Understanding reproductive development effects and transferable properties to next generation of zinc oxide nanoparticles is necessary for prevention of its potential risks. To accomplish this, rats were exposed to zinc oxide nanomaterials (500 mg/kg bw) of less than 100 nm beginning 2 weeks before mating to postnatal day 4. In addition, body distribution of zinc concentration was evaluated in dams and offspring. Rat treated with nano-zinc oxide showed reduced number of born/live pups, decreased body weights of pups and increased fetal resorption. Zinc oxide nanomaterials were also distributed to organs such as mammary tissue of dams and liver and kidney of pups. These results indicate that zinc oxide nanoparticles-exposure before and during pregnancy and lactation could pose health risks to pregnant women and their fetus.

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We performed repeated toxicity studies of benzalkonium chloride (BAK)-containing vehicles of ophthalmic solutions in monkeys and rabbits to assess the local toxicity of BAK after repeated applications on the ocular surface. Local toxicity of BAK was evaluated by toxicity studies in which a 0.01% BAK-containing vehicle was applied twice/day for 52 weeks, 4 times/day for 39 weeks, or 6 times/day for 13 weeks, or in which a 0.005% BAK-containing vehicle was applied 6 times/day for 52 weeks or twice/day for 4 weeks in monkeys. Local toxicity of BAK was also evaluated where a 0.01% BAK-containing vehicle was applied 6 times/day for 6 weeks, or a 0.005% BAK-containing vehicle was applied twice/day for 39 weeks or 8 times/day for 4 weeks in rabbits. These doses were chosen because BAK is generally used at concentrations up to 0.01% in ophthalmic solutions. The BAK-containing vehicle did not cause ophthalmological changes suggestive of irritation, allergy, or corneal damage. We also did not observe any histopathological changes in the eyeball, eyelid, lacrimal gland, and nasal cavity, with repeated applications of BAK for up to 52 weeks, up to 8 times/day, or at concentrations up to 0.01%, in monkeys and rabbits. Our results suggest that BAK in concentrations up to 0.01% in ophthalmic solution is non-toxic to the eyeball, its accessory organs, and the nasal cavity after long repeated applications.

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Bisphenol A (BPA) is one of the ‘environmental endocrine disrupters’ (EEDs) released by plastics and resin known to interfere with hormonal responses. In this study, female Wistar rats were exposed to low-dose BPA (24 μg/kg/day) during 7 days after giving birth. The male and female offspring, exposed to the BPA through lactation, were evaluated using an open field test (OFT) at the age of six weeks, an elevated plus maze test (EPM) at seven weeks, and a forced swimming test (FST) at nine weeks. The OFT indicated that females were more active than males, and that BPA selectively increased rearing duration in males, thereby eliminating the gender effect. The results of EPM showed that BPA did not enhance the anxiety-like action; rather, it was associated with an anxiolytic-like action in females. In the FST, not only there was an increase in the immobility time, but also there was reduction of latency observed in BPA rats. It indicated that the depression-like responses were clearly enhanced by the postnatal exposure. Altogether, these data suggest that low-dose BPA ingested by neonates through breastfeeding may cause persistent aberrant behaviors that are relevant to emotions.

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We reported that (–)-xanthatin, a xanthanolide sesquiterpene lactone present in the Cocklebur plant, exhibited potent anti-proliferative effects on human breast cancer cells, in which GADD45γ, a novel tumor suppressor gene, was induced. Mechanistically, topoisomerase IIα (Topo IIα) inhibition by (–)-xanthatin was shown to be the upstream trigger that stimulated the expression of GADD45γ mRNA and concomitantly produced reactive oxygen species (ROS) to maintain this expression. Since the anti-cancer drug etoposide, a selective Topo IIα inhibitor, has also been shown to induce intracellular ROS, (–)-xanthatin may exert its anti-proliferative effects on cancer cells in a similar manner to those of etoposide. In the present study, to generalize its applicability to cancer therapy, we further investigated the biological activities of (–)-xanthatin by comparing its activities to those of the established anti-cancer drug etoposide. After the exposure of breast cancer cells to (–)-xanthatin or etoposide, a prolonged and marked up-regulation in the expression of c-fos, a proapoptotic molecule, was detected together with GADD45γ; and the expression of these molecules was stabilized by ROS and abrogated by the pretreatment with N-acetyl-L-cysteine (NAC), a potent ROS scavenger. (–)-Xanthatin in particular exhibited stronger anti-proliferative potential than that of etoposide, which underlies the marked induction of c-fos/GADD45γ and ROS production.

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A 13-week repeated oral dose toxicity study of grape skin extract (GSE) was performed using F344 rats. Four groups of animals, each consisting of ten males and ten females, were fed a diet containing 0%, 0.2%, 1.0% or 5.0% GSE for 13 weeks. Throughout the experiment, there were no treatment-related changes in clinical signs, body weight or mean food intake in any of the treated groups of either gender. Hematological studies and serum biochemical analyses revealed no treatment-related changes in all groups in both genders. In the glandular epithelial cells of the parotid glands, diffuse hypertrophy and basophilia was observed in all animals in both 5.0% groups. Hypertrophy of the parotid glands was not detected in the 0.2% or the 1.0% dose groups. In female kidneys, slight calcification in the renal proximal tubules of the cortex and medulla was observed in all groups including controls. This is a common spontaneous change in female rats, and the incidence was comparable between controls and treated groups. However, the number of tubules with calcification was higher in the 5.0% group based on a semi-morphometric analysis. Based on the histopathology of the parotid glands and the minor change in the kidneys, the no observed adverse effect level (NOAEL) of GSE in the present study was a 1.0% treatment dose in both genders (males: 0.6 ± 0.2 g/kg body weight/day; females: 0.7 ± 0.1 g/kg body weight/day).

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T-cell dependent antibody response (TDAR) incorporating both primary and secondary responses to keyhole limpet hemocyanin (KLH) in canine models have not yet been fully understood. To develop a practical dog TDAR model, we characterized primary and secondary antibody responses by intravenous or intramuscular immunization of KLH twice at intervals of 8 days during a 28-day course of study. Primary immunization with KLH by both routes induced a maximum IgM response on 6 to 8 days after the treatment, whereas the IgG response started 6 to 8 days after the treatment with relatively low levels. Remarkable increases in anti-KLH IgG levels (about 10-times compared with the primary response) were produced 5 to 7 days after the secondary KLH immunization by both routes. These results indicate that IgM-predominant and IgG-predominant responses were respectively induced by the primary and secondary immunization. Furthermore, the intravenous route showed higher baseline titers of primary and secondary anti-KLH IgM responses, suggesting that intravenous immunization of KLH might be a more suitable method for immunotoxicity evaluation. No remarkable inter-individual variability was noted in our canine models. Treatment with cyclophosphamide at 2 mg/kg/day for a consecutive 28 days significantly suppressed primary and secondary anti-KLH IgM and IgG responses induced by KLH injection on Days 15 and 23 of CPA treatment. These results demonstrate that these experimental designs could provide valuable information about the influence on both the primary and secondary humoral immune responses in dogs when exposed to potential immunomodulatory drugs.

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The objective of this study was to elucidate the range of abilities of nonclinical safety assessment for predicting adverse drug reactions (ADRs) in humans. The dataset included 1256 ADRs with an incidence rate of 5% or more collected from 142 drugs approved in Japan from 2001 to 2010 (excluding anticancer agents and vaccines). Gastrointestinal, neurological and hepatobiliary ADRs were relatively common, followed by hematological, cutaneous, systemic and cardiovascular ADRs in the dataset. The analysis revealed that 48% of ADRs were predictable based on a comprehensive nonclinical safety assessment considering animal toxicity. Hematological and ocular ADRs, infection, and application site reactions showed a correlation of more than 70%, while musculoskeletal, respiratory and neurological ADRs showed a correlation of less than 30%. In addition to subjective patient perceptions, several laboratory parameters routinely monitored both in animals and humans showed a lower correlation, e.g., abnormalities in hepatobiliary and metabolic parameters, and blood pressure increase. Large molecule drugs showed lower correlation than small molecule drugs; ADRs were observed in various organs and consideration of pharmacological action did not significantly contribute to the prediction. It was also confirmed that the current standard of toxicology testing regarding dosing duration and dose level is adequate to detect concordant animal toxicity. This study collectively demonstrated a significant value of nonclinical safety assessment in predicting ADRs in humans. It also identified the subset of ADRs with poor predictability, highlighting the need for advanced testing that enables successful translation of animal toxicity to clinical settings with better accuracy and sensitivity.

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To meet the urgent need for a reliable alternative test for predicting skin sensitizing potential of many chemicals, we have developed a cell-based in vitro test, human Cell Line Activation Test (h-CLAT). However, the predictive performance for lipophilic chemicals in the h-CLAT still remains relatively unknown. Moreover, it’s suggested that low water solubility of chemicals might induce false negative outcomes. Thus, in this study, we tested relatively low water soluble 37 chemicals with log Kow values above and below 3.5 in the h-CLAT. The small-scale assessment resulted in nine false negative outcomes for chemicals with log Kow values greater than 3.5. We then created a dataset of 143 chemicals by combining the existing dataset of 106 chemicals and examined the predictive performance of the h-CLAT for chemicals with a log Kow of less than 3.5; a total of 112 chemicals from the 143 chemicals in the dataset. The sensitivity and overall accuracy for the 143 chemicals were 83% and 80%, respectively. In contrast, sensitivity and overall accuracy for the 112 chemicals with log Kow values below 3.5 improved to 94% and 88%, respectively. These data suggested that the h-CLAT could successfully detect sensitizers with log Kow values up to 3.5. When chemicals with log Kow values greater than 3.5 that were deemed positive by h-CLAT were included with the 112 chemicals, the sensitivity and accuracy in terms of the resulting applicable 128 chemicals out of the 143 chemicals became 95% and 88%, respectively. The use of log Kow values gave the h-CLAT a higher predictive performance. Our results demonstrated that the h-CLAT could predict sensitizing potential of various chemicals, which contain lipophilic chemicals using a large-scale chemical dataset.

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We have previously demonstrated super-induced expression of the Grin2c gene encoding the N-methyl-D-aspartate receptor 2C subunit during the process of liver enlargement induced by phenobarbital, clofibrate, piperonyl butoxide, or lead nitrate. In the present study, hepatic Grin2c gene expression levels were assessed by real-time RT-PCR in male F344 rats fed for 3 days, 4 weeks, and 13 weeks a diet containing either β-naphthoflavone (BNF) (5,000 ppm), indole-3-carbinol (I3C) (2,000 ppm), or acetaminophen (AA) (12,500 ppm until the first 14 days; 10,000 ppm from 15 days on), each of which is capable of inducing hepatocellular hypertrophy. Especially, either the 4-week or the 13-week treatment with each chemical, except for BNF, resulted in a drastic increase in the expression level of the Grin2c gene. DNA microarray analysis using RNAs of 13-week-treated rats showed that in the I3C- and AA-treated rats, the fold-increase rates of the Grin2c gene ranked second and first, respectively, among the genes analyzed. Histopathological analyses indicated that the slight hepatocellular hypertrophy in the periportal area and the hepatocellular necrosis in a portion of the centrilobular area developed in the BNF-treated and AA-treated rats, respectively. In addition, relative liver weight was significantly higher in the rats treated with BNF and I3C than in the control rats. The present findings suggest the possibility that the induction of Grin2c gene expression is not necessarily dependent on only the development of liver enlargement, although the significance of this induction remains unclear.

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A multi-wall carbon nanotube (MWCNT) product Mitsui MWNT-7 is a mixture of dispersed single fibers and their agglomerates/aggregates. In rodents, installation of such mixture induces inflammatory lesions triggered predominantly by the aggregates/agglomerates at the level of terminal bronchiole of the lungs. In human, however, pulmonary toxicity induced by dispersed single fibers that reached the lung alveoli is most important to assess. Therefore, a method to generate aerosol predominantly consisting of dispersed single fibers without changing their length and width is needed for inhalation studies. Here, we report a method (designated as Taquann method) to effectively remove the aggregate/agglomerates and enrich the well-dispersed singler fibers in dry state without dispersant and without changing the length and width distribution of the single fibers. This method is base on two major concept; liquid-phase fine filtration and critical point drying to avoid re-aggregation by surface tension. MWNT-7 was suspended in Tert-butyl alcohol, freeze-and-thawed, filtered by a vibrating 25 µm mesh Metallic Sieve, snap-frozen by liquid nitrogen, and vacuum-sublimated (an alternative method to carbon dioxide critical point drying). A newly designed direct injection system generated well-dispersed aerosol in an inhalation chamber. The lung of mice exposed to the aerosol contained single fibers with a length distribution similar to the original and the Taquann-treated sample. Taquann method utilizes inexpensive materials and equipments mostly found in common biological laboratories, and prepares dry powder ready to make well-dispersed aerosol. This method and the chamber with direct injection system would facilitate the inhalation toxicity studies more relevant to human exposure.

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Deltamethrin, a pyrethroid insecticide, used extensively for pest control has been reported to cause adverse health effects including carcinogenic/toxic effects in animals but the underlying mechanism remains elusive. In the present study, we investigated the effect of deltamethrin after short exposure on early protein expression changes involved in neoplastic transformation in mouse skin, validated the results in human keratinocyte HaCaT cells and thereby explore the possible underlying mechanism. Deltamethrin (4 mg/kg b.wt) and benzo[a]pyrene (B[a]P, 0.05 mg/kg b.wt) were topically applied on Swiss albino mice, respectively. The comparative protein expression profiles with vehicle control were generated by 2-dimensional gel electrophoresis (2-DE) and mass spectrometry. 2-DE maps of deltamethrin and B[a]P treated mouse skin showed 20 and 24 significant (2 fold change, p < 0.05) differentially expressed protein spots, against vehicle controls. However, comparison between them showed relatively similar expression level of 20 spots. Among them, 5 proteins (carbonic anhydrase III, peroxiredoxin-2, calcyclin, superoxide dismutase [Cu-Zn], ubiquitin) are of particular significance as these are involved in cancer-related key processes. Deregulation of these was confirmed at protein and mRNA levels by immunoblotting and RT-PCR in mouse skin and HaCaT cells. Therefore, we conclude that these preliminarily identified proteins might be responsible for the neoplastic transformation of mouse skin epidermal cells and HaCaT cells by deltamethrin. This study proposes complementary mechanism where inhibition of proteasome activator protein (PA200) is responsible for accumulation of ubiquitinated-calcyclin, regulates deltamethrin-induced neoplastic changes in mouse skin and HaCaT cells.

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Pentachlorophenol (PCP) was monitored for transcriptome responses in adult mouse liver at 2, 4, 8 and 24 hr after a single oral administration at four dose levels, 0, 10, 30 and 100 mg/kg. The expression data obtained using Affymetrix GeneChip MOE430 2.0 were absolutized by the Percellome method and expressed as three dimensional (3D) surface graphs with axes of time, dose and copy numbers of mRNA per cell. We developed the programs RSort, for comprehensive screening of the 3D surface data and PercellomeExploror for cross-referencing and confirmed the significant responses by visual inspection. In the first 8 hr, approximately 100 probe sets (PSs) related to PXR/SXR and Cyp2a4 and other metabolic enzymes were induced whereas Fos and JunB were suppressed. At 24 hr, about 1,200 PSs were strongly induced. We cross-referenced the Percellome database consisting of 111 chemicals on the liver transcriptome and found that about half of the PSs belonged to the metabolic pathways including Nrf2-mediated oxidative stress response networks shared with some of the 111 chemicals. The other half of the induced genes were interferon signaling network genes (ISG) and their induction was unique to PCP. Toll like receptors and other pattern recognition receptors, interferon regulatory factors and interferon alpha itself were included but inflammatory cytokines were not induced. In summary, these data indicated that functional symptoms of PCP treatment, such as hyperthermia and profuse sweating might be mediated by the ISG rather than the previously documented mitochondrial uncoupling mechanism. PCP might become a hint for developing low molecular weight orally available interferon mimetic drugs following imiquimod and RO4948191 as agonists of toll-like receptor and interferon receptor.

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