function of ABC transporters was enhanced, thereby improving the efficiency of material and energy exchange among microorganisms.

Biosynthesis of isonitrile lipopeptides.

Isonitrile lipopeptides discovered from Actinobacteria have attracted wide attention due to their fascinating biosynthetic pathways and relevance to the virulence of many human pathogens including Mycobacterium tuberculosis. Specifically, the identification of the new class of isonitrile-forming enzymes that belong to non-heme iron (II) and α-ketoglutarate dependent dioxygenases has intrigued several research groups to investigate their catalytic mechanism. Here we summarize the recent studies on the biosynthesis of isonitrile lipopeptides from Streptomyces and Mycobacterium. The latest research on the core and tailoring enzymes involved in the pathway as well as the isonitrile metabolic enzymes are discussed in this review.

The Circadian Clock Gene PHYTOCLOCK1 Mediates the Diurnal Emission of the Anti-Insect Volatile Benzyl Nitrile from Damaged Tea (Camellia sinensis) Plants.

Benzyl nitrile from tea plants attacked by various pests displays a diurnal pattern, which may be closely regulated by the endogenous circadian clock. However, the molecular mechanism by the circadian clock of tea plants that regulates the biosynthesis and release of volatiles remains unclear. In this study, the circadian clock gene CsPCL1 can activate both the expression of the benzyl nitrile biosynthesis-related gene CsCYP79 and the jasmonic acid signaling-related transcription factor CsMYC2 involved in upregulating CsCYP79 gene, thereby resulting in the accumulation and release of benzyl nitrile. Therefore, the anti-insect function of benzyl nitrile was explored in the laboratory. The application of slow-release beads of benzyl nitrile in tea plantations significantly reduced the number of tea geometrids and had positive effects on the yield of fresh tea leaves. These findings reveal the potential utility of herbivore-induced plant volatiles for the green control of pests in tea plantations.

Catalytic role of histidine-114 in the hydrolytic dehalogenation of chlorothalonil by Pseudomonas sp. CTN-3.

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile; TPN) is an environmentally persistent fungicide that sees heavy use in the USA and is highly toxic to aquatic species and birds, as well as a probable human carcinogen. The chlorothalonil dehalogenase from Pseudomonas sp. CTN-3 (Chd, UniProtKB C9EBR5) degrades TPN to its less toxic 4-OH-TPN analog making it an exciting candidate for the development of a bioremediation process for TPN; however, little is currently known about its catalytic mechanism. Therefore, an active site residue histidine-114 (His114) which forms a hydrogen bond with the Zn(II)-bound water/hydroxide and has been suggested to be the active site acid/base, was substituted by an Ala residue. Surprisingly, ChdH114A exhibited catalytic activity with a kcat value of 1.07 s-1, ~ 5% of wild-type (WT) Chd, and a KM of 32 µM. Thus, His114 is catalytically important but not essential. The electronic and structural aspects of the WT Chd and ChdH114A active sites were examined using UV-Vis and EPR spectroscopy on the catalytically competent Co(II)-substituted enzyme as well as all-atomistic molecular dynamics (MD) simulations. Combination of these data suggest His114 can quickly and reversibly move nearly 2 Å between one conformation that facilitates catalysis and another that enables product egress and active site recharge. In light of experimental and computational data on ChdH114A, Asn216 appears to play a role in substrate binding and preorganization of the transition-state while Asp116 likely facilitates the deprotonation of the Zn(II)-bound water in the absence of His114. Based on these data, an updated proposed catalytic mechanism for Chd is presented.

A Novel Approach to Integrate Human Biomonitoring Data with Model Predicted Dietary Exposures: A Crop Protection Chemical Case Study Using Lambda-Cyhalothrin.

The appropriate use of human biomonitoring data to model population chemical exposures is challenging, especially for rapidly metabolized chemicals, such as agricultural chemicals. The objective of this study is to demonstrate a novel approach integrating model predicted dietary exposures and biomonitoring data to potentially inform regulatory risk assessments. We use lambda-cyhalothrin as a case study, and for the same representative U.S. population in the National Health and Nutrition Examination Survey (NHANES), an integrated exposure and pharmacokinetic model predicted exposures are calibrated to measurements of the urinary metabolite 3-phenoxybenzoic acid (3PBA), using an approximate Bayesian computing (ABC) methodology. We demonstrate that the correlation between modeled urinary 3PBA and the NHANES 3PBA measurements more than doubled as ABC thresholding narrowed the acceptable tolerance range for predicted versus observed urinary measurements. The median predicted urinary concentrations were closer to the median measured value using ABC than using current regulatory Monte Carlo methods.