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Complement component 5 (C5), an important molecule in the complement cascade, blockade by antibodies shows clinical efficacy in treating complement-mediated disorders. However, insufficient blockading induced by single-nucleotide polymorphisms in the C5 protein or frequent development of "breakthrough" intravascular hemolysis in patients with paroxysmal nocturnal hemoglobinuria treated with eculizumab have been reported. Herein, we developed a lipid nanoparticle (LNP)-formulated siRNA targeting C5 that was efficiently delivered to the liver and silenced C5 expression. We identified a potent C5-siRNA with an in vitro IC 50 of 420 pM and in vivo ED 50 of 0.017 mg/kg following a single administration. Single or repeated administrations of the LNP-formulated C5-siRNA allowed robust and durable suppression of liver C5 expression in mice. Complement C5 silencing ameliorated C5b-dependent anti-acetylcholine receptor antibody-induced myasthenia gravis and C5a-dependent collagen-induced arthritis symptoms. Similarly, in nonhuman primates, a single administration of C5-siRNA/LNP-induced dose-dependent plasma C5 suppression and concomitantly inhibited serum complement activity; complement activity recovered to the pre-treatment levels at 65 days post administration, thus indicating that the complement activity can be controlled for a specific period. Our findings provide the foundation for further developing C5-siRNA delivered via LNPs as a potential therapeutic for complement-mediated diseases., Competing Interests: All authors were employees of the KAN Research Institute, Inc., and Eisai Co., Ltd., during the execution of this research project. KAN Research Institute, Inc., is a subsidiary of Eisai Co., Ltd. There are no conflicts of interest to declare., (© 2023 The Author(s).)

Background: Acetone is a common alternative solvent used in the Ames test when test chemicals are unstable or poorly soluble in water or dimethyl sulfoxide (DMSO). Yet, there is a very limited number of studies evaluating acetone as a solvent in the modified Ames test with preincubation (preincubation test)., Results: We evaluated the acetone as a solvent for the preincubation test. Fourteen mutagens dissolved in acetone was added each to the reaction mixture at 2 different volumes (25 or 50 μL) to examine mutagenicity using bacterial test strains recommended in the Organization for Economic Cooperation and Development (OECD) test guideline 471, and compared with DMSO (100 μL). Cytotoxicity of acetone was also examined in these bacterial strains. TA1537 was most sensitive to the cytotoxicity of acetone, the degree of which was moderate and similar to DMSO in TA1537 without S9 mix. In other strains, cytotoxicity was limited to a mild degree with or without S9 mix. Cytotoxicity of acetone did not affect detection of mutagenicity of any mutagens; many of them being comparable or less mutagenic than those with DMSO., Conclusions: These findings indicate that acetone is a viable candidate as a solvent for the preincubation test in the 5 bacterial strains., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Benzo[a]pyrene (BP) is highly mutagenic and yet does not lead to tumor development in the murine colon. We recently reported the generation of colonic tumors one week after treatment with BP followed by dextran sulfate sodium (DSS), a colitis-inducer. In this BP/DSS model, male CD2F1 mice were treated orally with BP at 125 mg/kg/day for 5 days, followed by 4% DSS in drinking water for one week. There has been no report so far on the molecular mechanisms involved in tumor development in this model. In the present study, we performed global gene expression analysis on the colonic mucosae obtained from BP-exposed mice one week after treatment with DSS and those treated with the vehicle, BP, or DSS alone. Global gene expression analysis revealed that there were 563 genes preferentially altered (≥2-fold vs vehicle group) in the colonic mucosae exposed to both BP and DSS. Furthermore, comparative gene expression analysis combined with Ingenuity Pathway Analysis™ identified 2 genes associated with Wnt/β-catenin signaling pathway that were preferentially up-regulated (≥2-fold vs vehicle group) when BP and DSS were treated in combination in the distal part (site of predilection for tumor induction) of the colonic mucosae, especially in colonic tumors: WNT inhibitory factor 1 (Wif1; 14.6-fold increase) and interferon induced membrane protein 3 (Ifitm3; 5.7-fold increase). In colonic tumors, expression of Wif1 and Ifitm3 proteins were both confirmed by western blot analysis. These findings suggest that these genes are associated with rapid induction of colonic tumors in mice after exposure to BP/DSS, providing insights into the mechanisms of the BP/DSS short-term colon carcinogenesis., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Dimethyl sulfoxide (DMSO) is a very common organic solvent used for dissolving lipophilic substances, for example for in vitro cell-based assays. At the same time, DMSO is known to be cytotoxic at high concentrations. Therefore, it is important to define threshold concentrations of DMSO for cells but relevant data at the molecular level are very limited. We have focused on conducting microarray analyses of human and rat hepatocytes treated with more than 100 chemicals in attempts to identify candidate biomarker genes. In the present study, the effects of DMSO on gene expression and cytotoxicity were assessed in human cryopreserved hepatocytes and rat primary cultured hepatocytes. A cytotoxicity test with lactate dehydrogenase (LDH) activity demonstrated DMSO to be noncytotoxic up to a concentration of 2% (v/v) in both cases and there were only few effects on the gene expression profiles up to 0.5% (v/v). The observed differences from controls were considered to be of little toxicological importance, but still need to be taken into account in interpretation of findings when DMSO is used at high concentration.

In order to verify the influence of the rat age on hepatotoxicity, male Sprague-Dawley rats of 6 (young) and 12 (adult) weeks of age were orally administered acetaminophen (APAP), isoniazid (INH), or carbon tetrachloride (CCl4). Liver samples were obtained in a time-course manner, and changes in gene expression examined by an Affymetrix GeneChip. APAP caused more prominent hepatic injury with respect to pathology and blood biochemistry in adults than in young rats, whereas no obvious age-related differences were observed in INH- or CCl4-treated rats. Comparing gene expression in control rats, CYP3A13 was higher and GSTY2c was lower in adults, suggesting that production of the active metabolite of APAP is higher and its detoxification is lower in adults. The total amount of glutathione and total SH in rat liver was found to be higher in adult rats whereas the extent of its reduction by APAP was larger in adults. A detailed analysis of genes showing age-related differences revealed that some of them were different not in their extent but in their time course, i.e., the stress responses occurred earlier in the young than in the adult, resulting in a difference at 24 hr after dosing. These results suggest that the age-related difference in toxicity would be attributed to a higher expression of CYP3A13, producing the active metabolite of APAP as well as the lower expression of the detoxification enzyme, GSTY2c, in adult rats. Furthermore, these differences affect the time course of APAP toxicity. The present study clearly depicts the advantage of the multi-time, multi-dose protocol employed in our project for analyzing the mechanism of toxicity by gene expression profiling.

The Toxicogenomics Project is a 5-year collaborative project by the Japanese government and pharmaceutical companies in 2002. Its aim is to construct a large-scale toxicology database of 150 compounds orally administered to rats. The test consists of a single administration test (3, 6, 9 and 24 h) and a repeated administration test (3, 7, 14 and 28 days), and the conventional toxicology data together with the gene expression data in liver as analyzed by using Affymetrix GeneChip are being accumulated. In the project, either methylcellulose or corn oil is employed as vehicle. We examined whether the vehicle itself affects the analysis of gene expression and found that corn oil alone affected the food consumption and biochemical parameters mainly related to lipid metabolism, and this accompanied typical changes in the gene expression. Most of the genes modulated by corn oil were related to cholesterol or fatty acid metabolism (e.g., CYP7A1, CYP8B1, 3-hydroxy-3-methylglutaryl-Coenzyme A reductase, squalene epoxidase, angiopoietin-like protein 4, fatty acid synthase, fatty acid binding proteins), suggesting that the response was physiologic to the oil intake. Many of the lipid-related genes showed circadian rhythm within a day, but the expression pattern of general clock genes (e.g., period 2, arylhydrocarbon nuclear receptor translocator-like, D site albumin promoter binding protein) were unaffected by corn oil, suggesting that the effects are specific for lipid metabolism. These results would be useful for usage of the database especially when drugs with different vehicle control are compared.

Sandwich-culturing is an excellent hepatocyte culturing method in drug metabolism studies, however, its advantages for gene expression of cytochrome P450 (CYP) have not been evaluated so far. The present study was undertaken to determine the utilities of sandwich-culturing hepatocytes for evaluation of CYP genes expression. Hepatocytes from male rats were cultured for 5 days between two layers of type-I collagen gel (sandwich-culturing) or over type-I collagen gel (single gel culturing). To determine the expression of CYP genes rapidly and accurately, the time course study using real-time RT-PCR quantification was conducted in the present study, and CYP2B1, CYP2B2, CYP3A2, CYP3A9 and CYP3A23 genes were measured. Albumin secretion was also measured by ELISA to evaluate cell viability. Higher expression and excellent maintenance of all CYP genes were confirmed in sandwich-culturing hepatocytes than those in single gel culturing. Particularly, significant difference in the amounts of CYP genes expression was observed between both methods after 3 days culturing. Albumin secretion was also higher in sandwich-culturing after 3 days culturing, suggesting that the cell viability of hepatocytes was maintained. These results indicate that sandwich-culturing method is much more advantageous than the ordinate method in maintaining the CYP gene expression and cell viability.

The mutagenic potential of gadobenate dimeglumine formulation (E7155) was studied by the micronucleus test in rats. Single intraperitoneal injection of E7155 to Sprague Dawley rats at the dose of 5295.2 mg/kg (5 mmol/kg) did not induce any statistically significant increase in the frequency of micronucleate cells in the bone marrow sampled after 18, 42 and 66 hr from time of administration.

The mutagenic potential of gadobenate dimeglumine formulation (E7155) was studied by the chromosome aberration test in cultured human lymphocytes. Human lymphocytes were exposed to E7155 at 0.078-10 mM both in the presence and absence of S9 mix derived from rat livers. Three dose levels (2.5-10 mM) were selected for the metaphase analysis. E7155 induced no increase in the incidence of aberrant cells or polyploid cells in any treatments both in the presence and absence of metabolic activation. Thus, it is concluded that E7155 has shown no evidence of clastogenic or polyploidy-inducing activity under these experimental conditions.

Effects of ER-40133, an inhibitor of angiotensin converting enzyme (ACE), on weight gain and sodium and potassium balance were studied in growing SD male rats. Thirty-two animals (seven weeks of age) were divided into two groups; one received a standard diet containing 0.227% sodium and the other a low (0.065%) sodium diet. They were divided into four subgroups; one control group and three treated groups receiving 3, 10 or 30 mg/kg of ER-40133, by gavage, once a day for five consecutive days. Body weight gain (average of the standard and low sodium diet groups) was -32% in the 3 mg/kg group,-74% in 10 mg/kg group and -99% in 30 mg/kg group, when compared with the control group. There was a highly linear correlation between suppression of body weight gain and reduction in sodium and potassium retention for both groups of animals given the standard and low sodium diet. The reduced sodium retention, the primary effect of ACE inhibitors, accounted for about 30% of suppressed weight gain, and the reduced potassium retention, the secondary effect of sodium deficiency, could account for the rest about 70% of weight suppression by ER-40133.

Two isozymes of nitric oxide reductase (Nor) from the denitrifying fungus Cylindrocarpon tonkinense (c.Nor1 and c.Nor2) are the heme-enzyme cytochrome P-450's (Usuda et al. (1995) Appl. Environ. Microbiol. 61, 883-889). However, they catalyze the NO reduction to N2O, but not the monooxygenation reaction using O2. We kinetically and spectrophotometrically studied the reactions of the two Nor's with NO and electron donor, NAD(P)H, using flash-photolysis and stopped-flow rapid scan methods. The enzyme in the Fe3+ state can bind NO to yield the Fe3+ NO complex. When the resultant Fe3+ NO complex reacted with the electron donor, it was converted to the Fe3+ enzyme via a transient formation of the characteristic intermediate (I). The spectroscopic results were essentially the same as those of the Nor from another denitrifying fungus Fusarium oxysporum (f.Nor), which we previously reported (Shiro et al. (1995) J. Biol. Chem. 270, 1617-1623), suggesting that these fungal Nor's catalyze the NO reduction by the same mechanism. Most probably, the Fe3+ NO complex of the Nor is reduced with two-electrons directly transferred from NAD(P)H to yield the intermediate I, and then the I reacts with another NO to generate N2O and the Fe3+ enzyme. However, the kinetic measurements showed that the reaction rate constant of each step was variable depending on the combination of the Nor and the electron donor; i.e., c.Nor1 + NADH, c.Nor2 + NADPH, c.Nor2 + NADH and f.Nor + NADH. In particular, the rate constant for the electron transfer step from the electron donor to the Fe3+ NO enzyme is dramatically different among these systems. On the other hand, we also measured paramagnetically shifted 1H-NMR spectra of c.Nor2 and f.Nor in the ferrous (reduced) state, where the iron-bound Cys beta-CH2 signal was observed at the same position (approximately 270 ppm) for c.Nor2 and f.Nor, indicating that the Cys thiolate (S-) coordinates to the heme iron in the same fashion in the Nor's. However, the heme peripheral proton signals were subtly but significantly different in their positions between the two enzymes. On the basis of these kinetic and spectroscopic data, we suggested that the Fe-S- binding character is not essential for the NO reduction reactivity, but that the subtle difference in interaction of their hemes with the surroundings is possibly responsible for the difference in the Nor reactivity, especially in the electron transfer step from NAD(P)H to the Fe3+ NO moiety.