A novel cascade catalysis for one-pot enzymatically modified isoquercitrin (EMIQ) conversion from rutin and sucrose using rationally designed gradient temperature control.

Enzymatically modified isoquercitrin (EMIQ) is a glyco-chemically modified flavonoid exhibiting notably high biological activity, such as antioxidant, anti-inflammatory and anti-allergic properties. However, the utilization of expensive substrates such as isoquercitrin and cyclodextrin in the conventional approach has hindered the industrial-scale production of EMIQ due to high cost and low yields. Hence, the development of a cost-effective and efficient method is crucial for the biological synthesis of EMIQ. In this study, a natural cascade catalytic reaction system was constructed with α-L-rhamnosidase and amylosucrase using the inexpensive substrates rutin and sucrose. Additionally, a novel approach integrating gradient temperature regulation into biological cascade reactions was implemented. Under the optimal conditions, the rutin conversion reached a remarkable 95.39% at 24 h. Meanwhile, the productivity of quercetin-3-O-tetraglucoside with the best bioavailability reached an impressive 41.69%. This study presents promising prospects for future mass production of EMIQ directly prepared from rutin and sucrose.

Enzymatically modified isoquercitrin and its protective effects against photoaging: In-vitro and clinical studies.

This research examines the anti-aging potential of the flavonoid derivative of isoquercitrin known as enzymatically modified isoquercitrin (EMIQ). Initial HPLC analyses showed that EMIQ used in the study contained 1-12 glucosides and 10.7% pentahydroxyflavonoids, promising potent antioxidant properties. In subsequent in-vitro studies with UVA-exposed human dermal fibroblasts (HDFa), EMIQ demonstrated protective properties by reducing collagen damage. It modulated both the TGFß/Smad pathway and the MMP1 pathway, contributing to collagen preservation. This protective effect was further confirmed using the T-Skin™ model, a reconstructed full-thickness human skin model, which illustrated that EMIQ could defend the physiological structures of both the epidermis and dermis against UV radiation. A 28-day clinical trial with 30 volunteers aged 31-55 years highlighted EMIQ's effectiveness. Participants using EMIQ-containing Essence displayed reduced facial trans-epidermal water loss and skin roughness, alongside improved skin elasticity. This study emphasizes EMIQ's potential as an anti-photoaging ingredient in cosmetics, warranting further research. The findings pave the way for developing innovative skincare products addressing photoaging effects.

Synergistic Effects of Heat-Treated Green Tea Extract and Enzymatically-Modified Isoquercitrin in Preventing Obesity.

Previous research has shown that both heat-treated green tea extract (HTGT) and enzymatically modified isoquercitrin (EMIQ) have anti-obesity effects. Given the absence of in vivo evidence demonstrating their synergistic effects, our study aimed to elucidate the combined obesity prevention potential of HTGT and EMIQ in mice. Mice were treated with these compounds for 8 weeks, while being fed a high-fat diet, to investigate their preventive anti-obesity effects. We demonstrated that the co-treatment of HTGT and EMIQ results in a synergistic anti-obesity effect, as determined by a Kruskal-Wallis test. Furthermore, the combined treatment of HTGT and EMIQ was more effective than orlistat in reducing body weight gain and adipocyte hypertrophy induced by high-fat diet. The co-treatment also significantly reduced total body fat mass and abdominal fat volume. Additionally, the group receiving the co-treatment exhibited increased energy expenditure and higher glucose intolerance. We observed a dose-dependent upregulation of genes associated with mitochondrial oxidative metabolism and PKA signaling, which is linked to lipolysis, in response to the co-treatment. The co-treatment group displayed elevated cAMP levels and AMPK activation in adipose tissue and increased excretion of fecal lipids. The results indicate that the co-treatment of HTGT and EMIQ holds the potential to be a promising combination therapy for combating obesity. To further validate the anti-obesity effect of the combined treatment of HTGT and EMIQ in human subjects, additional clinical studies are warranted.

Enzymatically modified isoquercitrin in soy protein temporarily enhanced the plasma amino-acid concentrations, antioxidant index, and plasma hormone levels: a randomized, double-blind cross-over trial.

This study investigated the effects of a dietary protein supplement containing enzymatically modified isoquercitrin (EMIQ) on plasma amino-acid levels in healthy people. A randomized double-blind cross-over trial (UMIN000044791) was conducted with a sample of nine healthy individuals. These participants ingested soy protein with or without 42 mg EMIQ for 7 days after performing mild exercise. Plasma amino-acid levels were measured before ingestion and at 15, 30, 45, 60, 90, 120, 180, and 240 min after ingestion on the last day. The concentrations of total amino acids at 0 and 120 min and easily oxidized amino acids at 120 min were significantly higher in the plasma of individuals who consumed 42 mg EMIQ. Oxidative stress levels were lower and plasma testosterone levels were higher in participants who ingested soy protein with 42 mg EMIQ than in those who did not. These results suggest that daily ingestion of soy protein with 42 mg EMIQ can be useful for effective protein absorption.

Sodium R-lipoate and enzymatically-modified isoquercitrin suppressed IgE-independent anaphylactic reactions and stress-induced gastric ulceration in mice.

Anaphylaxis is a life-threatening allergic reaction, for which the worldwide prevalence is rapidly increasing. The currently used synthetic antiallergic drugs have a high tendency to cause adverse effects, like gastric ulcers, in long-term use. Therefore, a great deal of attention has been given to develop new safer and more effective antiallergic agents from natural compounds that are chemically/enzymatically-modified. Here, we evaluated/compared the efficacy of two different doses (50 and 100 mg/kg body weight "b.w", given orally) of sodium R-lipoate (NaRLA) and enzymatically-modified isoquercitrin (EMIQ) in alleviating both local/systemic non-immunological anaphylactic reactions and stress-induced gastric ulceration in mice, in comparison with sulfasalazine (SSZ) as a reference drug. The results indicated that the pre-treatment of animals with NaRLA or EMIQ (especially at 100 mg/kg b.w) completely succeeded, as SSZ, in alleviating the hind paw edema induced by either histamine or compound 48/80 (Cpd 48/80). Furthermore, NaRLA and EMIQ prevented the mast cell degranulation and anaphylactic shock caused by Cpd 48/80 (in a dose-dependent manner) and reduced significantly (P < 0.001) the histamine release from the mouse peritoneal mast cells, like SSZ. Moreover, their use was associated with alleviating both gastric histopathological and biochemical alterations in the water-restraint stress (WRS) mice model towards the control values. They also decreased the percentage of degranulated mesenteric mast cells in the WRS mice model. In conclusion, our findings provide possibility that both NaRLA and EMIQ may serve as an effective therapeutic agents for mast cells-dependent anaphylactic reactions without risks of inducing gastric ulcers.

Anti-Obesity Effects of Soybean Embryo Extract and Enzymatically-Modified Isoquercitrin.

Soy isoflavones are bioactive phytoestrogens with known health benefits. Soybean embryo extract (SEE) has been consumed as a source of isoflavones, mainly daidzein, glycitein, and genistein. While previous studies have reported the anti-obesity effects of SEE, this study investigates their molecular mechanisms and the synergistic effects of co-treatment with SEE and enzymatically modified isoquercitrin (EMIQ). SEE upregulated genes involved in lipolysis and brown adipocyte markers and increased mitochondrial content in differentiated C3H10T1/2 adipocytes in vitro. Next, we use a high-fat diet-induced obesity mouse model to determine the anti-obesity effect of SEE. Two weeks of single or combined treatment with SEE and EMIQ significantly reduced body weight gain and improved glucose tolerance. Mechanistically, SEE treatment increased mitochondrial content and upregulated genes involved in lipolysis in adipose tissue through the cAMP/PKA-dependent signaling pathway. These effects required a cytosolic lipase adipose triglyceride lipase (ATGL) expression, confirmed by an adipocyte-specific ATGL knockout mouse study. Collectively, this study demonstrates that SEE exerts anti-obesity effects through the activation of adipose tissue metabolism and exhibits a synergistic effect of co-treatment with EMIQ. These results improve our understanding of the mechanisms underlying the anti-obesity effects of SEE related to adipose tissue metabolism.

Targeting Casein kinase 2 with quercetin or enzymatically modified isoquercitrin as a strategy for boosting the type 1 interferon response to viruses and promoting cardiovascular health.

The serine/threonine kinase CK2 has been shown to down-regulate the production of type 1 interferons in response to viral infections by conferring an inhibitory phosphorylation on RIG-I, which functions to detect double-stranded RNA generated during replication of RNA viruses. Quercetin and certain other planar flavones/flavonols can inhibit CK2 in high nanomolar concentrations; this may explain quercetin's ability to slow the proliferation of RNA viruses in cell cultures and in mice. Limited clinical evidence suggests that supplemental quercetin may decrease risk for upper respiratory infections in humans. Quercetin and enzymatically-modified isoquercitrin (EMIQ - a food additive/nutraceutical that upon oral administration achieves far higher plasma concentrations of quercetin than quercetin per se) also have exerted a range of vascular-protective effects clinically and in rodents - improving endothelial function, warding off atherosclerosis, lowering blood pressure, decreasing C-reactive protein, aiding glycemic control, stabilizing platelets - that might also, at least in part, reflect CK2 inhibition. The utility of quercetin, EMIQ, and other clinically feasible CK2 inhibitors for aiding control of viral infections and promoting vascular and metabolic health merits further evaluation.

Enzymatically modified isoquercitrin improves endothelial function in volunteers at risk of cardiovascular disease.

A higher intake of food rich in flavonoids such as quercetin can reduce the risk of CVD. Enzymatically modified isoquercitrin (EMIQ®) has a bioavailability 17-fold higher than quercetin aglycone and has shown potential CVD moderating effects in animal studies. The present study aimed to determine whether acute ingestion of EMIQ® improves endothelial function, blood pressure (BP) and cognitive function in human volunteers at risk of CVD. Twenty-five participants (twelve males and thirteen females) with at least one CVD risk factor completed this randomised, controlled, crossover study. In a random order, participants were given EMIQ® (2 mg aglycone equivalent)/kg body weight or placebo alongside a standard breakfast meal. Endothelial function, assessed by flow-mediated dilatation (FMD) of the brachial artery was measured before and 1·5 h after intervention. BP, arterial stiffness, cognitive function, BP during cognitive stress and measures of quercetin metabolites, oxidative stress and markers of nitric oxide (NO) production were assessed post-intervention. After adjustment for pre-treatment measurements and treatment order, EMIQ® treatment resulted in a significantly higher FMD response compared with the placebo (1·80 (95 % CI 0·23, 3·37) %; P = 0·025). Plasma concentrations of quercetin metabolites were significantly higher (P < 0·001) after EMIQ® treatment compared with the placebo. No changes in BP, arterial stiffness, cognitive function or biochemical parameters were observed. In this human intervention study, the acute administration of EMIQ® significantly increased circulating quercetin metabolites and improved endothelial function. Further clinical trials are required to assess whether health benefits are associated with long-term EMIQ® consumption.

High-efficiency enzymatic production of α-isoquercitrin glucosides by amylosucrase from Deinococcus geothermalis.

Isoquercitrin (IQ, quercetin-3-O-ß-d-glucopyranoside) has diverse biological functions, such as anti-oxidant and anti-cancer activity, but its use is limited by poor solubility and bioavailability. Enzymatically modified IQ (EMIQ) is a mixture of transglycosylated IQs that have better solubility and bioavailability than do quercetin and IQ. Two different enzymes, cyclodextrin glucanotransferase (CGTase) and amylosucrase (ASase), have the transglycosylation activity to produce EMIQ. Both enzymes produce a variety of EMIQs including IQ, IQ-glucoside (IQ-G1), IQ-diglucoside (IQ-G2), and IQ-triglucoside (IQ-G3). ASase had a higher bioconversion yield from IQ to EMIQ (97.6%) than did CGTase (76.8%). In addition, the yield of IQ-G3, which was the most bioavailable form, was higher with ASase (46%) than with CGTases (8%). Taken together, these results suggest that ASase can be used to synthesize EMIQ in a simple and specific process.

Comprehensive evaluation of the flavonol anti-oxidants, alpha-glycosyl isoquercitrin and isoquercitrin, for genotoxic potential.

Quercetin and its glycosides possess potential benefits to human health. Several flavonols are available to consumers as dietary supplements, promoted as anti-oxidants; however, incorporation of natural quercetin glycosides into food and beverage products has been limited by poor miscibility in water. Enzymatic conjugation of multiple glucose moieties to isoquercitrin to produce alpha-glycosyl isoquercitrin (AGIQ) enhances solubility and bioavailability. AGIQ is used in Japan as a food additive and has been granted generally recognized as safe (GRAS) status. However, although substantial genotoxicity data exist for quercetin, there is very little available data for AGIQ and isoquercitrin. To support expanded global marketing of food products containing AGIQ, comprehensive testing of genotoxic potential of AGIQ and isoquercitrin was conducted according to current regulatory test guidelines. Both chemicals tested positive in bacterial reverse mutation assays, and exposure to isoquercitrin resulted in chromosomal aberrations in CHO-WBL cells. All other in vitro mammalian micronucleus and chromosomal aberration assays, micronucleus and comet assays in male and female B6C3F1 mice and Sprague Dawley rats, and Muta™ Mouse mutation assays evaluating multiple potential target tissues, were negative for both chemicals. These results supplement existing toxicity data to further support the safe use of AGIQ in food and beverage products.

Enzymatically modified isoquercitrin supplementation intensifies plantaris muscle fiber hypertrophy in functionally overloaded mice.

BACKGROUND: Enzymatically modified isoquercitrin (EMIQ) is produced from rutin using enzymatic hydrolysis followed by treatment with glycosyltransferase in the presence of dextrin to add glucose residues. EMIQ is absorbed in the same way as quercetin, a powerful antioxidant reported to prevent disused muscle atrophy by targeting mitochondria and to have ergogenic effects. The present study investigated the effect of EMIQ on skeletal muscle hypertrophy induced by functional overload. METHODS: In Study 1, 6-week-old ICR male mice were divided into 4 groups: sham-operated control, sham-operated EMIQ, overload-operated control, and overload-operated EMIQ groups. In Study 2, mice were divided into 3 groups: overload-operated whey control, overload-operated whey/EMIQ (low dose), and overload-operated whey/EMIQ (high dose) groups. The functional overload of the plantaris muscle was induced by ablation of the synergist (gastrocnemius and soleus) muscles. EMIQ and whey protein were administered with food. Three weeks after the operation, the cross-sectional area and minimal fiber diameter of the plantaris muscle fibers were measured. RESULTS: In Study 1, functional overload increased the cross-sectional area and minimal fiber diameter of the plantaris muscle. EMIQ supplementation significantly increased the cross-sectional area and minimal fiber diameter of the plantaris muscle in both the sham-operated and overload-operated groups. In Study 2, EMIQ supplementation combined with whey protein administration significantly increased the cross-sectional area and minimal fiber diameter of the plantaris muscle. CONCLUSION: EMIQ, even when administered as an addition to whey protein supplementation, significantly intensified the fiber hypertrophy of the plantaris muscle in functionally overloaded mice. EMIQ supplementation also induced fiber hypertrophy of the plantaris in sham-operated mice.

Anti-inflammatory effects of the selective phosphodiesterase 3 inhibitor, cilostazol, and antioxidants, enzymatically-modified isoquercitrin and α-lipoic acid, reduce dextran sulphate sodium-induced colorectal mucosal injury in mice.

Developing effective treatments and preventing inflammatory bowel disease (IBD) are urgent challenges in improving patients' health. It has been suggested that platelet activation and reactive oxidative species generation are involved in the pathogenesis of IBD. We examined the inhibitory effects of a selective phosphodiesterase-3 inhibitor, cilostazol (CZ), and two antioxidants, enzymatically modified isoquercitrin (EMIQ) and α-lipoic acid (ALA), against dextran sulphate sodium (DSS)-induced colitis. BALB/c mice were treated with 0.3% CZ, 1.5% EMIQ, and 0.2% ALA in their feed. Colitis was induced by administering 5% DSS in drinking water for 8days. The inhibitory effects of these substances were evaluated by measuring relevant clinical symptoms (faecal blood, diarrhoea, and body weight loss), colon length, plasma cytokine and chemokine levels, whole genome gene expression, and histopathology. Diarrhoea was suppressed by each treatment, while CZ prevented shortening of the colon length. All treatment groups exhibited decreased plasma levels of interleukin (IL)-6 and tumour necrosis factor (TNF)-α compared with the DSS group. Microarray analysis showed that cell adhesion, cytoskeleton regulation, cell proliferation, and apoptosis, which might be related to inflammatory cell infiltration and mucosal healing, were affected in all the groups. DSS-induced mucosal injuries such as mucosal loss, submucosal oedema, and inflammatory cell infiltration in the distal colon were prevented by CZ or antioxidant treatment. These results suggest that anti-inflammatory effects of these agents reduced DSS-induced mucosal injuries in mice and, therefore, may provide therapeutic benefits in IBD.

Cilostazol and enzymatically modified isoquercitrin attenuate experimental colitis and colon cancer in mice by inhibiting cell proliferation and inflammation.

We previously reported the anti-inflammatory effects of cilostazol, a selective inhibitor of phosphodiesterase 3, and two antioxidants, enzymatically modified isoquercitrin and α-lipoic acid in a dextran sodium sulphate-induced colitis mouse model. We further examined the chemopreventive effects of these substances in a murine azoxymethane/dextran sodium sulphate -induced colorectal carcinoma model and compared the effects with those of the well-known anticancer natural plant pigment, anthocyanin. In addition, the effects on cell proliferation activity were evaluated in colon cancer cell lines and mucosal epithelial cells in a model of acute dextran sodium sulphate-induced colitis. Cilostazol and enzymatically modified isoquercitrin improved the outcome of azoxymethane/dextran sodium sulphate-induced colorectal cancer along with anthocyanin though inhibiting inflammation and cell proliferation, but the effect of α-lipoic acid was minimal. Inhibition of cell proliferation by cilostazol was confirmed in vitro. In the acute dextran sodium sulphate-induced colitis model, cilostazol and enzymatically modified isoquercitrin prevented the decrease in epithelial proliferative cells. These results indicate that cilostazol and enzymatically modified isoquercitrin first exhibited an anti-dextran sodium sulphate effect at the initial stage of colitis and then showed antitumour effects throughout subsequent inflammation-related cancer developmental stages.

Genetic and rat toxicity studies of cyclodextrin glucanotransferase.

INTRODUCTION: Microbiologically derived cyclodextrin glucanotransferase (CGTase) is used commercially as a processing agent in manufacture of food, pharmaceuticals, and cosmetics. Its toxic potential was evaluated in anticipation of use in the production of alpha-glycosyl isoquercitrin, a water-soluble form of quercetin. METHODS: Following OECD guidelines, CGTase, produced by Bacillus pseudalcaliphilus DK-1139, was evaluated in a genotoxicity battery consisting of a bacterial reverse mutation assay, an in vitro micronucleus (MN) assay and MN and comet assays using B6C3F1 male and female mice. These same genotoxicity assays were also conducted for sodium sulfate, a contaminant of CGTase preparation. In a 90-day Sprague Dawley rat toxicity study, CGTase was administered by gavage in water at daily doses of 0, 250, 500, and 1000 mg/kg/day. RESULTS: CGTase did not induce mutations with or without metabolic activation in the bacterial reverse mutation assay. Formation of micronuclei was not induced in either in vitro or in vivo MN assays with or without metabolic activation. No induction of DNA damage was detected in male or female mouse liver, stomach, or duodenum in the comet assay. Sodium sulfate also tested negative in these same genotoxicity assays. In the 90-day repeated dose rat study there were no treatment-related adverse clinical or pathological findings. CONCLUSION: The genotoxicity assays and repeated dose toxicity study support the safe use of CGTase in production of alpha-glycosyl isoquercitrin.

Tumor suppression effects of bilberry extracts and enzymatically modified isoquercitrin in early preneoplastic liver cell lesions induced by piperonyl butoxide promotion in a two-stage rat hepatocarcinogenesis model.

To investigate the protective effect of bilberry extracts (BBE) and enzymatically modified isoquercitrin (EMIQ) on the hepatocarcinogenic process involving oxidative stress responses, we used a two-stage hepatocarcinogenesis model in N-diethylnitrosamine-initiated and piperonyl butoxide (PBO)-promoted rats. We examined the modifying effect of co-administration with BBE or EMIQ on the liver tissue environment including oxidative stress responses, cell proliferation and apoptosis, and phosphatase and tensin homolog (PTEN)/Akt and transforming growth factor (TGF)-ß/Smad signalings on the induction mechanism of preneoplastic lesions during early stages of hepatocellular tumor promotion. PBO increased the numbers and area of glutathione S-transferase placental form (GST-P)(+) liver cell foci and the numbers of Ki-67(+) proliferating cells within GST-P(+) foci. Co-administration of BBE or EMIQ suppressed these effects with the reductions of GST-P(+) foci (area) to 48.9-49.4% and Ki-67(+) cells to 55.5-61.4% of the PBO-promoted cases. Neither BBE nor EMIQ decreased microsomal reactive oxygen species induced by PBO. However, only EMIQ suppressed the level of thiobarbituric acid-reactive substances to 78.4% of the PBO-promoted cases. PBO increased the incidences of phospho-PTEN(-) foci, phospho-Akt substrate(+) foci, phospho-Smad3(-) foci and Smad4(-) foci in GST-P(+) foci. Both BBE and EMIQ decreased the incidences of phospho-PTEN(-) foci in GST-P(+) foci to 59.8-72.2% and Smad4(-) foci to 62.4-71.5% of the PBO-promoted cases, and BBE also suppressed the incidence of phospho-Akt substrate(+) foci in GST-P(+) foci to 75.2-75.7% of the PBO-promoted cases. These results suggest that PBO-induced tumor promotion involves facilitation of PTEN/Akt and disruptive TGF-ß/Smad signalings without relation to oxidative stress responses, but this promotion was suppressed by co-treatment with BBE or EMIQ through suppression of cell proliferation activity of preneoplastic liver cells.

Isoquercitrin: pharmacology, toxicology, and metabolism.

The flavonoid isoquercitrin (quercetin-3-O-ß-d-glucopyranoside) is commonly found in medicinal herbs, fruits, vegetables and plant-derived foods and beverages. This article reviews the occurrence, preparation, bioavailability, pharmacokinetics, toxicology and biological activity of isoquercitrin and "enzymatically modified (α-glucosylated) isoquercitrin" (EMIQ). Pure isoquercitrin can now be obtained on a large scale by enzymatic rutin hydrolysis with α-l-rhamnosidase. Isoquercitrin has higher bioavailability than quercetin and displays a number of chemoprotective effects both in vitro and in vivo, against oxidative stress, cancer, cardiovascular disorders, diabetes and allergic reactions. Although small amounts of intact isoquercitrin can be found in plasma and tissues after oral application, it is extensively metabolized in the intestine and the liver. Biotransformation of isoquercitrin includes deglycosylation, followed by formation of conjugated and methylated derivatives of quercetin or degradation to phenolic acids and carbon dioxide. The acceptable daily intake of (95%) isoquercitrin and of EMIQ was estimated to be 5.4 and 4.9mg/kg/day, respectively. Adverse effects of higher doses in rats included mostly (benign) chromaturia; nevertheless some drug interactions may occur due to the modulation of the activity and/or expression of drug metabolizing/transporting systems. With respect to the safety, affordability and beneficial pharmacological activities, highly pure isoquercitrin is a prospective substance for food supplementation.

Ochratoxin A induces karyomegaly and cell cycle aberrations in renal tubular cells without relation to induction of oxidative stress responses in rats.

Ochratoxin A (OTA) is a renal carcinogen that induces karyomegaly in target renal tubular cells of the outer stripe of the outer medulla (OSOM). This study was performed to clarify the relationship between oxidative stress and the karyomegaly-inducing potential involving cell cycle aberration of OTA in the OSOM. Rats were treated with OTA for 28 days in combination with enzymatically modified isoquercitrin (EMIQ) or α-lipoic acid (ALA) as antioxidants. OTA increased the mRNA levels of the antioxidant enzyme-related genes Gpx1, Gpx2, Gstm1 and Nfe2l2, but did not increase the levels of Gsta5, Keap1, Nqo1, Hmox1, Aldh1a1, Por, Prdx1 and Txn1. OTA also did not change the levels of thiobarbituric acid-reactive substances, glutathione disulfide/reduced glutathione, and the immunoreactive tubular cell distribution of nuclear factor erythroid 2-related factor 2 in the OSOM. Co-treatment with EMIQ or ALA did not cause any changes in these parameters. As previously reported, OTA increased cell proliferation activity, apoptosis and immunohistochemical cellular distributions of molecules suggestive of induction of DNA damage and cell cycle aberrations involving spindle checkpoint disruption and cell cycle arrest. However, co-treatment with EMIQ or ALA did not suppress these changes, and ALA co-treatment increased the cell proliferation activity induced by OTA. These results suggest that OTA facilitates cell cycling involving cell cycle aberrations and apoptosis as a basis of the mechanism behind the development of karyomegaly and subsequent carcinogenicity targeting the OSOM, without relation to induction of oxidative stress. On the other hand, ALA may promote the OTA-induced proliferation of carcinogenic target cells.

Inhibitory effect of α-lipoic acid on thioacetamide-induced tumor promotion through suppression of inflammatory cell responses in a two-stage hepatocarcinogenesis model in rats.

To investigate the protective effect of α-lipoic acid (a-LA) on the hepatocarcinogenic process promoted by thioacetamide (TAA), we used a two-stage liver carcinogenesis model in N-diethylnitrosamine (DEN)-initiated and TAA-promoted rats. We examined the modifying effect of co-administered a-LA on the liver tissue environment surrounding preneoplastic hepatocellular lesions, with particular focus on hepatic macrophages and the mechanism behind the decrease in apoptosis of cells surrounding preneoplastic hepatocellular lesions during the early stages of hepatocellular tumor promotion. TAA increased the number and area of glutathione S-transferase placental form (GST-P)(+) liver cell foci and the numbers of proliferating and apoptotic cells in the liver. Co-administration with a-LA suppressed these effects. TAA also increased the numbers of ED2(+), cyclooxygenase-2(+), and heme oxygenase-1(+) hepatic macrophages as well as the number of CD3(+) lymphocytes. These effects were also suppressed by a-LA. Transcript levels of some inflammation-related genes were upregulated by TAA and downregulated by a-LA in real-time RT-PCR analysis. Outside the GST-P(+) foci, a-LA reduced the numbers of apoptotic cells, active caspase-8(+) cells and death receptor (DR)-5(+) cells. These results suggest that hepatic macrophages producing proinflammatory factors may be activated in TAA-induced tumor promotion. a-LA may suppress tumor-promoting activity by suppressing the activation of these macrophages and the subsequent inflammatory responses. Furthermore, a-LA may suppress tumor-promoting activity by suppressing the DR5-mediated extrinsic pathway of apoptosis and the subsequent regeneration of liver cells outside GST-P(+) foci.