Genotoxicity and subacute toxicity studies of a new astaxanthin-containing Phaffia rhodozyma extract.

Experimental and clinical studies demonstrate that astaxanthin (AXN), a xanthophyll carotenoid, has protective effects against oxidative damage. Because most of these studies assessed AXN derived from Haematococcus pluvialis that were cultivated at industrial scales, few studies have examined the toxicity of AXN derived from Phaffia rhodozyma. To evaluate the safety of astaxanthin-containing P. rhodozymaextract (AXN-PRE), genotoxicity was assessed in bacterial reverse mutation test and mouse bone marrow micronucleus test, and general toxicity was assessed in 4-week repeated oral toxicity study in rats. AXN-PRE did not induce reverse mutations in the Salmonella typhimurium strains TA98 or TA100 at concentrations of 5,000 µg/plate with or without S9 mix, and no chromosome damage was observed at a dose of 2,000 mg/kg in mouse micronucleus test. In the subacute toxicity study, male and female Sprague-Dawley rats were given AXN-PRE at doses of 0, 500, and 1,000 mg/kg by gavage for 4 weeks. Body weights, urinalysis, hematology, serum biochemistry, organ weights, or histopathological lesions indicated no distinct toxicity. In conclusion, AXN-PRE had no effect in bacterial reverse mutation test and mouse bone marrow micronucleus test. The no-observed-adverse-effect level for AXN-PRE in 4-week repeated oral toxicity study in rats was determined to be greater than 1,000 mg/kg (corresponding to dose of 50 mg/kg AXN) regardless of gender.

Ubiquinol-10 supplementation activates mitochondria functions to decelerate senescence in senescence-accelerated mice.

AIM: The present study was conducted to define the relationship between the anti-aging effect of ubiquinol-10 supplementation and mitochondrial activation in senescence-accelerated mouse prone 1 (SAMP1) mice. RESULTS: Here, we report that dietary supplementation with ubiquinol-10 prevents age-related decreases in the expression of sirtuin gene family members, which results in the activation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a major factor that controls mitochondrial biogenesis and respiration, as well as superoxide dismutase 2 (SOD2) and isocitrate dehydrogenase 2 (IDH2), which are major mitochondrial antioxidant enzymes. Ubiquinol-10 supplementation can also increase mitochondrial complex I activity and decrease levels of oxidative stress markers, including protein carbonyls, apurinic/apyrimidinic sites, malondialdehydes, and increase the reduced glutathione/oxidized glutathione ratio. Furthermore, ubiquinol-10 may activate Sirt1 and PGC-1α by increasing cyclic adenosine monophosphate (cAMP) levels that, in turn, activate cAMP response element-binding protein (CREB) and AMP-activated protein kinase (AMPK). INNOVATION AND CONCLUSION: These results show that ubiquinol-10 may enhance mitochondrial activity by increasing levels of SIRT1, PGC-1α, and SIRT3 that slow the rate of age-related hearing loss and protect against the progression of aging and symptoms of age-related diseases.

Novel medium-term carcinogenesis model for lung squamous cell carcinoma induced by N-nitroso-tris-chloroethylurea in mice.

Targeted treatments for lung cancer based on pathological diagnoses are required to enhance therapeutic efficacy. There are few well-established animal models for lung squamous cell carcinoma although several highly reproducible mouse models for lung adenoma and adenocarcinoma are available. This study was carried out to establish a new lung squamous cell carcinoma mouse model. In the first experiment, female A/J mice were painted topically on back skin twice weekly with 75 µL 0.013 M N-nitroso-tris-chloroethylurea for 2, 4, and 8 weeks (n = 15-20 per group) as initiation of lung lesions, and surviving mice were killed at 18 weeks. In the second experiment, mice were treated as above for 4 weeks and killed at 6, 12, or 18 weeks (n = 3 per group). Lung lobes were subjected to histopathological, immunohistochemical, immunoblotting, and ultrastructural analyses. In the case of treatment for 2, 4, and 8 weeks, incidences of lung squamous cell carcinoma were 25, 54, and 71%, respectively. Cytokeratin 5/6 and epidermal growth factor receptor were clearly expressed in dysplasia and squamous cell carcinoma. Desmosomes and tonofilaments developed in the squamous cell carcinoma. Considering the carcinogenesis model, we conclude that 2 or 4 weeks of N-nitroso-tris-chloroethylurea treatment may be suitable for investigating new chemicals for promotional or suppressive effects on lung squamous cell carcinoma.

Ubiquinol affects the expression of genes involved in PPARα signalling and lipid metabolism without changes in methylation of CpG promoter islands in the liver of mice.

Coenzyme Q(10) is an essential cofactor in the respiratory chain and serves as a potent antioxidant in biological membranes. Recent studies in vitro and in vivo provide evidence that Coenzyme Q(10) is involved in inflammatory processes and lipid metabolism via gene expression. To study these effects at the epigenomic level, C57BL6J mice were supplemented for one week with reduced Coenzyme Q(10) (ubiquinol). Afterwards, gene expression signatures and DNA promoter methylation patterns of selected genes were analysed. Genome-wide transcript profiling in the liver identified 1112 up-regulated and 571 down-regulated transcripts as differentially regulated between ubiquinol-treated and control animals. Text mining and GeneOntology analysis revealed that the "top 20" ubiquinol-regulated genes play a role in lipid metabolism and are functionally connected by the PPARα signalling pathway. With regard to the ubiquinol-induced changes in gene expression of about +3.14-fold (p≤0.05), +2.18-fold (p≤0.01), and -2.13-fold (p≤0.05) for ABCA1, ACYP1, and ACSL1 genes, respectively, hepatic DNA methylation analysis of 282 (sense orientation) and 271 (antisense) CpG units in the respective promoter islands revealed no significant effect of ubiquinol. In conclusion, ubiquinol affects the expression of genes involved in PPARα signalling and lipid metabolism without changing the promoter DNA methylation status in the liver of mice.

Oral repeated-dose toxicity studies of coenzyme Q10 in beagle dogs.

To support phase III testing of coenzyme Q10 (CoQ10) in humans, we conducted pharmacokinetic and toxicology studies in beagle dogs. Following single gavage administration of CoQ10 at 600, 1200, 1800, or 2400 mg/kg per d no obvious dose response was observed in maximum concentration (C(max)) or area under the curve (AUC) versus time curve at the 3 highest dosages. In a repeated-dose study of CoQ10 at 600, 1200, 1800, or 2400 mg/kg per d for 4 weeks, CoQ10 reached steady state in plasma by 2 weeks at all dosages. Both C (max) and AUC increased with increasing dosage of CoQ10. The highest plasma levels were recorded at 1800 mg/kg per d. In a 39-week chronic toxicity study of CoQ10 at 1200 and 1800 mg/kg per d or placebo, CoQ10 reached steady state in plasma by 13 weeks. Behaviors, blood chemistries, and detailed histopathology were normal. No deaths occurred. These results support the use of a 2400 mg/d dosage of CoQ10 in human clinical trials.

Inhibition by licorice flavonoid oil of glutathione S-transferase-positive foci in the medium-term rat hepatocarcinogenesis bioassay.

Licorice flavonoid oil (LFO) is a new functional food ingredient consisting of hydrophobic licorice polyphenols in medium-chain triglycerides. Recently, it was reported that licorice and its derivatives have anticarcinogenic activity in some types of tumors. However, the anticarcinogenic activity has not been identified in the liver, which is a major target organ for carcinogenesis in human. Therefore, we hypothesized that LFO has antihepatocarcinogenic activity, and we tested this hypothesis using the rat medium-term liver bioassay for carcinogens. Six-week-old male F344 rats (15 animals/group) received N-diethylnitrosamine (200 mg/kg by intraperitoneal injection) to initiate carcinogenesis. From the second week after initiation, animals received a 6-week regimen of either LFO concentrate solution (0, 150, 300, or 600 mg/kg) intragastrically or phenobarbital sodium salt in the diet (500 ppm) as a positive control. During the third week after initiation, animals were subjected to a two-thirds partial hepatectomy. During the eighth week of the treatment period, liver samples were taken from animals and examined immunohistochemically for expression of glutathione S-transferase placental form. No increase in the number of glutathione S-transferase placental form-positive liver foci was observed in all LFO groups compared with the negative control (solvent) group, and the number of foci in the 600 mg/kg LFO group was significantly lower than that in the negative control group. These results indicate that LFO concentrate solution has a significant inhibitory effect on liver carcinogenesis at 600 mg/kg.

Supplementation with the reduced form of Coenzyme Q10 decelerates phenotypic characteristics of senescence and induces a peroxisome proliferator-activated receptor-alpha gene expression signature in SAMP1 mice.

Our present study reveals significant decelerating effects on senescence processes in middle-aged SAMP1 mice supplemented for 6 or 14 months with the reduced form (Q(10)H(2), 500 mg/kg BW/day) of coenzyme Q(10) (CoQ(10)). To unravel molecular mechanisms of these CoQ(10) effects, a genome-wide transcript profiling in liver, heart, brain and kidney of SAMP1 mice supplemented with the reduced (Q(10)H(2)) or oxidized form of CoQ(10) (Q(10)) was performed. Liver seems to be the main target tissue of CoQ(10) intervention, followed by kidney, heart and brain. Stringent evaluation of the resulting data revealed that Q(10)H(2) has a stronger impact on gene expression than Q(10), primarily due to differences in the bioavailability. Indeed, Q(10)H(2) supplementation was more effective than Q(10) to increase levels of CoQ(10) in the liver of SAMP1 mice. To identify functional and regulatory connections of the "top 50" (p<0.05) Q(10)H(2)-sensitive transcripts in liver, text mining analysis was used. Hereby, we identified Q(10)H(2)-sensitive genes which are regulated by peroxisome proliferator-activated receptor-alpha and are primarily involved in cholesterol synthesis (e.g. HMGCS1, HMGCL and HMGCR), fat assimilation (FABP5), lipoprotein metabolism (PLTP) and inflammation (STAT-1). These data may explain, at least in part, the decelerating effects on degenerative processes observed in Q(10)H(2)-supplemented SAMP1 mice.

Potassium bromate enhances N-ethyl-N-hydroxyethylnitrosamine-induced kidney carcinogenesis only at high doses in Wistar rats: indication of the existence of an enhancement threshold.

As susceptibility to carcinogens varies considerably among different strains of experimental animals, evaluation of dose-response relationships for genotoxic carcinogen in different strains is indispensable for risk assessment. Potassium bromate (KBrO(3)) is a genotoxic carcinogen inducing kidney cancers at high doses in male F344 rats, but little is known about its carcinogenic effects in other strains of rats. The purpose of the present study was to determine dose-response relationships for carcinogenic effects of KBrO(3) on N-ethyl-N-hydroxyethylnitrosamine (EHEN)-induced kidney carcinogenesis in male Wistar rats. We found that KBrO(3) showed significant enhancement effects on EHEN-induced kidney carcinogenesis at above 250 ppm but not at doses of 125 ppm and below when evaluated in terms of induction of either preneoplastic lesions or tumors in male Wistar rats. Furthermore, KBrO(3) significantly increased the formation of oxidative DNA damage at doses of 125 and above but not at doses of 30 ppm and below in kidneys. These results demonstrated that low doses of KBrO(3) exert no effects on development of EHEN-initiated kidney lesions and induction of oxidative DNA damage. Taking account of previous similar findings in male F344 rats, it is strongly suggested that a threshold dose exists for enhancement effects of KBrO(3) on kidney carcinogenesis in rats.

Oral administration of lactic acid bacteria isolated from traditional South Asian fermented milk 'dahi' inhibits the development of atopic dermatitis in NC/Nga mice.

We investigated the suppressive effect of lactic acid bacteria (LAB) isolated from traditional South Asian fermented milk 'dahi' on the development of atopic dermatitis (AD) using NC/Nga AD model mice. In the initial evaluation, we confirmed the effect of LAB on serum total IgE using ovalbumin (OVA)-induced type 1 allergy model mice. Forty-one bacterial strains isolated from dahi were evaluated for their ability to induce interleukin (IL)-12 production and suppress IL-4 production in splenocytes obtained from OVA-sensitized mice. Of the 41 strains tested, Lactobacillus delbrueckii subsp. lactis R-037 exhibited the greatest IL-12 induction, suggesting that it is a potent Th1 inducer. Oral administration of heat-treated R-037 significantly suppressed the elevation of serum total IgE in OVA-induced type 1 allergy model mice. In NC/Nga AD model mice, oral administration of heat-treated R-037 reduced inflammatory auricular thickness and alleviated the AD clinical score although the effect on serum total IgE level was unclear. Histopathological findings showed a tendency toward improvement of inflammation. Hyperkeratosis in particular showed improvement in dermatitis skin lesions. These results suggest that oral administration of R-037 may alleviate AD.

Effects of ubiquinol-10 on microRNA-146a expression in vitro and in vivo.

MicroRNAs (miRs) are involved in key biological processes via suppression of gene expression at posttranscriptional levels. According to their superior functions, subtle modulation of miR expression by certain compounds or nutrients is desirable under particular conditions. Bacterial lipopolysaccharide (LPS) induces a reactive oxygen species-/NF-kappaB-dependent pathway which increases the expression of the anti-inflammatory miR-146a. We hypothesized that this induction could be modulated by the antioxidant ubiquinol-10. Preincubation of human monocytic THP-1 cells with ubiquinol-10 reduced the LPS-induced expression level of miR-146a to 78.9 +/- 13.22%. In liver samples of mice injected with LPS, supplementation with ubiquinol-10 leads to a reduction of LPS-induced miR-146a expression to 78.12 +/- 21.25%. From these consistent in vitro and in vivo data, we conclude that ubiquinol-10 may fine-tune the inflammatory response via moderate reduction of miR-146a expression.

Enhancement of preneoplastic lesion yield by Chios Mastic Gum in a rat liver medium-term carcinogenesis bioassay.

The mastic (Pistacia lentiscus var. chia) tree is native throughout the Mediterranean region and has long proved a source of food additives and medical treatments. To investigate the modifying effects of Chios Mastic Gum on rat liver carcinogenesis, 6-week-old male F344 rats were subjected to the established rat liver medium-term carcinogenesis bioassay (Ito-test). At the commencement, rats (groups 1-4) were intraperitoneally injected with 200 mg/kg body weight of diethylnitrosamine (DEN). After two weeks, mastic was added to CRF (Charles River Formula)-1 powdered basal diet at doses of 0, 0.01, 0.1 and 1% in groups 1-4, respectively. At week 3, all rats were underwent two-thirds partial hepatectomy. The experiment was terminated at week 8. As results show, liver weights were significantly increased in a mastic dose-dependent manner among groups 1-4. The numbers (/cm(2)) and the areas (mm(2)/cm(2)) of glutathione S-transferase placental form (GST-P)-positive cell foci (>or=0.2 mm in diameter) were significantly increased in the DEN-1% group compared to the DEN-alone group, along with the average areas per foci and larger-sized foci (>or=0.4 mm). 5-Bromo-2'-deoxyuridine (BrdU)+GST-P double-immunohistochemistry showed the highest BrdU-labeling indices within GST-P foci in the DEN-1% group. 8-hydroxydeoxyguanosine (8-OHdG) levels in liver DNA did not vary, while real-time quantitative polymerase chain reaction (PCR) analysis of livers revealed many up- or down-regulated genes in the DEN-1% group. In conclusion, this is the first report to display a promotion potential of Chios Mastic Gum on the formation of preneoplastic lesions in the established rat liver medium-term carcinogenesis bioassay.

Licorice flavonoid oil reduces total body fat and visceral fat in overweight subjects: A randomized, double-blind, placebo-controlled study.

OBJECTIVES: To evaluate effects of licorice flavonoid oil (LFO) on total body fat and visceral fat together with body weight, body mass index (BMI) and safety parameters in overweight subjects. METHODS: In this randomized, double-blind, placebo-controlled study, moderately overweight participants (56 males, 28 females, BMI 24-30 kg/m(2)) were assigned to four groups receiving a daily dose of either 0 (placebo), 300, 600, or 900 mg of LFO. Total body fat mass was measured by dual-energy X-ray absorptiometry (DXA) and visceral fat area by abdominal computed tomography (CT) scan at baseline and after 8 weeks of LFO ingestion. Body weight, BMI, and blood samples were examined at baseline and after 4 and 8 weeks of LFO ingestion. RESULTS: Although caloric intake was similar in all four groups, total body fat mass decreased significantly in the three LFO groups after 8 weeks of ingestion. LFO (900 mg/day) resulted in significant decreases from baseline levels in visceral fat area, body weight, BMI, and LDL-cholesterol. No significant adverse effects were observed.

Subchronic oral toxicity of ubiquinol in rats and dogs.

Ubiquinol is the two-electron reduction product of ubiquinone (coenzyme Q(10) or CoQ(10)) and functions as an antioxidant in both mitochondria and lipid membranes. In humans and most mammals, including dogs, the predominant form of coenzyme Q is coenzyme Q(10), whereas the primary form in rodents is coenzyme Q(9) (CoQ(9)). Therefore, the subchronic toxicity of ubiquinol was evaluated and compared in Sprague-Dawley rats and beagle dogs. In the initial rat study, males and females were given ubiquinol at doses of 0, 300, 600, or 1200 mg/kg or ubiquinone at 1200 mg/kg by gavage for 13 weeks. This was followed by the second study, where females were given with doses of 75, 150, 200, or 300 mg/kg/day in order to determine a no observed adverse effect level (NOAEL). In the dog study, the test material was administered to males and females at dose levels of 150, 300, and 600 mg/kg, and ubiquinone was included at 600 mg/kg. Clinical observations, mortality, body weights, food and water consumption, ophthalmoscopy, urinalysis, hematology, blood biochemistry, gross findings, organ weights, and histopathological findings were examined. In both species, determination of plasma and liver ubiquinol concentrations, measured as total coenzyme Q(10), were performed. There were no deaths or test article-related effects in body weight, food consumption, ophthalmology, urinalysis, or hematology in rats. Histopathological examinations revealed test article-related effects on the liver, spleen, and mesenteric lymph node in female rats but not in male rats. In the liver, fine vacuolation of hepatocytes was observed in the ubiquinol groups at 200 mg/kg and above. These changes were judged to be of no toxicological significance because they were not considered to induce cytotoxic changes. Microgranuloma and focal necrosis with accumulation of macrophages were observed in the ubiquinol groups at 300 mg/kg and above. These findings were accompanied by slight increases in blood chemistry enzymes (aspartate aminotransferase [AST], alanine aminotransferase [ALT], and lactate dehydrogenase [LDH]), which was suggestive of either potential hepatotoxicity or a normal physiological response to ubiguinol loading. Microgranuloma, and focal necrosis were judged to be only adverse effects induced by test article based on their incidence and pathological characteristics. These changes observed in liver were thought due to uptake of the administered ubiquinol by the liver as an adaptive response to xenobiotics, and the microgranulomas and focal necrosis were considered the results of excessive uptake of ubiquinol, which exceeded the capacity for adaptive response. Based on these findings the NOAEL in rats was conservatively estimated to be 600 mg/kg/day for males and 200 mg/kg/day for females. In dogs, there were no deaths or ubiquinol-related toxicity findings during the administration period. No test article-related effects were observed in body weight, food consumption, ophthalmology, electrocardiogram, urinalysis, hematology, or blood chemistry. Histopathological examination revealed no effects attributable to administration of ubiquinol or ubiquinone in any organs examined. Based on these findings, a NOAEL for ubiquinol in male and female dogs was estimated to be more than 600 mg/kg/day under the conditions of this study.

Evaluation of the mutagenic and genotoxic potential of ubiquinol.

Ubiquinol (the reduced form of coenzyme Q(10)) is the two-electron reduction product of ubiquinone (the oxidized form of coenzyme Q(10)), and has been shown to be an integral part of living cells, where it functions as an antioxidant in both mitochondria and lipid membranes. To provide information to enable a Generally Regarded as Safe (GRAS) evaluation for the use of ubiquinol in selected foods, a series of Organisation of Economic Cooperation and Development (OECD) and good laboratory practice (GLP) toxicological studies was conducted to evaluate the mutagenic and genotoxic potential of Kaneka QH brand of ubiquinol. Ubiquinol did not induce reverse mutations in Salmonella typhimurium strains TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2uvrA at concentrations up to 5000 mu g/plate, in either the absence and presence of exogenous metabolic activation by rat liver S9. Likewise, ubiquinol did not induce chromosome aberrations in Chinese hamster lung fibroblast (CHL/IU) cells in short-term (6-h) tests with or without rat liver S9 at concentrations up to 5000 mu g/ml or in a continuous (24-h) treatment test at concentrations up to 1201 mu g/ml. Finally, no mortalities, no abnormal clinical signs, and no significant increase in chromosome damage were observed in an in vivo micronucleus test when administered orally at doses up to 2000 mg/kg/day. Thus, ubiquinol was evaluated as negative in the bacterial reverse mutation, chromosomal aberration, and rat bone marrow micronucleus tests under the conditions of these assays.

Clinical safety of licorice flavonoid oil (LFO) and pharmacokinetics of glabridin in healthy humans.

OBJECTIVE: Licorice flavonoids have various physiological activities such as abdominal fat-lowering, hypoglycemic and antioxidant effects. Licorice flavonoid oil (LFO: Kaneka Glavonoid Rich Oil) is a new dietary ingredient containing licorice flavonoids dissolved in medium-chain triglycerides (MCT). Glabridin is one of the bioactive flavonoids included specifically in licorice Glycyrrhiza glabra L. and is the most abundant flavonoid in LFO. In this study, we assessed the safety of LFO in healthy humans and determined the plasma concentration profile of glabridin as a marker compound. METHODS: A single-dose and two multiple-dose studies at low (300 mg), moderate (600 mg) and high (1200 mg) daily doses of LFO were carried out using a placebo-controlled single-blind design. In each study the safety of LFO and the pharmacokinetics of glabridin were assessed. RESULTS: Pharmacokinetic analysis in the single-dose study with healthy male subjects (n = 5) showed that glabridin was absorbed and reached the maximum concentration (Cmax) after approximately 4 h (Tmax), and then eliminated relatively slowly in a single phase with a T1/2 of approximately 10 h at all doses. The Cmax and AUC(0-24 h) increased almost linearly with dose. The multiple-dose studies with healthy male and female subjects for 1 week and 4 weeks suggested that plasma glabridin reached steady state levels within 2 weeks with a single daily administration of 300 to 1200 mg/day LFO. In these human studies at three dose levels, there were no clinically noteworthy changes in hematological or related biochemical parameters. All clinical events observed were mild and considered to be unrelated to LFO administration even after repeated administration for 4 weeks. CONCLUSION: These studies demonstrated that LFO is safe when administered once daily up to 1200 mg/day. This is the first report on the safety of licorice flavonoids in an oil preparation and the first report on the pharmacokinetics of glabridin in human subjects.

Suppression by licorice flavonoids of abdominal fat accumulation and body weight gain in high-fat diet-induced obese C57BL/6J mice.

We applied licorice flavonoid oil (LFO) to high-fat diet-induced obese C57BL/6J mice and investigated its effect. LFO contains hydrophobic flavonoids obtained from licorice by extraction with ethanol. The oil is a mixture of medium-chain triglycerides, having glabridin, a major flavonoid of licorice, concentrated to 1.2% (w/w). Obese mice were fed on a high-fat diet containing LFO at 0 (control), 0.5%, 1.0%, or 2.0% for 8 weeks. Compared with mice in the control group, those in the 1% and 2% LFO groups efficiently reduced the weight of abdominal white adipose tissues and body weight gain. A histological examination revealed that the adipocytes became smaller and the fatty degenerative state of the hepatocytes was improved in the 2% LFO group. A DNA microarray analysis of the liver showed up-regulation of those genes for beta-oxidation and down-regulation of those for fatty acid synthesis in the 2% LFO group. These findings suggest that LFO prevented and ameliorated diet-induced obesity via the regulation of lipid metabolism-related gene expression in the liver.

Study on safety and bioavailability of ubiquinol (Kaneka QH) after single and 4-week multiple oral administration to healthy volunteers.

The safety and bioavailability of ubiquinol (the reduced form of coenzyme Q(10)), a naturally occurring lipid-soluble nutrient, were evaluated for the first time in single-blind, placebo-controlled studies with healthy subjects after administration of a single oral dose of 150 or 300 mg and after oral administration of 90, 150, or 300 mg for 4 weeks. No clinically relevant changes in results of standard laboratory tests, physical examination, vital signs, or ECG induced by ubiquinol were observed in any dosage groups. The C(max) and AUC(0-48 h) derived from the mean plasma ubiquinol concentration-time curves increased non-linearly with dose from 1.88 to 3.19 micro g/ml and from 74.61 to 91.76 micro g h/ml, respectively, after single administration. Trough concentrations had nearly plateaued at levels of 2.61 micro g/ml for 90 mg, 3.66 micro g/ml for 150 mg, and 6.53 micro g/ml for 300 mg at day 14, and increased non-linearly with dose in the 4-week study. In conclusion, following single or multiple-doses of ubiquinol in healthy volunteers, significant absorption of ubiquinol from the gastrointestinal tract was observed, and no safety concerns were noted on standard laboratory tests for safety or on assessment of adverse events for doses of up to 300 mg for up to 2 weeks after treatment completion.

Possible tumor development from double positive foci for TGF-alpha and GST-P observed in early stages on rat hepatocarcinogenesis.

Expression of TGF-alpha during promotion of neoplastic development from GST-P-positive foci in rat chemical hepatocarcinogenesis was investigated. One-hundred male F344 rats were given a single intraperitoneal injection of DEN (200 mg/kg bodyweight) and subjected to two-thirds partial hepatectomy at week 3. Commencing 2 weeks from the start, PB at doses of 0 or 500 p.p.m. was fed to the rats for 46 weeks. Groups of 10 rats were killed at weeks 4, 8, 16, 32, 48 and their livers were immunohistochemically examined for expression of GST-P and TGF-alpha. TGF-alpha-positive foci and single positive cells were observed from week 4, partially overlapping with GST-P-positive foci but being much fewer. Numbers of TGF-alpha-positive lesions did not increase from weeks 4-48, but their areas showed increment at weeks 32 and 48, especially with PB administration. Almost all of the tumors observed at weeks 16, 32 and 48 were positive for TGF-alpha (98%). In addition, epidermal growth factor receptor overexpression was observed in most TGF-alpha-positive lesions (foci and tumors). The proliferating cell nuclear antigen labeling index in double positive foci for GST-P and TGF-alpha was significantly higher than that in TGF-alpha-negative foci. In conclusion, TGF-alpha may be closely related with promotion from altered foci to neoplasms in rat hepatocarcinogenesis. Our data suggest that double positive foci for GST-P and TGF-alpha in the early stages of rat hepatocarcinogenesis may develop into tumors with promotion.

Effects of a new extracorporeal system using CTR on mortality and inflammatory responses to bacterial toxin-induced multiple organ dysfunction syndrome in rabbits.

BACKGROUND/AIMS: In the pathogenesis of multiple organ dysfunction syndrome (MODS) caused by bacterial infection, a complex series of systemically secreted bacterial toxins and cytokines are intensely associated. Our previous study demonstrated that a new adsorbent, CTR, was capable of removing cytokines and toxic shock syndrome toxin-1 (TSST-1) in vitro. Moreover, extracorporeal treatment with CTR reduced the high mortality rate and inhibited inflammatory responses in endotoxin-induced shock in rats. However, it is unclear whether CTR treatment will be an effective therapy for MODS. Here, we demonstrated the efficacy of a new extracorporeal system using CTR on MODS induced by bacterial toxins in rabbits. METHODS: Direct hemoperfusion (DHP) apheresis with or without CTR for 120 min was performed in rabbits that had been intravenously infused with endotoxin and TSST-1. The mean arterial pressure was recorded and the plasma toxin and cytokine concentrations were measured during the treatment period. Mortality was assessed up to 7 days after exposure to the toxins. In addition, tissues specimens were examined using microscopy. RESULTS: The mortality rates at 7 days after the injection of the toxins were 90 and 10% for the control and CTR groups, respectively. The plasma concentrations of TSST-1, tumor necrosis factor and interleukin-1 beta in the CTR group were significantly lower than those in the control group. Histopathological examination revealed that tissue damage, such as necrosis and depletion of lymphocytes in the spleen and mesenteric lymph node, was reduced in the CTR group, compared with that in the control group, at 24 h after toxin infusion. CONCLUSION: The new adsorbent CTR improved the mortality rate in a MODS rabbit model by adsorbing TSST-1 and cytokines. Further development of CTR may expand the scope of extracorporeal therapies for patients with MODS.

Suppression of azoxymethane-induced colonic premalignant lesion formation by coenzyme Q10 in rats.

Reactive oxygen species cause damage to proteins, lipids and DNA. Coenzyme Q10 (CoQ10) is a compound with mitochondrial bioenergetic functions. The reduced form of CoQ10 shows antioxidant activity. In the present study, effects of CoQ10 on development of azoxymethane (AOM)-induced aberrant crypt foci (ACF) and mucin-depleted foci (MDF) in F344 male rats were investigated. To induce ACF and MDF, 6-week old rats were given two weekly subcutaneous injections of AOM (15 mg/kg body weight) and also received a control diet or experimental diets containing CoQ10 (200 or 500 ppm) for 4 weeks, starting one day before the first dose of AOM. At 10 weeks of age, all animals were sacrificed and their colons were evaluated for numbers and sizes of ACF and MDF. Administration of 200 and 500 ppm CoQ10 resulted in reduction of ACF numbers, to 77% and 68% of the carcinogen control value, respectively. The percentages of ACF consisting of more than 4 crypts in these groups were also significantly lower than in the controls. Treatment with 500 ppm CoQ10 furthermore decreased the number of sialomucin-producing ACF and MDF per colon to 42% and 38% of the carcinogen control value without CoQ10, respectively. These results suggest that CoQ10 may be an effective chemopreventive agent against colon carcinogenesis.