Glomerular Damage in Trichloroethylene-Sensitized Mice: Targeting Cathepsin L-Induced Hyperactive mTOR Signaling.

Trichloroethylene (TCE) is a serious health hazard for workers with daily exposure, causing occupational medicamentosa-like dermatitis due to TCE (OMDT) and glomerular damage. Recent studies suggest that mTORC1 signaling is activated in various glomerular disorders; however, the role of mTORC1 signaling in TCE-induced glomerular damage remains to be explored. In the present study, 6 OMDT patients were enrolled and a TCE-sensitized mouse model was established to investigate molecular mechanisms underlying the glomerular damage associated with OMDT. Glomerular damage was assessed by levels of urine nephrin, H&E staining, and renal function test. Ultrastructural change of podocyte was investigated by transmission electron microscopy. The podocyte-related molecules including nephrin, α-actinin-4, and integrin ß1 were visualized by immunofluorescence. The activation of mTORC1 signaling was confirmed by Western blot. Glomerular apoptosis was examined by the TUNEL test and Western blotting. Expression and location of cathepsin L (CTSL) were assessed by RT-PCR and immunofluorescence. Our results showed that TCE sensitization caused damage to glomerular structural integrity and also increased the activation of mTORC1 signaling, which was accompanied by podocyte loss, hypertrophy, and glomerular apoptosis. Importantly, we also found that over-expressed CTSL was mainly located in podocyte and CTSL inhibition could partially block the activation of mTORC1 signaling. Thus, our findings suggested a novel mechanism whereby hyperactive mTOR signaling contributes to TCE sensitization-induced and immune-mediated glomerular damage via CTSL activation.

Altered levels of complement components associated with non-immediate drug hypersensitivity reactions.

Nonimmediate drug hypersensitivity reactions (niDHRs) range from mild-type maculopapular exanthema (MPE) to severe type Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) with unentirely clarified pathogenesis. This study sought to explore whether complement components participated in niDHRs. The participants comprised of three groups as follows: MPE (n = 65), SJS/TEN (n = 13, contains 7 SJS, 2 SJS-TEN overlap and 4 TEN), and equal healthy controls (n = 78). Skin pathological changes were confirmed by hematoxylin and eosin staining. The mRNA and protein levels of complement components were assessed. In the MPE group, there were no alterations in complement components at the protein and mRNA levels found except for a decrease in factor H mRNA. In the SJS/TEN group, up-regulated levels of C3aR and C5aR mRNA and down-regulated factor H mRNA levels in blood were noted. A lower plasma protein level of C3, Factor H and a higher level of C3a, C5, C5a, C5b-9, Factor B (p < 0.05) were found in the SJS/TEN group compared with in the control (p < 0.05). In SJS/TEN skin lesions, indirect immunofluorescence assays showed positive specific staining for C5b-9, but not C3. Both C3aR and C5aR were positive staining in the SJS/TEN samples, while staining for C1q, mannose-binding lectin (MBL), Factor B, and Factor H were only weak or negative. The findings reported here are the first to define the expression profiles/extent of the presence of various complement components at the mRNA and protein levels in niDHRs, especially in SJS/TEN. These altered complement components might, at least in part, be integral to the mechanisms underlying the pathogeneses of SJS and TEN.

Inflammatory kidney injury in trichloroethylene hypersensitivity syndrome mice: Possible role of C3a receptor in the accumulation of Th17 phenotype.

Trichloroethylene (TCE) is a common organic solvent which can cause TCE hypersensitivity syndrome (THS) in exposure workers. THS is an adverse skin disorder with severe inflammatory kidney damage. Complement C3a receptor (C3aR) acts as a specific receptor for the key complement cleavage product C3a and involves multiple inflammatory responses, but the role of C3aR in TCE induced kidney inflammatory injury remains unknown. In this study, BALB/c mouse model of skin sensitization induced by TCE was set up in the presence or absence of C3aR antagonist (C3aRA). Kidney pathology and renal function, expression of inflammatory mediators and C3aR, changes in Th17 cell numbers, and activation of signal transducer and activator of transcription 3 (STAT3) in the kidney were examined. TCE sensitization produced histopathological and functional damage to the kidney, accompanied by increased levels of interleukin (IL-) 1ß, IL-6, and IL-23. Local accumulation of Th17 cells and enhanced phosphorylation of STAT3 were also seen in the impaired kidney in TCE sensitization-positive mice. C3aR was mainly located in the impaired glomerulus and upregulated in TCE sensitization-positive mice. C3aRA pretreatment alleviated the structural and functional kidney damage and the inflammatory cytokine and Th17 responses by TCE sensitization, and specifically reduced the phosphorylation of STAT3. Together, our results demonstrate that C3aR signaling promotes the inflammatory responses and regulates the accumulation of Th17 phenotype via phosphorylation of STAT3 in TCE sensitization induced inflammatory kidney damage. C3aR may serve as a potential therapeutic target in TCE sensitization mediated kidney injury.

Terminal complement complex C5b-9 reduced megalin and cubilin-mediated tubule proteins uptake in a mouse model of trichloroethylene hypersensitivity syndrome.

Trichloroethylene (TCE), a commonly used industrial solvent and degreasing agent, is known to cause trichloroethylene hypersensitivity syndrome (THS) with multi-system damage, including skin, liver and kidney. Clinical evidence have shown that the kidney injury occurs in THS and our previous studies suggested that the terminal complement complex C5b-9 deposited in impaired renal tubules induced by TCE with unclear mechanisms. In the present study, we questioned whether activation of the complement system with renal deposition of C5b-9 contributes to TCE-induced kidney injury in THS. We established a BALB/c mouse model of TCE sensitization with or without pretreatment of exogenous CD59, a C5b-9 inhibitory protein. H&E staining, PAS staining, and biochemical detection of urinary proteins were performed to assess renal function. Deposition of C5b-9 and expression of CD59 were evaluated by immunohistochemistry. Sub-lytic effects of C5b-9 in tubular epithelial cells were assessed by lactate dehydrogenase (LDH) cytotoxicity assay. Expression of endocytosis receptors megalin and cubilin on proximal tubules were assessed by immunofluorescence and qRT-PCR. We found that TCE sensitization induced structural and functional changes of renal tubules in mice, associated with the deposition of sub-lytic C5b-9 on proximal tubular epithelial cells. TCE sensitization decreased proximal tubule uptake of filtered proteins and renal expression of megalin and cubilin, phenotypes that were attenuated by pretreatment with exogenous CD59. Overall, our findings reveal a novel mechanism underlying sub-lytic C5b-9 acting on megalin and cubilin, contributes to the renal tubules damage by TCE exposure.

The Effect of Intravenous Immunoglobulin Combined with Corticosteroid on the Progression of Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis: A Meta-Analysis.

BACKGROUND: Intravenous immunoglobulin (IVIG) treatment is commonly used to treat Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) with controversial therapeutic effect. METHODS: We conducted a comprehensive meta-analysis through combining the published eligible studies to evaluate the effectiveness of IVIG on SJS and TEN treatment. RESULTS: A total of 26 studies were selected from public available databases. The combination of IVIG and corticosteroid markedly reduced the recovery time (by 1.63 days, 95% CI: 0.83-2.43, P < 0.001), compared with solo corticosteroid group. The favorable effects were greater in Asian (2.19, 95% CI: 1.41-2.97, P < 0.001), TEN (2.56, 95% CI: 0.35-4.77, P = 0.023) and high-dose IVIG treated individuals (1.78, 95% CI: 0.42-3.14, P = 0.010). The hospitalization length reduced by 3.19 days (95% CI: 0.08-6.30, P = 0.045), though the outcome was proven to be unstable. We found heterogeneities, which sources were probably regional factors. Besides, IVIG was inclined to decrease SJS/TEN mortality (SMR: 0.84, 95% CI: 0.66-1.08, P = 0.178). This impact was possibly more profound when patients were treated with high dose IVIG (SMR: 0.74, 95% CI: 0.50-1.08, P = 0.116), or when patients were diagnosed as TEN (SMR: 0.68, 95% CI: 0.45-1.01, P = 0.058). CONCLUSIONS: Our current meta-analysis suggests that IVIG combined with corticosteroid could reduce recovery time for SJS and TEN. This effect is greater among Asian patients. Whereas, its impact on reducing mortality is not significant.

Plasma Kallikrein-Kinin system mediates immune-mediated renal injury in trichloroethylene-sensitized mice.

Trichloroethylene (TCE) is a major environmental pollutant. An immunological response is a newly-recognized mechanism for TCE-induced kidney damage. However, the role of the plasma kallikrein-kinin system (KKS) in immune-mediated kidney injury has never been examined. This study aimed to explore the role of the key components of the KKS, i.e. plasma kallikrein (PK), bradykinin (BK) and its receptors B1R and B2R, in TCE-induced kidney injury. A mouse model of skin sensitization was used to explore the mechanism of injury with or without a PK inhibitor PKSI. Kidney function was evaluated by measuring blood urea nitrogen (BUN) and creatinine (Cr) in conjunction with histopathologic characterization. Plasma BK was determined by ELISA; Renal C5b-9 membrane attack complex was evaluated by immunohistochemistry. Expression of BK and PK in the kidney was detected by immunofluorescence. mRNA and protein levels of B1R and B2R were assessed by real-time qPCR and Western blot. As expected, numerous inflammatory cell infiltration and tubular epithelial cell vacuolar degeneration were observed in TCE-sensitized mice. Moreover, serum BUN and Cr and plasma BK were increased. In addition, deposition of BK, PK and C5b-9 were observed and B1R and B2R mRNA and proteins levels were up-regulated. Pre-treatment with PKSI, a highly selective inhibitor of PK, alleviated TCE-induced renal damage. In addition, PKSI attenuated TCE-induced up-regulation of BK, PK and its receptors and C5b-9. These results provided the first evidence that activation of the KKS contributed to immune-mediated renal injury induced by TCE and also helped to identify the KKS as a potential therapeutic target for mitigating chemical sensitization-induced renal damage.

Complement C5a-C5aR interaction enhances MAPK signaling pathway activities to mediate renal injury in trichloroethylene sensitized BALB/c mice.

We have previously shown complement activation as a possible mechanism for trichloroethylene (TCE) sensitization, leading to multi-organ damage including the kidneys. In particular, excessive deposition of C5 and C5b-9-the membrane attack complex, which can generate significant tissue damage, was observed in the kidney tissue after TCE sensitization. The present study tested the hypothesis that anaphylatoxin C5a binding to its receptor C5aR mediates renal injury in TCE-sensitized BALB/c mice. BALB/c mice were sensitized through skin challenge with TCE, with or without pretreatment by the C5aR antagonist W54011. Kidney histopathology and the renal functional test were performed to assess renal injury, and immunohistochemistry and fluorescent labeling were carried out to assess C5a and C5aR expressions. TCE sensitization up-regulated C5a and C5aR expressions in kidney tissue, generated inflammatory infiltration, renal tubule damage, glomerular hypercellularity and impaired renal function. Antagonist pretreatment blocked C5a binding to C5aR and attenuated TCE-induced tissue damage and renal dysfunction. TCE sensitization also caused the deposition of major pro-inflammatory cytokines IL-2, TNF-α and IFN-γ in the kidney tissue (P < 0.05); this was accompanied by increased expression of P-p38, P-ERK and P-JNK proteins (P < 0.05). Pretreatment with the C5aR antagonist attenuated the increase of expression of P-p38, P-ERK and P-JNK proteins (P < 0.05) and also consistently reduced the TCE sensitization-induced increase of IL-2, TNF-α and IFN-γ (P < 0.05). These data identify C5a binding to C5aR, MAP kinase activation, and inflammatory cytokine release as a novel mechanism for complement-mediated renal injury by sensitization with TCE or other environmental chemicals.

Complement C3a binding to its receptor as a negative modulator of Th2 response in liver injury in trichloroethylene-sensitized mice.

Trichloroethylene (TCE) is a major occupational health hazard and causes occupational medicamentosa-like dermatitis (OMLDT) and liver damage. Recent evidence suggests immune response as a distinct mode of action for TCE-induced liver damage. This study aimed to explore the role of the key complement activation product C3a and its receptor C3aR in TCE-induced immune liver injury. A mouse model of skin sensitization was induced by TCE in the presence and absence of the C3aR antagonist SB 290157. Liver function was evaluated by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in conjunction with histopathological characterizations. C3a and C3aR were detected by immunohistochemistry and C5b-9 was assessed by immunofluorescence. IFN-γ and IL4 expressions were determined by flow cytometry and ELISA. The total sensitization rate was 44.1%. TCE sensitization caused liver cell necrosis and inflammatory infiltration, elevated serum ALT and AST, expression of C3a and C3aR, and deposition of C5b-9 in the liver. IFN-γ and IL-4 expressions were up-regulated in spleen mononuclear cells and their serum levels were also increased. Pretreatment with SB 290157 resulted in more inflammatory infiltration in the liver, higher levels of AST, reduced C3aR expression on Kupffer cells, and decreased IL-4 levels while IFN-γ remained unchanged. These data demonstrate that blocking of C3a binding to C3aR reduces IL4, shifts IFN-γ and IL-4 balance, and aggravates TCE-sensitization induced liver damage. These findings reveal a novel mechanism whereby modulation of Th2 response by C3a binding to C3a receptor contributes to immune-mediated liver damage by TCE exposure.

A cutoff point for arterial stiffness using the cardio-ankle vascular index based on carotid arteriosclerosis.

The cardio-ankle vascular index (CAVI) has been widely accepted as a good indicator of arteriosclerosis. However, the lack of a reliable diagnostic criterion for CAVI hampers the proper clinical screening for arteriosclerosis using CAVI and impedes the prompt treatment of cardiovascular disease (CVD). There is an urgent need to determine a criterion for CAVI in arteriosclerosis prevention. We conducted a cross-sectional study to determine this criterion based on receiver operating characteristic (ROC) analyses in a Chinese population consisting of 328 participants. CAVI was measured in duplicate, and carotid ultrasound detection was performed in a quiet environment by well-trained physicians. After multivariate adjustment, CAVI was positively associated with the risk of carotid arteriosclerosis. Compared with participants in the lowest tertile of CAVI (5.15-7.40), those in the medium (7.41-8.65) and highest (8.66-13.60) tertiles had odds ratios (95% confidence interval) of 2.2 (1.0, 4.9) and 4.4 (1.5, 13.3), respectively, for developing carotid arteriosclerosis (P trend=0.007). The areas under the ROC curve (AUC) of the male, female and pooled populations were 0.789, 0.897 and 0.856, respectively. The cutoff point of CAVI≥8.0 resulted in the largest sensitivity and specificity. Furthermore, CAVI and age acted synergistically to increase the risk of carotid arteriosclerosis. CAVI≥8.0 may be an optimal cutoff point for carotid arteriosclerosis prediction. The older population with higher CAVI scores had a higher risk of carotid arteriosclerosis. Additional large prospective studies are needed to confirm our findings.

[Changes of caspase-8 and caspase-9 activity during apoptosis of keratinocytes induced by trichloroethylene].

OBJECTIVE: To observe the change of caspase-8, caspase-9 activity and apoptosis rates in the process of trichloroethylene-induced damage in keratinocytes, and explore the tentative mechanism of apoptosis. METHODS: Human keratinocytes were exposed to 0.125, 0.250, 0.500, 1.000 and 2.000 mmol/L trichloroethylene for 4, 8, 12 and 24 h. The inhibitive groups were pretreated with 100 micromol/L Z-LEHD-FMK (a specific inhibitor of caspase-9) for 1 h, and were stimulated with 2.000 mmol/l TCE for 12 h. MTT assay was used to detect the viability of different cells; The activity of caspase were calculated according to spectrophotometry; Change of the apoptotic rates was assessed by flow cytometer (FCM) after double-stained with Annexin V-FITC and propidium iodide (PI). RESULTS: (1) The minimum effective concentration for cell viability reduction was 0.125 mmol/L at 12 h and the shortest time required to produce a change was 4 h at a concentration of 2.000 mmol/L (compared with control group, P < 0.01). Cell viability in all the groups markedly decreased from 12 h to 24 h (P < 0.05). (2) The activity of caspase-8 in the various dosage groups at different times had no statistical difference compared with the control group, P > 0.01. (3) At 8 h, 1.000 and 2.000 mmol/L TCE groups could significantly enhance caspase-9 activity (P < 0.05). The caspase-9 activity in all the groups showed differences and was significantly higher than those of control cells when time was over 12 h (P < 0.05). (4) After exposing to different dosages of TCE for 12 h, the rate of apoptosis rose to (80.43 +/- 4.21)% with the increase of dosage, compared with the control group, (9.40 +/- 2.98)%, which showed a dose-effect relationship. (5) The cells pre-treated with caspase-9 inhibitor resulted in a decrease in the caspase-9 activity and apoptosis rates (compared with 2.000 mmol/L TCE exposed group, P < 0.01). However, there was no statistical significance in comparison with the control group (P > 0.05). CONCLUSION: Caspase-9 may be an important mediator of apoptosis in keratinocytes induced by trichloroethylene.

[Mechanisms of apoptosis induced by trichloroethylene in normal human epidermis keratinocytes].

OBJECTIVE: To explore the potential mechanism of trichloroethylene (TCE)-induced apoptosis in normal human epidermis keratinocyte (NHEK) by assaying the Caspase activities, mitochondrial membrane potential (DeltaPsim) and apoptosis in vitro. METHODS: NHEK was exposed to TCE and Caspases-3, 8 and 9 activities were determined using a commercial assay kit. Apoptosis and DeltaPsim were detected by flow cytometry (FCM) after double-stained with annexin-V and PI, Rh123 and PI respectively. NHEK was pretreated with inhibitor of Caspase-3 or 9 to verify the activation of Caspases by TCE treatment. RESULTS: Various dose of TCE exposure could increase the Caspases-3 and 9 activities in dose- and time-dependent way. There was marked difference between TCE-treated group and control at 12 or 24 h. But no significant influence of Caspase-8 activity was evoked. 0.125, 0.25, 0.5, 1.0 and 2.0 mmol/L TCE treated NHEK 4 h then cultured for 12 h. Annexin-V(+)/PI(-) proportion were (20.1 +/- 4.1)%, (30.0 +/- 7.5)%, (42.1 +/- 8.2)%, (56.0 +/- 6.1)% and (79.1 +/- 4.3)% respectively. There was marked difference between TCE-treated group except for 0.125 mmol/L and control (9.4 +/- 3.0)% (all P < 0.05). FITC(+)/PI(-) proportion were marked positive correlation with Caspase-3 and 9 activities, r = 0.786, 0.736 (both P < 0.05). Caspase-3 activities had also a marked positive correlation with Caspase-9 activities, r = 0.845 (P < 0.05). There was no correlation with Caspase-8 activities. Pretreatment for 1 h with 100 micromol/L Z-DEVD-FMK decreased the Caspase-3 activities from (0.963 +/- 0.043) to (0.349 +/- 0.045) nmol pNA * min(-1) * ml(-1), annexin-V(+)/PI(-) proportion decreased from (80.0 +/- 5.5)% to (16.3 +/- 3.2)% in 2.0 mmol/L TCE treated NHEK with a significant difference (P < 0.01), but there was no change of Caspase-9 activities. 100 micromol/L Z-LEHD-FMK pretreatment decreased the Caspase-3 activities to (0.338 +/- 0.011) nmol pNA * min(-1) * ml(-1), annexin-V(+)/PI(-) proportion decreased to (16.1 +/- 1.7)% in 2.0 mmol/L TCE treated NHEK. And the Caspase-9 activities decreased from (0.821 +/- 0.031) to (0.240 +/- 0.043) nmol pNA * min(-1) * ml(-1) with a significant difference (P < 0.01). NHEK was cultured for 4, 8, 12, 24 h after a 4-hour treatment with 2.0 mmol/L TCE. Rhod123 fluorescence intensity (FI) were respectively with a marked decrease as compared with 0 h (18.7 +/- 0.5, all P < 0.01). At 0.125, 0.5 and 2.0 mmol/L TCE treated NHEK for 4 h then cultured 8 h, Rh123 FI were 16.1 +/- 0.5, 12.1 +/- 0.6 and 8.1 +/- 0.6 with a marked decrease as compared with control (18.1 +/- 0.5, all P < 0.01). CONCLUSION: TCE-induced NHEK apoptosis is mediated intrinsically through the mitochondrial pathway of the decrease of DeltaPsim and the Caspase-9 dependent activation of Caspase-3.

Trichloroethylene induce nitric oxide production and nitric oxide synthase mRNA expression in cultured normal human epidermal keratinocytes.

Trichloroethylene (TCE), a major chemical hazard during occupational exposure, can cause obvious skin lesions, including irritant reactions and dermatitis. Nitric oxide (NO) synthesized by nitric oxide synthase (NOS) is involved in a broad array of pathogenesis of skin inflammatory and immune responses. To understand the mechanisms of TCE-induced dermatoxicity, we investigated the effects of TCE on NO production and NOS mRNA expression in cultured normal human epidermal keratinocytes (NHEK). Cells were treated with TCE (0 mM, 0.125 mM, 0.25 mM, 0.5 mM, 1.0 mM, 2.0 mM) for 4 h, and then incubated for 12 h, 24 h, 48 h and 72 h. At each given time point, NO production were evaluated indirectly by measuring nitrite plus nitrate concentration in the culture medium using Griess reaction, as well as cell viability determined by MTT test, iNOS and cNOS activities assayed with a NOS activity detecting kit. The expression of iNOS and cNOS mRNA was detected using RT-PCR. TCE decreases cell viability and enhance NO production from NHEK in concentration- and time-dependent manner. Aminoguanidine (AG), an inhibitor of NOS, can prevent NO production and cell viability decrease in NHEK by TCE induced. Change to NO production was accompanied by increased activities of both types of NOS, but the iNOS activity accounted mainly for the TCE-induced NO production. RT-PCR detection showed that NHEK expressed both iNOS and cNOS mRNA by TCE exposure. Whereas a concentration- and time-dependent up-regulation of the mRNA expression was observed for iNOS and cNOS following TCE exposure, changes to iNOS were more marked. These results suggest that TCE caused increase in NO production, attributed to activation of iNOS as well as cNOS, and expression of iNOS and cNOS mRNA. These cellular changes may contribute to the pathological and physiological features of TCE-induced erythema and skin inflammation.

[Cytotoxicity of trichloroethylene in keratinocytes involving alterations of mitochondrial function and ultrastructure].

OBJECTIVE: To explore mechanism of dermal toxicity of trichloroethylene(TCE). METHODS: Normal human keratinocytes (KC) were isolated from foreskins of healthy donors undergoing circumcision by two-step trypsin digestion and cultured in serum-free medium. Cells were treated with medium, 1% acetone (volume fraction) 0.125, 0.500 or 2.000 mmol/L TCE for different time (4, 8, 12 or 24) hours. After treatment, MTT assay and ATPase activity detected, inhibition ratio of mitochondrial enzyme was calculated according to optical density (A) value of MTT assay. Mitochondrial membrane potential (MMP) was detected by flow cytometry FCM after being stained with Rhodamine123 (Rh123). Morphological changes were also observed through transmission electron microscope (TEM). RESULTS: Cellular viability and ATPase activity declined with dose of TCE, while inhibition ratio of mitochondrial enzyme increased with dose of TCE. FCM results showed that after treatment with 2.000 mmol/L TCE, fluorescence density of Rh123 decreased quickly from 18.73 +/- 0.45(0 h) to 8.20 +/- 0.66(8 h) (P < 0.01). After 8 h, fluorescence density maintained at the level equal to that of 8 h (fluorescence density of Rh123 were 8.20 +/- 0.36 and 8.20 +/- 0.40 for 12 and 24 h respectively, compared with that for 8 h group, P > 0.05). The results also showed that MMP diminished with dose of TCE. Under TEM, mitochondria in TCE-treated group appeared extensive swelling and vacuolar degeneration with less matrix and obscure or vanished mitochondria cristae but in control group, mitochondrial structure was integrated, with uniform matrix and visible mitochondria cristae. CONCLUSIONS: TCE could inhibit mitochondrial metabolic enzyme, reduce ATP production, diminish MMP, and destroy ultrastructure of mitochondria in KC, all these contributing to the cytotoxicity of TCE.