Introduction to the Forensic Research via Omics Markers in Environmental Health Vulnerable Areas (FROM) study.

Objectives: Exposure to humidifier disinfectants has been linked to respiratory diseases, including interstitial lung disease, asthma, and pneumonia. Consequently, numerous toxicological studies have explored respiratory damage as both a necessary and sufficient condition for these diseases. We systematically reviewed and integrated evidence from toxicological studies by applying the evidence integration method established in previous research to confirm the biological plausibility of the association between exposure and disease. Methods: We conducted a literature search focusing on polyhexamethylene guanidine phosphate (PHMG) and chloromethylisothiazolinone/methylisothiazolinone (CMIT/MIT), the primary ingredients in humidifier disinfectants. We selected relevant studies based on their quality and the population, exposure, comparator, outcome (PECO) statements. These studies were categorized into 3 lines of evidence: hazard information, animal studies, and mechanistic studies. Based on a systematic review, we integrated the evidence to develop an aggregate exposure pathway-adverse outcome pathway (AEP-AOP) model for respiratory damage. The reliability and relevance of our findings were assessed by comparing them with the hypothesized pathogenic mechanisms of respiratory diseases. Results: The integration of each AEP-AOP component for PHMG and CMIT/MIT led to the development of an AEP-AOP model, wherein disinfectants released from humidifiers in aerosol or gaseous form reached target sites, causing respiratory damage through molecular initiating events and key events. The model demonstrated high reliability and relevance to the pathogenesis of respiratory diseases. Conclusion: The AEP-AOP model developed in this study provides strong evidence that exposure to humidifier disinfectants causes respiratory diseases. This model demonstrates the pathways leading to respiratory damage, a hallmark of these conditions.

Palmitate induces lipoapoptosis in Schwann cells through ROS generation-mediated STAMP2 downregulation.

Free fatty acids (FFAs) are considered the principal inducers of lipotoxicity, leading to cell dysfunction and/or cell death. Lipotoxicity in Schwann cells (SCs) damages neurons, which may be associated with peripheral neuropathies and axon degeneration. However, the molecular mechanism by which FFAs exert lipotoxicity in SCs remains to be established. In the present study, we demonstrate that palmitate exerts lipotoxicity in SCs through apoptosis and that palmitate-induced lipotoxicity in SCs is mediated through reactive oxygen species (ROS) generation. We observed that the six-transmembrane protein of prostate 2 (STAMP2), which plays a pivotal role in lipid homeostasis, is expressed in SCs. We further demonstrate that palmitate induces lipoapoptosis in SCs through ROS generation-mediated STAMP2 downregulation and that STAMP2 depletion accelerates the palmitate-exerted lipoapoptosis in SCs, indicating that STAMP2 confers on SCs the ability to resist palmitate-induced lipotoxicity. In conclusion, palmitate induces lipoapoptosis in SCs through ROS generation-mediated STAMP2 downregulation. Our findings indicate that ROS and STAMP2 may represent suitable targets for pharmacological interventions targeting lipotoxicity-associated peripheral neuropathies and axon degeneration.

Dietary fat-associated osteoarthritic chondrocytes gain resistance to lipotoxicity through PKCK2/STAMP2/FSP27.

Free fatty acids (FFAs), which are elevated with metabolic syndrome, are considered the principal offender exerting lipotoxicity. Few previous studies have reported a causal relationship between FFAs and osteoarthritis pathogenesis. However, the molecular mechanism by which FFAs exert lipotoxicity and induce osteoarthritis remains largely unknown. We here observed that oleate at the usual clinical range does not exert lipotoxicity while oleate at high pathological ranges exerted lipotoxicity through apoptosis in articular chondrocytes. By investigating the differential effect of oleate at toxic and nontoxic concentrations, we revealed that lipid droplet (LD) accumulation confers articular chondrocytes, the resistance to lipotoxicity. Using high fat diet-induced osteoarthritis models and articular chondrocytes treated with oleate alone or oleate plus palmitate, we demonstrated that articular chondrocytes gain resistance to lipotoxicity through protein kinase casein kinase 2 (PKCK2)-six-transmembrane protein of prostate 2 (STAMP2)-and fat-specific protein 27 (FSP27)-mediated LD accumulation. We further observed that the exertion of FFAs-induced lipotoxicity was correlated with the increased concentration of cellular FFAs freed from LDs, whether FFAs are saturated or not. In conclusion, PKCK2/STAMP2/FSP27-mediated sequestration of FFAs in LD rescues osteoarthritic chondrocytes. PKCK2/STAMP2/FSP27 should be considered for interventions against metabolic OA.

Alpha B-Crystallin Protects Rat Articular Chondrocytes against Casein Kinase II Inhibition-Induced Apoptosis.

Although alpha (α)B-crystallin is expressed in articular chondrocytes, little is known about its role in these cells. Protein kinase casein kinase 2 (CK2) inhibition induces articular chondrocyte death. The present study examines whether αB-crystallin exerts anti-apoptotic activity in articular chondrocytes. Primary rat articular chondrocytes were isolated from knee joint slices. Cells were treated with CK2 inhibitors with or without αB-crystallin siRNA. To examine whether the silencing of αB-crystallin sensitizes rat articular chondrocytes to CK2 inhibition-induced apoptosis, we assessed apoptosis by performing viability assays, mitochondrial membrane potential measurements, flow cytometry, nuclear morphology observations, and western blot analysis. To investigate the mechanism by which αB-crystallin modulates the extent of CK2 inhibition-mediated chondrocyte death, we utilized confocal microscopy to observe the subcellular location of αB-crystallin and its phosphorylated forms and performed a co-immunoprecipitation assay to observe the interaction between αB-crystallin and CK2. Immunochemistry was employed to examine αB-crystallin expression in cartilage obtained from rats with experimentally induced osteoarthritis (OA). Our results demonstrated that silencing of αB-crystallin sensitized rat articular chondrocytes to CK2 inhibitor-induced apoptosis. Furthermore, CK2 inhibition modulated the expression and subcellular localization of αB-crystallin and its phosphorylated forms and dissociated αB-crystallin from CK2. The population of rat articular chondrocytes expressing αB-crystallin and its phosphorylated forms was reduced in an experimentally induced rat model of OA. In summary, αB-crystallin protects rat articular chondrocytes against CK2 inhibition-induced apoptosis. αB-crystallin may represent a suitable target for pharmacological interventions to prevent OA.

Targeted inhibition of mitochondrial Hsp90 induces mitochondrial elongation in Hep3B hepatocellular carcinoma cells undergoing apoptosis by increasing the ROS level.

Previous studies reported that a Gamitrinib variant containing triphenylphosphonium (G-TPP) binds to mitochondrial Hsp90 and rapidly inhibits its activity to induce apoptosis. We investigated the mechanisms underlying the antitumor activity of G-TPP in Hep3B hepatocellular carcinoma cells. Contrary to our predictions, we observed mitochondrial elongation in the G-TPP-treated Hep3B cells undergoing apoptosis. We found that the G-TPP-induced mitochondrial elongation in Hep3B cells was caused by a decrease in the mitochondrial fission-regulating protein Drp1 rather than by changes in the mitochondrial fusion machinery proteins Mfn1 and Opa1. Furthermore, G-TPP induced G2-M phase cell cycle arrest by reducing the interaction between CDK1 and cyclin B1. Additionally, reactive oxygen species (ROS) played a pivotal role in G-TPP-induced cell death and mitochondrial elongation in Hep3B cells, and these processes are mediated by the reduced association of CDK1 with cyclin B1 and the suppressed phosphorylation of Drp1 (Ser616). Thus, G-TPP induces cell death and causes Drp1-mediated mitochondrial elongation in Hep3B cells by increasing the ROS level.

Rosuvastatin attenuates inflammation, apoptosis and fibrosis in a rat model of cyclosporine-induced nephropathy.

BACKGROUND/AIM: Cyclosporine (CsA)-induced kidney injury is characterized by renal dysfunction with inflammatory cell infiltrations, apoptosis and fibrosis. Pleiotropic effects of statins may exert anti-inflammatory, antiapoptotic and antifibrotic actions beyond lipid control. The aim of this study is to investigate whether rosuvastatin (RUS) has anti-inflammatory, antiapoptotic and antifibrotic effects on chronic CsA-induced nephropathy in a rat model. METHODS: Male Sprague-Dawley rats fed a low-sodium diet were divided into three treatment groups: control (0.9% saline injection), CsA (15 mg/kg/day by subcutaneous injection), CsA + RUS (10 mg/kg/day by gastric gavage). Renal function, CsA level and lipid levels were measured at the end of 4 weeks. The expression of ED-1, transforming growth factor-ß(1) (TGF-ß(1)) and α-smooth muscle actin (α-SMA) for inflammation and fibrosis were examined by Western blot analysis. The expression levels of apoptosis-associated factors were examined by Western blot analysis. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase-mediated biotin nick end-labeling (TUNEL) method. RESULTS: Kidney function was decreased in CsA-treated rats compared with controls, which was attenuated by RUS. RUS did not affect the lipid level or the blood CsA level. TUNEL staining showed that RUS inhibited CsA-induced tubular apoptosis. RUS decreased CsA-induced increased expression of Bax/Bcl-2 ratio. The expressions of ED-1, α-SMA, TGF-ß(1), Smad2/3, Smad4 and p-JNK were increased in CsA-treated rats, which were attenuated by RUS. Tubular atrophy and interstitial fibrosis in CsA-treated rats were attenuated by RUS supplementation. CONCLUSION: RUS supplementation attenuates proinflammatory and apoptosis-related factors and inhibits the fibrotic pathways including the smad-dependent and smad-independent pathways in a rat model of CsA-induced nephropathy.

Cytoplasmic and nuclear anti-apoptotic roles of αB-crystallin in retinal pigment epithelial cells.

In addition to its well-characterized role in the lens, αB-crystallin performs other functions. Methylglyoxal (MGO) can alter the function of the basement membrane of retinal pigment epithelial (RPE) cells. Thus, if MGO is not efficiently detoxified, it can induce adverse reactions in RPE cells. In this study, we examined the mechanisms underlying the anti-apoptotic activity of αB-crystallin in the human retinal pigment epithelial cell line ARPE-19 following MGO treatment using various assays, including nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, western blot analysis, confocal microscopy and co-immunoprecipitation assays. To directly assess the role of phosphorylation of αB-crystallin, we used site-directed mutagenesis to convert relevant serine residues to alanine residues. Using these techniques, we demonstrated that MGO induces apoptosis in ARPE-19 cells. Silencing αB-crystallin sensitized ARPE-19 cells to MGO-induced apoptosis, indicating that αB-crystallin protects ARPE-19 cells from MGO-induced apoptosis. Furthermore, we found that αB-crystallin interacts with the caspase subtypes, caspase-2L, -2S, -3, -4, -7, -8, -9 and -12 in untreated control ARPE-19 cells and that MGO treatment caused the dissociation of these caspase subtypes from αB-crystallin; transfection of S19A, S45A or S59A mutants caused the depletion of αB-crystallin from the nuclei of untreated control RPE cells leading to the release of caspase subtypes. Additionally, transfection of these mutants enhanced MGO-induced apoptosis in ARPE-19 cells, indicating that phosphorylation of nuclear αB-crystallin on serine residues 19, 45 and 59 plays a pivotal role in preventing apoptosis in ARPE-19 cells. Taken together, these results suggest that αB-crystallin prevents caspase activation by physically interacting with caspase subtypes in the cytoplasm and nucleus, thereby protecting RPE cells from MGO-induced apoptosis.

The novel resveratrol analog HS-1793-induced polyploid LNCaP prostate cancer cells are vulnerable to downregulation of Bcl-xL.

Since resveratrol is not a potent cytotoxic compound when compared with other chemotherapeutic agents, several previous studies have been performed to obtain synthetic analogs of resveratrol with potent activity. Our previous study demonstrated that the resveratrol analog HS-1793 showed stronger antitumor activity than resveratrol in various cancer cells. We examined the antitumor activity exerted by HS-1793 in prostate cancer cells, and we observed that HS-1793 acts as a polyploidy inducer. Noticeably, multinucleation and polyploidization were induced in most LNCaP cells treated with HS-1793 at the dose causing a slight decline in cell viability. However, the induction of multinucleation and polyploidization was much lower in PC-3 prostate cancer cells treated with the same dose of HS-1793. Western blot and RT-PCR analyses showed that the expression of Aurora B was almost undetectable in LNCaP cells, but it was highly expressed in PC-3 cells. Further, silencing of Aurora B sensitized PC-3 cells to HS-1793-induced multi-nucleation. These results indicate that expression of Aurora B determines multinucleation in prostate cancer cells treated with HS-1793. Additional assays using multiple cancer cell lines show that the population of multinucleated cells induced by HS-1793 treatment is inversely proportional to Aurora B expression. We further elicited that the HS-1793-induced polyploid LNCaP cells are vulnerable to downregulation of Bcl-xL. Since the polyploidization in LNCaP induced by HS-1793 does not appear to cause definite commitment to apoptosis, the termination of polyploid cells by inhibition of Bcl-xL could provide an advantageous means to improve chemotherapeutic efficacy of HS-1793.

An experimental study of rabbit conjunctival epithelial toxicity using co-treatment with mitomycin-C and a histone deacetylase inhibitor.

This study was conducted to evaluate cytotoxicity due to co-treatment with low-dose Mitomycin-C (MMC) and the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA) for glaucoma filtration surgery. In this study, the effect of co-treatment with MMC and SAHA to induce apoptosis in cultured conjunctival epithelial cells (CEs) in rabbit was investigated. The cytotoxic potential following co-treatment with MMC and SAHA in CEs via assay for reactive oxygen species (ROS) and lactate dehydrogenase (LDH) was also examined. Co-treatment with MMC and SAHA did not induce apoptosis in CEs. In addition, co-treatment with MMC and SAHA caused no significant alteration of ROS and LDH levels in CEs. This study therefore shows that low-dose MMC could still be used as an antimetabolite in co-treatment with SAHA to effectively inhibit fibrosis of Tenon's capsule fibroblasts (TCFs) following glaucoma filtration surgery, while minimizing cytotoxicity in CEs.

Mutant p53 (G199V) gains antiapoptotic function through signal transducer and activator of transcription 3 in anaplastic thyroid cancer cells.

In the present study, we identified a missense mutation (G199V) in KAT-18 cell line established from primary cultures of anaplastic thyroid cancer (ATC). Notably, knockdown of this mutant (mt) p53 reduced cell viability and exerted antitumor activity equivalent to high doses of several chemotherapeutic agents. We showed that p53 knockdown had an antitumor effect via the induction of apoptosis. We further examined the underlying mechanism by which mt p53 (G199V) gains antiapoptotic function in KAT-18 cells. Microarray analysis revealed that p53 knockdown modified the expression of numerous apoptosis-related genes. Importantly, p53 knockdown led to downregulation of signal transducer and activator of transcription-3 (STAT3) gene expression. We further observed that p53 knockdown induced the downregulation of STAT3 protein. We also observed that a STAT3 inhibitor augmented the reduction of cell viability induced by p53 knockdown, whereas interleukin-6 treatment alleviated this effect. In addition, overexpression of STAT3 protected ATC cells against cell death induced by p53 knockdown. Taken together, these data show that mt p53 (G199V) gains antiapoptotic function mediated by STAT3 in ATC cells. Inhibition of the function of mt p53 (G199V) could be a novel and useful therapeutic strategy for decreasing the extent and severity of toxicity due to chemotherapeutic agents.

Sensitization of RPE cells by alphaB-crystallin siRNA to SAHA-induced stage 1 apoptosis through abolishing the association of alphaB-crystallin with HDAC1 in SC35 speckles.

PURPOSE: To better understand the mechanism underlying the anti-apoptotic activity of alphaB-crystallin in RPE cells. METHODS: Cells of the human retinal pigment epithelial line ARPE-19 were treated with a histone deacetylase inhibitor (HDACI), suberoylanilide hydroxamic acid (SAHA), with or without alphaB-crystallin siRNA. To examine the mechanism underlying the cell death induced in ARPE-19 cells, nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, Western blot analysis, confocal microscopy, and coimmunoprecipitation assay were undertaken. RESULTS: The present study demonstrated that an HDACI, SAHA, at the usual doses or the silencing of alphaB-crystallin by siRNA alone did not effectively induce apoptosis in ARPE-19 cells. Silencing of alphaB-crystallin likely abolishes the anti-apoptotic activity of alphaB-crystallin. The data indicated that silencing of alphaB-crystallin sensitizes ARPE19 cells to SAHA-induced apoptosis and leads them to stage 1 apoptosis. alphaB-Crystallin associates with HDAC1 on SC35 speckles, and silencing of alphaB-crystallin abolishes this association, resulting in the induction of apoptosis. The data indicated that the association between alphaB-crystallin and HDAC1 on SC35 speckles plays a pivotal role in anti-apoptotic activity. CONCLUSIONS: Knockout of alphaB-crystallin may be a promising new approach to enhance therapeutic potency for proliferative vitreoretinopathy without compromising efficacy.

Co-treatment of suberoylanilide hydroxamic acid and mitomycin-C induces the apoptosis of rabbit tenon's capsule fibroblast and improves the outcome of glaucoma filtration surgery.

PURPOSE: This study was undertaken to develop a new treatment modality that would be able to minimize fibrosis and provide better outcome with glaucoma filtration surgery (GFS). METHODS: We examined whether co-treatment with mitomycin-C (MMC) and histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) efficiently induces apoptosis on rabbit Tenon's capsule fibroblasts (TCF) in vitro. We further examined the effect of co-treatment with SAHA and MMC on the alteration of IOP and the bleb survival in rabbits following GFS. RESULTS: Co-treatment of MMC and SAHA efficiently induces apoptosis in TCFs via the up-regulation of p53 and increased phosphorylation of p53 on serine 15 and 392. Also, co-treatment of SAHA and low-dose MMC decreases IOP, prolongs bleb survival, and induces apoptosis of cells under the bleb area following GFS. CONCLUSION: This study shows that a co-treatment of SAHA and MMC could improve the outcome of GFS.

Application of newly developed amniotic membrane ointment for photorefractive keratectomy in rabbits.

We developed amniotic membrane ointment (AMO), and the effect of instilling the AMO after photorefractive keratectomy (PRK) was investigated with respect to inflammatory cell infiltration into the corneal stroma, apoptosis of keratocytes, and suppression of lipid peroxidation of cellular walls. The PRK procedure was performed on both eyes of 10 white rabbits. One eye of each rabbit (the experimental eye) was instilled with the AMO and the other eye of the rabbit (the control eye) with a base ointment 0, 8 and 16 h after the PRK procedure. Corneal specimens were collected 24 h after the PRK procedure. Hematoxylin-eosin stain and TUNEL assay were conducted to demonstrate polymorphonuclear and apoptotic cells, respectively. To assess lipid peroxidation, immunohistochemical staining with an antibody to malondialdehyde was undertaken. Compared to the control, the cornea instilled with the AMO had significantly less polymorphonuclear cells infiltrating into the corneal stroma as well as keratocytes subjected to apoptosis. These corneas also showed a significantly less extent of lipid peroxidation than the control. These data support that instillation of the AMO effectively reduced the recruitment of polymorphonuclear cells, the induction of apoptosis in keratocytes, and lipid peroxidation induced by PRK. Thus, this study could provide basic data on the clinical application of the AMO in the days ahead.

Doxorubicin induces apoptosis with profile of large-scale DNA fragmentation and without DNA ladder in anaplastic thyroid carcinoma cells via histone hyperacetylation.

Doxorubicin is known to be the most effective single cytotoxic drug against anaplastic thyroid carcinoma (ATC). Although doxorubicin has been shown to cause cell death, at least partly, by inducing apoptosis in ATC cells, the mechanism underlying its pharmacological efficacy has not been fully delineated. We, in this study, revealed that doxorubicin induced apoptosis in ATC cells by altering the acetylation state of histone. Doxorubicin reduced histone deacetylase activity and induced hyperacetylation of histone 3. Noticeably, ladder-like DNA fragments from their genomic DNA on agarose gel were not detected irrespective of several lines of evidence supporting the induction of apoptosis. Pulse field electrophoresis showed disintegration of nuclear DNA into giant fragments of 1-2 Mbp and high molecular-weight fragments of 100-1000 kbp. We next examined whether a histone deacetylase inhibitor trichostatin A (TsA) augmented doxorubicin-induced apoptosis in ATC cells. TSA potentiated doxorubicin-induced stage I apoptosis in ATC cells. Our study sheds light on the development of a new combination therapy strategy for more effective responses for ATC treatment.