Comparison of Toxicity and Recovery in the Duodenum of B6C3F1 Mice Following Treatment with Intestinal Carcinogens Captan, Folpet, and Hexavalent Chromium.

High concentrations of hexavalent chromium (Cr(VI)), captan, and folpet induce duodenal tumors in mice. Using standardized tissue collection procedures and diagnostic criteria, we compared the duodenal histopathology in B6C3F1 mice following exposure to these 3 carcinogens to determine whether they share similar histopathological characteristics. B6C3F1 mice ( n = 20 per group) were exposed to 180 ppm Cr(VI) in drinking water, 12,000 ppm captan in feed, or 16,000 ppm folpet in feed for 28 days. After 28 days of exposure, villous enterocyte hypertrophy and mild crypt epithelial hyperplasia were observed in all exposed mice. In a subset of mice allowed to recover for 28 days, duodenal samples were generally indistinguishable from those of unexposed mice. Changes in the villi and lack of observable damage to the crypt compartment suggest that toxicity was mediated in the villi, which is consistent with earlier studies on each chemical. These findings indicate that structurally diverse agents can induce similar (and reversible) phenotypic changes in the duodenum. These intestinal carcinogens likely converge on common pathways involving irritation and wounding of the villi leading to crypt regenerative hyperplasia that, under protracted high-dose exposure scenarios, increases the risk of spontaneous mutation and tumorigenesis.

Embryonic vascular disruption adverse outcomes: Linking high throughput signaling signatures with functional consequences.

Embryonic vascular disruption is an important adverse outcome pathway (AOP) as chemical disruption of cardiovascular development induces broad prenatal defects. High throughput screening (HTS) assays aid AOP development although linking in vitro data to in vivo apical endpoints remains challenging. This study evaluated two anti-angiogenic agents, 5HPP-33 and TNP-470, across the ToxCastDB HTS assay platform and anchored the results to complex in vitro functional assays: the rat aortic explant assay (AEA), rat whole embryo culture (WEC), and the zebrafish embryotoxicity (ZET) assay. Both were identified as putative vascular disruptive compounds (pVDCs) in ToxCastDB and disrupted angiogenesis and embryogenesis in the functional assays. Differences were observed in potency and adverse effects: 5HPP-33 was embryolethal (WEC and ZET); TNP-470 produced caudal defects at lower concentrations. This study demonstrates how a tiered approach using HTS signatures and complex functional in vitro assays might be used to prioritize further in vivo developmental toxicity testing.

Embryonic vascular disruption adverse outcomes: Linking high-throughput signaling signatures with functional consequences.

Embryonic vascular disruption is an important adverse outcome pathway (AOP) as chemical disruption of cardiovascular development induces broad prenatal defects. High-throughput screening (HTS) assays aid AOP development although linking in vitro data to in vivo apical endpoints remains challenging. This study evaluated two anti-angiogenic agents, 5HPP-33 and TNP-470, across the ToxCastDB HTS assay platform and anchored the results to complex in vitro functional assays: the rat aortic explant assay (AEA), rat whole embryo culture (WEC), and the zebrafish embryotoxicity (ZET) assay. Both were identified as putative vascular disruptive compounds (pVDCs) in ToxCastDB and disrupted angiogenesis and embryogenesis in the functional assays. Differences were observed in potency and adverse effects: 5HPP-33 was embryolethal (WEC and ZET); TNP-470 produced caudal defects at lower concentrations. This study demonstrates how a tiered approach using HTS signatures and complex functional in vitro assays might be used to prioritize further in vivo developmental toxicity testing.

Dietary Route of Exposure for Rabbit Developmental Toxicity Studies.

Dietary administration is a relevant route of oral exposure for regulatory toxicity studies of agrochemicals as it mimics potential human intake of the chemical via treated crops and commodities. Moreover, dietary administration of test compounds during a developmental toxicity study can deliver a prolonged and stable systemic exposure to the embryo or fetus at all stages of development. In this study, strategies were employed to optimize rabbit test material consumption via diet. Comparative toxicokinetic profiles of gavage versus dietary administration were evaluated in pregnant or non-pregnant New Zealand White rabbits for 2 novel agrochemicals with different plasma half-lives of elimination (sulfoxaflor, t½ = 13.5 h and halauxifen, t½ = 1 h). Dietary administration of sulfoxaflor resulted in stable 24-h plasma concentrations, whereas gavage administration resulted in a 3-fold fluctuation in plasma levels between Cmax and Cmin Dietary administration of sulfoxaflor resulted in a 2-fold higher nominal and diurnal systemic dose when compared with gavage dosing due to Cmax-related maternal toxicity following gavage. Results with the shorter half-life molecule, halauxifen, were more striking with a 6-fold diurnal fluctuation by the dietary route compared with a 368-fold fluctuation between Cmax and Cmin by gavage. Furthermore, plasma halauxifen was detectable only up to 12 h following gavage but up to 24 h following dietary administration. Finally, the presence of these compounds in fetal blood samples was demonstrated, confirming that dietary exposure is appropriate for achieving fetal exposure. Collectively, the results of these studies support the use of dietary exposure in rabbit developmental toxicity studies.

Implementing a framework for integrating toxicokinetics into human health risk assessment for agrochemicals.

A strategic and comprehensive program in which toxicokinetic (TK) measurements are made for all agrochemicals undergoing toxicity testing (both new compounds and compounds already registered for use) is described. This approach provides the data to more accurately assess the toxicokinetics of agrochemicals and their metabolites in laboratory animals and humans. Having this knowledge provides the ability to conduct more insightful toxicity studies, refine and interpret exposure assessments and reduce uncertainty in risk assessments. By developing a better understanding of TK across species, including humans via in vitro metabolism studies, any differences across species in TK can be identified early and the most relevant species can be selected for toxicity tests. It also provides the ability to identify any non-linearities in TK as a function of dose, which in turn can be used to identify a kinetically derived maximum dose (KMD) and avoid dosing inappropriately outside of the kinetic linear range. Measuring TK in key life stages also helps to identify changes in ADME parameters from in utero to adults. A robust TK database can also be used to set internal concentration based "Reference Concentrations" and Biomonitoring Equivalents (BE), and support selection of Chemical Specific Adjustment Factors (CSAF). All of these factors support the reduction of uncertainty throughout the entire risk assessment process. This paper outlines how a TK research strategy can be integrated into new agrochemical toxicity testing programs, together with a proposed Framework for future use.

Evaluation of metabolically stabilized angiotensin IV analogs as procognitive/antidementia agents.

Angiotensin IV (AngIV: VYIHPF)-related peptides have long been recognized as procognitive agents with potential as antidementia therapeutics. Their development as useful therapeutics, however, has been limited by physiochemical properties that make them susceptible to metabolic degradation and impermeable to gut and blood-brain barriers. A previous study demonstrated that the core structural information required to impart the procognitive activity of the AngIV analog, norleucine(1)-angiotensin IV, resides in its three N-terminal amino acids, Nle-Tyr-Ile. The goal of this project was to chemically modify this tripeptide in such a way to enhance its metabolic stability and barrier permeability to produce a drug candidate with potential clinical utility. Initial results demonstrated that several N- and C-terminal modifications lead to dramatically improved stability while maintaining the capability to reverse scopolamine-induced deficits in Morris water maze performance and augment hippocampal synaptogenesis. Subsequent chemical modifications, which were designed to increase hydrophobicity and decrease hydrogen bonding, yielded an orally active, blood-barrier permeant, metabolically stabilized analog, N-hexanoic-Tyr-Ile-(6) aminohexanoic amide (dihexa), that exhibits excellent antidementia activity in the scopolamine and aged rat models and marked synaptogenic activity. These data suggest that dihexa may have therapeutic potential as a treatment of disorders, such as Alzheimer's disease, where augmented synaptic connectivity may be beneficial.

TK Modeler version 1.0, a Microsoft® Excel®-based modeling software for the prediction of diurnal blood/plasma concentration for toxicokinetic use.

TK Modeler 1.0 is a Microsoft® Excel®-based pharmacokinetic (PK) modeling program created to aid in the design of toxicokinetic (TK) studies. TK Modeler 1.0 predicts the diurnal blood/plasma concentrations of a test material after single, multiple bolus or dietary dosing using known PK information. Fluctuations in blood/plasma concentrations based on test material kinetics are calculated using one- or two-compartment PK model equations and the principle of superposition. This information can be utilized for the determination of appropriate dosing regimens based on reaching a specific desired C(max), maintaining steady-state blood/plasma concentrations, or other exposure target. This program can also aid in the selection of sampling times for accurate calculation of AUC(24h) (diurnal area under the blood concentration time curve) using sparse-sampling methodologies (one, two or three samples). This paper describes the construction, use and validation of TK Modeler. TK Modeler accurately predicted blood/plasma concentrations of test materials and provided optimal sampling times for the calculation of AUC(24h) with improved accuracy using sparse-sampling methods. TK Modeler is therefore a validated, unique and simple modeling program that can aid in the design of toxicokinetic studies.

Ontogeny of mammalian metabolizing enzymes in humans and animals used in toxicological studies.

It is well recognized that expression of enzymes varies during development and growth. However, an in-depth review of this acquired knowledge is needed to translate the understanding of enzyme expression and activity into the prediction of change in effects (e.g. kinetics and toxicity) of xenobiotics with age. Age-related changes in metabolic capacity are critical for understanding and predicting the potential differences resulting from exposure. Such information may be especially useful in the evaluation of the risk of exposure to very low (µg/kg/day or ng/kg/day) levels of environmental chemicals. This review is to better understand the ontogeny of metabolizing enzymes in converting chemicals to either less-toxic metabolite(s) or more toxic products (e.g. reactive intermediate[s]) during stages before birth and during early development (neonate/infant/child). In this review, we evaluated the ontogeny of major "phase I" and "phase II" metabolizing enzymes in humans and commonly used experimental animals (e.g. mouse, rat, and others) in order to fill the information gap.

Assessment of diurnal systemic dose of agrochemicals in regulatory toxicity testing--an integrated approach without additional animal use.

Integrated toxicokinetics (TK) data provide information on the rate, extent and duration of systemic exposure across doses, species, strains, gender, and life stages within a toxicology program. While routine for pharmaceuticals, TK assessments of non-pharmaceuticals are still relatively rare, and have never before been included in a full range of guideline studies for a new agrochemical. In order to better understand the relationship between diurnal systemic dose (AUC(24h)) and toxicity of agrochemicals, TK analyses in the study animals is now included in all short- (excluding acute), medium- and long-term guideline mammalian toxicity studies including reproduction/developmental tests. This paper describes a detailed procedure for the implementation of TK in short-, medium- and long-term regulatory toxicity studies, without the use of satellite animals, conducted on three agrochemicals (X11422208, 2,4-D and X574175). In these studies, kinetically-derived maximum doses (KMD) from short-term studies instead of, or along with, maximum tolerated doses (MTD) were used for the selection of the high dose in subsequent longer-term studies. In addition to leveraging TK data to guide dose level selection, the integrated program was also used to select the most appropriate method of oral administration (i.e., gavage versus dietary) of test materials for rat and rabbit developmental toxicity studies. The integrated TK data obtained across toxicity studies (without the use of additional/satellite animals) provided data critical to understanding differences in response across doses, species, strains, sexes, and life stages. Such data should also be useful in mode of action studies and to improve human risk assessments.

Development of angiotensin IV analogs as hepatocyte growth factor/Met modifiers.

The 6-AH family [D-Nle-X-Ile-NH-(CH(2))(5)-CONH(2); where X = various amino acids] of angiotensin IV (Ang IV) analogs binds directly to hepatocyte growth factor (HGF) and inhibit HGF's ability to form functional dimers. The metabolically stabilized 6-AH family member, D-Nle-Tyr-Ile-NH-(CH(2))(5)-CONH(2,) had a t(1/2) in blood of 80 min compared with the parent compound norleual [Nle-Tyr-Leu-Ψ-(CH(2)-NH(2))(3-4)-His-Pro-Phe], which had a t(1/2) in blood of <5 min. 6-AH family members were found to act as mimics of the dimerization domain of HGF (hinge region) and inhibited the interaction of an HGF molecule with a (3)H-hinge region peptide resulting in an attenuated capacity of HGF to activate its receptor Met. This interference translated into inhibition of HGF-dependent signaling, proliferation, and scattering in multiple cell types at concentrations down into the low picomolar range. We also noted a significant correlation between the ability of the 6-AH family members to block HGF dimerization and inhibition of the cellular activity. Furthermore, a member of the 6-AH family with cysteine at position 2, was a particularly effective antagonist of HGF-dependent cellular activities. This compound suppressed pulmonary colonization by B16-F10 murine melanoma cells, which are characterized by an overactive HGF/Met system. Together, these data indicate that the 6-AH family of Ang IV analogs exerts its biological activity by modifying the activity of the HGF/Met system and offers the potential as therapeutic agents in disorders that are dependent on or possess an overactivation of the HGF/Met system.