The estrogenic activities of S-9 metabolites of benzophenone derivatives (benzophenone, 2-hydroxy-4-methoxybenzophenone, 2,2′-dihydroxy-4-methoxybenzophenone, 2-hydroxy-4-octyloxybenzophenone, 2,4-dihydroxybenzophenone and 2,3,4-trihydroxybenzophenone) and benzhydrol were examined with a yeast two-hybrid screening system. After chemicals were incubated in an S-9 mix at 37°C for 4 hr prior to their incubation with the yeast strain, the S-9 mix containing metabolites was assayed for the estrogenic activity by the yeast two-hybrid assay. Benzophenone, 2-hydroxy-4-methoxybenzophenone and 2,2′-dihydroxy-4-methoxybenzophenone exhibited estrogenic activities after incubation with the S-9 mix. The estrogenic metabolites of 2-hydroxy-4-methoxybenzophenone were fractionated by high-performance liquid chromatography, one of which was identified as 2,4-dihydroxybenzophenone. This assay will be a useful tool for detecting proestrogens.
The adverse effects of chemicals exerted via estrogen receptors (ER) and androgen receptors (AR) have been studied extensively in recent years. However, those occurring via thyroid hormone receptors (TR) have not been studied enough. We examined the TR-binding activities of thyronine derivatives and alkylphenol derivatives (bisphenol A, parabens and antioxidants with o-t-butylphenol residue) using a yeast two-hybrid assay. In this assay system, the TR-binding activity of 3,5,3′-triiodo-L-thyronine (T3) was detectable at concentrations as low as 3.0 × 10-8 M and reached a plateau at 1.0 × 10-6 M. The concentration of T3 producing 10% of the activity stimulated at 1.0 × 10-6 M (10% relative effective concentration, REC10) was 2.1 × 10-8 M. 3,5,3′,5′-tetraiodo-L-thyronine (T4), 3,3′,5′-triiodo-L-thyronine (rT3) and 3, 5-diiodo-L-thyronine (T2) also exhibited TR-binding activities. The REC10 values of these chemicals were 4.2 × 10-8, 5.0 × 10-7 and 1.0 × 10-5 M, respectively. o-Isopropylphenol and o-t-butylphenol exhibited TR-binding activities with REC10 values of 3.1 × 10-4 and 4.8 × 10-5 M, respectively, whereas t-butylbenzene, isopropylbenzene and the other chemicals tested had no detectable TR-bindng activity. These results suggest that a phenolic hydroxyl group and the ortho-substituents may play important roles in the TR-binding activities of these chemicals. This assay system will be a useful tool for screening the TR-binding activities of chemicals.
Analysis of the fatty acid composition in the microsomes of rats fed the oil rich in α-linolenic acid (n-3, perilla oil, Per) or linoleic acid (n-6, safflower oil, Saf) showed changes according to those compositions in the diets. The level of phase I enzyme in microsomes prepared from rat liver was measured by the Western blotting method. The levels of CYP1A1, 1A2 and 2E1 were similar between the two oil groups, Per and Saf. Meanwhile, the level of CYP4A1 in the microsomes of rats fed the Per diet was significantly higher than that from rats fed Saf. As a second step, we measured the level and activity of UDP-glucuronosyltransferase (UGT). The results showed that the level of UGT activity in microsomes from rats fed the Per diet was 2.4-fold higher than that from rats fed the Saf diet. The protein level of UGT in microsomes from rats fed the Per diet was also 1.7-fold greater than that from rats fed the Saf diet. The low level of protein amount and activity of UGT in rats fed Saf was recovered by the administration of the Per diet. Furthermore, the UGT activity in microsomes from rats fed high docosahexaenoic acid, another n-3 fatty acid, was higher than that in rats fed Per oil. These results suggest that the fatty acid composition, n-3 or n-6, in foods influences the level and activity of CYP4A and UGT. Thus, monitoring of the drug level in the blood of patients in consideration of oil intake should be undertaken.
Tumor necrosis factor-α (TNF-α) has been implicated in the pathogenesis of atherosclerosis through influence on vascular smooth muscle cell behavior. However, little is known about the expression and function of endogenous TNF-α in the cells. Dense and sparse cultures of bovine aortic smooth muscle cells were prepared and the expression of TNF-α and regulation of collagen synthesis by endogenous TNF-α were investigated. The results indicate that dense and sparse vascular smooth muscle cells express endogenous TNF-α that suppresses the synthesis of collagen (types I and III) only when the cell density is high; suppression was also observed in human and rabbit aortic smooth muscle cells. Transforming growth factor-β stimulated collagen synthesis in bovine aortic smooth muscle cells and the stimulation was significantly suppressed by endogenous TNF-α when cell density was high. The present data suggest that vascular smooth muscle cells express endogenous TNF-α that is involved in the suppressive regulation of collagen synthesis depending on the cell density.
The effect of wasabi leafstalk (Wasabia japonica MATTUM.) extract on bone components in the femoral-diaphyseal and -metaphyseal tissues of aged female rats in vitro and in vivo was investigated. Femoral-diaphyseal and -metaphyseal tissues were cultured for 48 hr in Dulbecco's modified Eagle's medium (serum free) containing either vehicle or wasabi leafstalk extract (10, 25 or 50 μg/ml of medium). The presence of wasabi leafstalk extract (50 μg/ml) caused a significant increase in calcium content, alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the diaphyseal and metaphyseal tissues in vitro. However, the effect of wasabi leafstalk extract (50 μg/ml) in increasing bone components was completely abolished in the presence of cycloheximide (10-6 M), an inhibitor of protein synthesis. Moreover, rats were orally administered wasabi leafstalk extract (10 or 20 mg/100 g body weight) once daily for 7 days. The calcium content, alkaline phosphatase activity and DNA content in the femoral-diaphyseal and -metaphyseal tissues of aged rats was significantly increased by the administration of wasabi leafstalk extract (10 or 20 mg/100 g) for 7 days in vivo. Meanwhile, body weight, serum calcium and inorganic phosphorus concentrations of female aged rats were not significantly altered by the administration of wasabi leafstalk extract (10 or 20 mg/100 g) for 7 days. The present study demonstrates that wasabi leafstalk extract has an anabolic effect on bone components in vitro and in vivo. The intake of wasabi leafstalk extract may have a preventive effect on bone loss with increasing age.
We have investigated 100 illegal amphetamine-type stimulant (ATS) tablets seized in Japan to obtain information about the nature of these tablets abused in Japan. For physical characterization, 15 items (logo, vertical view, colour, diameter, weight, smell, etc.) were measured and a photograph was taken. For chemical characterization, the components in the tablet were identified by GC-MS and HPLC, and quantified by HPLC using an ODS-type column. The maximum content of N-methyl-3,4-methylenedioxyamphetamine (MDMA) was 189 mg/tablet and that of 3,4-methylenedioxyamphetamine (MDA) was 87 mg/tablet. The detected components, other than MDMA and MDA, were N-ethyl-3,4-methylenedioxyamphetamine (MDEA), ephedrine, caffeine, ketamine, and methamphetamine. In this report, we propose a method for profiling ATS tablets.
A procedure for identifying surfactants was investigated by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) from the forensic toxicological point of view. The analysis of anionic, cationic, and nonionic surfactants in both negative and positive mode revealed that anionic and cationic surfactants are detected as M- ions in the negative mode and M+ ions in the positive mode, respectively, while [M+H]+ ions or [M+NH4]+ ions appeared for nonionic surfactants in the positive mode. Anionic surfactants (linear alkylbenzene sulfonate and polyoxyethylene alkyl ether sulfate) were successfully extracted using a weak anion exchange cartridge, with recovery rates of 53.8-76.7%. Cationic surfactant (benzalkonium) was also easily extracted using a weak cation exchange cartridge with a recovery rate of 107%. When aqueous solutions of cationic, anionic, and nonionic surfactants were concentrated to dryness, the recovery rates ranged from 65.8% to 124%. By analyzing anionic surfactants (alpha-olefine sulfate, polyoxyethylene alkyl ether sulfate, and linear alkylbenzene sulfonate) in the product ion scan mode in LC/ESI-MS/MS, it was possible to identify the structure of the hydrophilic group of surfactants, and MS/MS was confirmed to be a powerful technique for classification.
The effect of Sargassum horneri (S. horneri) extract on bone components in the femoral-diaphyseal and -metaphyseal tissues of streptozotocin (STZ)-diabetic rats was investigated. Rats received a single subcutaneous administration of STZ (6.0 mg/100 g body weight), and then the animals were orally administered water-solubilized extract (10 mg/100 g body weight) of S. horneri once daily for 14 or 21 days. The administration of STZ caused a significant decrease in body weight and a significant increase in serum glucose, triglyceride, and calcium levels, indicating a diabetic state. These alterations were significantly prevented by the administration of S. horneri extract for 14 or 21 days. The administration of S. horneri extract to normal rats for 14 or 21 days did not have a significant effect on body weight or serum glucose, triglyceride, and calcium levels. Calcium content, alkaline phosphatase activity, and DNA content in the femoral-diaphyseal and -metaphyseal tissues were significantly decreased in STZ-diabetic rats. These decreases were significantly prevented by the administration of S. horneri extract for 14 or 21 days. The administration of S. horneri extract to normal rats for 14 or 21 days caused a significant increase in calcium content, alkaline phosphatase activity, and DNA content in the femoral-diaphyseal and -metaphyseal tissues. Femoral-diaphyseal and -metaphyseal tissues obtained at 14 days after STZ administration were cultured for 48 hr in a medium containing either vehicle or S. horneri extract (10, 25, or 50 μg/ml of medium) in vitro. Calcium content and alkaline phosphatase activity in the femoral-diaphyseal and -metaphyseal tissues obtained from STZ-diabetic rats was significantly increased in the presence of S. horneri extract (10, 25, or 50 μg/ml). The present results demonstrate that the intake of S. horneri extract has a preventive effect on bone loss in STZ-diabetic rats.
We developed epitope-tagged metallothionein IIA cDNA by the addition of an 8-amino acid sequence, DYKDDDDK, at the N-terminus metallothionein using polymerase chain reaction. The fusion proteins were expressed by transfection in human embryonic kidney 293 (HEK-293) cells and were specifically recognized by commercially available antibody, an anti-Flag M2 monoclonal antibody. Furthermore, the fusion protein was detectable on a Western blot, whose migration was expectedly at 7000-8000 in an SDS-denatured form. Thus, availability of the epitope-tagged metallothionein will facilitate the investigation of cellular mechanisms of the protein such as its nuclear localization mechanism.
The effects of dietary protein and sulfur-containing amino acids on oxidative responses against N-nitrosodimethylamine (NDMA) treatment were investigated in rat liver, focusing on the mechanism of metallothionein (MT) induction. Rats were fed a 10% soybean protein isolate (SPI) diet or SPI supplemented with 0.3% L-methionine (SPI-Met) for 3 weeks after weaning. Rats fed on SPI showed lower glutathione (GSH) levels and higher MT levels in the liver than those fed on SPI-Met. After treatment with NDMA (20 mg/kg, i.p.), MT levels were elevated significantly in both groups, whereas GSH levels were only elevated in the SPI group. Pathologically, necrosis and hemorrhage were seen in the livers of NDMA-treated rats of both dietary groups, suggesting oxidative damage caused by NDMA treatment. Immunological histochemistry using anti-MT antibody showed that most of the MT was distributed in the cytosol region of all the rat groups. The ratios of the MT-positive cells were correlated to MT levels that were chemically analyzed. It was notable that the localization of MT staining in the hepatic lobules was altered after NDMA injection. The MT staining was principally observed around the central vein before injection, whereas it was more markedly observed at the peripheral region after NDMA treatment. These results indicate that the synthesis of hepatic MT basically occurs around the central vein with oxygen stress caused by insufficient oxygen supplement and/or stimulated oxygen consumption. NDMA could be oxidatively metabolized at the peripheral region with sufficient supplemental oxygen from the artery, resulting in oxidative stress and stimulation of MT synthesis there. Thus, distribution of tissue MT might vary depending on the site of oxidative stress.
The urinary and faecal metabolic profiles of 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in rat were investigated. Male Wistar rats were administrated 10 mg/kg of 2C-B hydrochloride orally, and the urine and faeces were collected 0-24 and 24-48 hr after administration. The samples were processed by liquid-liquid extraction, and the extracts analyzed by GC/MS, after derivatization. The major metabolite excreted into the urine was 5-O-desmethyl-N-acetyl-2C-B, comprising 13.2% of the administrated dose. Other metabolites detected in the urinary extracts were 2-O-desmethyl-N-acetyl-2C-B (5.8%), 2-O-desmethyl-2C-B (3.5%), carboxylic acid compound (1.9%), 5-O-desmethyl-2C-B (1.2%) and an alcoholic compound (0.8%). Only 0.2% of the administrated 2C-B was excreted into the urine in its original form. On the other hand, only 5-O-desmethyl-N-acetyl-2C-B and 2-O-desmethyl-N-acetyl-2C-B were detected in the faecal extracts. The major fraction of the urinary metabolites that contained a hydroxy group were recovered as conjugates.