Preclinical Evaluation of interferon-gamma primed human Wharton's jelly-derived mesenchymal stem cells.

The potential of human mesenchymal stem cells (MSCs) for cell therapy has been investigated in numerous immune-mediated conditions; MSCs are considered one of the most promising cellular therapeutics to treat intractable diseases. Recently, approaches to prime MSCs have been investigated, thereby generating cellular products with enhanced potential for a variety of clinical applications. Interferon-gamma (IFN-γ) priming is a current approach used to increase the therapeutic efficacy of MSCs. In this study, we determined the systemic toxicity, tumorigenicity and biodistribution of IFN-γ-primed Wharton's jelly-derived (WJ)-MSCs in male and female BALB/c-nu/nu mice. There were no deaths or pathologic lesions in the mice treated with 5 × 106 cells/kg IFN-γ-primed MSCs in the repeated dose study. In the tumorigenicity study, one of the subcutaneously treated mice showed bronchioloalveolar adenoma in the lung but tested negative for human-specific anti-mitochondrial antibody, suggesting the spontaneous murine origin of the adenoma. A biodistribution study using real-time quantitative polymerase chain reaction demonstrated the systemic IFN-γ-primed MSC clearance by day 28. Based on the toxicity, biodistribution, and tumorigenicity studies, we concluded that IFN-γ-primed MSCs at 5 × 106 cells/kg do not induce tumor formation and adverse changes.

Preclinical safety assessment of the suction-assisted intradermal injection of the SARS-CoV-2 DNA vaccine candidate pGO-1002 in white rabbit.

pGO-1002, a non-viral DNA vaccine that expresses both spike and ORF3a antigens of SARS-CoV-2, is undergoing phase 1 and phase 2a clinical trials in Korea and the US. A preclinical repeated-dose toxicity study in New Zealand white rabbits in compliance with Good Laboratory Practice (GLP) was conducted to assess the potential toxicity, local tolerance, and immunogenicity of the vaccine and GeneDerm suction device. The dose rate was 1.2 mg/head pGO-1002, and this was administered intradermally to a group of animals (eight animals/sex/group) three times at 2-week intervals, followed by a 4-week recovery period. After each administration, suction was applied to the injection site using the GeneDerm device. Mortality, clinical signs, body weight, food consumption, skin irritation, ophthalmology, body temperature, urinalysis, and clinical pathology were also monitored. Gross observations and histopathological evaluation were performed. Overall, pGO-1002 administration-related changes were confined to minor damage or changes at the injection site, increased spleen weight and minimal increased cellularity in white pulp. All changes of injection site were considered local inflammatory changes or pharmacological actions due to the vaccine with the changes in spleen considered consistent with vaccine-induced immune activation. All findings showed reversibility during the 4-week recovery period. Animals vaccinated with pGO-1002, administered by intradermal injection and followed by application of suction with GeneDerm, developed humoral and cellular responses against the SARS-CoV-2 antigens consistent with prior studies in rats. Collectively, it was concluded that the pGO-1002 vaccine was safe and effective under these experimental conditions and these data supported future human study of the vaccine, now known as GLS-5310, for clinical trial use.

Tumorigenicity Assessment of Human Cancer Cell Lines Xenografted on Immunodeficient Mice as Positive Controls of Tumorigenicity Testing.

Recent advances in human pluripotent stem cell (hPSC)-derived cell therapies and genome editing technologies such as CRISPR/Cas9 make regenerative medicines promising for curing diseases previously thought to be incurable. However, the possibility of off-target effects during genome editing and the nature of hPSCs, which can differentiate into any cell type and infinitely proliferate, inevitably raises concerns about tumorigenicity. Tumorigenicity acts as a major obstacle to the application of hPSC-derived and gene therapy products in clinical practice. Thus, regulatory authorities demand mandatory tumorigenicity testing as a key pre-clinical safety step for the products. In the tumorigenicity testing, regulatory guidelines request to include human cancer cell line injected positive control group (PC) animals, which must form tumors. As the validity of the whole test is determined by the tumor-forming rates (typically above 90%) of PC animals, establishing the stable tumorigenic condition of PC animals is critical for successful testing. We conducted several studies to establish the proper positive control conditions, including dose, administration routes, and the selection of cell lines, in compliance with Good Laboratory Practice (GLP) regulations and/or guidelines, which are essential for pre-clinical safety tests of therapeutic materials. We expect that our findings provide insights and practical information to create a successful tumorigenicity test and its guidelines.

Application of Hexanoyl Glycol Chitosan as a Non-cell Adhesive Polymer in Three-Dimensional Cell Culture.

Cell culture technology has evolved into three-dimensional (3D) artificial tissue models for better reproduction of human native tissues. However, there are some unresolved limitations that arise due to the adhesive properties of cells. In this study, we developed a hexanoyl glycol chitosan (HGC) as a non-cell adhesive polymer for scaffold-based and -free 3D culture. The uniform cell distribution in a porous scaffold was well maintained during the long culutre period on the HGC-coated substrate by preventing ectopic adhesion and migration of cells on the substrate. In addition, when culturing many spheroids in one dish, supplementation of the culture medium with HGC prevented the aggregation of spheroids and maintained the shape and size of spheroids for a long culture duration. Collectively, the use of HGC in 3D culture systems is expected to contribute greatly to creating excellent regenerative therapeutics and screening models of bioproducts.

Development of human pluripotent stem cell-derived hepatic organoids as an alternative model for drug safety assessment.

Human in vitro hepatic models that faithfully recapitulate liver function are essential for successful basic and translational research. A limitation of current in vitro models, which are extensively used for drug discovery and toxicity testing, is the loss of drug metabolic function due to the low expression and activity of cytochrome P450 (CYP450) enzymes. Here, we aimed to generate human pluripotent stem cell-derived hepatic organoids (hHOs) with a high drug metabolic ability. We established a two-step protocol to produce hHOs from human pluripotent stem cells for long-term expansion and drug testing. Fully differentiated hHOs had multicellular composition and exhibited cellular polarity and hepatobiliary structures. They also displayed remarkable CYP450 activity and recapitulated the metabolic clearance, CYP450-mediated drug toxicity, and metabolism. Furthermore, hHOs successfully modeled Wilson's disease in terms of Cu metabolism, drug responses, and diagnostic marker expression and secretion. In conclusion, hHOs exhibit high capacity for drug testing and disease modeling. Hence, this hepatic model system provides an advanced tool for studying hepatic drug metabolism and diseases.

Preclinical assessment of thrombin-preconditioned human Wharton's jelly-derived mesenchymal stem cells for neonatal hypoxic-ischaemic brain injury.

Hypoxic-ischaemic encephalopathy (HIE) is a type of brain injury affecting approximately 1 million newborn babies per year worldwide, the only treatment for which is therapeutic hypothermia. Thrombin-preconditioned mesenchymal stem cells (MSCs) exert neuroprotective effects by enriching cargo contents and boosting exosome biogenesis, thus showing promise as a new therapeutic strategy for HIE. This study was conducted to evaluate the tissue distribution and potential toxicity of thrombin-preconditioned human Wharton's jelly-derived mesenchymal stem cells (th-hWJMSCs) in animal models before the initiation of clinical trials. We investigated the biodistribution, tumorigenicity and general toxicity of th-hWJMSCs. MSCs were administered the maximum feasible dose (1 × 105 cells/10 µL/head) once, or at lower doses into the cerebral ventricle. To support the clinical use of th-hWJMSCs for treating brain injury, preclinical safety studies were conducted in newborn Sprague-Dawley rats and BALB/c nude mice. In addition, growth parameters were evaluated to assess the impact of th-hWJMSCs on the growth of newborn babies. Our results suggest that th-hWJMSCs are non-toxic and non-tumorigenic in rodent models, survive for up to 7 days in the brain and hold potential for HIE therapy.

Genotoxicity and subchronic general toxicity assessments of Lactobacillus curvatus WiKim 38 using Sprague-Dawley rats.

Lactobacillus curvatus WiKim 38 (LCW), isolated from kimchi, has shown novel immunomodulatory and anti-inflammatory properties. In the present study, to obtain data on the safety of LCW, we performed three genotoxicity (bacterial reverse mutation, chromosome aberration, and micronucleus) and two general toxicity (single-dosing and 13-week repeated-dosing) studies. In the genotoxicity assessment, LCW showed no increased reverse mutation for 4 strains of Salmonella typhimurium and a strain of Escherichia coli. In addition, LCW did not induce chromosome aberrations at concentrations up to 5000 µg/mL in cultured Chinese hamster lung (CHL) cells and did not induce an increased frequency of micronuclei in the bone marrow cells of rats at concentrations up to 2000 mg/kg. In the acute toxicity study using Sprague-Dawley (SD) rats, the approximate lethal dose of LCW was determined to be over 5000 mg/kg body weight (b.w.) in both sexes. Finally, in the subchronic toxicity study, no LCW-related adverse effects were observed at concentrations up to 5000 mg/kg b.w./day. Consequently, LCW is considered not to have mutagenic effects, and its no-observed-adverse-effect-level (NOAEL) is 5000 mg/kg b.w., equivalent to approximately 4.71 × 109 CFU/kg b.w., suggesting the LCW could be a potential probiotic for humans based on its safety profile.

Effect of PM10 on pulmonary immune response and fetus development.

Despite numerous reports that ambient particulate matter is a key determinant for human health, toxicity data produced based on physicochemical properties of particulate matters is very lack, suggesting lack of scientific evidence for regulation. In this study, we sampled inhalable particulate matters (PM10) in northern Seoul, Korea. PM10 showed atypical- and fiber-type particles with the average size and the surface charge of 1,598.1 ± 128.7 nm and -27.5 ± 2.8, respectively, and various toxic elements were detected in the water extract. On day 90 after the first pulmonary exposure, total cell number dose-dependently increased in the lungs of both sexes of mice. PM10 induced Th1-dominant immune response with pathological changes in both sexes of mice. Meanwhile, composition of total cells and expression of proteins which functions in cell-to-cell communication showed different trends between sexes. Following, male and female mice were mated to identify effects of PM10 to the next generation. PM10 remained in the lung of dams until day 21 after birth, and the levels of IgA and IgE increased in the blood of dams exposed to the maximum dose compared to control. In addition, the interval between births of fetuses, the number of offspring, the neonatal survival rate (day 4 after birth) and the sex ratio seemed to be affected at the maximum dose, and particularly, all offspring from one dam were stillborn. In addition, expression of HIF-1α protein increased in the lung tissue of dams exposed to PM10, and level of hypoxia-related proteins was notably enhanced in PM10-exposed bronchial epithelial cells compared to control. Taken together, we suggest that inhaled PM10 may induce Th1-shifting immune response in the lung, and that it may affect reproduction (fetus development) by causing lung hypoxia. Additionally, we propose that further study is needed to identify particle-size-dependent effects on development of the next generation.

Inhaled underground subway dusts may stimulate multiple pathways of cell death signals and disrupt immune balance.

In this study, we aimed to identify a toxic mechanism and the potential health effects of ambient dusts in an underground subway station. At 24 h exposure to human bronchial epithelial (BEAS-2B) cells (0, 2.5, 10, and 40 µg/mL), dusts located within autophagosome-like vacuoles, whereas a series of autophagic processes appeared to be blocked. The volume, potential and activity of mitochondria decreased in consistent with a condensed configuration, and the percentage of late apoptotic cells increased accompanying S phase arrest. While production of reactive oxygen species, expression of ferritin (heavy chain) protein, secretion of IL-6, IL-8 and matrix metalloproteinases, and the released LDH level notably increased in dust-treated cells (40 µg/mL), intracellular calcium level decreased. At day 14 after a single instillation to mice (0, 12.5, 50, and 200 µg/head), the total number of cells increased in the lungs of dust-treated mice with no significant change in cell composition. The pulmonary levels of TGF-ß, GM-CSF, IL-12 and IL-13 clearly increased following exposure to dusts, whereas that of CXCL-1 was dose-dependently inhibited. Additionally, the population of cytotoxic T cells in T lymphocytes in the spleen increased relative to that of helper T cells, and the levels of IgA and IgM in the bloodstream were significantly reduced in the dust-treated mice. Subsequently, to improve the possibility of extrapolating our findings to humans, we repeatedly instilled dusts (1 time/week, 4 weeks, 0.25 and 1.0 mg/head) to monkeys. The total number of cells, the relative portion of neutrophils, the level of TNF-α significantly increased in the lungs of dust-treated monkeys, and the expression of cytochrome C was enhanced in the lung tissues. Meanwhile, the pulmonary level of MIP-α was clearly reduced, and the expression of caveolin-1 was inhibited in the lung tissues. More importantly, inflammatory lesions, such as granuloma, were seen in both mice and monkeys instilled with dusts. Taken together, we conclude that dusts may impair the host's immune function against foreign bodies by inhibiting the capacity for production of antibodies. In addition, iron metabolism may be closely associated with dust-induced cell death and inflammatory response.

Four-week repeated dose oral toxicity study of KDS2010, a novel selective monoamine oxidase B inhibitor, in Sprague Dawley rats.

Repeated dose oral toxicity and toxicokinetic of KDS2010, a new drug for Parkinson's disease, was investigated after 4-week repeated oral administration at 30, 50, 75, or 100 mg/kg/day in rats. Body weight and body weight gain decreased in rats of both sexes in the 75 and 100 mg/kg groups, and food consumption was reduced in male rats of the 75 and 100 mg/kg male groups. Histological alterations were observed in the kidney (urothelial hyperplasia, inflammatory cell infiltration in the renal pelvis, tubular vacuolation/degeneration, basophilic tubules, and hyaline droplets in the proximal tubules) of the 75 and 100 mg/kg male groups and the 50 and 100 mg/kg female groups. The 75 and 100 mg/kg male groups showed adverse effect in the testes (degeneration/exfoliation of germ cells, seminiferous tubules atrophy) and epididymis (cellular debris, oligospermia). These changes were partially recovered after a 2-week recovery period. However, basophilic tubules and hyaline droplets in the proximal tubules in the kidney and germ cell degeneration/exfoliation in the testis were not recovered. In toxicokinetics study, systemic exposure to KDS2010 increased proportionally in both sexes by in a dose -dependent manner. In addition, repeated administration for 4 weeks led to increased tendency of systemic exposure in both sexes compared with that in Day 1. In conclusion, KDS2010 was shown to target the kidney and testis with a no-observed-adverse-effect level of 50 and 30 mg/kg/day for males and females, respectively.

Formation of lamellar body-like structure may be an initiator of didecyldimethylammonium chloride-induced toxic response.

Due to the pandemic of coronavirus disease 2019, the use of disinfectants is rapidly increasing worldwide. Didecyldimethylammonium chloride (DDAC) is an EPA-registered disinfectant, it was also a component in humidifier disinfectants that had caused idiopathic pulmonary diseases in Korea. In this study, we identified the possible pulmonary toxic response and mechanism using human bronchial epithelial (BEAS-2B) cells and mice. First, cell viability decreased sharply at a 4 µg/mL of concentration. The volume of intracellular organelles and the ROS level reduced, leading to the formation of apoptotic bodies and an increase of the LDH release. Secretion of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and matrix metalloproteinase-1 also significantly increased. More importantly, lamellar body-like structures were formed in both the cells and mice exposed to DDAC, and the expression of both the indicator proteins for lamellar body (ABCA3 and Rab11a) and surfactant proteins (A, B, and D) was clearly enhanced. In addition, chronic fibrotic pulmonary lesions were notably observed in mice instilled twice (weekly) with DDAC (500 µg), ultimately resulting in death. Taken together, we suggest that disruption of pulmonary surfactant homeostasis may contribute to DDAC-induced cell death and subsequent pathophysiology and that the formation of lamellar body-like structures may play a role as the trigger. In addition, we propose that the cause of sudden death of mice exposed to DDAC should be clearly elucidated for the safe application of DDAC.

KMRC011, an agonist of toll-like receptor 5, mitigates irradiation-induced tissue damage and mortality in cynomolgus monkeys.

In the study here, the potential applicability of KMRC011 - an agonist of toll-like receptor-5 - as a countermeasure for radiation toxicities was evaluated. Following a single 5.5 Gy total body irradiation (TBI, surface absorbed dose = 7 Gy) of Co60 γ-rays, mortality rates and degrees of pathological lesions that developed over 80 days were compared in monkeys that received TBI only and a group that was injected once with KMRC011 (10 µg/kg) after TBI. Compared to the TBI-only hosts (80%), the death rate was significantly improved by the use of KMRC011 (40%), all deaths in both groups occurred in the period from Days 19-24 post-TBI. Further analysis of monkeys that survived until the end of the experiment showed that AST and ALT levels were elevated only in the TBI group, and that radiation-induced tissue damage was alleviated by the KMRC011 injection. Additionally, expression of cell death-related proteins was lower in tissues from the KMRC011-treated hosts than in those in the TBI-only group. Other measured parameters, including body weight, food uptake, and hematological values did not significantly differ between the two groups over the entire period. The results of this study, thus demonstrate that KMRC011 could potentially be used as a medical countermeasure for the treatment of acute radiation exposure.

Repeated-oral dose toxicity of polyethylene microplastics and the possible implications on reproduction and development of the next generation.

With the increased distribution of microplastics in the environment, the potential for harmful effects on human health and ecosystems have become a global concern. Considering that polyethylene microplastics (PE-MPs) are among the most produced plastics worldwide, we administered PE-MPs (0.125, 0.5, 2 mg/day/mouse) by gavage to mice (10 mice/sex/dose) for 90 days. Compared to control, the body weight gain was significantly reduced in the male mice, and the proportion of neutrophils in the blood stream clearly increased in both sexes of mice. Persistence of a PE-MPs-like material and migration of granules to the mast cell membrane and accumulation of damaged organelles were observed in the stomachs and the spleens from the treated dams, respectively. Additionally, the IgA level in the blood stream was significantly elevated in the dams administered with PE-MPs compared to control, and the subpopulation of lymphocytes within the spleen was altered. Following, we performed an additional study to screen the effects of PE-MPs on reproduction and development (5 mice/sex/dose). Importantly, number of live births per dam, the sex ratio of pups, and body weight of pups was notably altered in groups treated with PE-MPs compared to the control group. Additionally, PE-MPs affected the subpopulation of lymphocytes within the spleen of the offspring, as did in the dams. Therefore, we propose that reproductive and developmental toxicity testing is warranted to evaluate the safety of microplastics. Additionally, we suggest that the IgA level may be used as a biomarker for harmful effects following exposure on microplastics.

In vitro skin irritation assessment using EpiDerm™: applicability for updating toxicity information of oxybenzone and N,N-diethyl-m-toluamide.

A skin irritation test using in vitro reconstructed human epidermis (RhE) models was established for hazard identification of irritant chemicals in accordance with UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) category. In this study, EpiDerm™ was used to assess skin irritation by oxybenzone and N,N-diethyl-m-toluamide (DEET), which are widely used sunscreen and insect repellent components, respectively. EpiDerm™ was applied with oxybenzone and DEET, combined and sequentially with each single dose. Epidermal morphology and differentiation/proliferation were examined microscopically. Oxybenzone and sequential administration groups were determined as nonirritant with cell viability >50% and the morphology was comparable to the human epidermis. Contrastingly, the DEET and coadministration groups exhibited cell viability <50% and poor epidermal morphology. Interleukin (IL)-1α release from substance-treated EpiDerm™ increased inversely to cell viability, suggesting the pro-inflammatory reaction was initiated by DEET. CK-10, E-cadherin, Ki-67, laminin, and ceramide were identified as relevant markers to assess oxybenzone- or DEET-induced epidermal injury. In conclusion, these results may indicate to be aware of the possible skin irritation by indiscriminate use of oxybenzone and DEET without animal testing.

Inhaled Kathon may induce eosinophilia-mediated disease in the lung.

In 2011, a link between humidifier disinfectants and patients with idiopathic pulmonary fibrosis was identified in Korea, and Kathon was suggested as one of the causative agents. In this study, Kathon induced apoptotic cell death along with membrane damage at 24 h post-exposure. Additionally, on day 14 after a single instillation with Kathon, the total number of pulmonary cells and the levels of TNF-α, IL-5, IL-13, MIP-1α, and MCP-1α clearly increased in the lung of mice. The proportion of natural killer cells and eosinophils were significantly elevated in the spleen and the bloodstream, respectively, and the level of immunoglobulin (Ig) A, but not IgG, IgM, and IgE, dose-dependently increased. Therefore, we suggest that inhaled Kathon may induce eosinophilia-mediated disease in the lung by disrupting homeostasis of pulmonary surfactants. Considering that eosinophilia is closely related to cancer and fibrosis, further studies are needed to understand the relationship between them.

Subacute dermal toxicity of perfluoroalkyl carboxylic acids: comparison with different carbon-chain lengths in human skin equivalents and systemic effects of perfluoroheptanoic acid in Sprague Dawley rats.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are used in various fields but raise concerns regarding human health and environmental consequences. Among PFASs, perfluorooctanoic acid (PFOA) and short-chain perfluoroalkyl carboxylic acids (SC PFCAs) are detectable in skin-contact consumer products and have dermal absorption potential. Here, we investigated the effects of dermal exposure to PFOA and SC PFCAs using in vitro and in vivo models. Human skin equivalents were topically treated with 0.25 mM and 2.5 mM PFOA and SC PFCAs (perfluoropentanoic acid, PFPeA; perfluorohexanoic acid, PFHxA; and perfluoroheptanoic acid, PFHpA) for 6 days, and cell viability, interleukin (IL)-1α, oxidative stress markers (malondialdehyde, MDA; and 8-hydroxydeoxyguanosine, 8-OHdG), and histopathology were examined. MDA levels were significantly higher in the PFASs groups than in controls. Compared with SC PFCAs, 2.5 mM PFOA caused more IL-1α (p < 0.001) release, decreased skin thickness and microscopic abnormalities. To evaluate systemic effects, Sprague Dawley (SD) rats were dermally treated with 250 and 1000 mg/kg PFHpA for 2 weeks and clinical and anatomic pathology were assessed. At 1000 mg/kg, 83% of the rats died, with severe ulcerative dermatitis at the application site. Adverse PFHpA-treated systemic changes were observed in the kidney, liver and testes, and histopathologic lesions such as renal tubular necrosis, hepatocellular necrosis, and germ cell degeneration were seen at 250 and 1000 mg/kg. Our study suggests that SC PFCAs have fewer effects on the skin than PFOA, but SC PFCAs can have adverse effects on major organs with systemic exposure at high concentrations.

Reproducible hindlimb ischemia model based on photochemically induced thrombosis to evaluate angiogenic effects.

Critical limb ischemia is one of the most common types of peripheral arterial disease. Preclinical development of ischemia therapeutics relies on the availability of a relevant and reproducible in vivo disease model. Thus, establishing appropriate animal disease models is essential for the development of new therapeutic strategies. Currently, the most commonly employed model of hindlimb ischemia is the surgical induction method with ligation of the femoral artery and its branches after skin incision. However, the efficiency of the method is highly variable depending on the availability of skilled technicians. In addition, after surgical procedures, animals can quickly and spontaneously recover from damage, limiting observations of the therapeutic effect of potential agents. The aim of this study was to develop a hindlimb ischemia mouse model with similarities to human ischemic disease. To that end, a photochemical reaction was used to induce thrombosis in the hindlimb. After the photochemical reaction was induced by light irradiation, thrombotic plugs and adjacent red blood cell stasis were observed in hindlimb vessels in the light-irradiated zone. Additionally, the photochemically induced thrombosis maintained the ischemic condition and did not cause notable side effects in mice.

A potential dermal substitute using decellularized dermis extracellular matrix derived bio-ink.

Upon bioprinting, cells are mixed with a biomaterial to fabricate a living tissue, thus emphasizing the importance of biomaterials. The biomaterial used in this study was a bio-ink prepared using skin decellularized extracellular matrix (dECM). Skin dECM was extracted by treating the dermis with chemicals and enzymes; the basic structural and functional proteins of the ECM, including collagen, glycosaminoglycans (GAGs), bioreactive materials and growth factors, were preserved, whereas the resident cells that might cause immune rejection or inflammatory responses were removed. The bio-ink based on dECM powder, together with human dermal fibroblasts (HDFs), was loaded into the nozzle of the 3D bioprinter to create the 3D construct. This construct underwent gelation with changing temperature while its shape was maintained for 7 days. The cells showed over 90% viability and proliferation. By analysing the gene expression pattern in the cells of the construct, the skin regenerative mechanism of the bio-ink was verified. Microarray results confirmed that the gene expression related to skin morphology and development had been enhanced because the bioreactive molecules and growth factors, in addition to residual ECM in dECM, provided an optimal condition for the HDFs.

Gene networking in colistin-induced nephrotoxicity reveals an adverse outcome pathway triggered by proteotoxic stress.

Colistin has been widely used for the treatment of infections of multidrug­resistant Gram­negative bacteria, despite the fact that it induces serious kidney injury as a side effect. To investigate the mechanism underlying its nephrotoxicity, colistin methanesulfonate sodium (CMS; 25 or 50 mg/kg) was administered via intraperitoneal injection to Sprague­Dawley rats daily over 7 days. Serum biochemistry and histopathology indicated that nephrotoxicity occurred in the rats administered with CMS. Whole­genome microarrays indicated 894 differentially expressed genes in the group treated with CMS (analysis of variance, false discovery rate <0.05, fold­change ≥1.3). Gene pathway and networking analyses revealed that genes associated with proteotoxic stress, including ribosome synthesis, protein translation, and protein folding, were significantly associated with the nephrotoxicity induced by CMS. It was found that colistin inhibited the expression of the target genes heat shock factor 1 and nuclear factor erythroid­2­related factor­2, which are associated with proteostasis, and that nephrotoxicity of CMS may be initiated by proteotoxic stress due to heat shock response inhibition, leading to oxidative stress, endoplasmic reticulum stress, cell cycle arrest and apoptosis, eventually leading to cell death. A putative adverse outcome pathway was constructed based on the integrated gene networking data, which may clarify the mode of action of colistin­induced nephrotoxicity.

Skin irritation and sensitization potential of oxidative hair dye substances evaluated with in vitro, in chemico and in silico test methods.

Permanent oxidative hair dyes are widely used but their toxicity is not well-established. Here we aimed to evaluate the skin sensitization and irritation of nine hair dye substances (MAP, MRP-N, RS, PAOX, 2,4-DAPE, 2,6-PYR, PPD, Grey HED and PM) permitted for use in EU and Korea, using in vitro and in chemico and in silico test methods. Skin sensitization was evaluated by the KeratinoSens™ assay, Direct Peptide Reactivity Assay (DPRA) and DEREK. Six of nine dyes tested were determined as sensitizers in common. However, the decision for MAP, RS or PAOX was diverged across assays showing 2 positives and 1 negative. Skin irritation of hair dye substances was assessed with or without 6% H2O2 on a reconstructed human epidermis, Epiderm™, which demonstrated that H2O2 increased the skin irritation potential of some hair dyes. PPD and PM were determined to be irritants with H2O2. Epidermal damages by hair dye and H2O2 could be further confirmed through the histology of tissue remaining after MTT assay. Collectively, our study demonstrated that hair dyes possess potential skin sensitization and irritation issues which could be further aggravated by H2O2.