Early-life perfluorooctanoic acid exposure disrupts the function of dopamine transporter protein with glycosylation changes implicating the links between decreased dopamine levels and disruptive behaviors in larval zebrafish.

Exposure to perfluorooctanoic acid (PFOA) during early embryonic development is associated with the increased risk of developmental neurotoxicity and neurobehavioral disorders in children. In our previous study, we demonstrated that exposure to PFOA affected locomotor activity and disrupted dopamine-related gene expression in zebrafish larvae. Consequently, we continue to study the dopaminergic system with a focus on dopamine levels and dopamine's effect on behaviors in relation to PFOA exposure. In the present study, we found a decrease in dopamine levels in larval zebrafish. We studied the dopamine transporter (DAT) protein, which is responsible for regulating dopamine levels through the reuptake of dopamine in neuronal cells. We demonstrated that exposure to PFOA disrupted the glycosylation process of DAT, inhibited its uptake function, and induced endoplasmic reticulum (ER) stress in dopaminergic cells. Besides, we conducted a light-dark preference test on larval zebrafish and observed anxiety/depressive-like behavioral changes following exposure to PFOA. Dopamine is one of the most prominent neurotransmitters that significantly influences human behavior, with low dopamine levels being associated with impairments such as anxiety and depression. The anxiety-like response in zebrafish larvae exposure to PFOA implies the link with the reduced dopamine levels. Taken together, we can deduce that glycosylation changes in DAT lead to dysfunction of DAT to regulate dopamine levels, which in turn alters behavior in larval zebrafish. Therefore, alternation in dopamine levels may play a pivotal role in the development of anxiety/depressive-like behavioral changes induced by PFOA.

NLRP3 Inflammasome-dependent Pathway is Involved in the Pathogenesis of Polycystic Ovary Syndrome.

Accumulating evidence has shown that inflammation is a key process in polycystic ovary syndrome (PCOS). Nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) inflammasomes play an essential role in inflammation. We investigated the expression of NLRP3 inflammasome in PCOS and its underlying mechanisms. Human granulosa cells (GCs) were isolated from patients with PCOS and control women who underwent in vitro fertilization and embryo transfer. Ovarian specimens were collected from mice with polycystic ovarian changes induced by a high-fat diet and letrozole. RNA sequencing (RNA-Seq) was performed on a granulosa cell line (KGN) overexpressing NLRP3. Polymerase chain reaction (PCR) was performed to quantify the differentially expressed genes of interest. NLRP3 and caspase-1 expression was significantly higher in GCs from patients with PCOS than in GCs from the control group. Increased NLRP3 and caspase-1 expression was also detected by immunohistochemistry in the GCs of a mouse model of polycystic ovarian changes. The serum IL-18 concentration in PCOS-like mice was significantly higher than that in control mice. Following NLRP3 overexpression in KGN cells, the genes involved in N-glycan processing, steroidogenesis, oocyte maturation, autophagy, and apoptosis were upregulated. The RT-qPCR results revealed that the expression levels of GANAB, ALG-5, HSD3B2, ULK1, PTK2B, and Casp7 in KGN cells after NLRP3 overexpression were significantly higher than those in control cells, which was consistent with the RNA-Seq results. Taken together, the NLRP3 inflammasome-dependent pathway is involved in the pathogenesis of PCOS not only by mediating pyroptosis, but also by regulating glycan synthesis, sex hormone synthesis, autophagy, and apoptosis in GCs.

Perfluorooctanoic acid disrupts thyroid-specific genes expression and regulation via the TSH-TSHR signaling pathway in thyroid cells.

Perfluorooctanoic acid (PFOA) is a highly persistent and widespread chemical in the environment with endocrine disruption effects. Although it has been reported that PFOA can affect multiple aspects of thyroid function, the exact mechanism by which it reduces thyroxine levels has not yet been elucidated. In this study, FRTL-5 rat thyroid follicular cells were used as a model to study the toxicity of PFOA to the genes related to thyroid hormone synthesis and their regulatory network. Our results reveal that PFOA interfered with the phosphorylation of the cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB) induced by thyroid-stimulating hormone (TSH), as well as the transcription levels of paired box 8 (PAX8), thyroid transcription factor 1 (TTF1), sodium/iodide cotransporter (NIS), thyroglobulin (TG), and thyroid peroxidase (TPO). However, the above outcomes can be alleviated by enhancing cAMP production with forskolin treatment. Further investigations showed that PFOA reduced the mRNA level of TSH receptor (TSHR) and impaired its N-glycosylation, suggesting that PFOA has disrupting effects on both transcriptional regulation and post-translational regulation. In addition, PFOA increased endoplasmic reticulum (ER) stress and decreased ER mass in FRTL-5 cells. Based on these findings, it can be inferred that PFOA disrupts the TSH-activated cAMP signaling pathway by inhibiting TSHR expression and its N-glycosylation. We propose that this mechanism may contribute to the decrease in thyroid hormone levels caused by PFOA. Our study sheds light on the molecular mechanism by which PFOA can disrupt thyroid function and provides new insights and potential targets for interventions to counteract the disruptive effects of PFOA.

Mitochondrial Glrx2 Knockout Augments Acetaminophen-Induced Hepatotoxicity in Mice.

Acetaminophen (APAP) is one of the most widely used drugs with antipyretic and analgesic effects, and thus hepatotoxicity from the overdose of APAP becomes one of the most common forms of drug-induced liver injury. The reaction towards thiol molecules, such as GSH by APAP metabolite, N-acetyl-p-benzo-quinonimine (NAPQI), is the main cause of APAP-induced hepatotoxicity. However, the role of many other thiol-related regulators in toxicity caused by APAP is still unclear. Here we have found that knockout of the Glrx2 gene, which encodes mitochondrial glutaredoxin2 (Grx2), sensitized mice to APAP-caused hepatotoxicity. Glrx2 deletion hindered Nrf2-mediated compensatory recovery of thiol-dependent redox systems after acetaminophen challenge, resulting in a more oxidized cellular state with a further decrease in GSH level, thioredoxin reductase activity, and GSH/GSSG ratio. The weakened feedback regulation capacity of the liver led to higher levels of protein glutathionylation and thioredoxin (both Trx1 and Trx2) oxidation in Glrx2-/- mice. Following the cellular environment oxidation, nuclear translocation of apoptosis-inducing factor (AIF) was elevated in the liver of Glrx2-/- mice. Taken together, these results demonstrated that mitochondrial Grx2 deficiency deteriorated APAP-induced hepatotoxicity by interrupting thiol-redox compensatory response, enhancing the AIF pathway-mediated oxidative damage.

Perfluorooctanoic acid exposure increases both proliferation and apoptosis of human placental trophoblast cells mediated by ER stress-induced ROS or UPR pathways.

Perfluorooctanoate acid (PFOA) is a highly persistent and widespread chemical in the environment. PFOA serum levels in pregnant women are positively associated with an increased risk of placenta-related disorders. However, the mechanism of PFOA cytotoxicity involved in placental cells and cellular responses such as ER stress remains poorly understood. In this study, we studied the cellular toxicity of PFOA with a focus on proliferation and apoptosis in a human placental trophoblast cell line. Cell viability, number, apoptosis, stress response, activation of the involved signaling pathways were assessed. Our results showed PFOA affected cell viability, proliferation and also resulted in apoptosis. Besides, both pro-proliferation and pro-apoptosis effects were attenuated by endoplasmic reticulum (ER) stress inhibitors. Further experiments demonstrated that two different signaling pathways were activated by PFOA-induced ER stress and involved in PFOA toxicity: the reactive oxygen species (ROS)-dependent ERK signaling triggered trophoblast proliferation, while the ATF4-dependent C/EBP homologous protein (CHOP) signaling was the trigger of apoptosis. We conclude that PFOA-induced ER stress is the trigger of proliferation and apoptosis of trophoblast via ROS or UPR signaling pathway, which leads to the altered balance critical to the normal development and function of the placenta.

Behavioral effects of early-life exposure to perfluorooctanoic acid might synthetically link to multiple aspects of dopaminergic neuron development and dopamine functions in zebrafish larvae.

Perfluorooctanoic acid (PFOA) is known as an environmental endocrine disruptor and has developmental neurotoxicity that could be associated with behavior changes in human and animal studies. Previous studies have shown that PFOA might affect the dopaminergic nervous system. However, the mode of action underlying the effects of PFOA remains poorly understood. Our study used zebrafish as an animal model to investigate the effects of early-life PFOA exposure on dopaminergic neuron development and dopamine functions in zebrafish larvae. Zebrafish fertilized eggs were exposed to different concentrations of PFOA (0, 10, 100, 1000 µg/L). After exposure to PFOA for 7 days, the locomotor activity of zebrafish was decreased; the mRNA levels of nuclear receptor subfamily 4 group a member 2b (nr4a2b), paired box 2 and 5 (pax2, pax5), tyrosine hydroxylase 1/2 (th1/th2) and dopamine transporter (dat) were increased; mRNA and protein level of mesencephalic astrocyte-derived neurotrophic factor (manf) were decreased. Neural cell proliferation in the preoptic area of hypothalamus was increased. In conclusion, dopaminergic neuron development might be one of the targets of early-life PFOA exposure. The neurobehavior changes induced by PFOA exposure might link to multiple aspects of dopaminergic neuron development and dopamine functions in zebrafish larvae.

The triclosan-induced shift from aerobic to anaerobic metabolism link to increased steroidogenesis in human ovarian granulosa cells.

Triclosan (TCS) is an endocrine-disrupting chemical (EDC), which is used ubiquitously as an antimicrobial ingredient in healthcare products and causes contamination in the environment such as air, water, and biosolid-amended soil. Exposure to TCS may increase the risk of reproduction diseases and health issues. Several groups, including ours, have proved that TCS increased the biosynthesis of steroid hormones in different types of steroidogenic cells. However, the precise mechanism of toxic action of TCS on increased steroidogenesis at a molecular level remains to be elucidated. In this study, we try to address the mode of action that TCS affects energy metabolism with increased steroidogenesis. We evaluated the adverse effects of TCS on energy metabolism and steroidogenesis in human ovarian granulosa cells. The goal is to elucidate how increased steroidogenesis can occur with a shortage of adenosine triphosphate (ATP) whereas mitochondria-based energy metabolism is impaired. Our results demonstrated TCS increased estradiol and progesterone levels with upregulated steroidogenesis gene expression at concentrations ranging from 0 to 10 µM. Besides, glucose consumption, lactate level, and pyruvate kinase transcription were increased. Interestingly, the lactate level was attenuated with increased steroidogenesis, suggesting that pyruvate fate was shifted away from the formation of lactate towards steroidogenesis. Our study is gathering evidence suggesting a mode of action that TCS changes energy metabolism by predominating glucose flow towards the biosynthesis of steroid hormones. To the best of our knowledge, this is the first report that TCS presents such toxic action in disrupting hormone homeostasis.

Nitric oxide mediates disruption of human placental trophoblast invasion induced by perfluorobutane sulfonate.

Perfluorobutane sulfonate (PFBS), an emerging pollutant, is associated with disruption of placental functions and adverse birth outcomes. However, the precise mechanism of this disruption remains unclear. Extravillous trophoblasts make up the majority of cells in the placenta, and have invasive abilities, which plays a critical role in a successful pregnancy. It has been reported that inducible nitric oxide (iNOS) and nitric oxide (NO) signaling is associated with trophoblast migration and invasion. In this study, PFBS exposure was found to enhance trophoblast invasion and increase matrix metalloproteinase 9 (MMP-9) levels. Additionally, PFBS upregulated iNOS levels and stimulated NO generation. iNOS inhibitor treatment attenuated the increased invasion of trophoblasts and MMP-9 expression induced by PFBS. Extracellular signal-regulated kinase (ERK) phosphorylation was also enhanced by PFBS exposure. In the presence of an ERK pathway inhibitor, however, the increases in trophoblast invasion, the induction of NO production, iNOS expression and MMP-9 expression induced by PFBS were attenuated. Taken together, these results suggest that iNOS/NO signaling is triggered by activation of the ERK signaling pathway, and that iNOS/NO signaling mediates PFBS-induced stimulation of trophoblast invasion.

Perfluoroalkyl and polyfluroalkyl substances and maternal thyroid hormones in early pregnancy.

BACKGROUND: The development of the embryo and fetal brain depends on maternal transfer of thyroid hormones (THs) in early pregnancy. Perfluoroalkyl and polyfluoralkyl substances (PFAS) may disrupt maternal TH homeostasis in pregnancy, but findings from epidemiologic studies were inconsistent. We aimed to assess this relationship in early pregnancy in a large prospective cohort study. METHODS: A total of 1885 pregnant women enrolled in the Shanghai Birth Cohort were used. Ten PFAS, free thyroxine (FT4), free triiodothyronine (FT3), thyroid stimulating hormone (TSH) and thyroid peroxidase antibody (TPOAb) were measured in maternal blood collected prior to 16 weeks of gestation. Multiple linear regression accompanied by restricted cubic spline was used to examine the association and the exposure-response relationship between each PFAS and TH in separate models. Possible effect modification by TPOAb status was also investigated. RESULTS: Perfluorooctanoic acid [PFOA, ß = 0.121, 95% confidence interval (CI): 0.015, 0.227] and perfluorohexane sulfonate (PFHxS, ß = 0.123, 95% CI: 0.024, 0.222) were positively associated with FT4. Perfluorononanoic acid (PFNA, ß = 0.179, 95% CI: 0.047, 0.311) and PFHxS (ß = 0.197, 95% CI: 0.054, 0.339) were positively associated with FT3, while PFHxS was negatively associated with TSH (ß = -0.115, 95%CI: 0.216, -0.014). TPOAb-positivity appeared to modify the associations between PFAS and THs. In TPOAb-positive women, several long-chain PFAS were positively associated with FT4 and/or FT3 and tended to be negatively associated with TSH. CONCLUSIONS: Several long-chain PFAS were associated with disrupted TH homeostasis in Chinese pregnant women, especially among TPOAb-positive women.

Higher DNA Yield for Epidemiological Studies: A Better Method for DNA Extraction from Blood Clot.

BACKGROUND: Blood clots can be used to extract DNA, but they are not as widely used as whole blood or buffy coats. This is due not only because of the relatively low DNA yields and quality obtained from blood clots, but also because sampling prior to DNA extraction is more difficult. METHODS: To solve these problems, we compared several clot liquefaction methods, determined the four most feasible methods, and subsequently performed a comparative analysis among them. We compared the yields and optical density ratios of the resulting DNA samples and assessed their integrity using agarose gel electrophoresis, polymerase chain reaction, and next-generation sequencing (NGS). RESULTS: Each of the four methods has advantages and disadvantages. But in general, higher yields of DNA with better quality and integrity were obtained using the high-shear homogenization method than using the other three methods. Additionally, this method is cost-effective and feasible at large operational scales. The DNA yields and A260/280 ratios were optimal and stable, the operation time and labor costs were acceptable, and the success rate of NGS applications was 99.74%. Furthermore, we developed a simple and rapid method for cleaning the homogenizer head to remove residual samples. According to our experimental results, our cleaning method effectively eliminated the risk of cross-contamination caused by the homogenizer head. CONCLUSION: We recommend high-shear homogenization as a superior method for clot liquefaction. We believe that this method is worthy of large-scale application as it can improve the efficiency of DNA extraction from clots, thus reducing labor and economic costs.

Oxidation of structural cysteine residues in thioredoxin 1 by aromatic arsenicals enhances cancer cell cytotoxicity caused by the inhibition of thioredoxin reductase 1.

Thioredoxin systems, composed of thioredoxin reductase (TrxR), thioredoxin (Trx) and NADPH, play important roles in maintaining cellular redox homeostasis and redox signaling. Recently the cytosolic Trx1 system has been shown to be a cellular target of arsenic containing compounds. To elucidate the relationship of the structure of arsenic compounds with their ability of inhibiting TrxR1 and Trx1, and cytotoxicity, we have investigated the reaction of Trx1 system with seven arsenic trithiolates: As(Cys)3, As(GS)3, As(Penicillamine)3, As(Mercaptoethanesulfonate)3, As(Mercaptopurine)3, As(2-mercaptopyridine)3 and As(2-mercaptopyridine N-oxide)3. The cytotoxicity of these arsenicals was consistent with their ability to inhibit TrxR1 in vitro and in cells. Unlike other arsenicals, As(Mercaptopurine)3 which did not show inhibitory effects on TrxR1 had very weak cytotoxicity, indicating that TrxR1 is a reliable drug target for arsenicals. Moreover, the two aromatic compounds As(2-mercaptopyridine)3 and As(2-mercaptopyridine N-oxide)3 showed stronger cytotoxicity than the others. As(2-mercaptopyridine)3 which selectively oxidized two structural cysteines (Cys62 and Cys69) in Trx1 showed mild improvement in cytotoxicity. As(2-mercaptopyridine N-oxide)3 oxidized all the Cys residues in Trx1, exhibiting the strongest cytotoxicity. Oxidation of Trx1 by As(2-mercaptopyridine)3 and As(2-mercaptopyridine N-oxide)3 affected electron transfer from NADPH and TrxR1 to peroxiredoxin 1 (Prx1), which could result in the reactive oxygen species elevation and trigger cell death process. These results suggest that oxidation of structural cysteine residues in Trx1 by aromatic group in TrxR1-targeting drugs may sensitize tumor cells to cell death, providing a novel approach to regulate cellular redox signaling and also a basis for rational design of new anticancer agents.

Glutaredoxin 2 reduces both thioredoxin 2 and thioredoxin 1 and protects cells from apoptosis induced by auranofin and 4-hydroxynonenal.

AIMS: Mitochondrial thioredoxin (Trx) is critical for defense against oxidative stress-induced cell apoptosis. To date, mitochondrial thioredoxin reductase (TrxR) is the only known enzyme catalyzing Trx2 reduction in mitochondria. However, TrxR is sensitive to inactivation by exo/endogenous electrophiles, for example, 4-hydroxynonenal (HNE). In this study, we characterized the mitochondrial glutaredoxin 2 (Grx2) system as a backup for the mitochondrial TrxR. Meanwhile, as Grx2 is also present in the cytosol/nucleus of certain cancer cell lines, the reducing activity of Grx2 on Trx1 was also tested. RESULTS: Glutathione alone could reduce oxidized Trx2, and the presence of physiological concentrations of Grx2 markedly increased the reaction rate. HeLa cells with Grx2 overexpression (particularly in the mitochondria) exhibited higher viabilities than the wild-type cells after treatment with TrxR inhibitors (Auranofin or HNE), whereas knockdown of Grx2 sensitized the cells to TrxR inhibitors. Accordingly, Grx2 overexpression in the mitochondria had protected Trx2 from oxidation by HNE treatment, whereas Grx2 knockdown had sensitized Trx2 to oxidation. On the other hand, Grx2 reduced Trx1 with similar activities as that of Trx2. Overexpression of Grx2 in the cytosol had protected Trx1 from oxidation, indicating a supportive role of Grx2 in the cytosolic redox balance of cancer cells. INNOVATION: This work explores the reductase activity of Grx2 on Trx2/1, and demonstrates the physiological importance of the activity by using in vivo redox western blot assays. CONCLUSION: Grx2 system could help to keep Trx2/1 reduced during an oxidative stress, thereby contributing to the anti-apoptotic signaling.

Plasma glutaredoxin activity in healthy subjects and patients with abnormal glucose levels or overt type 2 diabetes.

Oxidative stress induced by hyperglycemia is a key factor in the pathogenesis of diabetes complications. Glutaredoxin 1(Grx1) is a cytosolic redox protein that catalyzes GSH-dependent thiol redox reactions and reversible protein S-glutathionylation. In humans, Grx1 antigen has previously been detected in plasma; however, it has hitherto been unclear if plasma Grx1 is enzymatically active, which would indicate an extracellular function of the protein. Given that glucose overload damages cells through oxidative stress responses, we investigated whether postprandial hyperglycemia induces changes in extracellular Grx1 in patients with abnormal glucose tolerance and healthy subjects. Using a novel sensitive fluorescent substrate assay, we demonstrated that plasma Grx consists of active protein. Grx antigen, activity and total antioxidant capacity were significantly elevated in patients compared to healthy subjects. In response to oral glucose tolerance test, Grx activity and antioxidant capacity increased significantly in healthy volunteers, however, not to the high levels of the patients. In conclusion, these results indicate an extracellular function of plasma Grx in blood glucose metabolism. Thus, Grx may be a marker of increased oxidative stress during hyperglycemia in healthy subjects and may be a risk marker of progression toward diabetes onset.

Thioredoxin 1 is inactivated due to oxidation induced by peroxiredoxin under oxidative stress and reactivated by the glutaredoxin system.

The mammalian cytosolic thioredoxin system, comprising thioredoxin (Trx), Trx reductase, and NADPH, is the major protein-disulfide reductase of the cell and has numerous functions. Besides the active site thiols, human Trx1 contains three non-active site cysteine residues at positions 62, 69, and 73. A two-disulfide form of Trx1, containing an active site disulfide between Cys-32 and Cys-35 and a non-active site disulfide between Cys-62 and Cys-69, is inactive either as a disulfide reductase or as a substrate for Trx reductase. This could possibly provide a structural switch affecting Trx1 function during oxidative stress and redox signaling. We found that two-disulfide Trx1 was generated in A549 cells under oxidative stress. In vitro data showed that two-disulfide Trx1 was generated from oxidation of Trx1 catalyzed by peroxiredoxin 1 in the presence of H2O2. The redox Western blot data indicated that the glutaredoxin system protected Trx1 in HeLa cells from oxidation caused by ebselen, a superfast oxidant for Trx1. Our results also showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced the non-active site disulfide in vitro. This reaction was stimulated by glutaredoxin 1 via the so-called monothiol mechanism. In conclusion, reversible oxidation of the non-active site disulfide of Trx1 is suggested to play an important role in redox regulation and cell signaling via temporal inhibition of its protein-disulfide reductase activity for the transmission of oxidative signals under oxidative stress.

Glutathione and glutaredoxin act as a backup of human thioredoxin reductase 1 to reduce thioredoxin 1 preventing cell death by aurothioglucose.

Thioredoxin reductase 1 (TrxR1) in cytosol is the only known reductant of oxidized thioredoxin 1 (Trx1) in vivo so far. We and others found that aurothioglucose (ATG), a well known active-site inhibitor of TrxR1, inhibited TrxR1 activity in HeLa cell cytosol but had no effect on the viability of the cells. Using a redox Western blot analysis, no change was observed in redox state of Trx1, which was mainly fully reduced with five sulfhydryl groups. In contrast, auranofin killed cells and oxidized Trx1, also targeting mitochondrial TrxR2 and Trx2. Combining ATG with ebselen gave a strong synergistic effect, leading to Trx1 oxidation, reactive oxygen species accumulation, and cell death. We hypothesized that there should exist a backup system to reduce Trx1 when only TrxR1 activity was lost. Our results showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced Trx1 in vitro and that the reaction was strongly stimulated by glutaredoxin1. Simultaneous depletion of TrxR activity by ATG and glutathione by buthionine sulfoximine led to overoxidation of Trx1 and loss of HeLa cell viability. In conclusion, the glutaredoxin system and glutathione have a backup role to keep Trx1 reduced in cells with loss of TrxR1 activity. Monitoring the redox state of Trx1 shows that cell death occurs when Trx1 is oxidized, followed by general protein oxidation catalyzed by the disulfide form of thioredoxin.

Disruption of the mitochondrial thioredoxin system as a cell death mechanism of cationic triphenylmethanes.

Alterations in mitochondrial structure and function are a hallmark of cancer cells compared to normal cells and thus targeting mitochondria has emerged as an novel approach to cancer therapy. The mitochondrial thioredoxin 2 (Trx2) system is critical for cell viability, but its role in cancer biology is not well understood. Recently some cationic triphenylmethanes such as brilliant green (BG) and gentian violet were shown to have antitumor and antiangiogenic activity with unknown mechanisms. Here we demonstrate that BG killed cells at nanomolar concentrations and targeted mitochondrial Trx2, which was oxidized and degraded. HeLa cells were more sensitive to BG than fibroblasts. In HeLa cells, Trx2 down-regulation by siRNA resulted in increased sensitivity to BG, whereas for fibroblasts, the same treatments had no effect. BG was observed to accumulate in mitochondria and cause a rapid and dramatic decrease in mitochondrial Trx2 protein. With a redox Western blot method, we found that treatment with BG caused oxidation of both Trx1 and Trx2, followed by release of cytochrome c and apoptosis-inducing factor from the mitochondria into the cytosol. Moreover, this treatment resulted in an elevation of the mRNA level of Lon protease, a protein quality control enzyme in the mitochondrial matrix, suggesting that the oxidized Trx2 may be degraded by Lon protease.

Inhibition of mammalian thioredoxin reductase by black tea and its constituents: implications for anticancer actions.

Black tea is recently reported to have anti-carcinogenic effects through pro-oxidant property, but the underlying mechanisms remain unclear. Mammalian cytosolic thioredoxin reductase (TrxR1) is well -known for its anti-oxidation activity. In this study, we found that black tea extract (BTE) and theaflavins (TFs), the major black tea polyphenols, inhibited the purified TrxR1 with IC(50) 44 microg/ml and 21+/-1 microg/ml, respectively. Kinetics of TFs exhibited a mixed type of competitive and non-competitive inhibition, with K(is) 4+/-1 microg/ml and K(ii) 26+/-5 microg/ml against coenzyme NADPH, and with K(is) 12+/-3 microg/ml and K(ii) 27+/-5 microg/ml against substrate DTNB. In addition, TFs inhibited TrxR1 in a time-dependent manner. In an equilibrium step, a reversible TrxR1-TFs complex (E*I) forms, which is followed by a slow irreversible first-order inactivation step. Rate constant of the inactivation was 0.7 min(-1), and dissociation constant of E*I was 51.9 microg/ml. Treatment of NADPH-reduced TrxR1 with TFs decreased 5-(Iodoacetamido) fluorescein incorporation, a fluorescent thiol-reactive reagent, suggesting that Sec/Cys residue(s) in the active site may be involved in the binding of TFs. The inhibitory capacity of TFs depends on their structure. Among the TFs tested, gallated forms had strong inhibitory effects. The interactions between TFs and TrxR1 were investigated by molecular docking, which revealed important features of the binding mechanism of theaflavins. An inhibitory effect of BTE on viability of HeLa cells was observed with IC(50) 29 microg/ml. At 33 microg/ml of BTE, TrxR1 activity in HeLa cells was decreased by 73% at 22 h after BTE treatment. TFs inhibited cell viability with IC(50) 10+/-4 microg/ml for HeLa cells and with IC(50) 20+/-5 microg/ml for EAhy926 cells. The cell susceptibility to TFs was inversely correlated to cellular levels of TrxR1. The inhibitory actions of TFs on TrxR1 may be an important mechanism of their anti-cancer properties.

Inactivation mechanism of the beta-ketoacyl-[acyl carrier protein] reductase of bacterial type-II fatty acid synthase by epigallocatechin gallate.

Epigallocatechin gallate (EGCG), a major compound from green tea, reversibly inhibits beta-ketoacyl-[acyl carrier protein] reductase (FabG) from Escherichia coli. In this study, we found that EGCG exhibited an atypical time-dependent inhibition of FabG, which possibly resulted from the EGCG-induced aggregation of FabG. It was observed that FabG inactivation and aggregation occurred nearly simultaneously, with a lag time that decreased with increasing EGCG concentration. These results suggest that some chemical reactions, required for aggregation and inactivation, occurred during the lag time. Since EGC was detected by HPLC after the incubation of EGCG with FabG, EGCG probably covalently modified FabG. These further results showed that 1 tetramer of FabG must be modified by several, possibly 4, EGCG molecules before the formation of FabG aggregates. FabG aggregation was a first-order reaction independent of protein concentration. Due to an initial lag time, the first-order rate of aggregation gradually increased, reaching a maximal and constant value. The effect of increasing concentration of EGCG on the first-order rate constant for aggregation indicated that EGCG bound to FabG by affinity labeling. Based on the results, we propose a mechanism for the interaction of EGCG with FabG:EGCG first binds reversibly to each subunit of FabG, followed by covalent modification and then aggregation of the 4 EGCG-modified subunits.

Keemun black tea extract contains potent fatty acid synthase inhibitors and reduces food intake and body weight of rats via oral administration.

The inhibitory effects of a black tea extract on fatty acid synthase were measured through inhibition kinetics. The Keemun black tea extract showed more potent inhibitory activity on fatty acid synthase than green tea extract. Additionally, the inhibitory ability of the black tea extract depended on the extracting solvent and the conditions used. Only 10-23% of the inhibitory activity from the black tea was extracted by the general method of boiling with water. The results suggested that the main fatty acid synthase inhibitors in black tea might be theaflavins. Which were more potent than epigallocatechin gallate or C75. The reaction site on the fatty acid synthase and the inhibition kinetics for the extract were different from those of epigallocatechin gallate or C75. In addition, Keemun black tea extract significantly reduced food intake, body weight and blood triglyceride of diet-induced obesity SD rats via oral administration.