Thyrotoxic rubber antioxidants, 2-mercaptobenzimidazole and its methyl derivatives, cause both inhibition and induction of drug-metabolizing activity in rat liver microsomes after repeated oral administration.

We examined the effects of thyrotoxic rubber antioxidants, 2-mercaptobenzimidazole (MBI, 0.3 mmol/kg/day) and its methyl derivatives, methyl-MBIs [4-methyl-MBI (4-MeMBI, 0.6 mmol/kg/day), 5-methyl-MBI (5-MeMBI, 0.6 mmol/kg/day), and 4(or 5)-methyl-MBI (4(5)-MeMBI, 0.6 or 1.2 mmol/kg/day)], on the drug-metabolizing activity in male rat liver microsomes by 8-day repeated oral administration. The weight of liver and thyroid were increased by all the test chemicals; MBI was most potent, and there was no additive or synergistic effect between 4-MeMBI and 5-MeMBI. MBI decreased the cytochrome P450 (CYP) content, NADPH-cytochrome P450 reductase (POR) activity, 7-ethoxycoumarin O-deethylation (ECOD) activity, and flavin-containing monooxygenase (FMO) activity, but increased the 7-pentoxyresorufin O-depentylation (PROD) activity, suggesting inhibition of the drug-metabolizing activity on the whole but induce some activities such as the CYP2B activity. On the contrary, all the methyl-MBIs increased the CYP content, CYB5 content, ECOD activity, 7-ethoxyresorufin O-deethylation (EROD) activity, and PROD activity, indicating that they are mostly inducible of the CYP activity. However, the methyl-MBIs decreased the FMO activity, and 5-MeMBI and 4(5)-MeMBI appeared inhibitory for CYPs 2C11 and 2C13. Between 4-MeMBI and 5-MeMBI, there was no additive or synergistic effect on the drug-metabolizing activity, but was counteraction. It was concluded that MBI and methyl-MBIs had both inhibitory and inducible effects on the drug-metabolizing activity in rat liver microsomes at thyrotoxic doses. The effects of 4(5)-MeMBI indicated that the increased liver weight alone can be a hepatotoxic sign but not an adaptive no-adverse response in toxicity studies. The present results were related to the toxicokinetic profiles of MBI and 4(5)-MeMBI in the repeated toxicity studies.

Evaluation of synthetic naphthalene derivatives as novel chemical chaperones that mimic 4-phenylbutyric acid.

The chemical chaperone 4-phenylbutyric acid (4-PBA) has potential as an agent for the treatment of neurodegenerative diseases. However, the requirement of high concentrations warrants chemical optimization for clinical use. In this study, novel naphthalene derivatives with a greater chemical chaperone activity than 4-PBA were synthesized with analogy to the benzene ring. All novel compounds showed chemical chaperone activity, and 2 and 5 possessed high activity. In subsequent experiments, the protective effects of the compounds were examined in Parkinson's disease model cells, and low toxicity of 9 and 11 was related to amphiphilic substitution with naphthalene.

Proteomic analysis and in vitro developmental toxicity tests for mechanism-based safety evaluation of chemicals.

Mechanism-based safety evaluation and reduction of animal use are important issues in recent developmental toxicology. In vitro developmental toxicity tests with proteomic analysis are the most promising solution to these issues. Groebe et al. systematically applied proteomic analysis to the embryonic stem cell test, a validated in vitro developmental toxicity test, and found protein-expression changes induced by model test chemicals selected from various categories of toxicity. Cluster analysis of all the proteins with expression changes classified the test chemicals into two groups: highly embryotoxic chemicals and non- or weakly embryotoxic chemicals. In addition, some protein biomarker candidates that were known to be involved in normal development were identified. Although further mechanistic investigations are needed, the use of in vitro developmental toxicity tests with proteomic analysis will contribute to mechanism-based safety evaluation with minimal use of animals.

Inhibitory and inductive effects of 4- or 5-methyl-2-mercaptobenzimidazole, thyrotoxic and hepatotoxic rubber antioxidants, on several forms of cytochrome P450 in primary cultured rat and human hepatocytes.

Effects of 4-methyl-2-mercaptobenzimidazole (4-MeMBI) and 5-methyl-2- mercaptobenzimidazole (5-MeMBI) on cytochrome P450 (CYP) activity were examined in primary cultured rat hepatocytes. Hepatocytes from male Wistar rats were cultured in the presence of 4-MeMBI or 5-MeMBI (0-400 µM), and the activity of CYPs 3A2/4 (48 and 96 h) and 1A1/2 (48 h) was determined by measuring the activity of testosterone 6ß-hydroxylation and 7-ethoxyresorufin O-deethylation, respectively. As a result, 4-MeMBI and 5-MeMBI (≥12.5 µM) inhibited CYP3A2 activity. On the other hand, 4-MeMBI (≥25 µM) and 5-MeMBI (≥100 µM) induced CYP1A1/2 activity, being consistent with the previous in vivo results. In a comparative metabolism study using primary cultured human hepatocytes from two Caucasian donors, 4-MeMBI and 5-MeMBI induced the activity of CYPs 3A4 and 1A1/2 with individual variability. It was concluded from these results that 4-MeMBI, 5-MeMBI and MBI caused inhibition of CYP3A2 activity in primary cultured rat hepatocytes, suggesting their potential for metabolic drug-drug interactions. Primary cultured rat and human hepatocytes were considered to be useful for the evaluation of effects of the benzimidazole compounds on their inducibility and inhibitory activities of cytochrome P450 forms.

In vivo/in vitro study in rat embryos on indium-caused tail malformations.

Pathogenesis of indium-caused tail malformations was investigated by in vivo and in vitro experiments. In the in vivo experiment, pregnant Wistar rats received single intravenous administration of indium trichloride at 0.4 mg/kg on day 10 of gestation, and their embryos were examined on days 11, 12 and 13. Embryos in the indium group showed caudal hypoplasia from day 11. Increased apoptosis was observed in their tailbud on day 11. Similar effects were observed in the in vitro experiment, when day 10 rat embryos were cultured in the presence of indium trichloride at 50 microM for 24 h and for further 24 h in the absence of indium. It was considered from these results that caudal hypoplasia probably due to excessive cell loss by increased apoptosis in the tailbud accounted for indium-caused tail malformations in rat fetuses, and that indium-caused embryotoxic effects were direct effects on the conceptus.

Proteomic analysis of selenium embryotoxicity in cultured postimplantation rat embryos.

BACKGROUND: Developmental toxicity of selenium (Se) is a nutritional, environmental and medicinal concern. Here, we investigated Se embryotoxicity by proteomic analysis of cultured rat embryos. METHODS: Rat embryos at day 9.5 or 10.5 of gestation were cultured for 48 or 24 h, respectively, in the presence of sodium selenate (100 or 150 microM) or sodium selenite (20 or 30 microM). Proteins from the embryo proper and yolk sac membrane were analyzed by two-dimensional electrophoresis for quantitative changes from those in control embryos. Proteins with quantitative changes were identified by mass spectrometric analysis. RESULTS: Growth inhibition and morphological abnormalities of cultured embryos were observed in all the Se treatment groups. By the analysis of the embryo proper, actin-binding proteins were identified as proteins with quantitative changes by selenate: increased phosphorylated-cofilin 1, increased phosphorylated-destrin, decreased drebrin E, and decreased myosin light polypeptide 3. Many proteins showed similar changes between selenate and selenite, including increased ATP-synthase, decreased acidic ribosomal phosphoprotein P0, and decreased pyrroline-5-carboxylate reductase-like. In the yolk sac membrane, antioxidant proteins were identified for protein spots with quantitative changes by selenite: increased peroxiredoxin 1 and increased glutathione S-transferase. CONCLUSION: The identified proteins with quantitative changes by selenate or selenite were considered to be candidate proteins involved in Se embryotoxicity: the actin-binding proteins for selenate embryotoxicity, proteins with the similar changes for the common Se embryotoxicity and antioxidant proteins for modification of Se embryotoxicity by redox-related treatments. These proteins may also be used as biomarkers in developmental toxicity studies.

Two-dimensional electrophoresis of protein from cultured postimplantation rat embryos for developmental toxicity studies.

A simple method for two-dimensional electrophoresis (2-DE) of rat embryonic protein was described. Rat embryos cultured for 24h from day 10.5 of gestation were used as protein samples. Protein samples were lysed in rehydration buffer and separated by isoelectric focusing with immobilized pH gradient for the first dimension and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the second dimension. The use of the DeStreak Reagent as an antioxidant in the lysis buffer and electrode pads in the isoelectric focusing greatly improved the 2-DE pattern. When an embryo was used as a protein sample, about 800 protein spots were detected by silver staining in a 2-DE gel of the standard format. Eighty-one protein spots were identified by mass spectrometry for a primary 2-DE map. The same method could be applied to yolk sac membranes from the cultured embryos. The present method was considered to be suitable for a concomitant 2-DE analysis in in vitro developmental toxicity studies.

Effects of 13 developmentally toxic chemicals on the migration of rat cephalic neural crest cells in vitro.

The inhibition of neural crest cell (NCC) migration has been considered as a possible pathogenic mechanism underlying chemical developmental toxicity. In this study, we examined the effects of 13 developmentally toxic chemicals on the migration of rat cephalic NCCs (cNCCs) by using a simple in vitro assay. cNCCs were cultured for 48 h as emigrants from rhombencephalic neural tubes explanted from rat embryos at day 10.5 of gestation. The chemicals were added to the culture medium at 24 h of culture. Migration of cNCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of cNCCs between 24 and 48 h of culture. Of the chemicals examined, 13-cis-retinoic acid, ethanol, ibuprofen, lead acetate, salicylic acid, and selenate inhibited the migration of cNCCs at their embryotoxic concentrations; no effects were observed for acetaminophen, caffeine, indium, phenytoin, selenite, tributyltin, and valproic acid. In a cNCC proliferation assay, ethanol, ibuprofen, salicylic acid, selenate, and tributyltin inhibited cell proliferation, suggesting the contribution of the reduced cell number to the inhibited migration of cNCCs. It was determined that several developmentally toxic chemicals inhibited the migration of cNCCs, the effects of which were manifested as various craniofacial abnormalities.

Estrogen receptor binding assay of chemicals with a surface plasmon resonance biosensor.

We have developed a simple assay method for the evaluation of estrogen receptor (ER) binding capacity of chemicals without the use of radio- or fluorescence-labeled compounds. We used the solution competition assay by the BIACORE biosensor, a surface plasmon resonance biosensor, with estradiol as a ligand, human recombinant ER(alpha) (hrER(alpha)) as a high molecular weight (hmw) interactant and test chemicals as analytes. For the ligand, aminated estradiol with a spacer molecule (E2-17PeNH) was synthesized and immobilized on a carboxymethyl dextran-coated sensor chip by the amine coupling method. The injection of the hmw interactant hrER(alpha) to the biosensor raised the sensorgram, indicating its binding to the ligand E2-17PeNH. The binding of test chemicals to hrERalpha was determined as a reduction in the hrER(alpha) binding to E2-17PeNH. The dissociation constant for the binding to hrER(alpha) was calculated for estrone (4.29 x 10(-9)M), estradiol (4.04 x 10(-10)M), estriol (8.35 x 10(-10)M), tamoxifen (2.16 x 10(-8)M), diethylstilbestrol (1.46 x 10(-10)M), bisphenol A (1.35 x 10(-6)M) and 4-nonylphenol (7.49 x 10(-6)M), by plotting the data according to an equation based on mass action law. This method can also be used as a high throughput screening method.

Proteomic analysis of ethanol-induced embryotoxicity in cultured post-implantation rat embryos.

Protein expression changes were examined in day 10.5 rat embryos cultured for 24 hr in the presence of ethanol by using two-dimensional electrophoresis and mass spectrometry. Exposure to ethanol resulted in quantitative changes in many embryonic protein spots (16 decreased and 28 increased) at in vitro embryotoxic concentrations (130 and 195 mM); most changes occurred in a concentration-dependent manner. For these protein spots, 17 proteins were identified, including protein disulfide isomerase A3, alpha-fetoprotein, phosphorylated cofilin-1, and serum albumin. From the gene ontology classification and pathway mapping of the identified proteins, it was found that ethanol affected several biological processes involving oxidative stress and retinoid metabolism.

Simple in vitro migration assay for neural crest cells and the opposite effects of all-trans-retinoic acid on cephalic- and trunk-derived cells.

Here, we describe a simple in vitro neural crest cell (NCC) migration assay and the effects of all-trans-retinoic acid (RA) on NCCs. Neural tubes excised from the rhombencephalic or trunk region of day 10.5 rat embryos were cultured for 48 h to allow emigration and migration of NCCs. Migration of NCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of NCCs between 24 and 48 h of culture. RA was added to the culture medium after 24 h at embryotoxic concentrations determined by rat whole embryo culture. RA (10 µM) reduced the migration of cephalic NCCs, whereas it enhanced the migration of trunk NCCs, indicating that RA has opposite effects on these two types of NCCs.

Complement component C3 functions as an embryotrophic factor in early postimplantation rat embryos.

A presumed embryotrophic factor for early postimplantation rat embryos, partially purified from rat serum, was identified as complement component C3 (C3), the central component of the complement system, by sequence analysis of its N-terminal. Purified rat C3 showed embryotrophic activity for rat embryos cultured from day 9.5 of gestation for 48 h in the culture medium composed of rabbit serum. The maximum embryotrophic activity of C3 was observed around 0.5 mg/ml, a level which is lower than rat serum C3 levels. In the culture medium composed of rat serum, cultured rat embryos selectively consumed C3, and C3-depletion by cobra venom factor affected embryonic growth. Inactivation of the internal thiolester bond of C3, the critical functional site for its activity in the complement system, by methylamine had no effects on its embryotrophic activity. Purified rabbit C3 had only weak embryotrophic activity for cultured rat embryos, suggesting species specificity of the embryotrophic activity of C3. Immunochemical analyses showed the specific presence of C3 on the visceral yolk sac, but not on the embryo proper of day 9.5 or 10.5 rat embryos both in utero and in vitro. In analysis using fluorescein-labeled rat C3, unfragmented C3s bound to the visceral yolk sac stronger than C3b, the primary active fragment of C3 in the complement system. These results indicate that C3, which has always been considered to be detrimental to embryos, functions as an embryotrophic factor by novel mechanisms probably through the visceral yolk sac. The present study thus provides new insights into functions of C3 and postimplantation embryonic growth.

Sexing of postimplantation rat embryos in stored two-dimensional electrophoresis (2-DE) samples by polymerase chain reaction (PCR) of an Sry sequence.

Proteomic analysis of developmental toxicity by two-dimensional electrophoresis (2-DE) may detect gender-related toxic effects in embryos without visible gender characteristics. In the present study, we explored sexing of rat embryo stored in frozen 2-DE samples by polymerase chain reaction (PCR) of a male-specific gene sequence, sex determining region Y (Sry). The embryo proper and yolk sac membrane at gestation day 11 from Wistar rats were used for stored embryonic 2-DE samples. The embryonic 2-DE samples were desalted and their total DNA was extracted. The Sry sequence in the extracted DNA was amplified by PCR and the product was analyzed by agarose gel electrophoresis. The embryos with the PCR product of Sry were determined as male, and those without the product were determined as female. It was concluded that stored embryonic 2-DE samples could be used for retrospective examination of gender-related effects in proteomic analysis of developmental toxicity.

Proteomic analysis of indium embryotoxicity in cultured postimplantation rat embryos.

Indium embryotoxicity was investigated by proteomic analysis with two-dimensional electrophoresis of rat embryos cultured from day 10.5 of gestation for 24h in the presence of 50 microM indium trichloride. In the embryo proper, indium increased quantity of several protein spots including those identified as serum albumin, phosphorylated cofilin 1, phosphorylated destrin and tyrosyl-tRNA synthetase. The increased serum albumin, derived from the culture medium composed of rat serum, may decrease the toxicity of indium. The increase of phosphorylated cofilin 1 might be involved in dysmorphogenicity of indium through perturbation of actin functions. In the yolk sac membrane, indium induced quantitative and qualitative changes in the protein spots. Proteins from appeared spots included stress proteins, and those from decreased or disappeared spots included serum proteins, glycolytic pathway enzymes and cytoskeletal proteins, indicating yolk sac dysfunction. Thus, several candidate proteins that might be involved in indium embryotoxicity were identified.

Comparative proteome analysis of the embryo proper and yolk sac membrane of day 11.5 cultured rat embryos.

BACKGROUND: Proteomic analysis of cultured postimplantation rat embryos is expected to be useful for investigation into embryonic development. Here we analyzed protein expression in cultured postimplantation rat embryos by two-dimensional electrophoresis (2-DE) and mass-spectrometric protein identification. METHODS: Rat embryos were cultured from day 9.5 for 48 h or from day 10.5 for 24 h. Proteins of the embryo proper and yolk sac membrane were isolated by 2-DE and differentially analyzed with a 2-D analysis software. Selected protein spots in the 2-DE gels were identified by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometric analysis and protein database search. RESULTS: About 800 and 1,000 protein spots were matched through the replicate 2-DE gels each from one embryo in the embryo proper and yolk sac membrane, respectively, and virtually the same protein spots were observed irrespective to the length of culture period. From protein spots specific to the embryo proper (126 spots) and yolk sac membrane (304 spots), proteins involved in tissue-characteristic functions, such as morphogenesis and nutritional transfer, were identified: calponin, cellular retinoic acid binding protein, cofilin, myosin, and stathmin in the embryo proper, and Ash-m, dimerization cofactor of hepatocyte nuclear factor, ERM-binding phosphoprotein, cathepsin, and legumain in the yolk sac membrane. CONCLUSION: Proteomic analysis of cultured postimplantation rat embryos will be a new approach in developmental biology and toxicology at the protein level.

Structural mechanism and photoprotective function of water-soluble chlorophyll-binding protein.

A water-soluble chlorophyll-binding protein (WSCP) is the single known instance of a putative chlorophyll (Chl) carrier in green plants. Recently the photoprotective function of WSCP has been demonstrated by EPR measurements; the light-induced singlet-oxygen formation of Chl in the WSCP tetramer is about four times lower than that of unbound Chl. This paper describes the crystal structure of the WSCP-Chl complex purified from leaves of Lepidium virginicum (Virginia pepperweed) to clarify the mechanism of its photoprotective function. The WSCP-Chl complex is a homotetramer comprising four protein chains of 180 amino acids and four Chl molecules. At the center of the complex one hydrophobic cavity is formed in which all of the four Chl molecules are tightly packed and isolated from bulk solvent. With reference to the novel Chl-binding mode, we propose that the photoprotection mechanism may be based on the inhibition of physical contact between the Chl molecules and molecular oxygen.