Taking the leap toward human-specific nonanimal methodologies: The need for harmonizing global policies for microphysiological systems.

With a recent amendment, India joined other countries that have removed the legislative barrier toward the use of human-relevant methods in drug development. Here, global stakeholders weigh in on the urgent need to globally harmonize the guidelines toward the standardization of microphysiological systems. We discuss a possible framework for establishing scientific confidence and regulatory approval of these methods.

In vitro assays for characterization of distinct multiple catalytic activities of thyroid peroxidase using LC-MS/MS.

A diverse set of environmental contaminants have raised a concern about their potential adverse effects on endocrine signaling. Robust and widely accepted battery of in vitro assays is available to assess the disruption of androgenic and estrogenic pathways. However, such definitive systems to investigate effects on the disruption of thyroid pathways by the xenobiotics are not yet well established. One of the major "Molecular Initiating Events" (MIEs) in thyroid disruption involves targeting of thyroid peroxidase (TPO), a key enzyme involved in thyroid hormone synthesis. TPO catalyzes mono- and diiodination of L-Tyrosine (L-Tyr) to generate 3-Iodo-l-tyrosine (MIT) and 3,5-Diiodo-l-tyrosine (DIT), respectively, followed by the coupling of iodinated tyrosine rings to generate thyroid hormones, 3,3'5-Triiodo-l-thyronine (T3) and Levothyroxine (T4). We sought to develop a robust, sensitive, and rapid in vitro assay systems to evaluate the effects of test chemicals on the multiple catalytic activities of thyroid peroxidase. Simple in vitro assays were designed to study TPO mediated distinct reactions using a single LC-MS/MS method. Herein, we describe a battery of assays to investigate the iodination of L-Tyr to MIT and DIT, MIT to DIT as well as, T3 to T4 catalyzed by rat thyroid TPO. Importantly, two sequential reactions involving mono- and diiodination of L-Tyr could be analyzed in a single assay. The assay that monitors in vitro conversion of DIT to T4 was developed to study the coupling of tyrosine rings. Enzyme kinetics studies revealed distinct characteristics of multiple reactions catalyzed by TPO. Further, the known TPO inhibitors were used to assess their potency towards individual TPO substrates and reactions. The resultant half maximum inhibitory concentration (IC50) values highlighted differential targeting of TPO catalyzed reactions by the same inhibitor. Overall results underscore the need to develop more nuanced approaches that account for distinct multiple catalytic activities of TPO.

Comparison of in chemico skin sensitization methods and development of an in chemico skin photosensitization assay.

Chemical substances that induce an allergic response in skin upon contact are called skin allergens or sensitizers, while chemical substances that elicit an allergic response only in presence of light are called photoallergens or photo sensitizers. The Direct Peptide Reactivity Assay (DPRA, OECD N° 442C, 2015) and the Amino Acid Derivative Reactivity Assay (ADRA) are in chemico assays used to discriminate between allergens and non-allergens. The DPRA and the ADRA, respectively, monitor the depletion of model peptides and modified amino acids induced by crosslinking with test chemicals. In the current study we compared these two assays and analyzed their suitability to predict skin sensitization potential of several chemical substances.  In order to study the combined effect of a chemical compound and UV light, we modified DPRA (photo-DPRA) as well as ADRA (photo-ADRA) by introduction of a photo-irradiation parameter. Analysis using photo-DPRA and photo-ADRA correctly distinguished known photoallergens from non-photoallergens. Upon irradiation, photoallergens selectively showed higher depletion of model peptides or modified amino acids. Thus, photo-DPRA and/or photo-ADRA can serve as non-animal in vitro methods for the identification and assessment of photoallergens/ photosensitizers.

Attenuation of allergic contact dermatitis through the endocannabinoid system.

Allergic contact dermatitis affects about 5% of men and 11% of women in industrialized countries and is one of the leading causes for occupational diseases. In an animal model for cutaneous contact hypersensitivity, we show that mice lacking both known cannabinoid receptors display exacerbated allergic inflammation. In contrast, fatty acid amide hydrolase-deficient mice, which have increased levels of the endocannabinoid anandamide, displayed reduced allergic responses in the skin. Cannabinoid receptor antagonists exacerbated allergic inflammation, whereas receptor agonists attenuated inflammation. These results demonstrate a protective role of the endocannabinoid system in contact allergy in the skin and suggest a target for therapeutic intervention.

Oxidative modification of low-density lipoprotein: lipid peroxidation by myeloperoxidase in the presence of nitrite.

Oxidative modification of low-density lipoprotein (LDL) is a pivotal process in early atherogenesis and can be brought about by myeloperoxidase (MPO), which is capable of reacting with nitrite, a NO metabolite. We studied MPO-mediated formation of conjugated dienes in isolated human LDL in dependence on the concentrations of nitrite and chloride. This reaction was strongly stimulated by low concentrations (5-50 microM) of nitrite which corresponds to the reported concentration in the arterial vessel wall. Under these conditions no protein tyrosine nitration occurred; this reaction required much higher nitrite concentrations (100 microM-1 mM). Chloride neither supported lipid peroxidation alone nor was its presence mandatory for the effect of nitrite. We propose a prominent role of lipid peroxidation for the proatherogenic action of the MPO/nitrite system, whereas peroxynitrite may be competent for protein tyrosine nitration of LDL. Monomeric and oligomeric flavan-3-ols present in cocoa products effectively counteracted, at micromolar concentrations, the MPO/nitrite-mediated lipid peroxidation of LDL. Flavan-3-ols also suppressed protein tyrosine nitration induced by MPO/nitrite or peroxynitrite as well as Cu2+-mediated lipid peroxidation of LDL. This multi-site protection by (-)-epicatechin or other flavan-3-ols against proatherogenic modification of LDL may contribute to the purported beneficial effects of dietary flavan-3-ols for the cardiovascular system.