Local lymph node assay: 5-bromo-d-deoxyuridine-ELISA method for comparative study in assessing chemical potencies and skin sensitization in BALB/c and CBA/J strains.

Local lymph node assay (LLNA) is a predictive in vivo method to provide estimates of relative potency and to contribute to risk assessment/risk management regarding skin sensitizing potency of chemicals and formulations as a stand-alone alternative test. In addition, LLNA is relatively rapid and cost-effective compared to the Buehler method (Guinea pig test), and confers important animal welfare benefits. CBA/J and BALB/c strains are widely commercially available and have been evaluated by formal LLNA validation studies. However, the LLNA method using BrdU with ELISA, unlike other LLNA methods (OECD TG 429, 442 A, 442B), has not been previously validated. Therefore, in this study a validation method was performed to evaluate if the LLNA:BrdU-ELISA method could also be used to identify sensitizers among chemicals listed in OECD TG 429 using CBA/J and BALB/c strains. Here, we newly found that the LLNA:BrdU-ELISA validation method correctly identified 12 of 13 sensitizers in the BALB/c, 11 of 13 sensitizers in the CBA/J, and 3 of 5 non-sensitizers were identified in the two strains. Collectively, we found that the results of LLNA:BrdU-ELISA method provide a similar level of performance for accuracy and sensitivity in two mouse strains BALB/c and CBA/J.

Carfilzomib in Combination with Bortezomib Enhances Apoptotic Cell Death in B16-F1 Melanoma Cells.

Proteasome inhibitors, such as bortezomib (BZ) and carfilzomib (CFZ), have been suggested as treatments for various cancers. To utilize BZ and/or CFZ as effective therapeutics for treating melanoma, we studied their molecular mechanisms using B16-F1 melanoma cells. Flow cytometry of Annexin V-fluorescein isothiocyanate-labeled cells indicated apoptosis induction by treatment with BZ and CFZ. Apoptosis was evidenced by the activation of various caspases, including caspase 3, 8, 9, and 12. Treatment with BZ and CFZ induced endoplasmic reticulum (ER) stress, as indicated by an increase in eIF2α phosphorylation and the expression of ER stress-associated proteins, including GRP78, ATF6α, ATF4, XBP1, and CCAAT/enhancer-binding protein homologous protein. The effects of CFZ on ER stress and apoptosis were lower than that of BZ. Nevertheless, CFZ and BZ synergistically induced ER stress and apoptosis in B16-F1 cells. Furthermore, the combinational pharmacological interactions of BZ and CFZ against the growth of B16-F1 melanoma cells were assessed by calculating the combination index and dose-reduction index with the CompuSyn software. We found that the combination of CFZ and BZ at submaximal concentrations could obtain dose reduction by exerting synergistic inhibitory effects on cell growth. Moreover, this drug combination reduced tumor growth in C57BL/6 syngeneic mice. Taken together, these results suggest that CFZ in combination with BZ may be a beneficial and potential strategy for melanoma treatment.

Genotoxicity and acute toxicity evaluation of the three amino acid additives with Corynebacterium glutamicum biomass.

l-threonine, l-tryptophan and l-valine play a fundamental role in animal and human nutrition as essential amino acids required for normal growth. In addition, each amino acid is codified as a generally recognized as safe (GRAS) amino acid for the use in animal feed additives and presents no exposure risk from animal to humans consuming tissues or products from the target animal. Taking into account the important role of mutagenicity and genotoxicity in the risk of the three amino acid additives (l-threonine, l-tryptophan, and l-valine) fermentation products and other unknown impurities and derivatives from Corynebacterium glutamicum (C. glutamicum), the safety evaluation of these amino acid additives is not performed. Therefore, the purpose of this study is to evaluate toxicological effects, including Ames test, an in vitro mammalian chromosomal aberration test and an acute oral animal toxicity of the three amino acid additives in accordance with the Organisation for Economic Co-operation and Development (OECD) guidelines and the principles of Good Laboratory Practice (GLP). As a result, these amino acid additives were classified as non-mutagenic and non-clastogenic, and did not induce any toxicity in acute oral toxicity test. Collectively, these results suggest that the three amino acid additives are safe with no adverse effects, and able to be applied as an ingredient or other biological uses.

Effect of Early Sacral Neuromodulation on Bladder Function in a Rat Model of Incomplete Spinal Cord Injury Due to Focal Contusion.

OBJECTIVES: Incomplete spinal cord injury (SCI) accounts for two-thirds of all SCIs in clinical practice. Preclinical research on the effect of sacral neuromodulation (SNM) on bladder function, however, has been focused only on animal models of complete SCI. We aimed to evaluate the effect of early SNM on bladder responses in a rat model of incomplete SCI. MATERIALS AND METHODS: Altogether, 21 female Sprague-Dawley rats were equally assigned to control (CTR), SCI + sham stimulation (SHAM), and SCI + SNM (SNM) groups. In the SHAM and SNM groups, incomplete SCI was created by producing a moderate contusion with an NYU-MASCIS impactor at the T9-T10 level of the spine, with needle electrodes implanted bilaterally into the S2 or S3 sacral foramen. Only SNM group underwent electrical stimulation for 28 days, beginning on day 7 after SCI. Cystometry was performed 35 days after SCI. RESULTS: Although the interval between voiding contractions was significantly longer in the SHAM group than the CTR group (25.5 ± 1.4 vs. 12.5 ± 1.7 min; p < 0.05), there were no significant differences between the SNM group (16.5 ± 1.5 min) and the CTR group. Maximum voiding contraction pressure did not differ among the groups. The SNM group had a significantly lower frequency (3.5 ± 0.5 vs. 14.6 ± 2.0; p < 0.05) and maximum pressure (11.4 ± 6.2 vs. 21.3 ± 1.8 cmH2 O; p < 0.05) of nonvoiding contractions than the SHAM group. CONCLUSIONS: Our results provide experimental evidence that early SNM treatment may prevent or diminish bladder dysfunctions (e.g., detrusor overactivity, abnormal micturition reflex) in a clinical condition of incomplete SCI.

Outcomes of magnetic resonance imaging fusion-targeted biopsy of prostate imaging reporting and data system 3 lesions.

PURPOSE: To evaluate the characteristics and histological outcomes in patients with Prostate Imaging Reporting and Data System (PI-RADS) 3 lesions undergoing magnetic resonance imaging-guided fusion-targeted biopsy (MRIFTB). METHODS: We retrospectively reviewed 138 patients with PI-RADS category 3 lesions classified using multiparametric MRI who underwent MRIFTB between May 2016 and March 2018. The study population included biopsy-naïve and patients with prior negative biopsy. Univariate and multivariate analyzes were performed to determine significant predictors of prostate cancer (PCa) and clinically significant prostate cancer (csPCa). The definition of csPCa was set at Gleason score ≥ 3 + 4. RESULTS: Overall, 114 (82.6%) biopsied lesions were benign and 24 (17.4%) were identified as prostate cancer. Of these 24 lesions, 14 (58.3%) harbored csPCa. Peripheral zone (PZ) lesions were more likely to be associated with malignant disease than transition zone lesions (13.7 vs. 6.2%). Multivariate logistic analysis revealed that age, PZ location, and prostate-specific antigen (PSA) density (P < 0.05) were independent predictors of both PCa and csPCa. CONCLUSIONS: A non-negligible number of PI-RADS 3 patients harbor csPCa. Moreover, age, lesion location, and PSA density could be potential clinical predictors of PCa and csPCa. Physicians should be aware of the cancer prevalence of PI-RADS 3 lesions, as the use of the aforementioned factors can help in the decision-making process for these patients.

Outcomes of men aged ≤50 years treated with radical prostatectomy: a retrospective analysis.

Previous studies investigating prostate cancer (PCa) features in younger men have reported conflicting findings. This study aimed to investigate pathologic outcomes and biochemical recurrence (BCR) status in younger men who underwent radical prostatectomy (RP) for PCa. Records of 2057 patients who underwent RP at Seoul National University Bundang Hospital (Seongnam, Korea) between 2006 and 2015 were reviewed; patients were divided according to age into the younger and older groups (men aged ≤50 and >50 years, respectively). Postoperative BCR status and functional outcomes and clinicopathologic features were compared between both groups. All analyses were repeated after propensity score matching. Younger men were more likely to have low-risk disease (P < 0.001), lower pathologic Gleason score (P < 0.001) and pathologic stages (P < 0.001) than older men. The pathologic Gleason score (P = 0.002) and rates of extracapsular extension (P = 0.004) were lower in younger men after propensity score matching. In multivariate analysis, age at RP was not an independent predictor of BCR-free survival after RP (P = 0.669). Moreover, at 1 year after RP, younger men with preoperative 5-item International Index of Erectile Function score ≥22 (n = 228) showed more favorable results for urinary continence (defined as nonuse of pads daily) (99.4% vs 95%, P = 0.009) and erections sufficient for vaginal intercourse (81.8% vs 55.5%, P = 0.001). Younger men had more favorable clinicopathologic features at RP than their older counterparts. Although age was not an independent predictor of BCR status outcome, younger men had better functional outcomes following RP.

Phenyl 2-pyridyl ketoxime induces cellular senescence-like alterations via nitric oxide production in human diploid fibroblasts.

Phenyl-2-pyridyl ketoxime (PPKO) was found to be one of the small molecules enriched in the extracellular matrix of near-senescent human diploid fibroblasts (HDFs). Treatment of young HDFs with PPKO reduced the viability of young HDFs in a dose- and time-dependent manner and resulted in senescence-associated ß-galactosidase (SA-ß-gal) staining and G2/M cell cycle arrest. In addition, the levels of some senescence-associated proteins, such as phosphorylated ERK1/2, caveolin-1, p53, p16(ink4a), and p21(waf1), were elevated in PPKO-treated cells. To monitor the effect of PPKO on cell stress responses, reactive oxygen species (ROS) production was examined by flow cytometry. After PPKO treatment, ROS levels transiently increased at 30 min but then returned to baseline at 60 min. The levels of some antioxidant enzymes, such as catalase, peroxiredoxin II and glutathione peroxidase I, were transiently induced by PPKO treatment. SOD II levels increased gradually, whereas the SOD I and III levels were biphasic during the experimental periods after PPKO treatment. Cellular senescence induced by PPKO was suppressed by chemical antioxidants, such as N-acetylcysteine, 2,2,6,6-tetramethylpiperidinyloxy, and L-buthionine-(S,R)-sulfoximine. Furthermore, PPKO increased nitric oxide (NO) production via inducible NO synthase (iNOS) in HDFs. In the presence of NOS inhibitors, such as L-NG-nitroarginine methyl ester and L-NG-monomethylarginine, PPKO-induced transient NO production and SA-ß-gal staining were abrogated. Taken together, these results suggest that PPKO induces cellular senescence in association with transient ROS and NO production and the subsequent induction of senescence-associated proteins.