In repeated dose administration toxicity studies, which are regulatory requirements for the safety evaluation of drugs, pesticides, etc., the analysis of between-group differences is typically carried out using either parametric or nonparametric statistical methods. The choice of the method depends on whether the data distributions within the groups are normal or not, and or are homogeneous. In theory, testing for normality is important because many parametric tests, such as the t-test and ANOVA, assume that the data within each group follows a normal distribution. However, in repeated dose administration toxicity studies, the data are not always explicitly tested for normality. One reason for this is that there is no universally accepted threshold for deciding whether data is “normal enough” for parametric tests. Another reason is the power of normality tests varies depending on the sample size. In repeated dose administration studies, the number of animals in each group is often small (5–20). In such cases, normality tests may not provide meaningful results due to low statistical power, and the decision to use a parametric or nonparametric test often relies on other considerations, such as variance. It is a common practice to use equal-variance tests (for example Bartlett’s test, Levene’s test, etc.) to determine whether parametric or nonparametric methods should be used for analyzing data. In repeated dose administration toxicity studies for assessing normality the Shapiro-Wilk’s W (Shapiro-Wilk) test is recommended.

The prevalence of antibiotic residues and antibiotic-resistant bacteria in natural environments due to human and livestock waste emissions, pesticide runoff, and pharmaceutical effluents poses a serious threat to public health. Therefore, in this study, we aimed to examine the characteristics, including antibiotic susceptibility, biofilm formation ability, and virulence genes, of levofloxacin (LVFX)-resistant Escherichia coli isolated from the Tone River. Liquid chromatography-tandem mass spectrometry analysis revealed that the upstream, midstream, and downstream river water samples contained 2.2, 19.5, and 15.7 ng/L LVFX, respectively. Despite the very low concentration of LVFX, LVFX-resistant E. coli were isolated from the midstream and downstream regions. Based on the antibiotic sensitivity patterns, the following representative strains were selected: TLR101 and TLR104 (from midstream) and TLR105 and TLR108 (from downstream). These four isolates were LVFX and multidrug-resistant, with TLR101 showing high resistance to ampicillin and the others showing high resistance to chloramphenicol and tetracycline. Biofilm formation assays revealed that TLR104 and TLR108 exhibit very strong biofilm-forming abilities. One-step multiplex polymerase chain reaction (PCR) assays were used to detect various virulence genes, including eaeA, bfpA, invE, ipaH, stx1, stx2, elt, sth, aggR, and astA. A 100-bp astA gene product was detected in TLR104, and DNA sequence analysis confirmed this amplified PCR product, identifying it as a diarrheagenic E. coli strain. Overall, this study revealed the presence of multidrug-resistant diarrheagenic E. coli in the Tone River, underscoring the need for effective public health measures to prevent the release of untreated sewage and industrial waste into the river.

Volatile organic compound 2-ethyl-1-hexanol (2EH) causes the sick building syndrome. Inhalation exposure to 2EH causes olfactory epithelium (OE) degeneration and olfactory neuron loss in mice, which recover temporarily despite continued exposure but subsequently experience similar toxic effects. However, the exact course of recovery after 2EH cessation remains unknown. Therefore, in this study, we aimed to evaluate the histopathological changes in OE after 2EH inhalation cessation. Male ICR mice were exposed to 70 ppm 2EH for 8 hr daily, five days a week, for four weeks, followed by a recovery period of up to four weeks. Histopathological changes in mouse OE on the first (D1) and third (D3) days and first (W1), second (W2), and fourth (W4) weeks of the recovery period were analyzed. Notably, 2EH induced OE degeneration at W2, enlarged the Bowman’s glands at W1 and W2, and decreased the olfactory marker protein-positive cell proportions at W1 and W2. Total leukocytes, neutrophils, and lymphocytes were abundant at both W1 and W2, with no significant differences. Proliferating cell nuclear antigen-positive basal cell number increased; they were distributed throughout the OE on D1 but subsequently lined up the basement membrane and remained at low levels thereafter. Number of growth-associated protein-43-positive immature olfactory neurons increased from D3 to W2. In conclusion, OE was not immediately repaired; the toxic effects appeared 1–2 weeks after 2EH cessation, as indicated by the OE tissue damage with decreased olfactory marker protein-positive cell proportions, followed by proper recovery.

Methylmercury is an environmental pollutant that causes severe central nervous system damage. However, the mechanism involved in its toxicity remains unclear. In this study, expression of thioredoxin-interacting protein (TXNIP), which is involved in the regulation of intracellular redox status, was rapidly induced in mouse neuronal C17.2 cells in response to methylmercury exposure. In addition, C17.2 cells transfected with small interfering RNA against TXNIP mRNA showed greater resistance to methylmercury than control cells. These findings suggest that TXNIP is a novel factor involved in enhancing methylmercury toxicity and that methylmercury may cause cell death by inducing TXNIP expression.

The outer layer of the epidermis, called the stratum corneum (SC), comprises keratin-rich cells and intercorneal lipids. Staphylococcus aureus has been linked to the fragility of the stratum corneum and formation of atopic dermatitis (AD) lesions. Thus, binding of keratin to bacteria may reflect their binding to the AD-like stratum corneum. In this study, keratin films were prepared using keratin extracted from hair and their potential for bacterial removal and adhesion was investigated using chlorine dioxide, which exhibits bactericidal effects, and fucoidan, which is thought to inhibit bacterial adhesion. The results showed that chlorine dioxide was effective at the removal of S. aureus, whereas fucoidan effectively inhibited bacterial adhesion. Therefore, keratin films can be used to evaluate bacterial removal and inhibition of bacterial adhesion, and the results of this evaluation may reflect those of the AD-like stratum corneum. Using these methods, the effects of fucoidan on indigenous skin bacteria (Staphylococcus epidermidis and Cutibacterium acnes) and general bacteria (Escherichia coli) involved in moisturization were investigated. The results showed that fucoidan inhibited adhesion of S. epidermidis and S. aureus, but did not remove bacteria. Overall, the fucoidan (1700 kDa) used in this study has the potential to prevent and treat AD by inhibiting S. aureus adhesion and improving moisturization by maintaining C. acnes.

Based on that TRPM8 is a key factor of normal cell-carcinoma cell interaction and there is a tendency for proliferation of some cell types that are repressed in a high-density state rather than in a low-density state, we attempted to clarify the relationship between direct cell-cell interactions and expression of TRPM8, repressor of carcinoma cell proliferation. We examined TRPM8 and p21/Cip1 levels in normal and carcinoma epithelial cells at low and high cell density (confluent). TRPM8 and p21/Cip1 levels in confluent carcinoma cells markedly increased as compared with that of carcinoma cells in low density and normal cells in low density and confluent. When carcinoma cells cultured to confluent were re-seeded at low density, TRPM8 and p21/Cip1 levels decreased, indicating that the direct cell-cell interaction may regulate the expression of TRPM8 and p21/Cip1 in carcinoma cells. Totally, TRPM8 functions as repressor of cell proliferation in the condition which enables direct interaction between carcinoma cells occurs.

Fructose, a simple carbohydrate, is contained in fruits and honey. It is widely used as a commercial sweetener for beverages and foods and the average fructose intake in the United States was reported to be 49 g/day from 1999 to 2004. It has been reported that excessive intake of fructose induces developmental disorders of the offspring in rats, particularly abnormal glucose and lipid metabolism and neurodevelopment. However, it has not been reported whether excessive intake of fructose induces congenital morphological abnormalities in fetuses. Therefore, we investigated whether an excessive intake of fructose during pregnancy induces congenital morphological abnormalities in the fetus in rats. Pregnant rats were fed a diet containing 13 g, 26 g, 39 g and 65 g fructose / 66 g carbohydrate in 100 g diet, respectively, for the 13% to 65% Fru diet groups (equivalent to about 500, 1000, 1500 and 2500 g/man/day in humans) or control diet from the day after mating until the end of gestation. Caesarean section was performed on gestation day 20, and the fetuses were examined for caesarean section parameters (fetal viability, fetal body weight, placental weight) and fetal external, skeletal and visceral abnormalities. There were no changes/findings in the caesarean section parameters or fetal morphology in any of the fructose diet groups. In conclusion, excessive intake of fructose during gestation did not induce congenital morphological abnormalities in rat fetuses and did not affect fetal viability or development.

Lotus (Nelumbo nucifera) is an aquatic vegetable cultivated in Asia. Lotus rhizome rot is a sever disease that reduces productivity. We extracted and analyzed the compounds contained in the seed rhizomes of ‘Bicchu’ and ‘Lotus,’ cultivars that differ in resistance to rhizome rot. Interestingly, triglycerides were identified only in ‘Lotus,’ which exhibits a stronger disease resistance than ‘Bicchu.’ However, trilinolein, one of the major triglycerides in rhizomes, did not inhibit the growth of Fusarium commune isolated from rhizomes. Therefore, the differences in the rhizome lipid contents were unrelated to their resistance to rhizome rot. Although in this study we analyzed the major compounds in uninfected rhizomes, certain minor anti-fungal phytochemicals in disease-resistant rhizomes might be induced post-infection with pathogenic fungi. This cultivar comparison approach would be useful for further comprehensive studies on these phytochemicals.