3ßHSD activity saturates at physiological substrate concentrations in intact cells.

BACKGROUND: Conversion of adrenally produced dehydroepiandrosterone (DHEA) to the potent androgen dihydrotestosterone (DHT) is an important mechanism by which prostate cancer reaches castration resistance. At the start of this pathway is a branch point at which DHEA can be converted to Δ4 -androstenedione by the enzyme 3ß-hydroxysteroid dehydrogenase (3ßHSD) or to Δ5 -androstenediol by 17ßHSD. To better understand this process, we studied the kinetics of these reactions in cells. METHODS: Prostate cancer cells (LNCaP cell line) were incubated with steroids (DHEA and Δ5 -androstenediol) over a range of concentrations and the steroid metabolism reaction products were measured by mass spectrometry or by high-performance liquid chromatography to determine reaction kinetics. To confirm the generalizability of results, experiments were also performed in JEG-3 placental choriocarcinoma cells. RESULTS: The two reactions displayed very different saturation profiles, with only the 3ßHSD-catalyzed reaction beginning to saturate within a physiological substrate concentration range. Strikingly, incubating LNCaP cells with low (in the ~10 nM range) concentrations of DHEA resulted in a large majority of the DHEA undergoing 3ßHSD-catalyzed conversion to Δ4 -androstenedione, whereas high concentrations of DHEA (in the 100s of nM range) resulted in most of the DHEA undergoing 17ßHSD-catalyzed conversion to Δ5 -androstenediol. CONCLUSION: Contrary to expectations from previous studies that used purified enzyme, cellular metabolism of DHEA by 3ßHSD begins to saturate in the physiological concentration range, suggesting that fluctuations in DHEA concentrations could be buffered at the downstream active androgen level.

Effect of cx-DHED on Abnormal Glucose Transporter Expression Induced by AD Pathologies in the 5xFAD Mouse Model.

Alzheimer's disease (AD) is a form of dementia associated with abnormal glucose metabolism resulting from amyloid-beta (Aß) plaques and intracellular neurofibrillary tau protein tangles. In a previous study, we confirmed that carboxy-dehydroevodiamine∙HCl (cx-DHED), a derivative of DHED, was effective at improving cognitive impairment and reducing phosphorylated tau levels and synaptic loss in an AD mouse model. However, the specific mechanism of action of cx-DHED is unclear. In this study, we investigated how the cx-DHED attenuates AD pathologies in the 5xFAD mouse model, focusing particularly on abnormal glucose metabolism. We analyzed behavioral changes and AD pathologies in mice after intraperitoneal injection of cx-DHED for 2 months. As expected, cx-DHED reversed memory impairment and reduced Aß plaques and astrocyte overexpression in the brains of 5xFAD mice. Interestingly, cx-DHED reversed the abnormal expression of glucose transporters in the brains of 5xFAD mice. In addition, otherwise low O-GlcNac levels increased, and the overactivity of phosphorylated GSK-3ß decreased in the brains of cx-DHED-treated 5xFAD mice. Finally, the reduction in synaptic proteins was found to also improve by treatment with cx-DHED. Therefore, we specifically demonstrated the protective effects of cx-DHED against AD pathologies and suggest that cx-DHED may be a potential therapeutic drug for AD.

Amino Acids and Lipids Associated with Long-Term and Short-Term Red Meat Consumption in the Chinese Population: An Untargeted Metabolomics Study.

Red meat (RM) consumption is correlated with multiple health outcomes. This study aims to identify potential biomarkers of RM consumption in the Chinese population and evaluate their predictive ability. We selected 500 adults who participated in the 2015 China Health and Nutrition Survey and examined their overall metabolome differences by RM consumption by using elastic-net regression, then evaluate the predictivity of a combination of filtered metabolites; 1108 metabolites were detected. In the long-term RM consumption analysis 12,13-DiHOME, androstenediol (3α, 17α) monosulfate 2, and gamma-Glutamyl-2-aminobutyrate were positively associated, 2-naphthol sulfate and S-methylcysteine were negatively associated with long-term high RM consumption, the combination of metabolites prediction model evaluated by area under the receiver operating characteristic curve (AUC) was 70.4% (95% CI: 59.9-80.9%). In the short-term RM consumption analysis, asparagine, 4-hydroxyproline, and 3-hydroxyisobutyrate were positively associated, behenoyl sphingomyelin (d18:1/22:0) was negatively associated with short-term high RM consumption. Combination prediction model AUC was 75.6% (95% CI: 65.5-85.6%). We identified 10 and 11 serum metabolites that differed according to LT and ST RM consumption which mainly involved branch-chained amino acids, arginine and proline, urea cycle and polyunsaturated fatty acid metabolism. These metabolites may become a mediator of some chronic diseases among high RM consumers and provide new evidence for RM biomarkers.

Intranasal administration of DHED protects against exhaustive exercise-induced brain injury in rats.

DHED (10ß,17ß-dihydroxyestra-1,4-dien-3-one) is a brain-selective prodrug of 17ß-estradiol and has been reported to have a strong neuroprotective effect. In this study, the exhaustive swimming rat model was used to investigate the therapeutic effects and mechanisms of intranasal DHED treatment. Male eight-week-old healthy Sprague Dawley rats were randomly divided into three groups: control group (Cont), exhaustive swimming (ES), and DHED + exhaustive swimming (DHED). The open-field test and beam-walking test were performed to measure exploratory behavior and general activity in rats. Immunofluorescence staining, western blotting, ELISA analysis and related assay kits were applied to measure brain damage, inflammatory cytokines, and apoptosis pathways. Behavioral data shows that DHED intranasal administration can prevent neurobehavioral impairment caused by exhaustive swimming. Using a series of bioanalytical assays, we demonstrated that DHED markedly abated neuronal injury compared to the exhaustive swimming group, as evidenced by the reduced expression of apoptosis-regulated proteins, the improvement of neural survival, and the prevention of myelin loss. In addition, mitochondrial fission was attenuated distinctly, and a dynamic equilibrium was restored. Intranasal administration of DHED likewise significantly suppressed reactive gliosis and the release of inflammatory cytokines in the rat cerebral motor cortex. Consistent with previous reports, DHED treatment ameliorated changes of excitatory neurotransmitters. These results provide strong support for the promising therapeutic effects of DHED on neuroprotection during exhaustive swimming. The underlying mechanisms may rely on mitochondrial dynamics, neuroinflammation, and the balance of neurotransmitters.

Bioconversion of antifungal viridin to phytotoxin viridiol by environmental non-viridin producing microorganisms.

Biotransformation of viridin, an antifungal produced by biocontrol agent, with non-viridin producing microorganisms is studied. The results show that some environmental non-targeted microorganisms are able to reduce it in the known phytotoxin viridiol, and its 3-epimer. Consequently, this reduction, which happens in some cases by detoxification mechanism, could be disastrous for the plant in a biocontrol of plant disease. However, a process fermentation/biotransformation could be an efficient approach for the preparation of this phytotoxin.

Synthesis of human long-term metabolites of dehydrochloromethyltestosterone and oxymesterone.

We herein report the synthesis of the long-term metabolites "M4" (IUPAC: 4-chloro-17-hydroxymethyl-17-methyl-18-norandrosta-4,13-dien-3-ol) of dehydrochloromethyl-testosterone (DHCMT, Oral Turinabol) and "Oxy M9" (4-hydroxy-17ß-hydroxymethyl-17α-methyl-18-norandrosta-4,13-dien-3-one) of oxymesterone (Oranabol). Both compounds were derived from a common synthetic route starting from dehydroepiandrosterone acetate. Four different stereoisomers were evaluated for metabolite M4. The previously assigned structure could be corrected regarding the C-3 and C-17 stereocenters.

Molecular insights into how SHBG dimerization exerts changes on ligand molecular recognition.

Sex hormone binding globulin (SHBG) is a homodimeric glycoprotein and is the major carrier protein for sex steroids in plasma, regulating sex hormone availability in most vertebrate groups. Although it was initially thought that human dimeric SHBG bound a single ligand at the homodimer interface, studies demonstrated that dimeric SHBG binds a ligand to each subunit with similar affinity. In fact, the findings from recent experimental studies suggest that ligand binding to the SHBG dimer involves a complex allosteric mechanism involving conformational changes that limit observations of the presence of allosteric regulation. Therefore, we combined structural data with molecular dynamics simulations using Molecular Mechanics Generalized-Born Surface Area (MMGBSA) to dissect the structural and energetic basis for molecular recognition between five ligands whose affinities and binding positions on SHBG are known, i.e., 3ß,17α-diol; 3ß,17ß-diol; DHT; norgestrel (NOG); and estradiol (E2), and monomeric and dimeric SHBG. Protein-ligand complexes, involving dimeric SHBG saturated with two ligands on each subunit, reproduce the experimental affinity tendency and allow the observation that dimerization exerts disparate effects on binding affinity, characteristic of negative cooperativity for E2, DHT, and NOG, whereas 3ß-17α-diol and 3ß-17ß-diol lack allostery.

Hyfraxinic Acid, a Phytotoxic Tetrasubstituted Octanoic Acid, Produced by the Ash (Fraxinus excelsior L.) Pathogen Hymenoscyphus fraxineus Together with Viridiol and Some of Its Analogues.

A new tetrasubstituted octanoic acid, named hyfraxinic acid (1), was isolated together with known 1-deoxyviridiol (2), viridiol (3), nodulisporiviridin M (4), and demethoxyviridiol (5) from the organic extract of Hymenoscyphus fraxineus responsible for ash (Fraxinus excelsior L.) dieback in Europe. Hyfraxinic acid (1) was characterized, using spectroscopic methods, as 2,4-dihydroxy-7-methyl-6-methyleneoctanoic acid. Furthermore, the advanced Mosher method was used to determine the absolute configuration (3R) of 1-deoxyviridiol. Nodulisporiviridin M (4) was isolated for the first time from H. fraxineus. The phytotoxicity of each compound was tested by a leaf puncture assay on Celtis australis L., Quercus suber L., Hedera elix L., Juglans regia L., and Fraxinus angustifolia L. leaves. Compounds 1, 3, and 5 exhibited remarkable phytotoxicity on all plants tested, inducing necrotic lesions at concentrations of 1.0 and 0.5 mg/mL, while compounds 2 and 4 were found to be inactive in this bioassay. These results could contribute to a deeper understanding of the pathogenicity of H. fraxineus.

Gas Chromatography-Mass Spectrometry Analysis of Urinary Steroid Metabolomics for Detection of Early Signs of Adrenal Neoplasm Malignancy in Patients with Cushing's Syndrome.

The metabolomics of urinary steroids was studied by gas chromatography-mass spectrometry in 25 patients with Cushing's syndrome without malignant potential and in 12 patients with malignant potential of adrenal neoplasms (Weiss score 1-3). Patients with adrenocortical adenoma (N=24) constituted the control group. In patients with Cushing's syndrome and malignant potential, increased urinary excretion of 16-oxo-androstendiol, tetrahydro-11-deoxycortisol, and 16-hydroxypregnendiol, which had 100% specificity and sensitivity >90% for the diagnosis of malignant potential. Additionally, non-classical 5-ene-pregnenes (16-OHpregnenolone, 21-OH-pregnenolone, 3ß,16,20-pregnentriol, and 3ß,17,20-pregnentriol) were identified. The revealed changes in the metabolomics of steroids can be early signs of malignant potential in patients with Cushing's syndrome. In patients with malignant potential, three signs of reduced activity of 11ß-hydroxysteroid dehydrogenase type 2 were detected and in patients without malignant potential, one sign was found. In patients with and without malignant potential, three signs increased activity of 5ß-reductase were found.

Overexpression of cytochrome p450 125 in Mycobacterium: a rational strategy in the promotion of phytosterol biotransformation.

Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are generally produced by the biotransformation of phytosterols in Mycobacterium. The AD (D) production increases when the strain has high NAD+/NADH ratio. To enhance the AD (D) production in Mycobacterium neoaurum TCCC 11978 (MNR M3), a rational strategy was developed through overexpression of a gene involved in the phytosterol degradation pathway; NAD+ was generated as well. Proteomic analysis of MNR cultured with and without phytosterols showed that the steroid C27-monooxygenase (Cyp125-3), which performs sequential oxidations of the sterol side chain at the C27 position and has the oxidative cofactor of NAD+ generated, played an important role in the phytosterol biotransformation process of MNR M3. To improve the productivity of AD (D), the cyp125-3 gene was overexpressed in MNR M3. The specific activity of Cyp125-3 in the recombinant strain MNR M3C3 was improved by 22% than that in MNR M3. The NAD+/NADH ratio in MNR M3C3 was 131% higher than that in the parent strain. During phytosterol biotransformation, the conversion of sterols increased from 84 to 96%, and the yield of AD (D) by MNR M3C3 was increased by approximately 18% for 96 h fermentation. This rational strain modification strategy may also be applied to develop strains with important application values for efficient production of cofactor-dependent metabolites.

Enantioselective Chemical Syntheses of the Furanosteroids (-)-Viridin and (-)-Viridiol.

Herein we describe concise enantioselective chemical syntheses of (-)-viridin and (-)-viridiol. Our convergent approach couples two achiral fragments of similar complexity and employs an enantioselective intramolecular Heck reaction to set the absolute stereochemical configuration of an all-carbon quaternary stereocenter. To complete the syntheses of these base- and nucleophile-sensitive natural products, we conduct carefully orchestrated site- and diastereoselective oxidations and other transformations. Our work is the first to generate these targets as single enantiomers.

A sharp, robust, and quantitative method by liquid chromatography tandem mass spectrometry for the measurement of EAD for acute radiation syndrome and its application.

17-Ethinyl-3,17-dihydroxyandrost-5-ene (EAD) is an agent designed for the treatment of acute radiation syndrome (ARS). Given its vital role played in the prevention and mitigation of ARS, the development of a sharp, sensitive and robust liquid chromatography tandem mass spectrometry (LC-MS/MS) method to monitor the metabolism of EAD in vivo was crucial. A new method was constructed and validated for the determination of EAD with the internal standard of androst-5-ene-3ß,17ß-diol (5-AED). The blood samples were precipitated with methanol, centrifuged, from which the supernatant was separated on UPLC with C18 column and eluted in gradient with acetonitrile and Milli-Q water both containing 0.1% formic acid (FA). Quantification was performed by a triple quadrupole mass spectrometer with electro spray ionization (ESI) in multiple reactive monitoring (MRM) positive mode. A good linearity was obtained with R>0.99 for EAD within its calibration range from 5 to 1000ngmL-1 with a lowest limit of quantification (LLOQ) of 5ngmL-1. Inter- and intra-day accuracy and precision of three levels of quality control (QC) samples were within the range of 15%, while the LLOQ was within 20%. Samples were stable under the circumstances of the experiments. The method was simple, accurate and robust applied to determine the concentrations of EAD in Wistar rat after a single administration of EAD orally at the dose of 100mgkg-1.

Identification and characterization of novel long-term metabolites of oxymesterone and mesterolone in human urine by application of selected reaction monitoring GC-CI-MS/MS.

The search for metabolites with longer detection times remains an important task in, for example, toxicology and doping control. The impact of these long-term metabolites is highlighted by the high number of positive cases after reanalysis of samples that were stored for several years, e.g. samples of previous Olympic Games. A substantial number of previously alleged negative samples have now been declared positive due to the detection of various long-term steroid metabolites the existence of which was unknown during the Olympic Games of 2008 and 2012. In this work, the metabolism of oxymesterone and mesterolone, two anabolic androgenic steroids (AAS), was investigated by application of a selected reaction monitoring gas chromatography-chemical ionization-triple quadrupole mass spectrometry (GC-CI-MS/MS) protocol for metabolite detection and identification. Correlations between AAS structure and GC-CI-MS/MS fragmentation behaviour enabled the search for previously unknown but expected AAS metabolites by selection of theoretical transitions for expected metabolites. Use of different hydrolysis protocols allowed for evaluation of the detection window of both phase I and phase II metabolites. For oxymesterone, a new metabolite, 18-nor-17ß-hydroxymethyl-17α-methyl-4-hydroxy-androst-4,13-diene-3-one, was identified. It was detectable up to 46 days by using GC-CI-MS/MS, whereas with a traditional screening (detection of metabolite 17-epioxymesterone with electron ionization GC-MS/MS) oxymesterone administration was only detectable for 3.5 days. A new metabolite was also found for mesterolone. It was identified as 1α-methyl-5α-androstan-3,6,16-triol-17-one and its sulfate form after hydrolysis with Helix pomatia resulted in a prolonged detection time (up to 15 days) for mesterolone abuse. Copyright © 2017 John Wiley & Sons, Ltd.

A Metabolome-Wide Study of Dry Eye Disease Reveals Serum Androgens as Biomarkers.

PURPOSE: To test the association between serum metabolites and dry eye disease (DED) using a hypothesis-free metabolomics approach. DESIGN: Cross-sectional association study. PARTICIPANTS: A total of 2819 subjects from the population-representative TwinsUK cohort in the United Kingdom, with a mean age of 57 years (range, 17-82 years). METHODS: We tested associations between 222 known serum metabolites and DED. All subjects underwent nontargeted metabolomic analysis of plasma samples using gas and liquid chromatography in combination with mass spectrometry (Metabolon Inc., Durham, NC). Dry eye disease was defined from the validated Short Questionnaire for Dry Eye Syndrome (SQDES) as a previous diagnosis of DED by a clinician or "often" or "constant" symptoms of dryness and irritation. Analyses were performed with linear mixed effect models that included age, BMI, and sex as covariates, corrected for multiple testing. MAIN OUTCOME MEASURES: Primary outcome was DED as defined by the SQDES, and secondary outcomes were symptom score of DED and a clinical diagnosis of DED. RESULTS: Prevalence of DED as defined by the SQDES was 15.5% (n = 436). A strong and metabolome-wide significant association with DED was found with decreased levels of the metabolites androsterone sulfate (P = 0.00030) and epiandrosterone sulfate (P = 0.00036). Three other metabolites involved in androgen metabolism, 4-androsten-3beta,17beta-diol disulfate 1 and 2, and dehydroepiandrosterone sulfate, were the next most strongly associated of the 222 metabolites, but did not reach metabolome-wide significance. Dryness and irritation symptoms, as opposed to a clinical diagnosis, were particularly strongly associated with decreased androgen steroid metabolites, with all reaching metabolome-wide significance (androsterone sulfate, P = 0.000000029; epiandrosterone sulfate, P = 0.0000040; 4-androsten-3beta,17beta-diol disulfate 1, P = 0.000016; 4-androsten-3beta,17beta-diol disulfate 2, P = 0.000064; and dehydroepiandrosterone sulfate, P = 0.00011). Of these 5 androgens, epiandrosterone sulfate (P = 0.0076) was most associated with 2-year incidence of clinician-diagnosed DED. In addition, we found decreased glycerophosphocholines to be associated with DED, although not at metabolome-wide significance. CONCLUSIONS: This hypothesis-free metabolomic approach found decreased serum androgens to be highly associated with DED and adds important evidence to the growing body of research that links androgens to ocular surface disease and DED.

Neuroactive Steroids in Acute Ischemic Stroke: Association with Cognitive, Functional, and Neurological Outcomes.

Despite several scientific and technological advances, there is no single neuroprotective treatment that can reverse the brain damage after acute ischemic stroke (AIS). Neuroactive steroids are cholesterol-derived hormones that have the ability to modulate the normal and pathologic nervous system employing genomic and nongenomic mechanisms. In this work, we first investigated if AIS affects the plasma concentration of 5 neuroactive steroids (cortisol, estradiol, progesterone, testosterone, and 3α-androstenediol glucuronide). Second, we studied if levels of circulating steroids associate with neurological, cognitive, and functional outcome in a cohort of 60- to 90 year-old male and female patients with AIS. For this purpose, we recruited patients who were hospitalized at the Emergency Room of the Central Military Hospital within the first 24 h after stroke onset. We designed 2 experimental groups, each one composed of 30 control subjects and 30 AIS patients, both males and females. The assessment of neurological deficit was performed with the NIHSS and the tests used for the functional and cognitive status were: (1) modified Rankin Scale; (2) Photo test, and (3) abbreviated Pfeiffer's mental status questionnaire. We observed a significant difference in plasma concentration of cortisol and estradiol between both experimental groups. In the AIS group, higher levels of these neuroactive steroids were associated with more pronounced neurological, cognitive and functional deficits in women compared to men. We propose that in elderly patients, high levels of circulating neuroactive steroids like cortisol and estradiol could potentiate AIS-mediated neuropathology in the ischemic and penumbra areas.

New oxymesterone metabolites in human by gas chromatography-tandem mass spectrometry and their application for doping control.

Oxymesterone (17α-methyl-4, 17ß-dihydroxy-androst-4-ene-3-one) is one of the anabolic androgenic steroids (AAS) banned by the World Anti-Doping Agency (WADA). The biotransformation of oxymesterone is performed in vitro by human heptocytes and human urinary metabolic profiles are investigated after single dose of 20 mg to two adult males as well. Cell cultures and urine samples were hydrolyzed by ß-glucuronidase, extracted, and reacted with N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide (NH4 I), and dithioerythritol. After derivatization, a gas chromatography triple quadruple tandem mass spectrometry (GC-MS/MS) using full scan and MS/MS modes was applied. The total ion chromatographs of the blank and the positive samples are compared, and 7 new metabolites were found. In addition to the well-known 17-epioxymesterone, oxymesterone is metabolized by 4-ene-reduction, 3-keto-reduction, 11ß-hydroxylation, and 16ξ-hydroxylation. Based on the behavior of the MS/MS results of product ion and precursor ion modes, a GC-MS/MS method has been developed monitoring these metabolites. The structures of metabolite 2 and 4 are tentatively identified as 17α-methyl-3ß, 17ß-dihydroxy-5α-androstane-4-one and 17α-methyl-3α, 4ξ, 17ß-trihydroxy-5α-androstane, respectively. Detection of oxymesterone using new metabolites M2 and M4 can extend the detection window up to 4 days since the parent steroid was not detectable. Copyright © 2015 John Wiley & Sons, Ltd.

The prodrug DHED selectively delivers 17ß-estradiol to the brain for treating estrogen-responsive disorders.

Many neurological and psychiatric maladies originate from the deprivation of the human brain from estrogens. However, current hormone therapies cannot be used safely to treat these conditions commonly associated with menopause because of detrimental side effects in the periphery. The latter also prevents the use of the hormone for neuroprotection. We show that a small-molecule bioprecursor prodrug, 10ß,17ß-dihydroxyestra-1,4-dien-3-one (DHED), converts to 17ß-estradiol in the brain after systemic administration but remains inert in the rest of the body. The localized and rapid formation of estrogen from the prodrug was revealed by a series of in vivo bioanalytical assays and through in vivo imaging in rodents. DHED treatment efficiently alleviated symptoms that originated from brain estrogen deficiency in animal models of surgical menopause and provided neuroprotection in a rat stroke model. Concomitantly, we determined that 17ß-estradiol formed in the brain from DHED elicited changes in gene expression and neuronal morphology identical to those obtained after direct 17ß-estradiol treatment. Together, complementary functional and mechanistic data show that our approach is highly relevant therapeutically, because administration of the prodrug selectively produces estrogen in the brain independently from the route of administration and treatment regimen. Therefore, peripheral responses associated with the use of systemic estrogens, such as stimulation of the uterus and estrogen-responsive tumor growth, were absent. Collectively, our brain-selective prodrug approach may safely provide estrogen neuroprotection and medicate neurological and psychiatric symptoms developing from estrogen deficiency, particularly those encountered after surgical menopause, without the adverse side effects of current hormone therapies.

Synthesis and antiproliferative evaluation of some novel B-nor-D-homosteroids.

Using 3ß-hydroxy-5-androsten-17-one as a starting material, a series of novel nitrogen-containing B-nor-D-homosteroids were designed and synthesized by the oximation, Beckman rearrangement, ozonation, cyclization and condensation reaction. The structures of all new compounds were determined by analysis of their NMR, MS and IR spectra. The antiproliferative activity of compounds was evaluated against HT-29 (colonic carcinoma), HeLa (human cervical carcinoma) and Bel 7404 (human liver carcinoma) cells.

Genotypes of Fraxinus excelsior with different susceptibility to the ash dieback pathogen Hymenoscyphus pseudoalbidus and their response to the phytotoxin viridiol - a metabolomic and microscopic study.

Eight European ash (Fraxinus excelsior) genotypes with different known susceptibility to Hymenoscyphus pseudoalbidus were tested against the phytotoxin viridiol and their response described at the microscopic and metabolomic level. All ash genotypes were sensitive to the toxin and necrosis was detectable after 24h. Among the three viridiol concentrations used in the experiment, the lowest concentration (14.7µM) yielded markedly lower mean damage scores compared to those resulting from seedlings tested at higher dosages. The highest damage scores were associated with the susceptible ash clones S-101, S-106 and S-125, but also with resistant clone R-104. Three resistant clones (R-131, R-121, and R-118) had lower mean damage scores compared to susceptible clones. Wilting of leaves was more common 48h after treatment and more pronounced on seedlings with high damage scores. The resulting lesions generally lacked browning of tissue and displayed only surface disruption of cells in direct contact with the toxin. A delay in symptom development was evident on all five resistant clones tested with the two higher concentrations of viridiol. LC-HRMS and MS/MS analyses of ash seedling extracts suggest several secoiridoid compounds as well as compounds related to abscisic acid (ABA) to be produced in response to viridiol. ABA-cysteine and xanthoxin were found at significantly higher concentrations in susceptible clones compared to resistant clones after treatment with viridiol, suggesting a primary role of ABA in response to stress. The results observed in this study suggest that genetic resistance to H. pseudoalbidus among ash genotypes may be explained, in part, by the varied response to phytotoxins produced by the fungus.