Differences in endocrine and reproductive responses to substance exposure across generations: highlighting the importance of complementary findings.

This article analyzes the results from 112 Extended One-Generation Reproductive Toxicity studies. The objective was to determine if test animals show consistent endocrine and reproductive effects within the same and across different generations and life stages. The analysis, grounded in a comprehensive Binary Matrix, included 530 observed effects and 193 unique, statistically significant associations. Associations' strength was quantified using Jaccard (J) coefficients to measure effect co-occurrence in the same study. Associated effects co-occur infrequently across the whole dataset (median J = 0.231). However, specific patterns emerged: associations of same effects across generations exhibited a higher strength (median J = 0.400) compared to associations of different effects (median J = 0.222). Notably, associations with effects observed in both the parental animals of the adult first filial generation (P1) and developing second filial generations (dF2) demonstrated J coefficients (with medians ranging from 0.300 to 0.430) that were approximately twofold higher than those of other associations. Consistently, equivalent life stage associations across generations revealed statistically significant higher association strengths for the P1 and dF2 generations (medians of 0.375 and 0.333, respectively) compared to other generations (medians of 0.200 and 0.174), possibly due to longer exposure duration and altered cross-talk between pregnant P1 dam and its conceptus. Overall, it is concluded that co-occurrence of associated effects in the same study is rather infrequent and that associations with effects in P1 and dF2 are stronger than all other associations. In general, the findings underscore the importance of independently analyzing each effect per generation due to the generally low co-occurrence rates of associated effects, challenging traditional expectations of generational continuity in toxic effects.

Evaluating the conduct and reporting of the T-cell dependent antibody response in the Extended One-Generation Reproductive Toxicity study provided under the EU REACH regulation.

The European Union (EU) Chemicals Strategy for Sustainability regards chemicals that affect the immune system among the most harmful ones. The Extended One-Generation Reproductive Toxicity study (EOGRTS; Organisation for Economic Co-Operation and Development (OECD) Test Guideline (TG) 443), addresses, among others, potential effects of chemicals on development. In specific cases, the EOGRTS is performed with addition of a so-called cohort 3, that addresses potential effects on the developing immune system, by means of a central assay measuring the T-cell dependent antibody response (TDAR). This assay is based on an interplay of antigen presentation, T-cell help and antibody production by B-cells, and together comprises a functional immune response. In the context of the EOGRTS review project of the European Chemicals Agency (ECHA), we evaluated 15 available TDARs for compliance with conduct and reporting requirements. Collectively, the majority of the TDAR studies were considered to be adequately conducted. We however observed: (i) the protocols differed by the antigen used (sheep red blood cells (SRBC) or KLH), the route of administration (intravenous, intraperitoneal, or subcutaneous), prime or prime/boost immunizations, and whether IgG was measured. (ii) There was major variation in the effects of the positive control for immunosuppression, cyclophosphamide. (iii) Proficiency was not always shown. (iv) Statistical analysis was not always done or reported. (v) Results of effects on lymphocyte populations or other immunotoxicity observations obtained in cohort 1 (or 2) of the EOGRTS were not always discussed together with results of the TDAR. Taken together, next to an improved quality of reporting, this may suggest a need to better define the conduct of the TDAR in OECD TG 443 and OECD Guidance Document (GD) 151, at least for certain aspects.

A non-clinical and clinical IUCLID database for 530 pharmaceuticals (part I): Methodological aspects of its development.

A new IUCLID database is provided containing results from non-clinical animal studies and human information for 530 approved drugs. The database was developed by extracting data from pharmacological reviews of repeat-dose, carcinogenicity, developmental, and reproductive toxicity studies. In the database, observed and no-observed effects are linked to the respective effect levels, including information on severity/incidence and transiency/reversibility. It also includes some information on effects in humans, that were extracted from relevant sections of standard product labels of the approved drugs. The database is complemented with a specific ontology for reporting effects that was developed as an improved version of the Ontology Lookup Service's mammalian and human phenotype ontologies and includes different hierarchical levels. The developed ontology contains novel and unique standardized terms, including ontological terms for reproductive and endocrine effects. The database aims to facilitate correlation and concordance analyses based on the link between observed and no-observed effects and their respective effect levels. In addition, it offers a robust dataset on drug information for the pharmaceutical industry and research. The reported ontology supports the analyses of toxicological information, especially for reproductive and endocrine endpoints and can be used to encode legacy data or develop additional ontologies. The new database and ontology can be used to support the development of alternative non-animal approaches, to elucidate mechanisms of toxicity, and to analyse human relevance. The new IUCLID database is provided free of charge at https://iuclid6.echa.europa.eu/us-fda-toxicity-data.

UDP-glucuronosyltransferases (UGTs) 2B7 and UGT2B17 display converse specificity in testosterone and epitestosterone glucuronidation, whereas UGT2A1 conjugates both androgens similarly.

Testosterone and epitestosterone are endogenous steroids that differ in the configuration of the hydroxyl-bearing carbon at C-17. Testosterone is the predominant male sex hormone, whereas the role of epitestosterone is largely unclear. In humans, both androgens are excreted mainly as glucuronide conjugates and the urinary ratio of testosterone to epitestosterone (T/E), used to expose illicit testosterone abuse by male athletes, indicates the relative concentrations of the respective glucuronides. Some male athletes have T/E values greater than the accepted threshold value (4.0), even without testosterone abuse. We have analyzed athletes' urine samples and found that the main reason for such false-positive results in doping tests was a low epitestosterone glucuronide concentration not a high level of testosterone glucuronide. Sulfate conjugates of both testosterone and epitestosterone were also detected in the different urine samples. Glucuronidation assays with the 19 human UDP-glucuronosyltransferases (UGTs) of subfamilies UGT1A, UGT2A, and UGT2B revealed that UGT2B17 is the most active enzyme in testosterone glucuronidation. UGT2B17 does not glucuronidate epitestosterone, but inhibition studies revealed that it binds epitestosterone with affinity similar to that of testosterone. Epitestosterone glucuronidation is catalyzed mainly by UGT2B7, and the K(m) of this reaction is significantly lower than the K(m) of UGT2B17 for testosterone. Although UGT2B7 and UGT2B17 exhibited high, although converse, stereoselectivity in testosterone and epitestosterone glucuronidation, UGT2A1, an extrahepatic enzyme that is expressed mainly in the nasal epithelium, catalyzed the glucuronidation of both steroids at considerable rates and similar kinetics. The results shed new light on the substrate specificity and stereoselectivity of human UGTs.

Stereochemical and steric control of the UDP-glucuronosyltransferase-catalyzed conjugation reaction: a rational approach for the design of inhibitors for the human UGT2B7.

A set of 76 derivatives of the epimeric tricyclic sesquiterpenols longifolol and isolongifolol was subjected to inhibition and glucuronidation assays employing the human UDP-glucuronosyltransferase (UGT) 2B7. Detailed structure-activity relationships (SARs) with respect to functionality, stereochemical properties, and steric features were derived. To gain further insight into the SARs of UGT2B7 ligands herein, we have developed a 3D-quantitative structure-activity relationship (3D-QSAR) using Comparative Molecular Similarity Analysis (CoMSIA). The formation of the enzyme-inhibitor complex was predominantly controlled by spatially directed hydrophobic interactions. The glucuronidation rate was significantly influenced by the steric demand of substituents in proximity of the nucleophilic hydroxy group. The glucuronidation of the compounds was prevented by the introduction of bulky substituents such as isopropyl, tert-butyl, and phenyl groups. The epimeric longifolol derivatives of series D were the best inhibitors displaying IC(50) values as low as 4.6 nM. This study shows that high-potency substrates can be turned into potent inhibitors by addressing functional, stereochemical, and steric properties.

Eudismic analysis of tricyclic sesquiterpenoid alcohols: lead structures for the design of potent inhibitors of the human UDP-glucuronosyltransferase 2B7.

The epimeric tricyclic sesquiterpenoid alcohols globulol, epiglobulol, cedrol, epicedrol, longifolol, and isolongifolol were investigated in their ability to inhibit the recombinant human UDP-glucuronosyltransferase (UGT) 2B7. The stereoisomers displayed rapidly reversible competitive inhibition, which was substrate-independent. Longifolol and its stereoisomer isolongifolol displayed the lowest competitive inhibition constants (K(ic)) of 23 and 26 nM, respectively. The K(ic) values of cedrol and its epimer epicedrol were 0.15 and 0.21 microM, those of globulol and epiglobulol were 5.4 and 4.0 microM, respectively. The diastereomeric alcohols exhibited nearly identical affinities toward UGT2B7 indicating that the spatial arrangement of the hydroxy group had no influence on the dissociation of the enzyme-terpenoid complex. The high affinities stemmed presumably from mere hydrophobic interactions between the hydrocarbon scaffold of the terpenoid alcohol and the binding site of the enzyme. Glucuronidation assays revealed that there were large differences in the rates at which the epimeric alcohols were conjugated. Therefore, the spatial arrangement of the hydroxy group controlled the rate of the UGT2B7-catalyzed reaction. The introduction of a methyl group into the side chain of isolongifolol and longifolol increased the steric hindrance. As a result, the rate of the UGT2B7-catalyzed reaction was decreased by more than 88%. The findings indicated that the rate of the UGT2B7-catalyzed glucuronidation is significantly controlled by stereochemical and steric factors. Considering the high inhibition levels exerted by the tricyclic sesquiterpenoid alcohols, these compounds might serve as valuable lead structures for the design of potent inhibitors for UGT2B7.

Isoform-selective inhibition of the human UDP-glucuronosyltransferase 2B7 by isolongifolol derivatives.

A set of 48 derivatives of the tricyclic sesquiterpenol alcohol isolongifolol was synthesized. The set comprised homochiral and diastereomeric alcohols, amines, chlorohydrins, as well as carboxylic acids, phosphonic acids, and their corresponding esters. The absolute configuration of the epimeric compounds was assigned by 2D NMR experiments [gradient heteronuclear single quantum correlation (gHSQC) and gradient nuclear Overhauser enhancement spectroscopy (gNOESY)] in agreement with crystallographic data. The tricyclic derivatives were assessed as inhibitors of the human UDP-glucuronosyltransferase (UGT) 2B7. The phenyl-substituted secondary alcohol 26b was the best inhibitor in this series and its competitive inhibition constant was 18 nM. Compound 26b was not glucuronidated by UGT2B7 and other hepatic UGT enzymes, presumably due to the high steric hindrance exerted by its bulky phenyl substituent. Its inhibitory activity toward 14 other UGT isoforms of subfamily 1A and 2B was determined, and the data indicated that the tricyclic secondary alcohol 26b was highly selective for UGT2B7 (true selectivity >1000).

Potent inhibitors of the human UDP-glucuronosyltransferase 2B7 derived from the sesquiterpenoid alcohol longifolol.

The tricyclic sesquiterpenol (+)-longifolol served as a lead structure for the design of inhibitors of the human UDP-glucuronosyltransferase (UGT) 2B7. Twenty-four homochiral and epimeric longifolol derivatives were synthesized and screened for their ability to inhibit the enzyme. The absolute configuration at the stereogenic center C1' was determined by X-ray crystallography and 2D NMR spectroscopy (gHSQC, gNOESY). The phenyl-substituted secondary alcohol 16 b (beta-phenyllongifolol) displayed the highest affinity toward UGT2B7, and its inhibitory dissociation constant was 0.91 nM. The mode of inhibition was rapidly reversible and competitive. The inhibitor was not glucuronidated by UGT2B7 or other hepatic UGTs, presumably as a result of the high steric demand exerted by the phenyl group. Inhibition assays employing 14 other UGT isoforms suggested that inhibitor 16 b was highly selective for UGT2B7.

Chiral distinction between the enantiomers of bicyclic alcohols by UDP-glucuronosyltransferases 2B7 and 2B17.

The stereoselective binding and transformation of optically pure bicyclic alcohols by human UDP-glucuronosyltransferases from subfamily 2B were investigated. The enantiomers of 1-indanol, 1-tetralol, and 1-benzosuberol were synthesized by asymmetric Corey-Bakshi-Shibata reduction and subjected to glucuronidation assays. The alcohols studied were primarily glucuronidated by UGT2B7 and UGT2B17. The catalytic transformation by UGT2B17 was highly stereoselective, favoring conjugation of the (R)-enantiomers. UGT2B7, on the other hand, did not exhibit stereoselectivity toward 1-benzosuberol, the best substrate in this series. To assess binding affinities to the enzymes, the six different compounds were tested for their efficiency as inhibitors of either UGT2B7 or UGT2B17. The results of the latter analyses indicated that the affinities of both enantiomers of each pair towards UGT2B7 and UGT2B17 were of the same order of magnitude. Therefore, the findings of this study suggest that the spatial arrangement of the hydroxy group plays an important role in the glucuronic acid transfer reaction, but not necessarily in substrate binding to the UGTs.

Stereochemical sensitivity of the human UDP-glucuronosyltransferases 2B7 and 2B17.

A set of 28 enantiomers comprising rigid and flexible secondary alcohols was synthesized by the asymmetric Corey-Bakshi-Shibata reduction. The enantiomerically pure alcohols were subjected to enzymatic glucuronidation assays employing the human UDP-glucuronosyltransferases (UGTs) 2B7 and 2B17. Both UGTs displayed high levels of stereoselectivity, favoring the conjugation of the (R)-enantiomers over their respective (S)-stereoisomers at eudismic ratios up to 256. The spatial arrangement of the hydroxy group determined the diastereoselectivity of the UGT2B17-catalyzed reaction in agreement with Pfeiffer's rule (eudismic activity quotient = 0.83 +/- 0.14). Inhibition studies revealed that the enantiomers had similar affinities toward the enzymes. The diastereoselectivity of the UGT-catalyzed conjugation stemmed, therefore, from the arrangement of the substrates in the catalytic site, rather than from distinct affinities toward the enzymes. Taken together, this study showed that metabolic enzymes that are generally conceived to be rather "flexible" in nature are capable of displaying high levels of chiral distinction.