Molecular Cloning, Expression and Enzymatic Characterization of Tetrahymena thermophila Glutathione-S-Transferase Mu 34.

Glutathione-S-transferase enzymes (GSTs) are essential components of the phase II detoxification system and protect organisms from oxidative stress induced by xenobiotics and harmful toxins such as 1-chloro-2,4-dinitrobenzene (CDNB). In Tetrahymena thermophila, the TtGSTm34 gene was previously reported to be one of the most responsive GST genes to CDNB treatment (LD50 = 0.079 mM). This study aimed to determine the kinetic features of recombinantly expressed and purified TtGSTm34 with CDNB and glutathione (GSH). TtGSTm34-8xHis was recombinantly produced in T. thermophila as a 25-kDa protein after the cloning of the 660-bp full-length ORF of TtGSTm34 into the pIGF-1 vector. A three-dimensional model of the TtGSTm34 protein constructed by the AlphaFold and PyMOL programs confirmed that it has structurally conserved and folded GST domains. The recombinant production of TtGSTm34-8xHis was confirmed by SDS‒PAGE and Western blot analysis. A dual-affinity chromatography strategy helped to purify TtGSTm34-8xHis approximately 3166-fold. The purified recombinant TtGSTm34-8xHis exhibited significantly high enzyme activity with CDNB (190 µmol/min/mg) as substrate. Enzyme kinetic analysis revealed Km values of 0.68 mM with GSH and 0.40 mM with CDNB as substrates, confirming its expected high affinity for CDNB. The optimum pH and temperature were determined to be 7.0 and 25 °C, respectively. Ethacrynic acid inhibited fully TtGSTm34-8xHis enzyme activity. These results imply that TtGSTm34 of T. thermophila plays a major role in the detoxification of xenobiotics, such as CDNB, as a first line of defense in aquatic protists against oxidative damage.

Feature-Based Molecular Networking Combined with Multivariate Analysis for the Characterization of Glutathione Adducts as a Smoking Gun of Bioactivation.

Feature-based molecular networking (FBMN) is a powerful analytical tool for mass spectrometry (MS)-based untargeted metabolomics data analysis. FBMN plays an important role in drug metabolism studies, enabling the visualization of complex metabolomics data to achieve metabolite characterization. In this study, we propose a strategy for the characterization of glutathione (GSH) adducts formed via in vitro metabolic activation using FBMN assisted by multivariate analysis (MVA). Acetaminophen was used as a model substrate for method development, and the practical potential of the method was investigated by its application to 2-aminophenol (2-AP) and 2,4-dinitrochlorobenzene (DNCB). Two 2-AP GSH adducts and one DNCB GSH adduct were successfully characterized by forming networks with GSH even though the mass spectral information obtained for the parent compound was deficient. False positives were effectively filtered out by the variable influence on projection cutoff criteria obtained from orthogonal partial least-squares-discriminant analysis. The GSH adducts formed by enzymatic or nonenzymatic reactions were intuitively distinguished by the pie chart of FBMN results. In summary, our approach effectively characterizes GSH adducts, which serve as compelling evidence of bioactivation. It can be widely utilized to enhance risk assessment in the context of drug metabolism.

Heat shock protein 90 (Hsp90) inhibitor STA-9090 (Ganetespib) ameliorates inflammation in a mouse model of atopic dermatitis.

Molecular chaperones belonging to the heat shock protein 90 (Hsp90) family are implicated in inflammatory processes and described as potential novel therapeutic targets in autoimmune/inflammatory skin diseases. While the pathological role of circulating Hsp90 has been recently proposed in patients with atopic dermatitis (AD), a chronic inflammatory skin disease characterized by intense itching and recurrent skin lesions, studies aimed at investigating the role of Hsp90 as a potential target of AD therapy have not yet been conducted. Here, the effects of the Hsp90 blocker STA-9090 (Ganetespib) applied systemically or topically were determined in an experimental mouse model of dinitrochlorobenzene (DNCB)-induced AD. Intraperitoneal administration of STA-9090 ameliorated clinical disease severity, histological epidermal thickness, and dermal leukocyte infiltration in AD mice which was associated with reducing the scratching behavior in DNCB-treated animals. Additionally, topically applied STA-9090 led to lowered disease activity in AD mice, reduced serum levels of IgE, and up-regulated filaggrin expression in lesional skin samples. Our observations suggest that Hsp90 may be a promising therapeutic target in atopic dermatitis and potentially other inflammatory or autoimmune dermatoses.

Estrogen Receptor Alpha Gene (ESR1) Facilitates Th2-immune Response and Enhances Th2 Cytokines in Experimental Atopic Dermatitis Mice.

Background: Molecular markers are involved in atopic dermatitis (AD) pathogenesis. The estrogen receptor (ESR)-1 gene, encoding ERα, is reported to express aberrantly in AD patients. Objective: To detect the biological functions of ESR1 in 2,4 dinitrochlorobenzene (DNCB)-treated mice. Methods: The DNCB-treated mice received a topical application of emulsion containing the 1,3-bis(4 hydroxyphenyl)-4-methyl-5-[4-(2-piperidinyl ethoxy) phenol]-1H-pyrazole dihydrochloride (MPP; an ESR1-selective antagonist) to dorsal skins and ears. Then the dermatitis scores, histopathological changes, and cytokine levels were evaluated. Results: MPP specifically downregulated ESR1 expression in DNCB-applied mice. Functionally, application of MPP abolished the DNCB-induced promotion in dermatitis score. Additionally, MPP administration protected against DNCB-induced dermatitis severity, suppressed mast cell infiltration and reduced production of immunoglobulin E (IgE) and thymus and activation-regulated chemokine (TARC). Moreover, MPP treatment inhibited DNCB-induced production of Th2 cytokines and infiltration of CD4+ T cells. Conclusion: ESR1 facilitates Th2-immune response and enhances Th2 cytokines in AD mice.

Anti-Atopic Effect of Isatidis Folium Water Extract in TNF-α/IFN-γ-Induced HaCaT Cells and DNCB-Induced Atopic Dermatitis Mouse Model.

Isatidis folium or Isatis tinctoria L. is a flowering plant of the Brassicaceae family, commonly known as woad, with an ancient and well-documented history as an indigo dye and medicinal plant. This study aimed to evaluate the anti-atopic dermatitis (AD) effects of Isatidis folium water extract (WIF) using a 2,4-dinitrochlorobenzene (DNCB)-induced AD-like mouse model and to investigate the underlying mechanism using tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ)-activated HaCaT cells. Oral administration of WIF reduced spleen weight, decreased serum IgE and TNF-α levels, reduced epidermal and dermal thickness, and inhibited eosinophil and mast cell recruitment to the dermis compared to DNCB-induced control groups. Furthermore, oral WIF administration suppressed extracellular signal-regulated kinase and p38 mitogen-activated protein kinase protein expression levels, p65 translocation from the cytoplasm to the nucleus, and mRNA expression of TNF-α, IFN-γ, interleukin (IL)-6, and IL-13 in skin lesion tissues. In HaCaT cells, WIF suppressed the production of regulated upon activation, normal T cell expressed and secreted (RANTES), thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), MCP-1, and MIP-3a, which are inflammatory cytokines and chemokines related to AD, and inhibited the mRNA expression of RANTES, TARC, and MDC in TNF-α/IFN-γ-stimulated HaCaT cells. Overall, the results revealed that WIF ameliorated AD-like skin inflammation by suppressing proinflammatory cytokine and chemokine production via nuclear factor-κB pathway inhibition, suggesting WIF as a potential candidate for AD treatment.

Fulvic Acid Attenuates Atopic Dermatitis by Downregulating CCL17/22.

The main pathogenic factor in atopic dermatitis (AD) is Th2 inflammation, and levels of serum CCL17 and CCL22 are related to severity in AD patients. Fulvic acid (FA) is a kind of natural humic acid with anti-inflammatory, antibacterial, and immunomodulatory effects. Our experiments demonstrated the therapeutic effect of FA on AD mice and revealed some potential mechanisms. FA was shown to reduce TARC/CCL17 and MDC/CCL22 expression in HaCaT cells stimulated by TNF-α and IFN-γ. The inhibitors showed that FA inhibits CCL17 and CCL22 production by deactivating the p38 MAPK and JNK pathways. After 2,4-dinitrochlorobenzene (DNCB) induction in mice with atopic dermatitis, FA effectively reduced the symptoms and serum levels of CCL17 and CCL22. In conclusion, topical FA attenuated AD via downregulation of CCL17 and CCL22, via inhibition of P38 MAPK and JNK phosphorylation, and FA is a potential therapeutic agent for AD.

Ozonated oil alleviates dinitrochlorobenzene-induced allergic contact dermatitis via inhibiting the FcεRI/Syk signaling pathway. / 医用臭氧油通过抑制FcεRI/Syk信号通路缓解DNCB诱导的变应性接触性皮炎（英文）.

OBJECTIVES: Ozone is widely applied to treat allergic skin diseases such as eczema, atopic dermatitis, and contact dermatitis. However, the specific mechanism remains unclear. This study aims to investigate the effects of ozonated oil on treating 2,4-dinitrochlorobenzene (DNCB)-induced allergic contact dermatitis (ACD) and the underling mechanisms. METHODS: Besides the blank control (Ctrl) group, all other mice were treated with DNCB to establish an ACD-like mouse model and were randomized into following groups: a model group, a basal oil group, an ozonated oil group, a FcεRI-overexpressed plasmid (FcεRI-OE) group, and a FcεRI empty plasmid (FcεRI-NC) group. The basal oil group and the ozonated oil group were treated with basal oil and ozonated oil, respectively. The FcεRI-OE group and the FcεRI-NC group were intradermally injected 25 µg FcεRI overexpression plasmid and 25 µg FcεRI empty plasmid when treating with ozonated oil, respectively. We recorded skin lesions daily and used reflectance confocal microscope (RCM) to evaluate thickness and inflammatory changes of skin lesions. Hematoxylin-eosin (HE) staining, real-time PCR, RNA-sequencing (RNA-seq), and immunohistochemistry were performed to detct and analyze the skin lesions. RESULTS: Ozonated oil significantly alleviated DNCB-induced ACD-like dermatitis and reduced the expressions of IFN-γ, IL-17A, IL-1ß, TNF-α, and other related inflammatory factors (all P<0.05). RNA-seq analysis revealed that ozonated oil significantly inhibited the activation of the DNCB-induced FcεRI/Syk signaling pathway, confirmed by real-time PCR and immunohistochemistry (all P<0.05). Compared with the ozonated oil group and the FcεRI-NC group, the mRNA expression levels of IFN-γ, IL-17A, IL-1ß, IL-6, TNF-α, and other inflammatory genes in the FcεRI-OE group were significantly increased (all P<0.05), and the mRNA and protein expression levels of FcεRI and Syk were significantly elevated in the FcεRI-OE group as well (all P<0.05). CONCLUSIONS: Ozonated oil significantly improves ACD-like dermatitis and alleviated DNCB-induced ACD-like dermatitis via inhibiting the FcεRI/Syk signaling pathway.

Morinda officinalis extract exhibits protective effects against atopic dermatitis by regulating the MALAT1/miR-590-5p/CCR7 axis.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease with a genetic predisposition, and the traditional Chinese medicine Morinda officinalis and its roots are characterized with anti-inflammatory effects and have been used for the treatment of various disease. However, it is still largely unknown whether Morinda officinalis extract (MOE) can be used for the treatment of AD. OBJECTIVES: In our study we aimed to determine whether MOE could ameliorate 2,4-dinitrochlorobenzene (DNCB)-induced AD and elucidate molecular mechanisms. METHODS: We established an AD mouse model by using DNCB. Skin pathological analysis and ELISA assay were used to detect the effect of MOE on the inflammation of AD model mouse skin and the expression changes of inflammatory factors, and further functional verification was performed in TNF-α/IFN-γ-induced HaCaT cells. RESULTS: Our in vivo experiments confirmed that MOE remarkably reduced DNCB-induced AD lesions and symptoms, such as epidermal and dermal thickness and mast cell infiltration and inflammatory cytokines secretion in the mice models. In addition, the underlying mechanisms by which MOE ameliorated AD had been uncovered, and we verified that MOE inhibited MALAT1 expression in AD, resulting in attenuated expression of C-C chemokine receptor type 7 (CCR7) regulated by MALAT1-sponge miR-590-5p in a competing endogenous RNA (ceRNA) mechanisms-dependent manner, thereby inhibiting TNF-α/IFN-γ-induced cellular proliferation and inflammation.

Protective Effects of Topical Administration of Laminarin in Oxazolone-Induced Atopic Dermatitis-like Skin Lesions.

Laminarin is a polysaccharide isolated from brown marine algae and has a wide range of bioactivities, including immunoregulatory and anti-inflammatory properties. However, the effects of laminarin on atopic dermatitis have not been demonstrated. This study investigated the potential effects of topical administration of laminarin using a Balb/c mouse model of oxazolone-induced atopic dermatitis-like skin lesions. Our results showed that topical administration of laminarin to the ear of the mice improved the severity of the dermatitis, including swelling. Histological analysis revealed that topical laminarin significantly decreased the thickening of the epidermis and dermis and the infiltration of mast cells in the skin lesion. Serum immunoglobulin E levels were also significantly decreased by topical laminarin. Additionally, topical laminarin significantly suppressed protein levels of oxazolone-induced proinflammatory cytokines, such as interleukin-1ß, tumor necrosis factor-α, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1α in the skin lesion. These results indicate that topical administration of laminarin can alleviate oxazolone-induced atopic dermatitis by inhibiting hyperproduction of IgE, mast cell infiltration, and expressions of proinflammatory cytokines. Based on these findings, we propose that laminarin can be a useful candidate for the treatment of atopic dermatitis.

(-)-α-Bisabolol Alleviates Atopic Dermatitis by Inhibiting MAPK and NF-κB Signaling in Mast Cell.

(-)-α-Bisabolol (BIS) is a sesquiterpene alcohol derived mostly from Matricaria recutita L., which is a traditional herb and exhibits multiple biologic activities. BIS has been reported for treatment of skin disorders, but the effect of BIS on anti-atopic dermatitis (AD) remains unclear. Therefore, we investigated the effects of BIS on 2,4-dinitrochlorobenzene (DNCB)-induced AD in BALB/c mice and the underlying mechanism in Bone Marrow-Derived Mast Cells (BMMCs). Topical BIS treatment reduced AD-like symptoms and the release of interleukin (IL)-4 without immunoglobulin (Ig)-E production in DNCB-induced BALB/c mice. Histopathological examination revealed that BIS reduced epidermal thickness and inhibited mast cells in the AD-like lesions skin. Oral administration of BIS effectively and dose-dependently suppressed mast-cell-mediated passive cutaneous anaphylaxis. In IgE-mediated BMMCs, the levels of ß-hexosaminidase (ß-hex), histamine, and tumor necrosis factor (TNF)-α were reduced by blocking the activation of nuclear factor-қB (NF-қB) and c-Jun N-terminal kinase (JNK) without P38 mitogen activated protein (P38) and extracellular regulated protein kinases (Erk1/2). Taken together, our experimental results indicated BIS suppresses AD by inhibiting the activation of JNK and NF-κB in mast cells. BIS may be a promising therapeutic agent for atopic dermatitis and other mast-cell-related diseases.

Inhibitory Effect of Bisdemethoxycurcumin on DNCB-Induced Atopic Dermatitis in Mice.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Bisdemethoxycurcumin (BDMC) is an ingredient from the rhizome of the traditional Chinese herbal medicine turmeric. BDMC has been reported to have important pharmacological properties, such as anti-inflammatory, antioxidant, antitumor and antiproliferative activities. However, its effect on atopic dermatitis has not been reported. The purpose of our study was to demonstrate the effectiveness of BDMC on TNF-α/IFNγ-stimulated HaCaT cells and on 2,4-dinitrochlorobenzene (DNCB)-induced AD mice. Our studies showed in vitro that BDMC was able to significantly inhibit the mRNA expression of chemokines and cytokines in TNF-α/IFN-γ-stimulated HaCaT cells and alleviate their inflammatory response. Our studies found in vivo that BDMC was able to significantly improve the symptoms of DNCB-induced AD skin lesions, decrease the number of scratches, ear thickness, and spleen index, improve inflammatory cells and mast cell infiltration and decrease skin thickness. Moreover, it was also able to inhibit the mRNA expression levels of chemokines and inflammatory cytokines and the activation of the MAPK and NF-κB signaling pathways. Thus, the results indicated that BDMC can improve atopic dermatitis in mice and that further clinical studies are warranted on its treatment of AD.

Artemisia anomala Herba Alleviates 2,4-Dinitrochlorobenzene-Induced Atopic Dermatitis-Like Skin Lesions in Mice and the Production of Pro-Inflammatory Mediators in Tumor Necrosis Factor Alpha-/Interferon Gamma-Induced HaCaT Cells.

Artemisia anomala S. Moore is a perennial herbaceous plant classified as Asteraceae of the genus Artemisia. Many species of Artemisia have been used as medicinal materials. Artemisia anomala S. Moore has been widely used in China to treat inflammatory diseases. However, the mechanism of its action on the keratinocyte inflammatory response is poorly understood. Here, we investigated the anti-inflammatory reaction of Artemisia anomala S. Moore ethanol extract (EAA) using human keratinocyte (HaCaT) cells, which involved investigating the nuclear factor kappa B (NF-κB), signal transducer, and activator of transcription-1 (STAT-1), as well as mitogen-activated protein kinase (MAPK) signaling pathways and atopic dermatitis-like skin lesions in mice. We elucidated the anti-inflammatory effects of EAA on tumor necrosis factor-α/interferon-γ (TNF-α/IFN-γ)-treated human keratinocyte cells and 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD)-like mice. The levels of chemokines and cytokines (IL-8, IL-6, TARC, and RANTES) were determined by an enzyme-linked immunosorbent assay. The NF-κB, STAT-1, and MAPK signaling pathways in HaCaT cells were analyzed by western blotting. Thickening of the mice dorsal and ear skin was measured and inflammatory cell infiltration was observed by hematoxylin and eosin staining. Results showed that EAA suppressed IL-8, IL-6, TARC, and RANTES production. EAA inhibited nuclear translocation of NFκB and STAT-1, as well as reduced the levels of phosphorylated ERK MAPKs. EAA improved AD-like skin lesions in DNCB-treated mice. These findings suggest that EAA possesses stronger anti-inflammatory properties and can be useful as a functional food or candidate agent for AD.

Investigation of the Substrate-Binding Site of a Prostaglandin E Synthase in Bombyx mori.

Prostaglandin E synthase (PGES) catalyzes the conversion of prostaglandin H2 to prostaglandin E2 in the presence of glutathione (GSH) in mammals. Amid the limited knowledge on prostaglandin and its related enzymes in insects, we recently identified PGES from the silkworm Bombyx mori (bmPGES) and determined its crystal structure complexed with GSH. In the current study, we investigated the substrate-binding site of bmPGES by site-directed mutagenesis and X-ray crystallography. We found that the residues Tyr107, Val155, Met159, and Glu203 are located in the catalytic pockets of bmPGES, and mutagenesis of each residue reduced the bmPGES activity. Our results suggest that these four residues contribute to the catalytic activity of bmPGES. Overall, this structure-function study holds implications in controlling pests by designing rational and efficient pesticides.

Biotransformation Capacity of Zebrafish (Danio rerio) Early Life Stages: Functionality of the Mercapturic Acid Pathway.

Zebrafish (Danio rerio) early life stages offer a versatile model system to study the efficacy and safety of drugs or other chemicals with regard to human and environmental health. This is because, aside from the well-characterized genome of zebrafish and the availability of a broad range of experimental and computational research tools, they are exceptionally well suited for high-throughput approaches. Yet, one important pharmacokinetic aspect is thus far only poorly understood in zebrafish embryo and early larvae: their biotransformation capacity. Especially, biotransformation of electrophilic compounds is a critical pathway because they easily react with nucleophile molecules, such as DNA or proteins, potentially inducing adverse health effects. To combat such adverse effects, conjugation reactions with glutathione and further processing within the mercapturic acid pathway have evolved. We here explore the functionality of this pathway in zebrafish early life stages using a reference substrate (1-chloro-2,4-dinitrobenzene, CDNB). With this work, we show that zebrafish embryos can biotransform CDNB to the respective glutathione conjugate as early as 4 h postfertilization. At all examined life stages, the glutathione conjugate is further biotransformed to the last metabolite of the mercapturic acid pathway, the mercapturate, which is slowly excreted. Being able to biotransform electrophiles within the mercapturic acid pathway shows that zebrafish early life stages possess the potential to process xenobiotic compounds through glutathione conjugation and the formation of mercapturates. The presence of this chemical biotransformation and clearance route in zebrafish early life stages supports the application of this model in toxicology and chemical hazard assessment.

Selective Disruption of Mitochondrial Thiol Redox State in Cells and In Vivo.

Mitochondrial glutathione (GSH) and thioredoxin (Trx) systems function independently of the rest of the cell. While maintenance of mitochondrial thiol redox state is thought vital for cell survival, this was not testable due to the difficulty of manipulating the organelle's thiol systems independently of those in other cell compartments. To overcome this constraint we modified the glutathione S-transferase substrate and Trx reductase (TrxR) inhibitor, 1-chloro-2,4-dinitrobenzene (CDNB) by conjugation to the mitochondria-targeting triphenylphosphonium cation. The result, MitoCDNB, is taken up by mitochondria where it selectively depletes the mitochondrial GSH pool, catalyzed by glutathione S-transferases, and directly inhibits mitochondrial TrxR2 and peroxiredoxin 3, a peroxidase. Importantly, MitoCDNB inactivates mitochondrial thiol redox homeostasis in isolated cells and in vivo, without affecting that of the cytosol. Consequently, MitoCDNB enables assessment of the biomedical importance of mitochondrial thiol homeostasis in reactive oxygen species production, organelle dynamics, redox signaling, and cell death in cells and in vivo.

A study of inter-individual variability in the Phase II metabolism of xenobiotics in human skin.

Understanding skin metabolism is key to improve in vitro to in vivo extrapolations used to inform risk assessments of topically applied products. However, published literature is scarce and usually covers a limited and non-representative number of donors. We developed a protocol to handle and store ex vivo skin samples post-surgery and prepare skin S9 fractions to measure the metabolic activity of Phase II enzymes. Preincubation of an excess of cofactors at 37 °C for fifteen minutes in the S9 before introduction of the testing probe, greatly increased the stability of the enzymes. Using this standardised assay, the rates of sulphation (SULT) and glucuronidation (UGT) of 7-hydroxycoumarin, methylation (COMT) of dopamine and N-acetylation (NAT) of procainamide were measured in the ng/mg protein/h (converted to ng/cm2/h) range in eighty-seven individuals. Glutathione conjugation (GST) of 1-chloro-2,4-dinitrobenzene was assessed in a smaller pool of fifty donors; the metabolic rate was much faster and measured over six minutes using a different methodology to express rates in µg/mg protein/min (converted to µg/cm2/min). A comprehensive statistical analysis of these results was carried out, separating donors by age, gender and metabolic rate measured.

Assessment of the inhibitory activity of the pyrethroid pesticide deltamethrin against human placental glutathione transferase P1-1: A combined kinetic and docking study.

Deltamethrin (DEL), which is a synthetic pyrethroid insecticide, has been used successfully all over the world to treat mosquito nets for the control of malaria. Glutathione S-transferases (GSTs; EC 2.5.1.18) catalyze the conjugation of reduced glutathione (GSH) to a variety of xenobiotics and are normally recognized as detoxification enzymes. Here, we used a colorimetric assay based on the human placental GSTP1-1 (hpGSTP1-1)-catalyzed reaction between GSH and the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) as well as molecular docking to investigate the mechanistic and structural aspects of hpGSTP1-1 inhibition by DEL. We show that DEL is a potent, noncompetitive inhibitor of hpGSTP1-1 with an IC50 value of 6.1 µM and Ki values of 5.61 ±â€¯0.32 µM and 7.96 ±â€¯0.97 µM at fixed [CDNB]-varied [GSH] and fixed [GSH]-varied [CDNB], respectively. DEL appears to be accommodated well in an eccentric cavity located at the interface of the hpGSTP1-1 homodimer, presumably causing conformational changes to the enzyme's substrate-binding sites such that the enzyme is no longer able to transform GSH and CDNB effectively. Correspondingly, considerable maternal exposure to and subsequent accumulation of DEL may interfere with the proper development of the vulnerable fetus, possibly increasing the risk of developing congenital defects.

Mechanistic insights into EgGST1, a Mu class glutathione S-transferase from the cestode parasite Echinococcus granulosus.

Glutathione transferases (GSTs) comprise a major detoxification system in helminth parasites, displaying both catalytic and non-catalytic activities. The kinetic mechanism of these enzymes is complex and depends on the isoenzyme which is being analyzed. Here, we characterized the kinetic mechanism of rEgGST1, a recombinant form of a cytosolic GST from Echinococcus granulosus (EgGST1), which is related to the Mu-class of mammalian enzymes, using the canonical substrates glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Initial rate and product inhibition studies were consistent with a steady-state random sequential mechanism, where both substrates are bound to the enzyme before the products are released. Kinetic constants were also determined (pH 6.5 and 30 °C). Moreover, rEgGST1 lowered the pKa of GSH from 8.71 ± 0.07 to 6.77 ± 0.08, and enzyme-bound GSH reacted with CDNB 1 × 105 times faster than free GSH at pH 7.4. Finally, the dissociation of the enzyme-GSH complex was studied by means of intrinsic fluorescence, as well as that of the complex with the anthelminth drug mebendazole. This is the first report on mechanistic issues related to a helminth parasitic GST.

Identification of a diazinon-metabolizing glutathione S-transferase in the silkworm, Bombyx mori.

The glutathione S-transferase superfamily play key roles in the metabolism of numerous xenobiotics. We report herein the identification and characterization of a novel glutathione S-transferase in the silkworm, Bombyx mori. The enzyme (bmGSTu2) conjugates glutathione to 1-chloro-2,4-dinitrobenzene, as well as metabolizing diazinon, one of the organophosphate insecticides. Quantitative reverse transcription-polymerase chain reaction analysis of transcripts demonstrated that bmGSTu2 expression was induced 1.7-fold in a resistant strain of B. mori. Mutagenesis of putative amino acid residues in the glutathione-binding site revealed that Ile54, Glu66, Ser67, and Asn68 are crucial for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTu2 and into the detoxification of organophosphate insecticides.

Gills as a glutathione-dependent metabolic barrier in Pacific oysters Crassostrea gigas: Absorption, metabolism and excretion of a model electrophile.

The mercapturic acid pathway (MAP) is a major phase II detoxification route, comprising the conjugation of electrophilic substances to glutathione (GSH) in a reaction catalyzed by glutathione S-transferase (GST) enzymes. In mammals, GSH-conjugates are exported from cells, and the GSH-constituent amino acids (Glu/Gly) are subsequently removed by ectopeptidases. The resulting Cys-conjugates are reabsorbed and, finally, a mercapturic acid is generated through N-acetylation. This pathway, though very well characterized in mammals, is poorly studied in non-mammalian biological models, such as bivalve mollusks, which are key organisms in aquatic ecosystems, aquaculture activities and environmental studies. In the present work, the compound 1-chloro-2,4-dinitrobenzene (CDNB) was used as a model electrophile to study the MAP in Pacific oysters Crassostrea gigas. Animals were exposed to 10µM CDNB and MAP metabolites were followed over 24h in the seawater and in oyster tissues (gills, digestive gland and hemolymph). A rapid decay was detected for CDNB in the seawater (half-life 1.7h), and MAP metabolites peaked in oyster tissues as soon as 15min for the GSH-conjugate, 1h for the Cys-conjugate, and 4h for the final metabolite (mercapturic acid). Biokinetic modeling of the MAP supports the fast CDNB uptake and metabolism, and indicated that while gills are a key organ for absorption, initial biotransformation, and likely metabolite excretion, hemolymph is a possible milieu for metabolite transport along different tissues. CDNB-induced GSH depletion (4h) was followed by increased GST activity (24h) in the gills, but not in the digestive gland. Furthermore, the transcript levels of glutamate-cysteine ligase, coding for the rate limiting enzyme in GSH synthesis, and two phase II biotransformation genes (GSTpi and GSTo), presented a fast (4h) and robust (∼6-70 fold) increase in the gills. Waterborne exposure to electrophilic compounds affected gills, but not digestive gland, while intramuscular exposure was able to modulate biochemical parameters in both tissues. This study is the first evidence of a fully functional and interorgan MAP pathway in bivalves. Hemolymph was shown to be responsible for the metabolic interplay among tissues, and gills, acting as a powerful GSH-dependent metabolic barrier against waterborne electrophilic substances, possibly also participating in metabolite excretion into the sea water. Altogether, experimental and modeled data fully agree with the existence of a classical mechanism for phase II xenobiotic metabolism and excretion in bivalves.