The role of TNF-α/TNFR1 inhibiting autophagy of M1-type Kupffer cells in liver injury in trichloroethylene-sensitized mice / 中华劳动卫生职业病杂志

Objective: To detect the expression levels of M1-type polarization and autophagy-related indicators in the liver of trichloroethylene (TCE) -sensitized mice, and to explore the role of liver tumor necrosis factor-α (TNF-α) and tumor necrosis factor receptor 1 (TNFR1) in regulating M1-type Kupffer cells autophagy in liver injury in TCE-sensitized mice. Methods: In November 2019, according to simple random grouping, 45 SPF grade BALB/c female mice (6-8 weeks old) were divided into 4 groups: blank control group (n=5) , solvent control group (n=5) , TCE treatment group (n=18) , TCE+R7050 (inhibitor) treatment group (n=17) . Transdermally sensitized mice, 24 h after the last challenge, the mice were divided into TCE sensitized group and TCE non-sensitized group according to the skin reaction score. The livers of mice were harvested, and the pathological changes of the livers were observed under light and electron microscopes. Western blotting was used to detect the expressions of TNF-α, TNFR1 and autophagy-related indexes. The expression of inducible nitric oxide synthase (iNOS) , a marker of M1-type Kupffer cells, was detected by immunohistochemistry, and the occurrence of autophagy in M1-type Kupffer cells was detected by immunofluorescence double-labeling method. Results: The sensitization rate of TCE treatment group was 38.9% (7/18) , and TCE+R7050 treatment group was 35.3% (6/17) , with no significant difference between the two groups (P=1.000) . Compared with the blank control group, mice in the TCE sensitized group had abnormal liver ocytes, obvious liver injury, reduced mitochondria and broken endoplasmic reticulum. Western blotting results showed that the expressions of TNF-α and TNFR1 protein in the liver of the mice in the TCE sensitized group increased, the expression of iNOS protein in M1-type Kupffer cells increased, and the expressions of autophagic microtubule-associated protein 1 light-chain 3 (LC3B) and Beclin1 protein were decreased (P<0.05) . The results of immunohistochemistry showed that iNOS was not significantly expressed in the blank control group and solvent control group, and a small amount of expression was found in the TCE non-sensitized group, the positive staining area was obvious in TCE sensitized group, and the expression of iNOS was significantly increased (P<0.05) . Immunofluorescence results showed that the iNOS protein levels in the blank control group, solvent control group and TCE non-sensitized group were lower, and only partially colocalized with P62; the colocalization of iNOS with P62 in the TCE sensitized group was significantly increased. Conclusion: TNF-α/TNFR1 signaling pathway may promote liver injury in TCE-sensitized mice by inhibiting autophagy of M1-type Kupffer cells.

The role of ROS/TXNIP/NLRP3 pathway in the skin injury of trichloroethylene sensitized mice / 中华劳动卫生职业病杂志

Objective: To explore the mechanism of reactive oxygen species/thioredoxin-interacting protein/nucleotide-binding oligomerization domain-like receptor 3 (ROS/TXNIP/NLRP3) pathway in the skin injury of trichloroethylene (TCE) sensitized mice. Methods: In August 2020, 40 female BALB/c mice were randomly divided into control group (n=5) , solvent control group (n=5) , TCE treatment group (n=15) and TCE+(2-(2, 2, 6, 6-Tetrameyhylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (Mito TEMPO) treatment group (n=15) . The TCE sensitization model was established. Mice in the TCE treatment group and TCE+Mito TEMPO treatment group were divided into the sensitized positive group and the sensitized negative group according to the skin erythema and edema reactions on the back of the mice 24 h after the last stimulation. The mice were sacrificed 72 h after the last stimulation, the back skin of the mice was taken, and the skin lesions were observed. Immunohistochemistry (IHC) was used to detect the expression level of NLRP3, and the Western Blot was performed to detect the expression levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC) , cysteinyl aspartate specific proteinase 1 (Caspase 1) , Interleukin-1β (IL-1β) and TXNIP proteins in the skin of the mice, the reactive oxygen species (ROS) kit was used to detect the level of intracellular ROS in the back skin tissue. Results: The sensitization rates of TCE treatment group and TCE+Mito TEMPO treatment group were 40.0% (6/15) and 33.3% (5/15) , respectively, and there was no significant difference between the two groups (P>0.05) . The back skin of the mice in the TCE sensitized positive group was thickened and infiltrated by a large number of inflammatory cells. The number of mitochondria in the epidermis cells was significantly reduced, the mitochondrial crest disappeared and vacuolar degeneration occurred. TCE+Mito TEMPO sensitized positive group had less damage, more mitochondria and relatively normal cell structure. Compared with the solvent control group and corresponding sensitized negative groups, the expression levels of NLRP3, ASC, Caspase 1, IL-1β, TXNIP proteins and the content of ROS in the TCE sensitized positive group and TCE+Mito TEMPO sensitized positive group were significantly increased (P<0.05) . Compared with TCE sensitized positive group, the expression levels of NLRP3, ASC, Caspase 1, IL-1β, TXNIP proteins and the content of ROS in the TCE+Mito TEMPO sensitized positive group were significantly decreased (P<0.05) . Conclusion: ROS/TXNIP/NLRP3 pathway was activated and then encouraged the release of IL-1β, finally aggravated the TCE-induced skin injury.

Effect of trichloroethylene intake via drinking water on Th17 cells in BALB/c mice / 中华预防医学杂志

<p><b>OBJECTIVE</b>This study aimed to investigate the effect of trichloroethylene (TCE) intake via drinking water on Th17 cells in mice.</p><p><b>METHODS</b>Forty eight six weeks old female BALB/c mice were divided into blank control, vehicle control, 2.5 mg/ml TCE and 5.0 mg/ml TCE groups by random number table (12 mice each group), and exposed to TCE by drinking water. On the 14(th), 28(th), 56(th), 84(th) days, blood were collected and assayed for IL-17, IL-6, and TGF-β concentration in serum through ELISA. Animals were killed and spleen biopsies were taken sterility. The proportion of Th17 cells among CD4(+) T cells and RORγt mRNA expression level in spleen were measured by FCM and real-time PCR.</p><p><b>RESULTS</b>In 2.5 mg/ml TCE and 5.0 mg/ml TCE group mice, Th17 cells/CD4(+) T cells in spleen were (3.46 ± 0.32)% and (5.45 ± 0.45)% on day 14, (3.47 ± 0.33)% and (4.10 ± 0.39)% on day 84, which were significantly higher than those for solvent control group at the same time point ((2.15 ± 0.20)%, (2.16 ± 0.35)%, respectively) (P < 0.01). RORγt mRNA expression levels were (1.870 ± 0.084) and (1.965 ± 0.060) on 14 day, (1.998 ± 0.079) and (2.028 ± 0.073) on day 56, which were also significantly higher than those for solvent control group at the same time point (1.77 ± 0.04 and 1.75 ± 0.09, respectively) (P < 0.05). IL-17 concentrations in serum were (32.28 ± 5.38) and (34.47 ± 5.02) pg/ml on day 14, and (34.87 ± 5.48) and (41.94 ± 6.19) pg/ml on day 28, which were significantly higher than those for solvent control group at the same time point((21.57 ± 5.23), (22.11 ± 5.11) pg/ml). IL-6 concentration in serum were (43.07 ± 6.71) and (47.86 ± 8.52) pg/ml on day14, (41.32 ± 7.04) and (46.74 ± 9.33) pg/ml on day 56, which were significantly higher than solvent control group at the same time point ((7.56 ± 7.71) and (28.26 ± 7.22) pg/ml). TGF-β concentration were (17.48 ± 3.06) and (18.93 ± 3.12) pg/ml on day 14, which did not show significant difference from solvent control group ((15.25 ± 2.95) pg/ml). Correlation analysis showed that IL-6 in serum were significantly positively correlated with the proportion of Th17 cells among CD4(+) T cells and RORγt expression level in spleen (r = 0.741, 0.765, P < 0.01).</p><p><b>CONCLUSION</b>TCE might promote the differentiation of Th17 cells and increase IL-17 secretion by inducing IL-6 and up-regulating RORγt expression together with TGF-β.</p>

Changes in humoral immunity in sensitized guinea pigs exposed to trichloroethylene / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the changes of serum complement C and immunoglobulin (Ig) in sensitized guinea pigs exposed to trichloroethylene.</p><p><b>METHODS</b>Thirty six white female guinea pigs (250 ∼ 300 g) were randomly divided into blank control group (5 guinea pig), solvent (olive oil) control group (5 guinea pig) and TCE treatment group (26 guinea pig). According to guinea pig maximization test (GPMT), guinea pigs were exposed to TCE. After stimulating contact for 24 h, the skin reactions of guinea pig back test area were recorded and scored. According to Skin sensitization integral, the guinea pigs treated with TCE were divided into the sensitized group (score ≥ 1) and un-sensitized group (score 0). The concentrations of serum C3, C4, IgA, IgG and IgM were detected in 24 and 72 h, respectively after the experiment.</p><p><b>RESULTS</b>The sensitization rates of group treated by TCE was 65.38%. The serum C3 levels of groups sensitized to TCE for 24 and 73h were 99.75 ± 1.45 and 93.28 ± 3.61g/ml, respectively, which were significantly lower than that (112.30 ± 9.10 g/ml) of solvent control group (P < 0.05). Also The serum C4 levels of groups sensitized to TCE for 24 and 73 h were 34.63 ± 2.53 and 33.82 ± 2.76g/ml, respectively, which were significantly lower than that (43.87 ± 3.65 g/ml) of solvent control group (P < 0.05). The serum IgA and IgM levels of groups sensitized to TCE and unsensitized to TCE for 24 and 72 h were significantly lower than those of solvent group (P < 0.05). as compared with unsensitized groups, the serum IgA levels of the groups sensitized to TCE for 24 and 72 h significantly decreased (P < 0.05).</p><p><b>CONCLUSION</b>After the guinea pig skin was sensitized to TCE, the serum C3, C4 levels decreased, the immune function disordered.</p>

Beta-arrestin and NF-kappaB, AP-1 activity in peripheral blood mononuclear cells of guinea pigs sensitized by trichloroethylene / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To explore the regulatory mechanism of immune response of guinea pigs sensitized by trichloroethylene (TCE), and the expression level of 3-arrestin, and the activity of NF-kappaB and AP-1 in peripheral blood mononuclear cells (PBMC) of guinea pigs sensitized by TCE.</p><p><b>METHODS</b>Guinea pigs were treated with TCE based on the guinea pig maximum response test (GPMT); Blank control group and DNCB positive control group were established. Scores of skin reaction were evaluated and used to determine whether or not allergy in guinea pig. Then TCE treated group was divided into sensitized group or un-sensitized group. The expression levels of beta-arrestin protein, activity of NF-kappaB and AP-1 in PBMC were detected by Western Blotting and EMSA, respectively. TNF-alpha level in serum was detected by ELISA kits.</p><p><b>RESULTS</b>No erythema or edema was found in the control group; part of guinea pigs treated with TCE developed erythema and edema, while obvious erythema and edema could be found in DNCB group. The sensitization rates were 71.4% and 100% in TCE and DNCB group, respectively. Compared with TCE un-sensitized group, expression of beta-arrestin and AP-1 activity were not significantly different in TCE sensitized group (P > 0.05). While the NF-kappaB activity was elevated obviously (P < 0.05). Compared with blank control groups [(32.118 +/- 12.550) pg/ml], serum TNF-alpha levels in TCE sensitized groups [(55.485 +/- 8.732) pg/ml] significantly elevated (P < 0.05);</p><p><b>CONCLUSION</b>In guinea pigs, beta-arrestin and AP-1 may not be activated, while the NF-kappaB activation is significant, and plays a immune regulatory role in the immune reaction of allergy induced by TCE.</p>

Role of apoptosis in trichloroethylene induced irritant injury in the skin / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the expression of apoptosis and caspase-8, cyt c in the skin of the BALB/c mice exposed to trichloroethylene (TCE).</p><p><b>METHODS</b>30 BALB/c mice were divided in random into the solvent control group, 10% TCE group, 20% TCE group, 40% TCE group, 80% TCE group and 100% TCE group. Apoptotic cells were detected by TUNEL and EM. The expressions of caspase-8 and cyt c were detected with immunohistochemical method.</p><p><b>RESULTS</b>EM showed that the apoptosis of cells was found in the high dosage groups. The immunohistochemical results showed that there were significant differences in the apoptosis rate and the activity of cyt c between the different dosage groups. There was the significant difference in the apoptosis rate between the 40%, 80%, 100% TCE groups the control group (P < 0.01). There was the significant difference in the expression of cyt c between the 20%, 40%, 80%, 100% TCE groups [(2.60 +/- 0.54), (3.42 +/- 0.56), (5.81 +/- 1.30) and (6.00 +/- 0.70), respectively] and the control group (P < 0.01). The expressions of caspase-8 had no significant differences (P > 0.05).</p><p><b>CONCLUSION</b>Apoptosis plays an important role in trichloroethylene induced irritant injury in skin and the apoptosis may be related with the mitochondrial injury.</p>

Changes of caspase-8 and caspase-9 activity during apoptosis of keratinocytes induced by trichloroethylene / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>(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).</p><p><b>CONCLUSION</b>Caspase-9 may be an important mediator of apoptosis in keratinocytes induced by trichloroethylene.</p>

Changes of nitric oxide after trichloroethylene irritation in hairless mice skin and protection of ginkgo biloba extract and vitamin E / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the changes of nitric oxide (NO) in the BALB/c hairless mice skin after trichloroethylene (TCE) irritation and the protection of ginkgo biloba extract (GbE) and vitamin E (VE).</p><p><b>METHODS</b>132 BALB/c hairless mice were randomly divided into blank control group, solvent group (olive oil), TCE groups (20%TCE, 40%TCE, 80%TCE and 100%TCE), GbE groups (0.1%GbE, 1%GbE and 10%GbE) and VE groups (5%VE, 10% VE and 20% VE), with 11 animals in each group, 5 for acute irritation test and 6 for the cumulative irritation test. The skin irritation was observed, and the levels of NO in the dorsal skin of BALB/C hairless mice were detected. The kit of NO was used to detect the levels of NO in the dorsal skin of BALB/c hairless mice.</p><p><b>RESULTS</b>(1) The skin presented erythema and edema after TCE irritation both in acute irritation and cumulative irritation test and the skin inflammation showed time-dose effect relationship; the mice skin was protected in GbE or VE groups. (2) In the acute stimulation test, the levels of NO in 80%TCE group (69.895 +/- 9.605 micromol/mg pro) and 100%TCE group (77.273 +/- 9.290 micromol/mg pro) were significantly different compared with blank control group and solvent control group (P < 0.05 or P < 0.01). In the protection group, the NO level were reduced, with the statistically significant differences. (3) In acute irritation test, the levels of NO in 80%TCE group (60.362 +/- 9.817 micromol/mg pro) and 100%TCE group (68.027 +/- 9.354 micromol/mg pro) were significantly different compared with blank control group and solvent control group, (P < 0.05 or P < 0.01); In the protection group, 1% GbE, 10% GbE, 10% VE and 20%VE could reduce the levels of NO, with statistically significant differences.</p><p><b>CONCLUSION</b>TCE can produce the irritation on the dorsal skin of BALB/c hairless mice and induce the significant increase of the NO levels. GbE and VE can protect the skin from TCE irritation damage.</p>

Cytotoxicity of trichloroethylene in keratinocytes involving alterations of mitochondrial function and ultrastructure / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To explore mechanism of dermal toxicity of trichloroethylene(TCE).</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Effects of TCE and PCE on cultured human keratinocyte lipid peroxidation and protective effect of vitamin E on it / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To explore the different concentrations of trichloroethylene (TCE) or perchloroethylene (PCE) induced cultured normal human epidermal keratinocyte (KC) lipid peroxidation and protective effect of Vitamin E on it.</p><p><b>METHODS</b>KC derived from 3 or more donors were pooled together and cultured with K-SFM. Neutral Red Uptake Assay was used to determine the IC50 of TCE or PCE, and then different concentrations of TCE or PCE were administered for culturing KC; 0.5 mmol/L TCE or 0.2 mmol/L PCE and different concentrations of Vitamin E were used to determine the protective effect of Vitamin E. After 4 hours' culture, kits were used to determine cellular MDA, SOD and ROS level.</p><p><b>RESULTS</b>Treatment of KC with different concentrations of TCE or PCE showed significant dose-related variations in lipid peroxidation, with the higher concentration, higher level of MDA, ROS and lower activity of SOD displayed in this study. Vitamin E 10 - 200 mmol/L dose-dependently attenuated MDA and ROS level, and increased SOD activities.</p><p><b>CONCLUSION</b>TCE or PCE can induce the lipid peroxidation in cultured KC and Vitamin E protects it from TCE- or PCE-induced peroxidation.</p>

Study of trichloroethylene-induced apoptosis in normal human epidermis keratinocytes in vitro / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the apoptosis-inducing effect of trichloroethylene (TCE) on cultured normal human epidermis keratinocytes (NHEK) in vitro.</p><p><b>METHODS</b>NR(50) values (the concentration of neutral red absorbed is reduced to 50%) of TCE on NHEK were assayed by neutral red uptake (NRU), and the administered dose of TCE was determined. Lipid peroxidation (LPO) and oxidative stress were assessed by measurement of malondialdehyde (MDA) contens and superoxide dismutase (SOD) activity. Transmission electron microscope (TEM) were used to observe morphologic changes, flow cytometer (FCM) was used to measure DNA contents and calculate cell apoptosis rate and proliferation index (PI).</p><p><b>RESULTS</b>NR(50) values of TCE on NHEK was found to be 4.53 mmol/L (95% CI: 3.92-5.13 mmol/L). The increase in MDA content and inhibition of SOD activity in a concentration-dependent manner were shown after NHEK was treated with a series of dose of TCE 4 h later, and typical morphologic changes of apoptosis were also observed by TEM examination. FCM analysis revealed a sub-G(1) peak in the apoptotic cells. The apoptotic rate in TCE 0.125, 0.500, 2.000 mmol/L exposed groups (31.83%, 38.63%, 44.35%, respectively) were significantly higher than that in blank control (18.42%), while PI in TCE 0.125, 0.500, 2.000 mmol/L group (3.26%, 2.48%, 2.07%, respectively) were significantly lower than that in blank control (4.99%).</p><p><b>CONCLUSION</b>TCE may induce apoptosis of cultured NHEK in vitro, and inhibit cell proliferation through lipid peroxidation and oxidative stress.</p>

Studies on fluoride concentration and cholinesterase activity in rat hippocampus / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the accumulation of fluoride in rat hippocampus and its effect on cholinesterase activity.</p><p><b>METHODS</b>Rats were subchronically exposed to NaF, and fluoride concentration and cholinesterase activity in rat hippocampus were determined.</p><p><b>RESULTS</b>Fluoride concentration in rat hippocampus was significantly correlated with the dosage of fluoride, and there were significant differences among high dosage group [(13.03 +/- 1.79) micro g/g], low dosage group [(9.83 +/- 0.92) micro g/g] and control [(8.27 +/- 1.11) micro g/g], P < 0.01. Acetylcholinesterase activities among three groups [(0.111 +/- 0.031) micro mol/mg, (0.143 +/- 0.025) micro mol/mg, (0.183 +/- 0.027) micro mol/mg] were also significantly different (P < 0.01), which was negatively correlated with fluoride concentration in rat hippocampus (r = -0.700, P < 0.01). The activity of butylcholinesterase in high dosage group [(0.041 +/- 0.010) micro mol/mg] was different from that of control [(0.067 +/- 0.025) micro mol/mg, P < 0.05], but the activity was not significantly related with fluoride concentration in rat hippocampus (r = -0.317, P = 0.094).</p><p><b>CONCLUSION</b>Fluoride may go through the blood-brain barrier and accumulate in rat hippocampus, and inhibit the activity of cholinesterase.</p>