Jae-Woo Cho

Dose-response Effects of Bleomycin on Inflammation and Pulmonary Fibrosis in Mice

Many studies have reported that bleomycin, anti-cancer drug, induces pulmonary fibrosis as a side effect. However, few investigations have focused on the dose-response effects of bleomycin on pulmonary fibrosis. Therefore, in the present study, we investigated the effects of different doses of bleomycin in male mice. ICR mice were given 3 consecutive doses of bleomycin: 1, 2, or 4 mg/kg in bleomycin-treated (BT) groups and saline only in vehicle control (VC) groups. The animals were sacrificed at 7 and 24 days postinstillation. The severity of pulmonary fibrosis was evaluated according to inflammatory cell count and lactate dehydrogenase (LDH) activity in the broncho alveolar lavage fluid (BALF) , and lung tissues were histologically evaluated after hematoxylin and eosin (H&E) , and Masson’s trichrome staining. BT groups exhibited changed cellular profiles in BAL fluid compared to the VC group, which had an increased number of total cells, neutrophils, and lymphocytes and a modest increase in the number of macrophages at 7 days post-bleomycin instillation. Moreover, BT groups showed a dose-dependent increase in LDH levels and inflammatory cell counts. However, at 24 days after treatment, collagen deposition, interstitial thickening, and granulomatous lesions were observed in the alveolar spaces in addition to a decrease in inflammatory cells. These results indicate that pulmonary fibrosis induced by 4 mg/kg bleomycin was more severe than that induced by 1 or 2 mg/kg. These data will be utilized in experimental animal models and as basic data to evaluate therapeutic candidates through non-invasive monitoring using the pulmonary fibrosis mouse model established in this study.

Studies on N-Ethyl-N-nitrosourea Mutagenesis in BALB/c Mice

N-ethyl-N-nitrosoures (ENU) is effective in inducing hypermorphic mutation as well as hypomorphic and antimorphic mutations. Therefore, this mutagen is used to the production of mutant in the mice. In order to perform an effective ENU mutagenesis using BALB/cAnN mice, determination of optimal dosage and dosage regimen of ENU is necessary. And this study tried to develop a suitable screening method and searched for novel and various mutants as model animals in phenotypedriven ENU mutagenesis. We have carried out dosage regimen for mutagenizing dose of 200 mg/kg ENU in the BALB/c mice. Total screened mice were 30,133. As the results of Esaki and Cho’s Phenotype Screening, we got 2,516 phenotypic and behavior abnormalities in G1, G2 and G3 mice. One hundred thirty five G1 phenodeviants were tested for inheritance and 16 dominant mutants were discovered. Forty two recessive mutants were also found in tested 201 micropedigrees. Early-onset mutant mice included the dysmorphology of face, eye, tail, limb, skin, and foot and abnormal behavior like circling, swimming, head tossing, stiff-walking, high cholesterol level, and tremor etc. In this study we could effectively screen G3 recessive mutants. The frequent and concise early-onset screening before weaning will be available for ENU mutagenesis.

Studies on the Small Body Size Mouse Developed by Mutagen N-Ethyl-N-nitrosourea

Mutant mouse which show dwarfism has been developed by N-ethyl-N-nitrosourea (ENU) mutagenesis using BALB/c mice. The mutant mouse was inherited as autosomal recessive trait and named Small Body Size (SBS) mouse. The phenotype of SBS mouse was not apparent at birth, but it was possible to distinguish mutant phenotype from normal mice 1 week after birth. In this study, we examined body weight changes and bone mineral density (BMD), and we also carried out genetic linkage analysis to map the causative gene(s) of SBS mouse. Body weight changes were observed from birth to 14 weeks of age in both affected (n = 30) and normal mice (n = 24). BMD was examined in each five SBS and normal mice between 3 and 6 weeks of age, respectively. For the linkage analysis, we produced backcross progeny [(SBS × C57BL/6J) F1 × SBS] N2 mice (n = 142), and seventy-four microsatellite markers were used for primary linkage analysis. Body weight of affected mice was consistently lower than that of the normal mice, and was 43.7% less than that of normal mice at 3 weeks of age (P < 0.001). As compared with normal mice at 3 and 6 weeks of age, BMD of the SBS mice was significantly low. The results showed 15.5% and 14.1% lower in total body BMD, 15.3% and 8.7% lower in forearm BMD, and 29.7% and 20.1% lower in femur BMD, respectively. The causative gene was mapped on chromosome 10. The map order and the distance between markers were D10Mit248 - 2.1 cM - D10Mit51 - 4.2 cM - sbs - 0.7 cM - D10Mit283 - 1.4 cM - D10Mit106 - 11.2 cM - D10Mit170.

Genomic and proteomic analyses of 1,3-dinitrobenzene-induced testicular toxicity in Sprague–Dawley rats

1,3-Dinitrobenzene (DNB) is an industrial intermediate and testicular toxicant that has been shown to target Sertoli cells. The mechanism of action of DNB in the testis, however, is unclear. To investigate global alterations in gene or protein expression during testicular toxicity, testes from rats treated orally with DNB were subjected to microarray and two-dimensional gel electrophoresis (2-DE) analyses. Histopathological abnormalities were detected in the testes of the DNB-treated rats. Microarray analysis revealed that, during early testicular toxicity, several genes involved in apoptosis, germ cell/Sertoli cell junction, and tight junction signaling pathways were differentially expressed. Based on 2-DE analysis, 36 protein spots showing significantly different expression during early testicular toxicity were selected and identified. Network analysis of the identified proteins revealed that these proteins are associated with cellular development or reproductive system diseases. Collectively, these data will help clarify the molecular mechanism underlying testicular toxicity in DNB-exposed rats.

Pulmonary Toxicity and Recovery from Inhalation of Manual Metal Arc Stainless Steel Welding Fumes in Rats

The objectives of this study were to examine the lung injury and inflammation caused by manual metal arc stainless steel (MMA-SS) welding fume inhalation and to evaluate the recovery process. Sprague-Dawley rats were exposed to MMA-SS welding fumes for 2 h per day in a whole-body exposure chamber, with a total suspended particulate (TSP) concentration of 51.4 ± 2.8 mg/m3 (low dose) or 84.6 ± 2.9 mg/m3 (high dose) for 30 days. The animals were sacrificed after 30 days of exposure as well as after a 30-day recovery period. To assess the inflammatory or injury responses, cellular and biochemical parameters as well as cytokines were assayed in the bronchoalveolar lavage fluid (BALF). MMA-SS welding fume exposure led to a significant elevation in the number of alveolar macrophages (AM) and polymorphonuclear cells (PMN). Additionary, the values of β-nacetyl glucosaminidase (β-NAG) and lactate dehydrogenase (LDH) in the BALF were increased in the exposed group when compared to controls. After 30 days of recovery from exposure, a significant reduction in inflammatory parameters of BALF was observed between the exposed and recovered groups. Slight, but significant elevations were noted in the number of AM and PMN in the recovered groups, and AM that had been ingested fume particles still remain in the lungs. In conclusion, these results indicated that welding fumes induced inflammatory responses and cytotoxicity in the lungs of exposed rats. Fume particles were not fully cleared from lungs even after a 30-day recovery period.

Analysis of Gene Expression in 4,4'-Methylenedianiline-induced Acute Hepatotoxicity

4,4′-Methylenedianiline (MDA) is an aromatic amine that is widely used in the industrial synthetic process. Genotoxic MDA forms DNA adducts in the liver and is known to induce liver damage in human and rats. To elucidate the molecular mechanisms associated with MDA-induced hepatotoxicity, we have identified genes differentially expressed by microarray approach. BALB/c male mice were treated once daily with MDA (20 mg/kg) up to 7 days via intraperitoneal injection (i.p.) and hepatic damages were revealed by histopathological observation and elevation of serum marker enzymes such as AST, ALT, ALP, cholesterol, DBIL, and TBIL. Microarray analysis showed that 952 genes were differentially expressed in the liver of MDA-treated mice and their biological functions and canonical pathways were further analyzed using Ingenuity Pathways Analysis (IPA). Toxicological functional analysis showed that genes related to hepatotoxicity such hyperplasia/hyperproliferation (Timpl), necrosis/cell death (Cd14, Mt1f, Timpl, and Pmaipl), hemorrhaging (Mt1f), cholestasis (Akr1c3, Hpx, and Slc10a2), and inflammation (Cd14 and Hpx) were differentially expressed in MDA-treated group. This gene expression profiling should be useful for elucidating the genetic events associated with aromatic amine-induced hepatotoxicity and for discovering the potential biomarkers for hepatotoxicity.

Characteristics of Ethylnitrosourea-Induced Cataracts

Purpose: This study analyzed genes associated with the morphology and regulation of ethylnitrosourea (ENU)-induced cataract mouse. Materials and Methods: Immunohistochemistry analysis using anti-crystallins and PCNA antibody revealed that the localization pattern of these specific markers differed between the cataractous and wild-type lens epithelium. Two-dimensional electrophoresis and microarray techniques were used to identify the proteins and genes related to ENU-induced cataract. Results: A novel ENU-induced mutation in the mouse led to nuclear and cortical opacity of the eye lens at 5 weeks postnatal. This cataract phenotype was similar to that of the zonular-pulverulent type of human cataract. Crystallin proteins and gap-junction genes have relations to the formation of cataract. Conclusions: Together, the results suggest that various proteins affect the formation and specific phenotypes of ENU-induced cataract mouse.

Higher incidence of sperm granuloma in the epididymis of C57BL/6N mice

C57BL/6N mice are inbred strains widely used in biomedical research. Hence, a large amount of basic data has been accumulated. However, in the field of histopathology, spontaneous data for relatively younger mice that are used more frequently are not yet abundant, in contrast to data for older mice and their neoplastic lesions. To acquire the essential background data required by various research and toxicological assessments, 120 mice of the C57BL/6N strain (10 and 13 weeks of age) were collected from two institutions (From Korea and Japan) and subjected to histopathological analyses of the major organs (liver, spleen, kidney, thymus, heart, testis, epididymis). The results showed significantly higher incidence of sperm granulomas in the epididymides (10-56%) of these mice, compared with that in other strains or species of lab animals. Upon closer inspection, oligospermia/clear cell hyperplasia, cellular debris, and tubular vacuolation were also observed in the epididymides with sperm granulomas. Moreover, diseased organs were significantly heavier than healthy ones. Immunohistochemical staining showed a significant increase in the chromatic figures of cysteine-dependent aspartate-directed proteases-3 (caspase-3) and cleaved-poly(ADP-ribose) polymerase (c-PARP), and damages to the tubule due to spontaneous apoptosis, which may have led to the sperms leaking out of the tubule, causing the granuloma. To conclude, spontaneous sperm granuloma can occur in 10- and 13-week-old C57BL/6N mice and may thus affect the results of various studies using these mice. Therefore, sperm granuloma in epididymis needs to be carefully considered as an important factor when design the study using C57BL/6N.

Low-power and low-drug-dose photodynamic chemotherapy via the breakdown of tumor-targeted micelles by reactive oxygen species

ABSTRACT Tumor‐targeted delivery of anticancer agents using nanocarriers has been explored to increase the therapeutic index of cancer chemotherapy. However, only a few nanocarriers are clinically available because the physiological complexity often compromises their ability to target, penetrate, and control the release of drugs. Here, we report a method which dramatically increases in vivo therapeutic drug efficacy levels through the photodynamic degradation of tumor‐targeted nanocarriers. Folate‐decorated poly(ethylene glycol)‐polythioketal micelles are prepared to encapsulate paclitaxel and porphyrins. Photo‐excitation generates reactive oxygen species within the micelles to cleave the polythioketal backbone efficiently and facilitate drug release only at the illuminated tumor site. Intravenous injection of a murine xenograft model with a low dose of paclitaxel within the micelles, one‐milligram drug per kg (mouse), corresponding to an amount less than that of Taxol by one order of magnitude, induces dramatic tumor regression without any acute systemic inflammation responses or organ toxicity under low‐power irradiation (55 mW cm−2) at 650 nm. Graphical abstract Figure. No caption available. HighlightsPhotodynamically‐induced drug delivery under low light intensity and low drug doseROS‐scavenging micelles releasing paclitaxel under 650 nm light irradiationDramatic reduction of IC50 of paclitaxel to 4.27% of the reported valueEfficient ROS consumption by PPADT micellesOnly 5% of maximum tolerated dose of paclitaxel for tumor suppression

Tannin–Titanium Oxide Multilayer as a Photochemically Suppressed Ultraviolet Filter

UV filters can initiate redox reactions of oxygen and water when exposed to sunlight, generating reactive oxygen species (ROS) that deteriorate the products containing them and cause biological damages. This photochemical reactivity originates from the high chemical potential of UV filters, which also determines the optical properties desirable for sunscreen applications. We hypothesize that this dilemma can be alleviated if the photochemical pathway of UV filters is altered to coupling with redox active molecules. Here, we employ tannic acid (TA) as a key molecule for controlling the photochemical properties of titanium dioxide nanoparticles (TiO2 NPs). TA provides an unusual way for layer-by-layer assembly of TiO2 NPs by the formation of a ligand-to-metal charge transfer complex that alters the nature of UV absorption of TiO2 NPs. The galloyl moieties of TA efficiently scavenge ROS due to the stabilization of ROS by intramolecular hydrogen bonding while facilitating UV screening through direct charge injection from TA to the conduction band of TiO2. The TiO2-TA multilayers assembled in open porous polymer microspheres substantially increased sun protection while dramatically reducing ROS under UV exposure. The assembled structure exhibits excellent in vivo anti-UV skin protection against epidermal hyperplasia, inflammation, and keratinocyte apoptosis without long-term toxicity.