- Since our previous study demonstrated the exacerbation of acute myocardial ischemia/reperfusion (AMIR)-related arrhythmia by intratracheal instillation (IT) of diesel exhaust particles (DEP), the influence of IT with extracts of DEP in organic solvents on AMIR-related arrhythmia was examined in rats. Oxidative activity in a non-biological assay system and proinflammatory activity in mice of DEP extracts were examined. The dichloromethane-soluble fraction (DMSF) of DEP was further fractionated into n-hexane-soluble (n-HSF) and n-hexane-insoluble (n-HISF) fractions. The oxidative activities of the fractions evaluated by dithiothreitol assay were ranked as follows: n-HISF>DMSF>n-HSF. Twenty-one to 34 hr after IT, the AMIR experiment was performed. Exacerbation of AMIR-related arrhythmia and increased reperfusion-related mortality were observed only in rats treated with DMSF. In fact, n-HSF and n-HISF did not affect arrhythmia up to 5 mg/kg. Twelve hr after IT, a significant increase in neutrophil count was observed only with DMSF. The levels of granulocyte colony-stimulating factor and interleukin-6 in bronchoalveolar lavage fluid were significantly elevated in the group treated with DMSF, while neither, n-HSF nor n-HISF, affected the level of cytokines up to 5 mg/kg. In fact, tumor necrosis factor-α, IL-10 and granulocyte-macrophage colony-stimulating factor were unchanged with any of the fractions. In conclusion, exacerbation of AMIR-related arrhythmia by DMSF suggests the contribution of non-particle components of DEP to arrhythmia while the component contributed to the effects did not become clear. Furthermore, it is confirmed that exacerbation of AMIR-related arrhythmia is accompanied by an increased neutrophil count in the circulatory blood.
To evaluate the developmental effects of exposure to acrylamide (ACR) on the nervous and male reproductive systems, pregnant Sprague-Dawley rats were given ACR at 0, 50, 100 or 200 ppm in the drinking water from gestational day 10 to postnatal day 21 and histopathological assessment of offspring was performed at weaning and postnatal week 11. Neurotoxicity was quantitatively assessed with reference to nerve fiber density, percentages of degenerated and small caliber axons in the sciatic nerves, and numbers of aberrant dot-like structures immunoreactive for synaptophysin in the cerebellar molecular layer. Although maternal neurotoxicity was evident from 100 ppm, no changes suggestive of neurotoxicity or testicular toxicity were observed in offspring. However, lowering of body weights was dose-dependently observed from birth at the dose levels of ≥50 ppm in males and ≥100 ppm in females. Maternal malnutrition was apparent at ≥100 ppm during the lactation period. Therefore, poor lactational ACR-exposure due to maternal toxicity might account for the lack of ACR-induced offspring toxicity other than retarded body growth.
Carbendazim is a systemic broad-spectrum fungicide controlling a wide range of pathogens. It is also used as a preservative in paint, papermaking and leather industry, and as a preservative of fruits. In the present study, low dose intracellular effect of carbendazim was investigated employing 5, 10, 25 and 50 mM of the compound administered to male rats intradermally. Blood and liver of each animal was collected 6 hrs later to analyze serum and tissue enzyme activities, tissue lipid peroxidation and hematological and biochemical parameters. The experimental results of low dosage carbendazim use indicated augmentation of investigated parameters. However, the higher dosage of carbendazim use resulted in renormalization of investigated parameters to control levels or to values below control, providing a U-shaped hormesis type dose-response profile. Histopathological sections revealed portal vein congestion, mononuclear cell infiltration and hydropic degeneration of the liver tissue. These results indicated that carbendazim even at low dose exhibited toxicity, affected the liver and also caused specific changes in hematological and biochemical parameters in the rat.
Aldehyde dehydrogenase-2 (ALDH2) metabolizes acetaldehyde produced from ethanol into acetate and plays a major role in the oxidation of acetaldehyde in vivo. About half of all Japanese people have inactive ALDH2. We generated homozygous Aldh2 null (Aldh2-/-) mice by gene targeting knockout as a model of ALDH2-deficient humans. To investigate the mutagenicity of acetaldehyde, a micronucleus assay and a T-cell receptor (TCR) gene mutation assay were performed in Aldh2-/- mice and wild-type (Aldh2 +/+) mice exposed to acetaldehyde. The mice were continuously exposed to 125 and 500 ppm of acetaldehyde vapor for 2 weeks. Another group was orally administered 100 mg/kg once a day for 2 weeks continuously. The mice were killed after 2 weeks of exposure to acetaldehyde, and the frequency of micronucleated reticulocytes was measured by flow cytometry. We also observed the incidence of TCR gene mutations in T-lymphocytes by measuring the variant CD3−CD4+ expression by flow cytometry. The frequency of micronucleated reticulocytes induced by acetaldehyde was significantly increased in Aldh2 −/− mice, but not in Aldh2 +/+ mice. TCR mutant frequency was also associated with acetaldehyde exposure in Aldh2−/ − mice, especially after oral administration; however, it was not associated with acetaldehyde exposure in Aldh2 +/+ mice. In conclusion, Aldh2 −/− mice showed high sensitivity in the micronuclei and TCR mutation assays compared with Aldh2 +/+ mice after exposure to acetaldehyde.
The present study was conducted as a model case of the toxicogenomics approach for analyzing toxicological mechanisms and toxicity assessments in the early stage of drug development by comparing with classical toxicology data. Methapyrilene (MP) 100 mg/kg produced obvious histopathological changes in liver of rats by single or repeated dose up to 28 days with significant elevation of ALT and AST. In the middle dose groups (30 mg/kg MP), no apparent changes were noted in blood biochemical data by single dosing or repeated dosing up to one week, and no obvious histopathological changes were observed except a slight hypertrophy in the hepatocytes. Comprehensive gene expression changes were analyzed using Affymetrix GeneChip® and differentially expressed probe sets were statistically extracted. These contained many genes related to "glutathione metabolism", "apoptosis", "MAPK signaling pathway" and "regulation of cell cycle", which were all thought to be involved in the development of presently observed phenotypes. In the high dose groups, TGP1 scores (developed in our system in order to overview the responsiveness of drugs to multiple marker gene lists) for these categories were markedly increased from the early time point after single dose and kept their high expression throughout the repeated dose period. In the middle dose groups, the increment of the scores were noted not only at the time points when apparent pathological changes emerged, but also at the earlier stage of repeated dosing and even after single dosing. We conclude that toxicogenomics would enable a more sensitive assessment at the earlier time point than classical toxicology evaluation.
The activation of dendritic cells (DC), including Langerhans cells (LC) that reside within the epidermis, is a critical event in the induction phase of allergic contact hypersensitivity. Although recently, p38 mitogen-activated protein kinase (MAPK) has been reported to play a role in the activation of DC induced by allergens, the signaling pathways involved in this process have yet to be determined. We previously found that THP-1 cells have a high capacity to induce TNF-α release and CD86, CD54, and CD40 expression following allergen treatment; reflecting in vitro allergen-induced DC activation during skin sensitization. In this study, we investigated the signaling pathways in THP-1 cells activated by two representative allergens, DNCB and NiSO4. We found that DNCB and NiSO4 induced phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK activation selectively blocked DNCB-induced TNF-α release, but not NiSO4-induced release. In contrast, inhibition of ERK pathways selectively suppressed NiSO4-induced TNF-α release but not DNCB-induced release. In addition, we found that the inhibition of p38 MAPK and ERK pathways caused a selective inhibition of CD86, CD54, and/or CD40 expression following treatment with DNCB or NiSO4. In particular, inhibition of p38 MAPK suppressed CD86, CD54, and CD40 expression induced by DNCB and CD86 expression induced by NiSO4 while inhibition of ERK pathways suppressed CD86, CD54 and CD40 expression induced by DNCB and NiSO4. These data indicate that both DNCB and NiSO4 activate p38 MAPK and ERK, and thereby stimulate TNF-α release and phenotypic changes through the different signal transduction pathways.
The present study was designed to investigate testicular effects of Acyclovir [9-(2-hydroxyethoxymethyl)-9H−guanine] in mouse. Swiss albino male mice (N=6/group/dose/sample time) were treated (i. p.) every day with 4, 16, 32, and 48 mg/kg body weight of Acyclovir for 15 d. The testis was examined for histopathological changes and the seminiferous tubular diameter (STD) and epithelial height (SE) were measured. The sperm count, sperm motility and sperm morphology assay in caudae epididymes, and intra-testicular levels lactate dehydrogenase (LDH) and testosterone were estimated on 7 d, 14 d, 21 d, 28 d, 35 d, and 70 d, following the last exposure. Acyclovir did not affect the body weight, but decreased the testis weight on 21 d and 28 d at two higher dose-levels, and on 35 d at all dose-levels. The STD was decreased on 21 d at 16-48 mg/kg dose-levels (p<0.01), on 28 d and 35 d, at all dose-levels (p<0.01-0.001). The SE was decreased on 14 d at two higher dose-levels (p<0.01), thereafter up to 35 d at all dose-levels (p<0.001). Acyclovir decreased the sperm count from 7-35 d (p<0.001) with a recovery observed on 70 d, except at 48 mg/kg dose-level (p<0.05), and inhibited the sperm motility (p<0.001) from 7-35 d with a maximum effect on 28-35 d. On the other hand, sperm abnormalities increased on 21 d, 28 d and 35 d at 16-48 mg/kg, at 32-48 mg/kg, and at 32 mg/kg dose-levels, respectively (P<0.05). LDH activity was increased (p<0.05-0.001) from 7 d (except at 4 mg/kg) to 35 d. Only 48 mg/kg dose group showed increase in LDH concentration on 70 d. In conclusion, Acyclovir is cytotoxic to germ cells inducing the cellular destruction, and reducing the sperm count and motility, and increasing sperm abnormalities. Further, Acyclovir also caused cellular destruction thus releasing LDH from the cells, and affecting the Leydig cell function. All adverse effects of Acycovir are reversible by 70 d, except the sperm count and LDH level, which appear to be affected over an extended period of time at a higher dose-level.
Dendritic cells (DCs), including Langerhans cells (LCs), play a critical role in the induction phase of allergic contact hypersensitivity. Following exposure to chemical allergens in the skin, LCs undergo a maturation process leading to the up-regulation of expression of co-stimulatory molecules, such as CD86, CD54 and CD40. Our previous study revealed that chemical allergens induce phenotype alterations (e.g., CD86, CD54 and CD40) and cytokine production (TNF-α and IL-8) in THP-1 cells that possibly reflect the maturation of dendritic cells during skin sensitization. However, the physiological signals for phenotypic alterations by chemical allergens are still not fully understood. Therefore, in this study, we investigated the effect of TNF-α and extracellular ATP on THP-1 cell activation induced by chemical allergens. Kinetic studies revealed that TNF-α and IL-8 release occurred in a time-dependent manner with release of two cytokines beginning at 3 hr post-exposure to well-known haptens, DNCB and NiSO4. While recombinant human TNF-α augmented CD54 and CD40 expression in a dose-dependent manner, rhTNF-α did not increase CD86 expression. Furthermore, neutralization of TNF-α activity strongly inhibited CD54 and CD40 expression induced by allergens. On the contrary, extracellular ATP induced the up-regulation of both CD86 and CD54 expression. In the presence of the P2 receptor antagonist suramin, the up-regulation of CD86 and CD54 expression by allergens was in part suppressed. Therefore, we postulate that not only TNF-α but also extracellular ATP may contribute to cell activation following allergen stimulation, which might reflect the mechanism by which DCs respond to allergens.
Gentamycin (GS) is an aminoglycoside antibiotic used to treat infections of various Gram-negative organisms. The present study was designed to investigate the effects of GS on oxidative stress, antioxidant levels, testicular structure and sperm parameters in the rat. Adult Wistar rats (12 week old; N=7/group) were treated (i. p.) with 0 mg/kg, 3 mg/kg and 5 mg/kg for 10 days at an interval of 24 hr between subsequent treatments. Animals were sacrificed on days 1 and 35 after the last treatment, and the reproductive organs were removed and weights of testis and seminal vesicle were recorded. The right testis was processed for light microscopic analysis. The left testis was homogenized and step 19 spermatids were counted to determine the daily sperm production (DSP) and daily abnormal sperm production (DASP). The sperm count, sperm motility and incidence of abnormal sperms were estimated in the epididymis. In testicular sections, along with the evaluation of qualitative changes, the seminiferous tubule diameter (STD) and the epithelial height (SE) were measured. The incidence of stage XIV tubules in testicular sections, meiotic figures and step 14 spermatids/stage XIV tubule, and step 19 spermatids/stage VII tubule were estimated. Intra-testicular levels of superoxide anion, lipid peroxidation and antioxidants-superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and ascorbic acid were measured. GS did not affect the body weight, but the testis weight and DSP were decreased at 5 mg/kg dose-level on both days (p<0.05), and the weight of seminal vesicle decreased on day 35 at both dose-levels. The DASP was increased in a dose-dependent manner (p<0.05) on days 1 and 35 at both dose-levels. The sperm count was decreased at both dose-levels on day 35, whereas the sperm motility was decreased and sperm abnormality was increased on day 1 at 5 mg/kg and on day 35 at both dose-levels. GS induced structural changes such as sloughing of seminiferous epithelium, vacuoles and gaps in the epithelium, nuclear pyknosis and atrophic changes in a few tubules. The tubular shrinkage was observed as indicated by decreased STD and SE on both days at 5 mg/kg dose-level. Incidence of stage XIV tubules and step 19 spermatids/stage VII tubule decreased on all time points at all dose-levels, whereas the step 14 spermatids and meiotic figures decreased on day 35 at both dose-levels (p<0.05). The free radical- superoxide anion concentration was significantly increased on day 1 in a dose-dependent pattern (p<0.05). However, activities of all 3 enzymatic antioxidants and ascorbic acid level decreased in a dose-dependent pattern on day 1 (p<0.05), except the GPx, which was also decreased on day 35 at 5 mg/kg dose-level. There was a significant rise in the thiobarbituric acid reactive substances on day 1 indicating increased lipid peroxidation in the testis. In conclusion, GS induces an oxidative stress-status in the testis by increasing free radical formation and lipid peroxidation, and by decreasing the antioxidant reserves. These biochemical changes manifest as structural and cytotoxic changes in the testis. Further, GS also affects the spermatozoa by affecting their number, motility and morphology.
In order to know the different statistical tools used to analyze the data obtained from twenty-eight-day repeated dose oral toxicity studies with rodents and the impact of these statistical tools on interpretation of data obtained from the studies, study reports of 122 numbers of twenty-eight-day repeated dose oral toxicity studies conducted in rats were examined. It was found that both complex and easy routes of decision trees were followed for the analysis of the quantitative data. These tools include Scheffe's test, non-parametric type Dunnett's and Scheffe's tests with very low power. Few studies used the non-parametric Dunnett type test and Mann-Whitney's U test. Though Chi-square and Fisher's tests are widely used for analysis of qualitative data, their sensitivity to detect a treatment-related effect is questionable. Mann-Whitney's U test has better sensitivity to analyze qualitative data than the chi-square and Fisher's tests. We propose Dunnett's test for analysis of quantitative data obtained from twenty-eight-day repeated dose oral toxicity tests and for qualitative data, Mann-Whitney's U test. For both tests, one-sided test with p=0.05 may be applied.
Nanomaterials of carbon origin tend to form various shapes of particles in micrometer dimensions. Among them, multi-wall carbon nanotubes (MWCNT) form fibrous or rod-shaped particles of length around 10 to 20 micrometers with an aspect ratio of more than three. Fibrous particles of this dimension including asbestos and some man-made fibers are reported to be carcinogenic, typically inducing mesothelioma. Here we report that MWCNT induces mesothelioma along with a positive control, crocidolite (blue asbestos), when administered intraperitoneally to p53 heterozygous mice that have been reported to be sensitive to asbestos. Our results point out the possibility that carbon-made fibrous or rod-shaped micrometer particles may share the carcinogenic mechanisms postulated for asbestos. To maintain sound activity of industrialization of nanomaterials, it would be prudent to implement strategies to keep good control of exposure to fibrous or rod-shaped carbon materials both in the workplace and in the future market until the biological/ carcinogenic properties, especially of their long-term biodurability, are fully assessed.
When human neuroblastoma cells (SH-SY5Y) were exposed to 0.5 - 5 mM acrylamide for 18 hr, the levels of heat shock proteins (HSPs) of 90, 70 and 27 kDa (Hsp90, Hsp70, and Hsp27, respectively) were elevated in the incubation media depending on the dose of acrylamide whereas only the Hsp70 level increased within cells. U0126, a specific inhibitor of extracellular signal-regulated protein kinase kinase and a potent suppressor of the cytotoxicity of acrylamide, suppressed the increase in the levels of all HSPs in the incubation media but not their expression within cells. Total protein concentrations in the incubation media increased depending on the dose of acrylamide, and this increase was associated with the increasing number of bands detected by silver staining after SDS-polyacrylamide gel electrophoresis. One of the clearest bands was identified as Hsp90 by peptide mass fingerprinting. Thus, acrylamide causes release of proteins, including that of HSPs, from SH-SY5Y cells. HSP in extracellular fluid may be a good indicator of cytotoxicity of acrylamide.