Antidepressant-like Effect of l-perillaldehyde in Stress-induced Depression-like Model Mice through Regulation of the Olfactory Nervous System.

Perillae Herba (a leaf of Perilla frutescens) has been prescribed as one of the component herbs in certain Kampo (Japanese herbal) medicines that are used clinically for the improvement of depressive mood. l-Perillaldehyde (PAH) is a major component in the essential oil containing in Perillae Herba, but its antidepressant-like effect has not been reported. To clarify the antidepressant-like effect of PAH, the inhaled effect of PAH on stress-induced depression-like model mice prepared by subjection to a combination of forced swimming and chronic mild stresses was investigated. The degree of the depression-like state was measured by the animal's duration of immobility using a forced swimming test. Inhalation of PAH (0.0965 and 0.965 mg/mouse/day, 9 days) significantly shortened the duration of immobility of the depression-like model mice and did not affect locomotor activity. However, another odor substance, cinnamaldehyde containing in Cinnamomi Cortex, exhibited no reduction in the immobility. The reduction in the immobility induced by the inhalation of PAH was prevented on anosmia-induced mice prepared by intranasal irrigation with zinc sulfate. These results suggest that the inhalation of PAH shows antidepressant-like activity through the olfactory nervous function.

Ferulic acid induces neural progenitor cell proliferation in vitro and in vivo.

Ferulic acid (4-hydroxy-3-methoxycinnamic acid; FA) is a plant constituent and is contained in several medicinal plants for clinical use. In this paper, we investigated the effects of FA on the proliferation of neural stem/progenitor cells (NSC/NPCs) in vitro and in vivo. FA significantly increased the proliferation of NSC/NPCs cultured from the telencephalon of embryonic day-14 rats, and increased the number and size of secondary formed neurospheres. An in vitro differentiation assay showed that FA did not affect the percentage of either neuron-specific class III beta-tubulin (Tuj-1)-positive cells or glial fibrillary acidic protein (GFAP)-positive cells in the total cell population. Oral administration of FA increased the number of newly generated cells in the dentate gyrus (DG) of the hippocampus of corticosterone (CORT)-treated mice, indicating that FA enhances the proliferation of adult NSC/NPCs in vivo. We also found that oral administration of FA increased cAMP response element binding protein (CREB) phosphorylation and brain-derived neurotrophic factor (BDNF) mRNA level in the hippocampus of CORT-treated mice, and ameliorated the stress-induced depression-like behavior of mice. These novel pharmacological effects of FA may be useful for the treatment of mood disorders such as depression.

Phytoestrogens levels determination in the cord blood from Malaysia rural and urban populations.

This study is a result of an analysis of free and conjugated phytoestrogens daidzein, genistein, daidzin, genistin and coumesterol in human cord blood plasma using LCMS. Cord blood was collected from urban and rural populations of Malaysia (n=300) to establish a simple preliminary database on the levels of the analyzed compounds in the collected samples. The study also aimed to look at the levels of phytoestrogens in babies during birth as this may have a profound effect on the developmental process. The sample clean up was carried out by solid-phase extraction using C18 column and passed through DEAE sephadex gel before analysis by LCMS. The mean concentrations of total phytoestrogens were daidzein (1.4+/-2.9 ng/ml), genistein (3.7+/-2.8 ng/ml), daidzin (3.5+/-3.1 ng/ml), genistin (19.5+/-4.2 ng/ml) and coumesterol (3.3+/-3.3 ng/ml). Distribution of phytoestrogen was found to be higher in samples collected from rural areas compared to that of urban areas.

Antidepressant-like activity of a Kampo (Japanese herbal) medicine, Koso-san (Xiang-Su-San), and its mode of action via the hypothalamic-pituitary-adrenal axis.

Koso-san (Xiang-Su-San in Chinese), a Kampo (Japanese herbal) medicine, is used clinically in East Asia for the treatment of depression-like symptoms associated with the initial stage of the common cold, allergic urticaria due to food ingestion, irritable bowel syndrome, chronic fatigue syndrome, insomnia, and autonomic imbalance. However, the antidepressant-like activity of Koso-san has never been evaluated scientifically. In this study, ddY mice subjected to a combination of forced swimming and chronic mild stresses were termed depression-like model mice. The degree of the depression-like state was measured by the animal's duration of immobility using the forced swimming test (FST). Oral administration of Koso-san (1.0 g/kg/body wt./day, 9 days) significantly shortened the duration of immobility of the depression-like model mice in the FST; however, locomotor activity was not affected. Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis plays an important role in the pathophysiology of depression. Levels of corticotropin-releasing hormone mRNA expression in the hypothalamus and proopiomelanocortin mRNA expression in the pituitary were significantly increased, and glucocorticoid receptor protein expression in the hypothalamus paraventricular nucleus was downregulated in the depression-like model mice. However, Koso-san ameliorated these alterations to the normal conditions. The results of this study suggest that Koso-san shows the antidepressant-like effect through suppressing the hyperactivity of the HPA axis in depression-like model mice.

A novel CLCN1 mutation: P480T in a Japanese family with Thomsen's myotonia congenita.

At least 50 disease-causing mutations in the skeletal muscle voltage-gated chloride channel gene (CLCN1), almost all of which originate from Caucasian families, have been identified. We investigated a Japanese family with Thomsen's myotonia congenita that included 16 affected individuals (8 men and 8 women) through five generations. Polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) screening of 11 members showed an aberrant conformer in exon 13 of CLCN1 complementary DNA (cDNA) in 8 affected and 1 unaffected members. By sequence analysis, we identified a C-to-A transition at nucleotide position 1438, resulting in a substitution of proline for threonine at amino acid position 480 (P480T), the same position of the original mutation (P480L) in Thomsen's disease. The P480T mutation was novel and absent in 100 normal controls. Seven of the 8 affected individuals were heterozygous; another, from affected parents, was homozygous. Clinically, myotonia in the homozygous patient was more severe than that in heterozygous patients, probably due to the gene dosage effect. On a long-train nerve-stimulation test at a rate of 3 Hz, M-wave responses in the homozygous patient showed marked decrement followed by recovery. In contrast, the heterozygous patients showed just a slight decrement or no changes, and none of 2 patients with myotonic muscular dystrophy or 2 normal controls revealed any decrement. Thus, the long-train nerve-stimulation test at a low stimulus frequency may be a useful tool to assess the disease-severity/genotype relationship in myotonia congenita.

Effect of pravastatin on survival in patients with advanced hepatocellular carcinoma. A randomized controlled trial.

Chemotherapy is not effective for hepatocellular carcinoma (HCC). HMG-CoA redutase inhibitors have cytostatic activity for cancer cells, but their clinical usefulness is unknown. To investigate whether pravastatin, a potent HMG-CoA reductase inhibitor, prolongs survival in patients with advanced HCC, this randomized controlled trial was conducted between February 1990 and February 1998 at Osaka University Hospital. 91 consecutive patients <71 years old (mean age 62) with unresectable HCC were enroled in this study. 8 patients were withdrawn because of progressive liver dysfunction; 83 patients were randomized to standard treatment with or without pravastatin. All patients underwent transcatheter arterial embolization (TAE) followed by oral 5-FU 200 mg(-1)d for 2 months. Patients were then randomly assigned to control (n = 42) and pravastatin (n = 41) groups. Pravastatin was administered at a daily dose of 40 mg. The effect of pravastatin on tumour growth was assessed by ultrasonography. Primary endpoint was death due to progression of HCC. The duration of pravastatin administration was 16.5 +/- 9.8 months (mean +/- SD). No patients in either group were lost to follow-up. Median survival was 18 months in the pravastatin group versus 9 months in controls (P = 0.006). The Cox proportional hazards model showed that pravastatin was a significant factor contributing to survival. Pravastatin prolonged the survival of patients with advanced HCC, suggesting its value for adjuvant treatment.

Enhancing effects of Thai edible plants on 2-amino-3, 8-dimethylimidazo(4,5-f)quinoxaline-hepatocarcinogenesis in a rat medium-term bioassay.

Boesenbergia pandurata (Zingiberaceae), Languas galanga (Zingiberaceae) and Citrus hystrix (Rutaceae) are edible plants that are commonly used as flavors or condiments in various Thai food dishes. They are known to exert strong anti-promoting activity in a test of tumor promoter-induced Epstein-Barr virus (EBV) activation. In the present study their effects on hepatocarcinogenesis were investigated in a medium-term bioassay using F344 male rats. C. hystrix significantly enhanced 2-amino-3,8-dimethylimidazo(4, 5-f)quinoxaline-associated preneoplastic liver cell focus development while B. pandurata and L. galanga had borderline effects. The results suggest that C. hystrix as well as B. pandurata and L. galanga may contain agents augmenting the hepatocarcinogenicity of 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline.

Organ-dependent modifying effects of caffeine, and two naturally occurring antioxidants alpha-tocopherol and n-tritriacontane-16,18-dione, on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary and colonic carcinogenesis in female F344 rats.

Modifying effects of caffeine, alpha-tocopherol, and n-tritriacontane-16,18-dione (TTAD) on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary and colonic carcinogenesis were investigated in female F344 rats. Groups of 20 rats, 6 weeks old, were given 0.02% PhIP (in diet) alone, or together with 0.1% caffeine (in drinking water), 0.5% alpha-tocopherol (in diet) or 0.1% TTAD (in diet) for up to 54 weeks. Groups of 10 females receiving basal diet or one of the test chemicals without PhIP supplementation were also maintained. The final combined incidences (adenomas plus adenocarcinomas) and multiplicity (No./rat) of mammary adenomas and adenocarcinomas were significantly lowered in the PhIP plus caffeine group (10%, 0.10) as compared to the PhIP alone value (40%, (1.50). Incidences of mammary tumors in the PhIP plus alpha-tocopherol or TTAD groups tended to be decreased while their multiplicities were significantly lowered. With regard to colon tumor development, on the other hand, rats given PhIP plus caffeine exhibited an elevated incidence (75% versus 15% in the control), whereas alpha-tocopherol and TTAD had no effect. Surprisingly, metabolic activation of PhIP was inhibited by addition of caffeine in an in vitro assay. The results indicate that caffeine exerts a potent chemopreventive action against PhIP-induced mammary carcinogenesis, but acts as a co-carcinogen for PhIP-induced colonic carcinogenesis.

Modifying effects of phytic acid and gamma-oryzanol on the promotion stage of rat carcinogenesis.

The modifying effects of phytic acid and gamma-oryzanol on the promotion stage of carcinogenesis were investigated using several two stage carcinogenesis models in rats. In a multi-organ carcinogenesis model, male F344 rats were given combined treatment with 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN), N-ethyl-N-hydroxyethylnitrosamine (EHEN) and 3,2'-dimethyl-4-aminobiphenyl (DMAB) during the initial 3 weeks as initiators, and then treated with dietary 2% phytic acid (50% in water), 1% gamma-oryzanol or basal diet alone for 32 weeks. Although the appearance of hepatic tumors was suppressed, the incidence of urinary bladder papillomas was increased by phytic acid. In addition, the incidence and multiplicity of lung tumors were significantly increased by gamma-oryzanol. Esophagus, colon, pancreas, kidney and thyroid lesion development was not influenced by these compounds. In a gamma-oryzanol dose response experiment using DHPN in the drinking water as an initiator, enhancing effects on lung were observed at a dose of 1% but not at 0.5% or lower. When the modifying effects of phytic acid, and its sodium (Na-PA), potassium (K-PA) and magnesium (Mg-PA) salt were further examined in rats pretreated with the bladder carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), a clear increase in the incidences of bladder tumors was noted, with only Na-PA, phytic acid itself being without effect. Finally, examination of the modifying potential of phytic acid and gamma-oxyzanol on mammary carcinogenesis in female Sprague Dawley rats pretreated with a single intragastric dose of 7,12-dimethylbenz(a)anthracene (DMBA) revealed no significant differences in the final incidences and multiplicities of mammary tumors, but the average tumor diameter was significantly reduced and the average survival time was increased with phytic acid. gamma-Oryzanol tended to decrease the size of the tumor but without significant difference. These results indicate that phytic acid inhibits hepatic and mammary carcinogenesis, while its Na-salt is a promoter of bladder carcinogenesis. The effect of phytic acid itself on urinary bladder carcinogenesis is equivocal. gamma-Oryzanol is a promoter of lung carcinogenesis but its effect is weak and exerted only at a very high dose level.

Effect of intracameral anesthesia on the corneal endothelium.

PURPOSE: To evaluate the effects of intracameral anesthesia on the corneal endothelium. SETTING: Department of Ophthalmology, Yokohama City University School of Medicine, Yokohama, Japan. METHODS: This study comprised 24 eyes of 12 white rabbits. One eye of 3 rabbits each was injected with preservative-free lidocaine at concentrations of 0.02, 0.2, or 2% and the fellow eye injected with balanced salt solution (BSS) as a control. The anesthetic agent was injected into the anterior chamber using a bimanual technique. Immediately after enucleation, the cornea was examined by scanning electron microscopy. RESULTS: Scanning electron microscopy revealed no abnormal findings in the eyes injected with lidocaine 0.02 or 0.2% when compared with eyes in the control group. Scanning electron microscopy of the eyes injected with lidocaine 2% showed irregular hexagonal endothelial cells and a significant loss of microvilli. CONCLUSION: Intracameral anesthesia with high concentrations of lidocaine risks corneal endothelial damage but at the low concentration usually used in cataract surgery did not appear to have an adverse effect.

Antitussive effect of azelastine hydrochloride in patients with bronchial asthma.

To investigate the efficacy of azelastine hydrochloride (azelastine, CAS 79307-93-0, Azeptin) in suppressing cough, 22 bronchial asthma patients complaining mainly of cough were given the drug for four weeks. Peak flow rates (PEFR), pulmonary function tests, capsaicin cough threshold, and bronchial hyperresponsiveness were compared pre- and post-administration. After four-week's administration of azelastine (2 mg twice daily), cough decreased as demonstrated in a significant progressive improvement of cough points. The morning PEFR (1/min) was improved significantly at one week and two weeks post-administration. Changes were from 434 +/- 26.4 pre-administration to 461 +/- 25.8 at Week 1 (p < 0.05), 462 +/- 26.7 at Week 2 (p < 0.05), 452 +/- 22.5 at Week 3, and 462 +/- 20.8 at Week 4. The evening PEFR (1/min) showed 439 +/- 22.2 pre-administration, 454 +/- 21.4 at Week 1, 464 +/- 22.4 at Week 2, 457 +/- 19.3 at Week 3 and 467 +/- 17.8 at Week 4, improvement being significant at Week 1 (p < 0.05). Regarding pulmonary function tests no significant changes were observed. FVC (liter), FEV1 (liter), and FEV1/FVC (%) were 3.45 +/- 0.86, 2.68 +/- 0.52, and 83.6 +/- 5.93 pre-administration; and 3.48 +/- 0.21, 2.72 +/- 0.65, and 84.1 +/- 6.21 post-administration, respectively. The capsaicin cough threshold [Ccap (mumol/l)] showed significant improvement, changing from 5.95 (0.016-50.0) pre-administration to 19.7 (0.08-50.0) post-administration (p < 0.05). Conversely, an index of bronchial hyperresponsiveness, Dmin (mg/dl;U), showed no significant changes (14.9 +/- 5.2 vs. 19.7 +/- 5.3). These results suggest that azelastine inhibits cough in patients with bronchial asthma by increasing the level of the cough threshold without changing bronchial hyperresponsiveness.

Modulation of dihydroxy-di-n-propylnitrosamine-induced liver lesion development in Opisthorchis-infected Syrian hamsters by praziquantel treatment in association with butylated hydroxyanisole or dehydroepiandrosterone administration.

The effects of praziquantel coupled with dehydroepiandrosterone (DHEA) or butylated hydroxyanisole (BHA) administration 16 weeks subsequent to dihydroxy-di-n-propylnitrosamine (DHPN) treatment and infection with Opisthorchis viverrini (OV) on lesion development in the liver of Syrian hamsters were investigated. Animals were given 80 OV metacercariae and then two i.p. injections of DHPN (500 mg/kg body weight) 4 and 5 weeks thereafter. At week 16, groups received praziquantel (250 mg/kg, i.g.) and were placed on normal diet or diet supplemented with BHA (1%) or DHEA (0.6%) until they were killed at week 24. Histopathological assessment revealed that, whereas antihelminthic treatment alone resulted in a clear reduction in hepatocellular lesion development, effects on cholangiocellular lesions were equivocal. BHA and DHEA, in contrast, were both associated with a significant reduction in frequency of cholangiofibrosis and cholangiocellular carcinoma. The former chemical, however, increased the numbers of liver nodules while the hormone brought about a decrease as well as a shift in the phenotype of the lesions. The results thus indicate that although cholangiocellular lesion development may, unlike generation of hepatocellular nodules, be to a certain extent independent of the continued presence of parasite, it can be influenced by exogenous treatments.

[Successful treatment of intraoperative myocardial ischemia with nicorandil].

We report five patients who developed intraoperative myocardial ischemia but were treated successfully with nicorandil. Case 1; An 84 year-old male underwent emergent laparotomy and ileolysis under inhalational plus thoracic epidural anesthesia. During his emergence from anesthesia, arterial pressure and heart rate increased abruptly due to excitement, leading to ST-T depression on V5 lead. Bradycardia and hypotension developed subsequently. Immediately after i.v. injections of nicorandil 4 mg and atropine 0.3 mg, ST-T change and hemodynamics improved dramatically. Case 2; A 67 year-old male underwent esophagectomy under inhalational plus thoracic epidural anesthesia. Following the completion of surgery, elevation of ST-T developed suddenly on lead II, though hemodynamics were not compromised. ST-T elevation disappeared immediately after nicorandil 6 mg and continuous infusion of nitroglycerin (TNG) was initiated. Case 3; A 71 year-old female underwent aortic valve replacement under high-dose fentanyl anesthesia. Shortly after starting cardiopulmonary bypass (CPB), ST-T segment on leads II and V5 was elevated suddenly. This was accompanied by severe pulmonary hypertension suggestive of severe left ventricular failure. Shortly after nicorandil 4 mg via a pulmonary artery (PA) catheter, ST-T segment returned to the baseline and pulmonary arterial pressure was normalized. Case 4; A 61 year-old male underwent coronary revascularization under high-dose fentanyl anesthesia. During weaning from CPB, elevation of ST-T segment occurred on leads II and V5. ST change improved, responding to nicorandil 6 mg en bolus via a PA catheter. Case 5; A 67 year-old male underwent coronary revascularization under high-dose fentanyl anesthesia. He was unable to be weaned from CPB for several hours because of frequent and repeated attacks of ventricular tachycardia and ventricular fibrillation. The arrhythmia did not respond to various kinds of treatments including intra-aortic balloon pumping and continuous infusions of inotropes, anti-arrhythmic drugs and anti-anginal drugs. In spite of repeated intracoronary injections of TNG, graft flow to the left anterior descending branch remained low at 40 ml.min-1. After an intracoronary injection of nicorandil 1 mg, however, blood flow increased to 100 ml.min-1, resulting in a marked reduction in frequency of ventricular arrhythmia. The patient came off bypass successfully. In each case, intraoperative myocardial ischemia was treated successfully with nicorandil. Neither hypotension nor arrhythmia resulted from its bolus injection. Nicorandil might be a useful therapeutic tool for myocardial ischemia during anesthesia.

Effects of muscle relaxation therapy using specially designed plates in patients with pulmonary emphysema.

It has been suggested that respiratory muscle dysfunction plays a major role in the development of acute ventilatory failure in patients with chronic obstructive pulmonary disease (COPD). In this study, we devised a respiratory muscle relaxation maneuver using wedge-shaped wooden plates, with which pressure was exerted on the intercostal and accessory respiratory muscles by hand, or by tapping with a wooden hammer, for 15-20 minutes twice a day. The muscle relaxation maneuver was performed in 5 moderate to severe pulmonary emphysema patients for 4 weeks and in 7 emphysema patients for 6 weeks, and the effects on pulmonary function were examined. After the therapy, inspiratory capacity (IC) and vital capacity (VC) in both the 4 weeks-and 6 weeks-treated groups, and the forced expiratory volume in 1 second (FEV 1.0) in the 6 weeks-treated group were significantly increased, and CO2 retention had also improved. Daily peak expiratory flow (PEF) showed significant increases from 2 weeks after the initiation of the therapy. These results suggest that the respiratory muscle relaxation maneuver is effective for improving the pulmonary function of pulmonary emphysema patients.

Measurement of beta 2-microglobulin in bovine serum and urine by radioimmunoassay.

A radioimmunoassay (RIA) system for quantification of bovine beta 2-microglobulin (beta 2-M) in serum and urine was developed. The protein isolated from bovine colostrum showed a single band in SDS-PAGE, and its molecular weight was approximately 11,600. Amino acid sequences for the first 24 residues and the amino acid composition of the protein were in agreement with those in the bovine beta 2-M of previous research works. In an Ouchterlony test, a single precipitation line was formed between the protein and the antiserum made by the protein. From these results, it was confirmed that the protein isolated from the colostrum was pure bovine beta 2-M. For creation of an RIA calibration curve for urine, a urine void of beta 2-M, as much as possible (beta 2-M-free urine), and a PBS were used as diluents. Intraassay (n = 10) and interassay (n = 3) variances were 1.7-4.6% and 7.1-11.5% in the PBS dilute method, and were 1.4-5.1% and 12.3-13.5% in the beta 2-M-free urine dilute method, respectively. Mean recoveries were 160 +/- 19% (mean +/- SD) and 98.4 +/- 7.9% in PBS and beta-M-free urine, respectively. It was found that the method using the beta 2-M-free urine as a diluent was more accurate than using PBS. The beta 2-M concentrations in serum and urine of healthy Holstein cows measured by this RIA system showed a logarithmic normal distribution for urine and a normal distribution for serum. The mean beta 2-M concentrations were 0.0305(+0.04443)(-0.0210) mg/l (Geometric mean +/- S.D., n = 43) in urine and 2.87 +/- 0.45 mg/l (Arithmetic mean +/- S.D., n = 26) in sera. Further, we could not observe the particular tendency of daily variation in urinary beta 2-M concentrations of healthy cows (Holstein, n = 3 x 2 days).

Cough threshold for capsaicin increases by azelastine in patients with cough-variant asthma.

To assess the effects of azelastine in patients with cough-variant asthma, we measured the cough threshold for capsaicin (the concentration required to elicit more than five coughs) in 16 patients with cough-variant asthma before and after 4 weeks of treatment with azelastine (2 mg; b.i.d.) or placebo. After treatment, coughing decreased in all patients and the cough threshold for capsaicin increased significantly, from 0.67 +/- 0.30 microM to 4.76 +/- 1.55 microM (P < 0.01) in the azelastine group. However, the cough threshold for capsaicin did not increase significantly, from 0.86 +/- 0.33 microM to 1.11 +/- 0.35 microM (P > 0.10) in the placebo group. These results suggest that azelastine inhibits coughing in patients with cough-variant asthma.

Inhibitory effects of 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), green tea catechins and other antioxidants on 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1)-induced rat hepatocarcinogenesis and dose-dependent inhibition by HTHQ of lesion induction by Glu-P-1 or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx).

The effects of 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), green tea catechins (GTC), alpha-tocopherol, beta-carotene, chlorophyllin, phenylethylisothiocyanate (PEITC), 3-O-ethylascorbic acid (EAsA), 3-O-dodecylcarbomethyl ascorbic acid (DAsA), n-tritriacontane-16,18-dione (TTAD) and d-limonene on 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1)- or dimethylnitrosamine (DMN)-induced hepatocarcinogenesis, and the dose dependence of HTHQ inhibition of Glu-P-1- or 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx)-influence on lesion development were examined in a rat medium-term liver bioassay system featuring diethylnitrosamine initiation and partial hepatectomy. At the end of week 8, the number and total area of glutathione S-transferase placental form (GST-P) positive liver foci in rats treated with 0.03% Glu-P-1 alone were increased significantly (46.8 +/- 11.0 and 12.0 +/- 5.6 respectively) as compared to the control values (3.8 +/- 1.6 and 0.4 +/- 0.2). Combined treatment with 1% HTHQ remarkably reduced both of these parameters (8.1 +/- 2.1 and 0.6 +/- 0.2). GTC (1%), PEITC (0.1%), beta-carotene (0.1%) and DAsA (1%) also demonstrated inhibition but less than HTHQ. On the other hand, these antioxidants did not influence development of foci initiated by 0.002% DMN. In the dose-response study, up to 0.125% HTHQ significantly reduced the effects of 0.02% Glu-P-1 or 0.03% MeIQx on the number and area of foci. These results indicate that several antioxidants exert chemopreventive effects against heterocyclic amine (HCA)-induced hepatocarcinogenesis, and particularly HTHQ which thus deserves further attention as a chemopreventor in the contest of the environmentally important HCA group of carcinogens.

Enhanced levels of lipoperoxides in low density lipoprotein incubated with murine fibroblast expressing high levels of human 15-lipoxygenase.

There is strong experimental evidence that oxidized low density lipoprotein (Ox-LDL) plays an important role in atherosclerosis. However, the mechanisms by which Ox-LDL is formed in vivo are unknown. To test whether 15-lipoxygenase (15-LO) could play a role in oxidation of LDL by cells, we expressed 15-LO activity in murine fibroblasts, which do not normally have 15-LO activity, and tested their ability to modify LDL. Using a retroviral vector, we prepared fibroblasts that expressed 2- to 20-fold more 15-LO activity than control fibroblasts infected with a vector containing beta-galactosidase (lacZ). Compared with LDL incubated with lacZ cells, LDL incubated with 15-LO-containing cells were enriched with lipid hydroperoxides. When these LDL samples were subsequently subjected to oxidative stress, they were more susceptible to further oxidative modification, as judged by increased conjugated diene formation and by increased ability to compete with 125I-Ox-LDL for uptake by macrophages. These findings establish that cellular 15-LO can contribute to oxidative modification of LDL, but the quantitative significance of these findings to the in vivo oxidation of LDL remains to be established.

Chemoprevention of 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP)-induced mammary gland carcinogenesis by antioxidants in F344 female rats.

Chemopreventive effects of the antioxidants 1-O-hexyl-2,3,5- trimethylhydroquinone (HTHQ), 3-O-ethylascorbic acid (EAsA), 3-O-dodecylcarbomethylascorbic acid (DAsA), green tea catechins (GTC) and ellagic acid on 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis were examined in female F344 rats. Groups of 20-21 6-week-old rats were maintained on a powdered diet containing 0.02% PhIP alone, PhIP together with 0.5% HTHQ, 1% EAsA, 1% DAsA, 1% GTC or 0.1% ellagic acid, these antioxidants alone or basal diet alone without supplement for 52 weeks. The survival rates of PhIP plus antioxidant groups at the end of the experiment were higher than that of the PhIP alone group. Sequential observation of palpable mammary tumors demonstrated only one tumor by week 52 in the PhIP plus HTHQ group, whereas 40% of the rats receiving PhIP alone had tumors by this time point. The final incidence of mammary adenocarcinomas was significantly decreased in the PhIP plus HTHQ group (4.8%, P < 0.01) as compared to the PhIP alone value (40%). Although statistically not significant, incidences of adenocarcinomas in the other antioxidant-treated groups (23.8-28.6%) were also lower than in the PhIP alone group. Furthermore, the incidence of large intestinal tumors in the PhIP plus HTHQ group (0%) showed a tendency to decrease relative to the PhIP alone group (16.7%). These results indicate that antioxidants, particularly HTHQ, exert a potent chemopreventive action against PhIP-induced carcinogenesis.

Analysis of the potential carcinogenicity of coffee and its related compounds in a medium-term liver bioassay of rats.

The potential carcinogenicity of coffee and related compounds was examined using a medium-term liver bioassay based on the induction of glutathione S-transferase placental form (GST-P)-positive foci in F344 rats. A total of 230 males were initially injected with diethylnitrosamine (200 mg/kg body weight, ip) or saline as controls and 2 wk later were fed on diet or drinking water supplemented as follows for 6 wk: 5% regular instant coffee; 5% decaffeinated instant coffee; freshly brewed coffee, 8 g in 140 ml water; 0.1% caffeine, 0.2% methylglyoxal, 0.2% glyoxal; or 0.3% theophylline in the drinking water (w/v); and 0.4% theobromine in the diet (w/w). All rats were subjected to two-thirds partial hepatectomy at wk 3 and killed at wk 8. The resultant values for GST-P-positive hepatic focus induction were slightly increased with methylglyoxal and decreased with glyoxal and theobromine compared with the corresponding controls. Although the increase in number of foci for methylglyoxal was statistically significant at P < 0.05, the value was within the historical control levels. Regular and decaffeinated instant coffee as well as fresh-brewed coffee, caffeine and theophylline exerted no effects on focus development. Thus, the coffee-related compounds examined demonstrated no obvious enhancing potential, and it is therefore concluded that coffee and its main constituents are not carcinogenic for the rat liver.