Eicosapentaenoic acid inhibits voltage-gated sodium channels and invasiveness in prostate cancer cells.

BACKGROUND AND PURPOSE: The voltage-gated Na(+) channels (Na(v)) and their corresponding current (I(Na)) are involved in several cellular processes, crucial to metastasis of cancer cells. We investigated the effects of eicosapentaenoic (EPA), an omega-3 polyunsaturated fatty acid, on I(Na) and metastatic functions (cell proliferation, endocytosis and invasion) in human and rat prostate cancer cell lines (PC-3 and Mat-LyLu cells). EXPERIMENTAL APPROACH: The whole-cell voltage clamp technique and conventional/quantitative real-time reverse transcriptase polymerase chain reaction analysis were used. The presence of Na(v) proteins was shown by immunohistochemical methods. Alterations in the fatty acid composition of phospholipids after treatment with EPA and metastatic functions were also examined. KEY RESULTS: A transient inward Na(+) current (I(Na)), highly sensitive to tetrodotoxin, and Na(V) proteins were found in these cells. Expression of Na(V)1.6 and Na(V)1.7 transcripts (SCN8A and SCN9A) was predominant in PC-3 cells, while Na(V)1.7 transcript (SCN9A) was the major component in Mat-LyLu cells. Tetrodotoxin or synthetic small interfering RNA targeted for SCN8A and SCN9A inhibited metastatic functions (endocytosis and invasion), but failed to inhibit proliferation in PC-3 cells. Exposure to EPA produced a rapid and concentration-dependent suppression of I(Na). In cells chronically treated (up to 72h) with EPA, the EPA content of cell lipids increased time-dependently, while arachidonic acid content decreased. Treatment of PC-3 cells with EPA decreased levels of mRNA for SCN9A and SCN8A, cell proliferation, invasion and endocytosis. CONCLUSION AND IMPLICATIONS: Treatment with EPA inhibited I(Na) directly and also indirectly, by down-regulation of Na(v) mRNA expression in prostate cancer cells, thus inhibiting their metastatic potential.

Spontaneous intraocular pressure reduction in normal-tension glaucoma and associated clinical factors.

PURPOSE: To investigate the intraocular pressure (IOP) reduction in certain normal-tension glaucoma (NTG) patients and clinical factors associated with this reduction. METHODS: Fifty-four NTG patients who met the following enrollment criteria were selected: IOP <21 mm Hg during a 24-hour pressure curve and throughout the subsequent 12 months; examined every 1 to 4 months for at least 3 years with no ocular hypotensive therapy. For each patient, the eye with the higher mean IOP during the 24-hour pressure curve was selected for this study. RESULTS: Six patients had an IOP reduction which was defined as a significant decrease (P <.05) of IOP over time, determined by the Spearman rank correlation coefficient method. These 6 eyes were rated positive for subsequent IOP reduction. The IOP reduction was correlated to clinical factors by means of a logistic multiple regression analysis (LOGIST procedure using PC-SAS), which demonstrated that the larger difference between the maximum IOP and the minimum IOP during the initial 24-hour pressure curve and the absence of disc hemorrhage showed significant correlation with IOP reduction (P =.026 and P =.013, respectively). The odds ratios were 2.05 per 1 mm Hg increase of difference between the maximum IOP and the minimum IOP during the initial 24-hour pressure curve and 1.13 for the absence of disc hemorrhage. CONCLUSIONS: The current study demonstrated that a significant reduction of IOP over time is not uncommon in NTG patients. One ninth of the NTG patients in this study showed a significant IOP reduction during a 3-year follow-up period.

Association between watershed zone and visual field defect in normal tension glaucoma.

PURPOSE: To evaluate the association between the watershed zone and glaucomatous optic nerve head (ONH) damage. METHODS: We performed indocyanine green fluorescence angiography with a scanning laser ophthalmoscope in 54 eyes of 27 patients with normal tension glaucoma (NTG). RESULTS: We identified 7 eyes of 8 patients (14.8%) with a watershed zone not including the ONH, 32 eyes of 20 patients (59.3%) with the watershed zone partially including the ONH, and 10 eyes of 14 NTG patients (25.9%) with the watershed zone including the ONH. Of the 27 NTG patients, 10 patients (37.0%) had different types in each eye. CONCLUSIONS: In these patients, the mean deviation of visual field indices was greater in the eye with the watershed zone, which included a larger part of the ONH than in the contralateral eye. Conversely, the eye with the greater mean deviation had a watershed zone that included a larger part of the ONH. The location of the watershed zone appeared to influence the progression of the visual field defect.

Mutagenic activity of 2-phenylbenzotriazole derivatives related to a mutagen, PBTA-1, in river water.

A mutagen, 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]5-ami no-7-bromo-4-chloro-2H-benzotriiazole (PBTA-1), isolated from water of the Nishitakase River in Kyoto exhibits potent mutagenic activity in Salmonella typhimurium TA98 with S9 mix and has characteristic moieties, including bromo, chloro, acetylamino, bis(2-methoxyethyl)amino and primary amino groups on a 2-phenylbenzotriazole skeleton. The mutagenicities of PBTA-1, its congeners and five related 2-phenylbenzotriazoles were examined in S. typhimurium TA98 with S9 mix in order to elucidate the structure-activity relationships. The data obtained suggest that a primary amino group plays an essential role in the mutagenic activity as do aromatic amines including heterocyclic amines in cooked foods. The effect of planarity of the 2-phenylbenzotriazole ring was significant, and in addition, halogen groups of PBTA-1 influenced the enhancement of the mutagenic activity.

Eos: a novel member of the Ikaros gene family expressed predominantly in the developing nervous system.

We identified a novel member of the Ikaros gene family, which has critical roles in the development of lymphoid lineages. This gene, which we named Eos, was expressed predominantly in the developing central and peripheral nervous system. Eos protein could interact with itself and Ikaros protein through its C-terminal portion in the yeast two hybrid assay. These findings suggested that Eos may have important roles in neural development similarly to the Ikaros family in the development of hemolymphoid tissue.

Identification of a 2-phenylbenzotriazole (PBTA)-type mutagen, PBTA-2, in water from the Nishitakase River in Kyoto.

We previously isolated five mutagens, compounds I-V, in blue rayon-adsorbed materials from the Nishitakase River in Kyoto. The chemical structure of compound I, a major mutagen that accounted for 21% of the total mutagenicity, was determined to be 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-am ino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1). Compound II was also a major mutagen and accounted for 17% of the total mutagenicity. In this study, a large quantity (1.2 mg) of compound II was isolated from adsorbate to 27 kg of blue cotton, and its UV, mass, and 1H NMR spectra were analyzed. On the basis of the spectral data, compound II was deduced to be the PBTA-1 analogue 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5- amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2). As with PBTA-1, PBTA-2 was synthesized from an azo dye by reduction and chlorination. Since all of the spectra of PBTA-2 coincided with those of compound II obtained from river water, compound II was concluded to be PBTA-2. PBTA-2 is a newly identified potent mutagen, which induces 93 000 and 3 200 000 revertants of Salmonella typhimurium TA98 and YG1024 per microgram, respectively, in the presence of S9 mix. Like PBTA-1, PBTA-2 may also be produced from an azo dye during industrial processes in dyeing factories and treatment at sewage plants.

Transscleral cyclophotocoagulation with the diode laser for neovascular glaucoma.

BACKGROUND AND OBJECTIVE: To compare the outcome of transscleral cyclophotocoagulation (TSCPC) using a diode laser with that of TSCPC using an Nd:YAG laser in neovascular glaucoma. PATIENTS AND METHODS: The surgical outcome of diode laser TSCPC was retrospectively compared with that of free-running mode Nd:YAG laser (FR-YAG) TSCPC and continuous-wave mode Nd:YAG laser (CW-YAG) TSCPC. Twenty-one eyes of 21 patients in the diode laser group, 9 eyes of 9 patients in the FR-YAG group, and 9 eyes of 9 patients in the CW-YAG group were treated. RESULTS: The Kaplan-Meier life-table analysis revealed that the probability (mean +/- standard error) of successful intraocular pressure control with diode laser TSCPC at 3 years postoperatively was 47.2 +/- 12.6% per operation and 55.9 +/- 16.3% per eye. Compared with the CW-YAG TSCPC, the diode laser TSCPC had a significantly higher probability of success throughout the follow-up period. Diode laser TSCPC was associated with improvements or preservation of visual acuity in 16 of 21 eyes (76%), and was the best of the three laser sources. Postoperative complications were minor following diode laser TSCPC. CONCLUSION: Diode laser TSCPC appears to be as effective as FR-YAG TSCPC and better than CW-YAG TSCPC for treating neovascular glaucoma.

[Association between watershed zone and visual field defect in normal tension glaucoma].

In order to evaluate the association between the watershed zone and glaucomatous optic damage, we performed indocyanine green fluorescence angiography with a scanning laser ophthalmoscope in 54 eyes of 27 patients with normal tension glaucoma. The visual field indices were measured with a Humphrey Field Analyzer. We identified 8 eyes (14.8%) of 7 patients with a watershed zone not including the optic nerve head (type I), 32 eyes (59.3%) of 20 patients with the zone partially including the optic nerve head (type II), and 14 eyes (26.0%) of 10 patients with the zone including the optic nerve head (type III). Of the total of 27 patients, 10 patients (37.0%) had different types in each eye. In these patients, the mean deviation (MD) of visual field indices was worse in the eye with the watershed zone which included a larger part of the optic disc than in the contralateral eye (p < 0.05). Conversely, the eye with worse MD than the contralateral eye had a watershed zone which included a larger part of the optic disc than the other eye (p < 0.05). The location of watershed zone appeared to influence the progression of the visual field defect.

Inhibitory effects of antioxidants on formation of heterocyclic amines.

It is important to search for effective antioxidants to suppress formation of mutagenic and carcinogenic heterocyclic amines (HCAs), like 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), because these HCAs are considered to be probable human carcinogens. The effects of various food-derived antioxidants on MeIQx formation were examined by their addition (0.2 mmol each) to mixtures of creatine (0.4 mmol), glycine (0.4 mmol) and glucose (0.2 mmol), and heating at 128 degreesC for 2 h. Glycine was replaced by l-phenylalanine in the case of PhIP formation. Among the 14 kinds of antioxidants tested, green tea catechins and the major component [(-)-epigallocatechin gallate], two flavonoids (luteolin and quercetin) and caffeic acid were found to clearly suppress the formation of both MeIQx and PhIP, being 3.2-75% of the level of the controls. These phenolic antioxidants also reduced the total mutagenicity of the heated mixtures. The results suggest that foodstuffs containing catechins, flavonoids and caffeic acid may suppress the formation of HCAs in cooked foods.

Chemical synthesis of a novel aromatic amine mutagen isolated from water of the Nishitakase River in Kyoto and a possible route of its formation.

Among five mutagenic compounds isolated from water samples, taken at sites below the sewage plants of the Nishitakase River in Kyoto, Japan, the structure of compound I has been determined to be 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-am ino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1). Since this novel aromatic amine mutagen has characteristic substituents in its molecule, it is postulated that the azo dye, 2-[(2-bromo-4, 6-dinitrophenyl)azo]-4-methoxy-5-[bis(2-methoxyethyl)amino]acetoanili de (AZO DYE-1), used as an industrial material, is converted to the corresponding 2-phenylbenzotriazole derivative with a reducing reagent and subsequently to PBTA-1 by chlorination. In fact, AZO DYE-1 changed to the dechlorinated derivative of PBTA-1 (deClPBTA-1) on treatment with sodium hydrosulfite, and this reacted with sodium hypochlorite to produce PBTA-1. Moreover, the presence of deClPBTA-1 was confirmed in a river water sample, along with PBTA-1. PBTA-1 showed potent mutagenic activities in Salmonella typhimurium TA98 and YG1024, inducing 88 000 and 3 000 000 revertants, respectively, per microg, with S9 mix. deClPBTA-1 was also mutagenic, but less potent. From these observations, it is suggested that PBTA-1 is produced from AZO DYE-1 through deClPBTA-1, during industrial processes at dyeing factories and the treatment of wastewater at sewage plants.

Incidence of elevation of intraocular pressure over time and associated factors in normal-tension glaucoma.

PURPOSE: The authors determine whether intraocular pressure (IOP) increases to levels that challenge the diagnosis of normal-tension glaucoma (NTG) and determine clinical factors associated with the IOP elevation. METHODS: Forty patients with NTG who met the following enrollment criteria were selected: IOPs less than 21 mm Hg during initial 24-hour pressure curve and throughout the subsequent 12 months; examined every 1 to 4 months for at least 4 years; and no ocular hypotensive treatment. The eye with the higher mean IOP during the initial 24-hour pressure curve was selected from each patient. The observation period ranged from 4.0 years to 7.8 years (mean, 5.2 years). Significant IOP elevation was defined as a significant increase (p < 0.05) of IOP over time, determined by Spearman rank correlation coefficient method. The IOP elevation was correlated to clinical factors by means of a logistic multiple regression analysis (LOGIST procedure using PC-SAS, SAS Institute, Inc. Cary, NC, U.S.A.). RESULTS: Eleven eyes were rated positive for IOP elevation. The regression model demonstrated that maximum IOP during the initial 24-hour pressure curve and the development of disc hemorrhage (DH) were significantly correlated with IOP elevation (p = 0.006 and p = 0.049, respectively). The odds ratios calculated were 1.98 per 1 mm Hg rise of maximum IOP during the initial 24-hour pressure curve and 6.54 for positive DH. CONCLUSIONS: Intraocular pressure might increase in NTG eyes following the initial diagnosis. A higher maximum IOP during initial 24-hour pressure curve and the development of DH during follow-up was significantly associated with subsequent IOP elevation in NTG patients.

Reduction in formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced aberrant crypt foci in the rat colon by docosahexaenoic acid (DHA).

Docosahexaenoic acid (DHA), a major component of fish oil, suppresses the formation and growth of aberrant crypt foci induced by 1,2-dimethylhydrazine and azoxymethane. In the present study we examined the effects of intragastric gavage administration of DHA on the yield of rat colonic aberrant crypt foci due to treatment with a heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which induces colon cancer in male F344 rats and is considered to be a possible human colon carcinogen. Male F344 rats were given a standard diet (AIN-76A) and received 10 doses of PhIP (75 mg/kg body wt, by intragastric intubation, on days 1-5 and 8-12) with or without intragastric application of 1 ml DHA 4 h prior to each carcinogen treatment, followed by further DHA dosing. The numbers of PhIP-induced aberrant crypt foci per colon after 4 and 12 weeks DHA administration were significantly reduced to 47 and 38% respectively of the values obtained when PhIP alone was used. The mean number of aberrant crypts per focus was also decreased by DHA treatment. At week 4 the PhIP-DNA adduct levels in the colon of rats from the PhIP+DHA group were approximately two thirds of the PhIP group value. The results thus suggest that DHA exerts a preventive effect on PhIP-induced colon carcinogenesis.

Human exposure to mutagenic/carcinogenic heterocyclic amines and comutagenic beta-carbolines.

Various kinds of mutagenic and carcinogenic heterocyclic amines (HCAs) are produced by heating protein-rich foods, such as meat and fish. To evaluate the risk of these HCAs in terms of human cancer development, exposure levels must be measured. We therefore analyzed their amounts in various kinds of cooked foods and in urine samples of healthy volunteers living in Tokyo. Based on the obtained quantitative data, daily exposure levels to 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were calculated to be 0.3-3.9 and 0.005-0.3 microgram per person, respectively. Moreover, human DNA samples were analyzed with the 32P-postlabeling method, and colon, rectum and kidney tissues were found to contain an adduct spot corresponding to the standard 5'-pdG-C8-MeIQx by TLC and HPLC, at levels of 14, 18 and 1.8 per 10(10) nucleotides, respectively. The beta-carboline compound, norharman, is produced by heating L-tryptophan, and is known to be present in cooked foods and in cigarette smoke at higher levels than mutagenic and carcinogenic HCAs. While norharman is not itself mutagenic to Salmonella, it does become mutagenic to S. typhimurium TA98 with S9 mix in the presence of non-mutagenic aromatic amines like aniline and o-toluidine. When we examined whether DNA adducts are formed in the DNA of S. typhimurium TA98 by treatment with norharman and aromatic amines using 32P-postlabeling analysis, DNA adduct formation by norharman with aromatic amines was found to be related to the appearance of mutagenicity by norharman with aromatic amines.

Mitomycin C dissolved in a reversible thermosetting gel: target tissue concentrations in the rabbit eye.

AIMS: To determine whether a new, reversible thermosetting gel enhances mitomycin C transfer to target ocular tissues in the rabbit eye. METHODS: A 0.1 ml solution of mitomycin C containing 0.22 microgram, 2.9 micrograms, or 28 micrograms of the agent dissolved in a reversible thermosetting gel consisting of methylcellulose, citric acid, and polyethylene glycol was injected subconjunctivally in 30 New Zealand albino rabbits. Scleral and conjunctival tissues were excised at 0.5, 1, 2, 4, or 24 hours after the injection and mitomycin C concentrations in these tissues were determined by high performance liquid chromatography. The concentration over time was approximated to a single exponential curve, and initial mitomycin C concentrations, time constants, and half life values were determined. Finally, the areas under the curves (AUCs) between 0.5 and 24 hours were calculated. RESULTS: The mitomycin C concentrations in the target tissues were dose dependent and decreased rapidly over 24 hours. Both the initial mitomycin C concentrations as well as AUCs in these eyes treated with mitomycin C, dissolved in a reversible thermosetting gel, were higher than those in eyes treated similarly in a previous study in which the gel was not used. CONCLUSION: Applied subconjunctivally in the rabbit eye, mitomycin C dissolved in the reversible thermosetting gel enhanced transfer of the agent to the sclera and the conjunctiva.

Isolation and chemical-structural determination of a novel aromatic amine mutagen in water from the Nishitakase River in Kyoto.

Water samples from the Nishitakase River in Kyoto, Japan, especially taken at sites below sewage plants, show significantly high mutagenicity in the Ames test. In the present study, mutagens in the river water were adsorbed to 24 g of blue rayon, extracted, and separated by HPLC on ODS columns. Five mutagenic compounds (I-V) were isolated, and they accounted for 21%, 17%, 11%, 12%, and 6%, respectively, of the total mutagenicity of the blue rayon-adsorbed materials. With compound I obtained from adsorbate to 24 g of blue rayon as a marker, a large quantity (1.1 mg) of mutagenic compound I was isolated by Sephadex LH-20 column chromatography and HPLC on ODS columns from material adsorbed to 27 kg of blue cotton. X-ray crystal analysis was carried out with the debrominated derivative of compound I. Based on this X-ray crystallography data and the UV, mass, and 1H-NMR spectra of both the derivative and compound I, the structure of compound I was determined to be 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino - 7-bromo-4-chloro-2H-benzotriazole (PBTA-1). PBTA-1 is a newly identified potent mutagen, inducing 1,200,000 revertants of Salmonella typhimurium YG1024 per microgram in the presence of S9 mix.

Extrahepatic portosystemic venous shunt without portal hypertension.

A 72-year-old woman was admitted for recurrent episodes of encephalopathy. Laboratory data showed mild liver dysfunction and hyperammonemia, while she had neither anemia nor splenomegaly. The dilated inferior mesenteric vein (IMV) was opacified retrogradely from the superior mesenteric vein by superior mesenteric arteriography, and IMV was found to connect with the inferior vena cava (IVC) through a torturous shunt. No obstruction of the extrahepatic portal vein or hepatic vein was observed by arteriography. Histological evaluation of the liver biopsy indicated remarkable fatty change without cirrhosis. Finally, we diagnosed this case as extrahepatic portosystemic venous shunt without portal hypertension.

Detection of mutagenicity of diphenyl ether herbicides in Salmonella typhimurium YG1026 and YG1021.

Three kinds of diphenyl ether herbicides, 4-nitrophenyl 2,4,6-trichlorophenyl ether (CNP, chlornitrofen), 2,4-dichlorophenyl 3-methoxy-4-nitrophenyl ether (chlomethoxynil) and 2,4-dichlorophenyl 3-methoxycarbonyl-4-nitrophenyl ether (bifenox), were tested for mutagenicity in Salmonella typhimurium YG1026 and YG1021, which have high nitroreductase activity, and also in S. typhimurium TA100 and TA98. CNP and chlomethoxynil showed mutagenicity in S. typhimurium YG1026, without S9 mix, inducing 50 and 304 revertants per microgram. These mutagenicities were suppressed by the addition of S9 mix. CNP and chlomethoxynil were also mutagenic to YG1021 with and without S9 mix, and their mutagenicities were lower than those to YG1026. On the other hand, bifenox was mutagenic to YG1026 only with S9 mix, inducing 3.0 revertants per micrograms. These three herbicides showed no mutagenicity in S. typhimurium TA100 and TA98 either with or without S9 mix.