ATG7 upregulation contributes to malignant transformation of human bronchial epithelial cells by B[a]PDE via DNMT3B protein degradation and miR-494 promoter methylation.

Lung cancer primarily arises from exposure to various environmental factors, particularly airborne pollutants. Among the various lung carcinogens, benzo(a)pyrene and its metabolite B[a]PDE are the strongest ones that actively contribute to lung cancer development. ATG7 is an E1-like activating enzyme and contributes to activating autophagic responses in mammal cells. However, the potential alterations of ATG7 and its role in B[a]PDE-caused lung carcinogenesis remain unknown. Here, we found that B[a]PDE exposure promoted ATG7 expression in mouse lung tissues, while B[a]PDE exposure resulted in ATG7 induction in human normal bronchial epithelial cells. Our studies also demonstrated a significant correlation between high ATG7 expression levels and poor overall survival in lung cancer patients. ATG7 knockdown significantly repressed Beas-2B cell transformation upon B[a]PDE exposure, and such promotive effect of ATG7 on cell transformation mediated the p27 translation inhibition. Further studies revealed that miR-373 inhibition was required to stabilize ATG7 mRNA, therefore increasing ATG7 expression following B[a]PDE exposure, while ATG7 induction led to the autophagic degradation of the DNA methyltransferase 3 Beta (DNMT3B) protein, in turn promoted miR-494 transcription via its promoter region methylation status suppression. We also found that the miR-494 upregulation inhibited p27 protein translation and promoted bronchial epithelial cell transformation via its directly targeting p27 mRNA 3'-UTR region. Current studies, to the best of our knowledge, are for the first time to identify that ATG7 induction and its mediated autophagy is critical for B[a]PDE-induced transformation of human normal epithelial cells.

Endoplasmic reticulum stress mediates nickel chloride-induced epithelial­mesenchymal transition and migration of human lung cancer A549 cells through Smad2/3 and p38 MAPK activation.

BACKGROUND: The endoplasmic reticulum (ER) is a cellular membrane-bound organelle whereby proteins are synthesized, folded and glycosylated. Due to intrinsic (e.g., genetic) and extrinsic (e.g., environmental stressors) perturbations, ER proteostasis can be deregulated within cells which triggers unfolded protein response (UPR) as an adaptive stress response that may impact the migration and invasion properties of cancer cells. However, the mechanisms underlying the nickel compounds on lung cancer cell migration and invasion remain uncertain. OBJECTIVE: We aimed to study whether Nickel chloride (NiCl2) induces ER stress in lung cancer cells, and whether ER stress is involved in modulating epithelial-mesenchymal transition (EMT) and migration by Smads and MAPKs pathways activation following NiCl2 treatment. METHODS: A549 cells were treated with NiCl2 to determine the cell viability using MTT assay. The wound healing assay was used to evaluate cell migration ability. ER ultrastructure was observed by transmission electron microscopy. Western blotting assay was performed to evaluate the protein levels of BIP, PERK, IRE-1α, XBP-1 s, and ATF6 for ER stress and UPR, E-cadherin and Vimentin for EMT, p-Smad2/3, p-ERK, p-JNK, and p-P38 for activation of Smads and MAPKs signaling pathways. RESULTS: The expression levels of BIP, PERK, IRE-1α, XBP-1 s, and ATF6 were significantly increased following treatment with NiCl2 in time- and dose-effect relationship. The ER stress inhibitor 4-PBA downregulated the expression levels of the above five proteins, and reversed the decrease in E-cadherin protein level and the increase in vimentin protein expression and cell migration abilities caused by NiCl2. Furthermore, 4-PBA significantly reduced nickel chloride-induced Smad2/3 and p38 MAPK pathway activation, while not affected ERK and JNK MAPK pathways. CONCLUSION: NiCl2 triggers ER stress and UPR in A549 cells. Moreover, 4-PBA alleviates NiCl2-induced EMT and migration ability of A549 cells possibly through the Smad2/3 and p38 MAPK pathways activation, rather than ERK and JNK MAPK pathways.

MCLR induces dysregulation of calcium homeostasis and endoplasmic reticulum stress resulting in apoptosis in Sertoli cells.

Microcystins-LR (MCLR) is a potent reproductive system toxin. We have previously shown that MCLR induced endoplasmic reticulum (ER) stress and apoptosis in testis. ER is the main calcium storage site in cells, and its calcium homeostasis plays an important role in the regulation of apoptosis. Hence, in the present study, we have investigated the role of calcium (Ca2+) in inducing apoptosis and how it affect the mitochondria and endoplasmic reticulum in TM4 cells. Our study found that MCLR induced an increase in Ca2+ concentration in TM4 cells. Compared to the controls, MCLR induced phosphorylation of calmodulin-dependent protein kinase II (CaMKII) which was involved in MAPKs activation, resulting in the induction of mitochondrial apoptosis pathways. Ca2+ chelator Bapta-AM partially reversed MCLR-induced apoptosis, confirming the possible involvement of calcium homeostasis disruption after MCLR exposure. Meanwhile, MCLR activated unfolded protein response and activated the ER apoptotic pathway by activating caspase-12. In addition, exposure to MCLR causes mitochondrial defects and increased apoptosis by up-regulating caspase 3 and cytosol cytochrome c expression. Collectively, these results demonstrated that MCLR disturbed calcium homeostasis, which caused ER-mitochondria dysfunction, ultimately promoted cell apoptosis in Sertoli cells.

Maternal vitamin D supplementation inhibits bisphenol A-induced proliferation of Th17 cells in adult offspring.

Bisphenol A (BPA) exposure can increase the risk of immune-related diseases in later life. Vitamin D3 (Vit D3) has been shown to have multiple immunomodulatory actions and has been used to treat immune diseases. However, the potential beneficial effects of Vit D3 on BPA-induced adverse effects in the immune system have not explored. We hypothesize that VitD3 may ameliorate BPA-induced side effects in the immune system, even in offspring of VitD3-supplemented mothers. Here, we established our experimental model by exposing pregnant dams with 1000 nM BPA with or without VitD3 (0.25 µg/kg, 1 µg/kg and 4 µg/kg) treatment. We show that mother's exposure to BPA increases proliferation of the spleen T helper 17 (Th17) cells and serum protein level of IL-17 in the offspring; however, VitD3 supplementation in mothers dose-dependently ameliorated these BPA-induced side effects on the immune system in the offspring as evidenced by attenuated upregulation of Th17 proliferation, and RORγt, IL-17, IL-6, and IL-23 expressions in the offspring. Our data provide the first evidence that maternal VitD3 supplementation offers benefits to the offspring by attenuating BPA-induced side effects on the immune system through vitamin D receptor (VDR)-dependent regulation of transcription factors and cytokines, suggesting its translational potential.

Exogenous hydrogen sulfide donor NaHS alleviates nickel-induced epithelial-mesenchymal transition and the migration of A549 cells by regulating TGF-ß1/Smad2/Smad3 signaling.

Nickel compounds are known to be common environmental and occupational carcinogens which also promote the migration of lung cancer cells. However, the molecular mechanism yet remains to be clarified. Hydrogen sulfide (H2S) is involved in cancer biological processes. However, the exact effect and functionality of H2S on nickel, towards the promotion of the migration ability of lung cancer cells, remains to be unknown. In this study, we have found that the nickel chloride (NiCl2) treatment significantly downregulates the protein levels of endogenous H2S enzyme cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-Mercaptopyruvate sulfurtransferase (3-MST). A correlation between NiCl2-induced epithelial-mesenchymal transition (EMT) and the migration ability of lung cancer A549 cells has been observed. Exogenous H2S donor, sodium hydrogen sulfide (NaHS) (100 µmol/L), can reverse NiCl2-induced EMT as well as the migration ability of A549 cells. NiCl2 treatment is able to upregulate the protein level of transforming growth factor-ß1 (TGF-ß1), p-Smad2, p-Smad3, p-JNK, p-ERK and p-P38 in a time-dependent fashion, indicating that both TGF-ß1/Smad2/Smad3 and mitogen-activated protein kinase (MAPK) signaling cascades (a non-Smad pathway) may play essential roles in NiCl2-dependent EMT as well as cell migration of human lung cancer cells. Furthermore, exogenous NaHS alleviates the NiCl2-induced EMT and the migration ability of A549 cells only by regulating TGF-ß1/Smad2/Smad3, rather than the MAPK, signaling pathway. These results indicate that the exogenous administration of NaHS might be a potential therapeutic strategy against nickel-induced lung cancer progression.

Bisphenol A promotes macrophage proinflammatory subtype polarization via upregulation of IRF5 expression in vitro.

Exposure to environmental endocrine-disrupting chemical Bisphenol-A (BPA) is closely associated with an imbalance of immune homeostasis, but the underlying mechanisms are not fully understood. In the present study, the effects of BPA on the polarization of mouse peritoneal macrophages were investigated in vitro. Environmentally relevant low concentrations of BPA treatment under M1 type polarization conditions increased the number of M1 subtype macrophages, the gene expression of M1 phenotypic marker CD11c and the activity and gene expression of M1 functional marker iNOS, as well as the production of pro-inflammatory cytokines. However, The same dose BPA treatment under M2 type polarization conditions reduced the number of M2 subtype macrophages, the gene expression of M2 phenotypic marker CD206 and the activity and gene expression of M2 functional marker Arg-1, along with the production of anti-inflammatory cytokines. We also identified that the expression of transcription factor IRF5 was upregulated by BPA exposure in M1 macrophages under M1 type polarization conditions. Our results demonstrate that BPA promotes macrophage polarization toward proinflammatory M1 subtype and M1 activity, associated with upregulated expression of IRF5, while BPA inhibits macrophage toward anti-inflammatory M2 subtype polarization. These findings provide new insight into the link between exposure to BPA and impairment of immune functions.

Hydrogen sulfide attenuates paraquat-induced epithelial-mesenchymal transition of human alveolar epithelial cells through regulating transforming growth factor-ß1/Smad2/3 signaling pathway.

Exogenous H2 S donor, sodium hydrosulfide (NaHS), can influence the bleomycin-induced pulmonary fibrosis by attenuating the epithelial-mesenchymal transition (EMT) of alveolar epithelial cells, but whether NaHS affects paraquat (PQ)-induced EMT and the molecular mechanisms remain unclarified. The aim of the present study is to examine the effect of exogenous NaHS on PQ-induced EMT in human alveolar epithelial cells (A549 cells) and assess if this effect occurs through regulating transforming growth factor (TGF)-ß1/Smad2/3 signaling pathway. The expressions of endogenous H2 S producing enzymes, namely cystathionine ß-synthase, cystathionine γ-lyase and 3-mercaptopyruvate sulfur transferase, were detected by reverse transcription-polymerase chain reaction and western blotting. The induced EMT was assessed by morphological and phenotypic characterizations, and the protein level of E-cadherin and vimentin were detected by western blotting. To investigate the effect of NaHS on PQ-induced EMT and potential mechanism, A549 cells were pretreated with NaHS before incubating with PQ and then evaluated by morphological changes, cell migration ability, the expression of EMT markers and TGF-ß1/Smad2/3 signaling pathway related proteins. PQ significantly downregulated the expression levels of cystathionine ß-synthase and cystathionine γ-lyase, but not 3-mercaptopyruvate sulfur transferase, in a time-dependent manner in A549 cells. Exogenous NaHS could significantly retard PQ-induced morphological changes and cell migration ability. Furthermore, exogenous NaHS significantly upregulated the expression of E-cadherin, whereas it downregulated the expression of vimentin. In addition, exogenous NaHS could also significantly attenuates PQ-induced TGF-ß1, phosphorylated Smad2/3 proteins expression, which induced by PQ in a time-dependent manner. This study provides the first evidence that exogenous NaHS attenuates PQ-induced EMT and migration of human alveolar epithelial cells through regulating the TGF-ß1/Smad2/3 signaling pathway.

Viral mimic polyinosine-polycytidylic acid potentiates liver injury in trichloroethylene-sensitized mice - Viral-chemical interaction as a novel mechanism.

Occupational trichloroethylene (TCE) exposure can induce hypersensitivity dermatitis and severe liver injury. Recently, several clinical investigations indicate that viral infection, such as human herpesvirus-6, is associated with hepatic dysfunction in patients with TCE-related generalized skin disorders. However, whether viral infection potentiates TCE-induced liver injury remains unknown. This study aimed to explore the contribution of viral infection to the development of TCE-sensitization-induced liver injury in BALB/c mice. Female BALB/c mice were randomly assigned into four groups: solvent control group (n = 20), TCE group (n = 80), poly(I:C) group (n = 20) and combination of TCE and poly(I:C) (poly(I:C)+TCE) group (n = 80). Poly(I:C) (50 µg) was i.p. administrated. TCE and poly(I:C)+TCE groups were further divided into sensitization and non-sensitization subgroup. Complement 3 and C3a protein levels, and complement factors were measured. Combination treatment significantly enhanced TCE-induced liver injury, decreased complement 3, but increased C3a in serum and liver tissues in sensitization group. These changes were not correlated with the hepatic complement 3 transcription. Moreover, combination treatment specifically promoted complement factor B, but not factor D and factor H expressions. These data provide first evidence that poly(I:C) potentiates liver injury in BALB/c mouse model of TCE-sensitization. Upregulated C3a and factor B contributes to the poly(I:C) action in TCE-induced liver injury. This new mode of action may explain increased risk of chemical-sensitization induced tissue damage by viral infection.

Prognostic value of hematological parameters in patients with paraquat poisoning.

Paraquat (PQ) is a non-selective contact herbicide, and acute PQ poisoning has a high mortality. The aim of the present study is to evaluate the prognostic value of hematological parameters in patients with acute PQ poisoning. We retrospectively reviewed the records of patients with acute PQ poisoning from January 2010 to December 2015 at the First Affiliated Hospital, Anhui Medical University (Hefei, China). A total of 202 patients were included in the study, and the 30-day mortality was 51.98%. Leukocyte, neutrophil counts and neutrophil-lymphocyte ratio (NLR) were significantly higher in non-survivors than in survivors. In the receiver operating characteristic (ROC) curve analysis, the NLR had an area of 0.916(95%CI, 0.877-0.954) and the optimal cut-off value was 10.57 (sensitivity, 86.70%; specificity, 83.51%; Youden's index, 0.702). The leukocyte counts had an area of 0.849(95%CI, 0.796-0.902) and the optimal cut-off value was 13.15 × 103/mm3 (sensitivity, 77.10%; specificity, 83.50%; Youden's index, 0.606). The neutrophil counts had an area of 0.878(95%CI, 0.830-0.925) and the optimal cut-off value was 10.10 × 103/mm3 (sensitivity, 83.80%; specificity, 79.38%; Youden's index, 0.632). NLR, leukocyte and neutrophil counts are associated with the 30-day mortality, which may be useful and simple parameters in predicting the prognosis of PQ poisoning.

Association of glutathione S-transferase pi (GSTP1) Ile105Val polymorphism with the risk of skin cancer: a meta-analysis.

Numerous epidemiological studies have evaluated the association of Glutathione S-transferase P1 (GSTP1) Ile105Val polymorphism with the risk of skin cancer. However, the results remain inconclusive. To derive a more precise estimation of the association between the GSTP1 Ile105Val polymorphism and skin cancer risk, a meta-analysis was performed. A comprehensive search was conducted to identify the eligible studies. We used odds ratios (ORs) with 95 % confidence intervals (CIs) to assess the association of GSTP1 Ile105Val polymorphism with skin cancer risk. Thirteen case-control studies in nine articles, which included a total of 1504 cases and 2243 controls. Overall, we found that GSTP1 Ile105Val polymorphism was not associated with skin cancer risk. Furthermore, subgroup analysis by histological types showed that GSTP1 Ile105Val polymorphism was associated with risks of malignant melanoma under the dominant model (Val/Val + Val/Ile vs. Ile/Ile: OR 1.230, 95 % CI 1.017-1.488, P = 0.033). However, lack of association between GSTP1 Ile105Val polymorphism and BCC and SCC risk in all genetic models. Our meta-analysis suggested that the GSTP1 Ile105Val polymorphism might be associated with increased risk of malignant melanoma in Caucasian population.

Association of a disintegrin and metalloprotease 33 (ADAM33) gene polymorphisms with the risk of COPD: an updated meta-analysis of 2,644 cases and 4,804 controls.

A series of observational studies have been made to investigate the association of the ADAM33 gene polymorphisms with the risk of COPD, but their results were conflicting. Therefore, we performed an updated meta-analysis to quantitatively summarize the associations of ADAM33 gene polymorphisms with the risk of COPD. Thirteen case-control studies referring to nine SNPs were identified: V4 (rs2787094), T+1 (rs2280089), T2 (rs2280090), T1 (rs2280091), S2 (rs528557), S1 (rs3918396), Q-1 (rs612709), F+1 (rs511898) and ST+5 (rs597980). A dominant model (AA+Aa vs. aa), recessive model (AA vs. Aa+aa), additive model (AA vs. aa) and allelic model (A vs. a) were used to evaluate the association of ADAM33 polymorphism with the risk of COPD. The results indicated that significant associations were found for ADAM33 T1, T2, S1, Q-1, F+1 and ST+5 polymorphisms associated with the risk of COPD in different populations. However, no significant associations were found for V4, T+1 and S2 polymorphisms with the risk of COPD in all genetic models, even in the subgroup analysis by ethnicity. This meta-analysis provided evidence that the ADAM33 T1, T2, S1, Q-1, F+1 and ST+5 six locus polymorphisms association with the risk of COPD. Furthermore, T2, Q-1 and ST+5 indicated an association with the risk of COPD in the European populations, whereas T1, T2, S1, F+1 and Q-1 indicated an association with the risk of COPD in the Asian populations.

Bleomycin-induced epithelial-mesenchymal transition in sclerotic skin of mice: possible role of oxidative stress in the pathogenesis.

Epithelial-mesenchymal transition (EMT) derived myofibroblasts are partly responsible for the increased collagen synthesis and deposition that occur in tissue fibrosis; however EMT occurrence in skin fibrosis and its mechanism remain unknown. The aim of this study was to investigate whether epithelial cells undergo EMT and determine the role of oxidative stress in this process. BALB/c mice were subcutaneously injected with bleomycin (BLM) or phosphate buffer saline (PBS) into the shaved back daily for 2, 3, and 4weeks. Skin collagen deposition was evaluated by histopathology and Western blotting. EMT characteristics in the skin were determined by histopathology and immunofluorescent staining for E-cadherin and vimentin, which were further evaluated by Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR). To investigate the role of oxidative stress in EMT, the antioxidant N-acetylcysteine (NAC) was intraperitoneally (100mg/kg body weight/day) injected daily for 3weeks. The epithelial suprabasal cells were detached from the basement membrane zone (BMZ) in the sclerotic skin treated with BLM. Immunofluorescent staining indicated vimentin-positive epithelial cells frequently occurring in the thickened epidermis of BLM-treated mice. Western blotting and RT-PCR showed that the expression of E-cadherin was significantly decreased but that of vimentin significantly increased in the skin treated with BLM. NAC attenuated BLM induced oxidative damage, changes in E-cadherin and vimentin expressions and collagen deposition in the sclerotic skin of mice. This study provides the first evidence that BLM induces the EMT of the epithelial cells superficial to the basement membrane zone in the skin fibrosis. Oxidative stress may contribute, at least in part, to BLM induced EMT and skin fibrosis in mice.

Complement activation and liver impairment in trichloroethylene-sensitized BALB/c mice.

Our recent studies have shown that trichloroethylene (TCE) was able to induce multisystem injuries in the form of occupational medicamentosa-like dermatitis, including skin, kidney, and liver damages. However, the role of complement activation in the immune-mediated liver injury is not known. This study examined the role of complement activation in the liver injury in a mouse model of TCE-induced sensitization. Treatment of female BALB/c mice with TCE under specific dosing protocols resulted in skin inflammation and sensitization. Skin edema and erythema occurred in TCE-sensitized groups. Trichloroethylene sensitization produced liver histopathological lesions, increased serum alanine aminotransferase, aspartate transaminase activities, and the relative liver weight. The concentrations of serum complement components C3a-desArg, C5a-desArg, and C5b-9 were significantly increased in 24-hour, 48-hour, and 72-hour sensitization-positive groups treated with TCE and peaked in the 72-hour sensitization-positive group. Depositions of C3a, C5a, and C5b-9 into the liver tissue were also revealed by immunohistochemistry. Immunofluorescence further verified high C5b-9 expression in 24-hour, 48-hour, and 72-hour sensitization-positive groups in response to TCE treatment. Reverse transcription-polymerase chain reaction detected C3 messenger RNA expression in the liver, and this was significantly increased in 24-hour and 48-hour sensitization-positive groups with a transient reduction at 72 hours. These results provide the first experimental evidence that complement activation may play a key role in the generation and progression of immune-mediated hepatic injury by exposure to TCE.

Possible role of complement activation in renal impairment in trichloroethylene-sensitized guinea pigs.

Recent studies have revealed that trichloroethylene (TCE) can induce occupational medicamentosa-like dermatitis (OMLD) with multi-system injuries, including liver, kidney and skin injuries, which can subsequently cause multiple organ failure later. But the mechanism of immune dysfunction leading to organ injury was rarely clarified. The present study was initiated to analyze the influence of trichloroethylene on renal injury and study the relevant mechanism in guinea pigs. Guinea pig maximization test (GPMT) was carried out. Inflammation on the guinea pigs' skin was scored. Kidney function, urine protein and ultra-structural change of kidney were determined by biochemical detection and electron microscope. Deposition of complement 3 and membrane attack complex (MAC, C5b-9) were determined by immunohistochemistry. Erythema and edema of skin impairment were observed in TCE sensitized groups, and sensitization rate was 63.16%. Through electron microscope, tubular epithelial cell mitochondrial swelling, vacuolar degeneration and atrophy of microvillus were observed in TCE sensitized groups. The parameters of urease and urinary protein elevated markedly, and a high degree of C3 and MAC deposition was found in the renal tubular epithelial cells in TCE sensitized groups. By demonstrating that TCE and its metabolites can cause the deposition of C3 and MAC in renal epithelial cells, we found that activated complement system may be the mechanism of the acceleration and the development of TCE-induced kidney disease.

[Changes of nitric oxide after trichloroethylene irritation in hairless mice skin and protection of ginkgo biloba extract and vitamin E].

OBJECTIVE: 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). METHODS: 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. RESULTS: (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. CONCLUSION: 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.

[Relationship of nitric oxide with the apoptosis of keratinocytes].

Nitric oxide (NO) is a kind of free radical, also is a signaling molecule, which universally distributed in the tissues of organism. NO play important role in the dermato-, cardia-, encephalo-, hepat- and immunological regulation on the physio- and patho- situation. Recently studying show that NO can bifunctionally regulate the apoptosis of keratinocytes, which play an important role in the occurrence and development of many skin diseases. However, the regulatory mechanism still can't be completely elucidated at present time. In this review, we are reviewing the recent investigation progress on the apoptosis of keratinocytes and some diseases of skin that related to the regulating disturbance of NO.

Kupffer cells and reactive oxygen species partially mediate lipopolysaccharide-induced downregulation of nuclear receptor pregnane x receptor and its target gene CYP3a in mouse liver.

Pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that regulates target gene transcription in a ligand-dependent manner. The in vivo effects of lipopolysaccharide (LPS) on expression of PXR and its target gene cytochrome P450 3A (CYP3A) in mouse liver were investigated in this study. Mice were injected intraperitoneally with different doses of LPS (0.1-5.0 mg/kg). PXR and CYP3A11 mRNA levels were measured using reverse transcription polymerase chain reaction. Results indicate that LPS significantly inhibits the expression of PXR mRNA in a dose-dependent manner, followed by suppression of CYP3A11 mRNA in mouse liver. LPS also represses the upregulation of CYP3A11 mRNA levels and erythromycin N-demethylase (ERND) catalytic activity in mice pretreated with PXR ligands dexamethasone, rifampicin, mifepristone, and phenobarbital. LPS-induced downregulation of PXR and CYP3A11 mRNA in liver was significantly attenuated in mice pretreated with gadolinium chloride, a selective Kupffer cell toxicant. Pretreatment with a single dose of gadolinium chloride (10 mg/kg) also significantly attenuated LPS-induced downregulation of dexamethasone-, rifampicim-, mifepristone-, and phenobarbital-inducible, CYP3A11 mRNA expression and ERND activity in mouse liver. Furthermore, LPS-induced downregulation of PXR and CYP3A11 mRNA was significantly attenuated in mice pretreated with allopurinol, an inhibitor of xanthine oxidase, and diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. Allopurinol and diphenyleneiodonium chloride pretreatment also attenuated the repressive effects of LPS on dexamethasone-, rifampicin-, mifepristone-, and phenobarbital-inducible CYP3A11 mRNA expression and ERND catalytic activity in mouse liver. However, aminoguanidine, a selective inhibitor of inducible nitric oxide synthase, has no effect on LPS-induced downregulation of PXR and CYP3A11 mRNA. Finally, LPS-induced downregulation of PXR and CYP3A11 mRNA was prevented in mice pretreated with either N-acetylcysteine or ascorbic acid. These antioxidants also prevented the repressive effects of LPS on dexamethasone-, rifampicin-, mifepristone-, and phenobarbital-inducible CYP3A11 mRNA expression and ERND catalytic activity in mouse liver. These results indicate that Kupffer cells contribute to LPS-induced downregulation of PXR and CYP3A in mouse liver. Reactive oxygen species, produced possibly by NADPH oxidase and perhaps by xanthine oxidase, are involved in LPS-induced downregulation of nuclear receptor PXR and its target gene CYP3A in mouse liver.