Search

Extracellular vesicles (EVs), a class of heterogeneous membrane vesicles, are generally divided into exosomes and microvesicles on basis of their origination from the endosomal membrane or the plasma membrane, respectively. EV-mediated bidirectional communication among various cell types supports cancer cell growth and metastasis. EVs derived from different cell types and status have been shown to have distinct RNA profiles, comprising messenger RNAs and non-coding RNAs (ncRNAs). Recently, ncRNAs have attracted great interests in the field of EV-RNA research, and growing numbers of ncRNAs ranging from microRNAs to long ncRNAs have been investigated to reveal their specific functions and underlying mechanisms in the tumor microenvironment and premetastatic niches. Emerging evidence has indicated that EV-RNAs are essential functional cargoes in modulating hallmarks of cancers and in reciprocal crosstalk within tumor cells and between tumor and stromal cells over short and long distance, thereby regulating the initiation, development and progression of cancers. In this review, we discuss current findings regarding EV biogenesis, release and interaction with target cells as well as EV-RNA sorting, and highlight biological roles and molecular mechanisms of EV-ncRNAs in cancer biology.

Anti-Müllerian hormone (AMH) downregulates the level of stem cell factor (SCF) via the cAMP/PKA signaling pathway in human granulosa cells (GCs). Little information is available on the molecular mechanism underlying the interaction. This study is aimed at determining whether AMH regulates expression of SCF via the cAMP-PKA-CREB signaling pathway in human GCs. In the present study, we verified the binding of cAMP-response element-binding protein (CREB) to promoter of SCF in human GCs. Furthermore, the effect of CREB was tested on the SCF promoter, and the site of CREB binding to SCF promoter was identified using truncations as well as assays of SCF-promoted mutation and CREB mutation. To investigate the correlation among AMH, SCF promoter, and CREB, pGL-Basic-SCF+CREB was transfected into overexpressed AMH GCs (AMH-high GCs), low expressed AMH GCs (AMH-low GCs), and normal GCs (GCs), respectively. Finally, immunofluorescence, double immunostaining, and Western blot were carried out in AMH-high and AMH-low GCs to confirm the AMH-mediated regulation of SCF expression by inhibiting the phosphorylation of CREB (pCREB) in GCs. Results indicated CREB interacted with SCF promoter and significantly enhanced the transcription level of SCF. The CREB binding site was localized at 318-321 bp of SCF gene promote. AMH inhibits the expression of SCF by phosphorylation of CREB via the PKA signaling pathway in GCs. These findings provide an in-depth understanding of the molecular mechanism underlying AMH suppressing the follicle growth, which would aid in the development of a novel therapy.

Chronic infection with liver flukes (such as Clonorchis sinensis) can induce severe biliary injuries, which can cause cholangitis, biliary fibrosis, and even cholangiocarcinoma. The release of extracellular vesicles by C. sinensis (CsEVs) is of importance in the long-distance communication between the hosts and worms. However, the biological effects of EVs from liver fluke on biliary injuries and the underlying molecular mechanisms remain poorly characterized. In the present study, we found that CsEVs induced M1-like activation. In addition, the mice that were administrated with CsEVs showed severe biliary injuries associated with remarkable activation of M1-like macrophages. We further characterized the signatures of miRNAs packaged in CsEVs and identified a miRNA Csi-let-7a-5p, which was highly enriched. Further study showed that Csi-let-7a-5p facilitated the activation of M1-like macrophages by targeting Socs1 and Clec7a; however, CsEVs with silencing Csi-let-7a-5p showed a decrease in proinflammatory responses and biliary injuries, which involved in the Socs1- and Clec7a-regulated NF-κB signaling pathway. Our study demonstrates that Csi-let-7a-5p delivered by CsEVs plays a critical role in the activation of M1-like macrophages and contributes to the biliary injuries by targeting the Socs1- and Clec7a-mediated NF-κB signaling pathway, which indicates a mechanism contributing to biliary injuries caused by fluke infection. However, molecules other than Csi-let-7a-5p from CsEVs that may also promote M1-like polarization and exacerbate biliary injuries are not excluded.

Background Various stimuli, including Clonorchis sinensis infection, can cause liver fibrosis. Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs) with massive production of extracellular matrix (ECM). Our previous study showed that the TGF-β1-induced Smad signaling pathway played a critical role in the activation of HSCs during liver fibrosis induced by worm infection; however, the mechanisms that modulate the TGF-β/Smad signaling pathway are still poorly understood. Accumulating evidence demonstrates that miRNAs act as an important regulator of activation of HSCs during liver fibrosis. Methods The target of miR-497 was determined by bioinformatics analysis combined with a dual-luciferase activity assay. LX-2 cells were transfected with miR-497 inhibitor and then stimulated with TGF-β1 or excretory/secretory products of C. sinensis (CsESPs), and activation of LX-2 was assessed using qPCR or western blot. In vivo, the mice treated with CCl4 were intravenously injected with a single dose of adeno-associated virus serotype 8 (AAV8) that overexpressed anti-miR-497 sequences or their scramble control for 6 weeks. Liver fibrosis and damage were assessed by hematoxylin and eosin (H&E) staining, Masson staining, and qPCR; the activation of the TGF-β/Smad signaling pathway was detected by qPCR or western blot. Results In the present study, the expression of miR-497 was increased in HSCs activated by TGF-β1 or ESPs of C. sinensis. We identified that Smad7 was the target of miR-497 using combined bioinformatics analysis with luciferase activity assays. Transfection of anti-miR-497 into HSCs upregulated the expression of Smad7, leading to a decrease in the level of p-Smad2/3 and subsequent suppression of the activation of HSCs induced by TGF-β1 or CsESPs. Furthermore, miR-497 inhibitor delivered by highly-hepatotropic (rAAV8) inhibited TGF-β/smads signaling pathway by targeting at Smad7 to ameliorate CCL4-induced liver fibrosis. Conclusions The present study demonstrates that miR-497 promotes liver fibrogenesis by targeting Smad7 to promote TGF-β/Smad signaling pathway transduction both in vivo and in vitro, which provides a promising therapeutic strategy using anti-miR-497 against liver fibrosis. Graphical Abstract

AimsSerous cystadenomata (SCAs) are benign pancreatic cystic neoplasms that present a diagnostic challenge despite many investigational approaches. Notwithstanding the promise of molecular diagnostics, these tests have limited accessibility in day-to-day surgical pathology practices. We aim to corroborate and build on recent evidence which suggests that positive α-inhibin immunohistochemistry (IHC) is a helpful adjunct in the biopsy confirmation of pancreatic SCA.MethodsWe retrospectively reviewed 22 fine-needle aspirates/biopsies from 14 patients (mean age 65 years, 47–83 years) with pancreatic multicystic lesions radiologically suspicious for SCA (location: 6 body, 2 head, 4 tail, 1 neck, 1 uncinate; cyst size: mean 3.7 cm, 2.0–7.6 cm), as well as an additional 10 pancreatic resection specimens with confirmed SCA; α-inhibin IHC was performed on all cell blocks, biopsy slides and representative resection specimen sections. Where available, associated cyst fluid was analysed for correlative vascular endothelial growth factor A (VEGF-A) and carcinoembryonic antigen levels.ResultsAn α-inhibin IHC sensitivity of 80% was observed in the cases with resection confirmed SCA. Of the fine-needle aspirate/biopsy specimens, 59% (13/22) contained epithelial cells strongly positive for α-inhibin. When selecting for specimens that exhibited distinct strips of epithelium, the α-inhibin strong positivity rate increased to 73% (8/11). VEGF-A values were supportive of false-negative α-inhibin IHC in three cases and true-negative α-inhibin IHC in one case.ConclusionThis study postulates a diagnostic algorithm to confirm pancreatic SCA which may help to decrease unnecessary follow-up endoscopy/surgical resection and would decrease the associated morbidity, mortality and financial costs in patients with this otherwise benign condition.

Background The diagnosis of alpha-1-antitrypsin (A1AT) deficiency has been hindered by obscurity concerning the testing process and treatment implications. In this study, we aimed to identify regional differences in the diagnostic rates for A1AT deficiency in the western Canadian provinces of British Columbia (BC) and Alberta (AB). Methods The number of A1AT deficiency variant genotype (ZZ, SZ, MZ, SS, and MS) diagnoses were reviewed for BC and AB. The regional diagnostic rates for A1AT deficiency variants in these two provinces, normalized for the predicted population prevalence of each variant genotype, was defined as the annual provincial diagnostic rate (APDR) for a given variant genotype. Sex specific variations in the mean age at diagnosis for the five variant genotypes were compared both within and between provinces. Results The SZ and MZ genotype APDRs were significantly increased in the AB population compared to the BC population. The SS and MS APDRs were similar between AB and BC. There was a significantly decreased mean age of diagnosis for AB males, as compared to BC males (for the SZ, MS, and MZ genotypes) and as compared to AB females (for the MS, MZ, and SS genotypes). There were no significant differences in the mean age of diagnosis between the females and males in BC, or between females in AB and BC, for any genotype. Conclusion The notably higher APDR for more severe A1AT deficiency genotypes, and lower mean age of diagnosis for most variant genotypes in AB males, deserves further investigation to determine the explanation(s) for these differences.

Mice with different genetic backgrounds have various susceptibilities to infection with Clonorchis sinensis, although the mechanisms underlying are largely unknown. Toll-like receptor 4 (TLR4) as one of the most important pattern recognition receptors (PPRs) is essential for the invasion, survival, pathogenesis, and elimination of worms. The roles played by TLR4 in C. sinensis infection may vary due to the different genetic backgrounds of mice. In the present study, a relatively resistant mouse strain-C57BL/10 to C. sinensis was used for investigation on the possible roles of TLR4 in the biliary injuries and peribiliary fibrosis. TLR4 wild type (TLR4wild) and TLR4 defective (TLR4def) mice were orally infected with 45 metacercariae of C. sinensis, and all C. sinensis-infected mice and non-infected groups were anesthetized on day 28 post-infection. The liver and serum from each mouse were collected for assessment of the biliary injuries and biliary fibrosis. Meanwhile, hepatic leukocytes were isolated and detected for the activation of M1 or M2 macrophage using flow cytometry. The hepatic type 1 immune response and type 2 immune responses -relative molecules were also evaluated using ELISA and quantitative PCR. The data showed that TLR4def aggravated liver inflammatory cell infiltrations, bile duct proliferation, biliary and hepatocellular injuries, and ECM deposition in C. sinensis-infected mice, compared with TLR4wild mice when they were intragastrically administered with the same amounts of C. sinensis metacercaria. Furthermore, the M2-like macrophages and type 2 immune responses were significantly predominant induced in TLR4def mice, compared with that of TLR4wild mice following C. sinensis infection. But the type 1 immune response were significantly decreased in TLR4def mice, compared with TLR4wild mice after C. sinensis infection. These data demonstrate that TLR4 deficiency exacerbates biliary injuries and peribiliary fibrosis caused by C. sinensis in C57BL/10 strain mice, which is contributed by augments of type 2 immune responses and decrease pro-inflammatory responses.

Repeated implantation failure (RIF) is a common endocrine disease that causes female infertility and the etiology is unknown. The abnormal expression of key proteins and hormones at the maternal-fetal interface affected the maternal-fetal communication and leads to adverse pregnancy outcomes. The expression of anti-Mullerian hormone (AMH) and AMH receptor II (AMHRII) was observed in the endometrium. This study aimed to investigate the expression of AMH and AMHRII at the human endometrium, decidual tissue, and blastocyst. Furthermore, the expression of AMH and AMHRII were examined in the RIF patients using immunohistochemistry and quantitative real-time PCR to test the AMHRII expression. The results demonstrated that AMH and AMHRII were present in healthy endometrium and AMHRII was highly expressed in mid-luteal phase. In addition, AMHRII expression was detected throughout the pregnancy and AMHRII's highest expression was in the second trimester. AMHRII was expressed in the blastocysts; however, AMH was not observed. The positive expression rate for AMHRII was significantly higher in the endometrium from RIF. Estrogen receptor (ER), insulin-like growth factor binding protein 1(IGFBP1), and prolactin (PRL) were significantly less expressed in RIF with high expression of AMHRII. The apoptosis was significantly higher in patients with high expression of AMHRII than in patients with normal expression of AMHRII. Our data suggests that AMHRII had an effect on RIF via the AMH and AMHRII signaling pathway. It participated in the development of RIF by interfering with endometrial decidualization and apoptosis.

Oxidative stress aggravates mitochondrial injuries and accelerates the proliferation of vascular smooth muscle cells (VSMCs), which are important mechanisms contributing to vascular remodeling in hypertension. We put forward the hypothesis that Astaxanthin (ATX), known to possess strong features of antioxidant, could attenuate vascular remodeling by inhibiting VSMC proliferation and improving mitochondrial function. The potential effects of ATX were tested on spontaneously hypertensive rats (SHRs) and cultured VSMCs that injured by angiotensin II (Ang II). The results showed that ATX lowered blood pressure, reduced aortic wall thickness and fibrosis, and decreased the level of reactive oxygen species (ROS) and H2O2 in tunica media. Moreover, ATX decreased the expression of proliferating cell nuclear antigen (PCNA) and ki67 in aortic VSMCs. In vitro, ATX mitigated VSMC proliferation and migration, decreased the level of cellular ROS, and balanced the activities of ROS-related enzymes including NADPH oxidase, xanthine oxidase, and superoxide dismutase (SOD). Besides, ATX mitigated Ca2+ overload, the overproduction of mitochondrial ROS (mtROS), mitochondrial dysfunction, mitochondrial fission, and Drp1 phosphorylation at Ser616. In addition, ATX enhanced mitophagy and mitochondrial biosynthesis by increasing the expression of PINK, parkin, mtDNA, mitochondrial transcription factor A (Tfam), and PGC-1α. The present study indicated that ATX could efficiently treat vascular remodeling through restraining VSMC proliferation and restoring mitochondrial function. Inhibiting mitochondrial fission by decreasing the phosphorylation of Drp1 and stimulating mitochondrial autophagy and biosynthesis via increasing the expression of PINK, parkin, Tfam, and PGC-1α may be part of its underlying mechanisms.

Due to limitations in early diagnosis and treatments of Parkinson's disease (PD), it is necessary to explore the neuropathological changes that occur early in PD progression and to design neuroprotective therapies to prevent or delay the ongoing degeneration process. Metabotropic glutamate receptor 5 (mGlu5) has shown both diagnostic and therapeutic potential in preclinical studies on PD. Clinical trials using mGlu5 negative allosteric modulators to treat PD have, however, raised limitations about the neuroprotective role of mGlu5. It is likely that mGlu5 has different regulatory roles in different stages of PD. Here, we investigated a protective role of cystic fibrosis transmembrane conductance regulator-associated ligand (CAL) in the progression of PD by differential regulation of mGlu5 expression and activity to protect against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity. Following treatment with 6-OHDA, mGlu5 and CAL expressions were elevated in the early stage and reduced in the late stage, both in vitro and in vivo. Activation of mGlu5 in the early stage by (RS)-2-chloro-5-hydroxyphenylglycine, or blocking mGlu5 in the late stage by 2-methyl-6-(phenylethynyl) pyridine, increased cell survival and inhibited apoptosis, but these effects were significantly weakened by knockdown of CAL. CAL alleviated 6-OHDA-induced neurotoxicity by regulating mGlu5-mediated signaling pathways, thereby maintaining the physiological function of mGlu5 in different disease stages. In PD rat model, CAL deficiency aggravated 6-OHDA toxicity on dopaminergic neurons and increased motor dysfunction because of lack of regulation of mGlu5 activity. These data reveal a potential mechanism by which CAL specifically regulates the opposite activity of mGlu5 in progression of PD to protect against neurotoxicity, suggesting that CAL is a favorable endogenous target for the treatment of PD.

Metabotropic glutamate receptor (mGlu)5 regulates microglia activation, which contributes to inflammation. However, the role of mGlu5 in neuroinflammation associated with Parkinson's disease (PD) remains unclear. Triptolide (T10) exerts potent immunosuppressive and anti-inflammatory effects and protects neurons by inhibiting microglia activation. In this study, we investigated the role of mGlu5 in the anti-inflammatory effect of T10 in a lipopolysaccharide (LPS)-induced PD model. In cultured BV2 cells and primary microglia, blocking mGlu5 activity or knocking down its expression abolished T10-inhibited release of proinflammatory cytokines induced by LPS. Moreover, T10 up-regulated mGlu5 expression decreased by LPS through enhancing mRNA expression and protein stability. T10 also reversed the reduction in mGlu5 membrane localization and modulated receptor-mediated mitogen-activated protein kinase activity induced by LPS. Pharmacological inhibition of signaling molecules increased nitric oxide level and inducible nitric oxide synthase (iNOS), tumor necrosis factor-α, and interleukin (IL)-1β and -6 transcript levels that were downregulated by treatment with T10. Consistent with these in vitro findings, blocking mGlu5 attenuated the anti-inflammatory effects of T10 in an LPS-induced PD model and blocked the decreases in the number and morphology of ionized calcium binding adaptor molecule 1-positive microglia and LPS-induced iNOS protein expression caused by T10 treatment. Besides, mGlu5 mediated the effect of T10 on microglia-induced astrocyte activation in vitro and in vivo. The findings provide evidence for a novel mechanism by which mGlu5 regulates T10-inhibited microglia activation via modulating protein expression of the receptor and its intracellular signaling. The study might contribute to the biological effects of Chinese herbs as an approach for protecting against neurotoxicity in PD.

Six undescribed pentacyclic triterpenoids including four triterpenoid aglycones, 1β,2a,3β,23-tetrahydroxyurs-12-en-28-ursolic acid, 2a,3a,6β,19α,23-pentahydroxyurs-12-en-28-ursolic acid, 2α,3α,20β,23-tetrahydroxyurs-12-en-28-ursolic acid and 1β,2a,3β,23-tetrahydroxyurs-12,20(30)-dien-28-ursolic acid, and two triterpenoid glucosides, 2a,3a,23-trihydroxy-12,20(30)-dien-28-ursolic acid 28-O-β-d-glucopyranoside and 1-oxo-3β,23-dihydroxyolean-12-en-28-oic acid 28-O-β-d-xylopyranoside, along with 5 known triterpenoids were isolated from a CH3Cl-soluble extract of the leaves of Cyclocarya paliurus. Their structures were established on the basis of chemical and spectroscopic approaches. These compounds were assessed for their antioxidant effects on FFA-induced hepatic steatosis in HepG2 cells. The results revealed that three saponins and two aglycones markedly increased SOD activity and reduced MDA level.

Four new C-11 monosaccharide attached dammarane triterpenoid glycosides cypaliurusides S V (1–4), along with nine known dammarane triterpenoid glycosides (5–13) were isolated from a CHCl3-soluble extract of the leaves of Cyclocarya paliurus. All characterized compounds were assayed for their cytotoxicities against HepG2 cells and 10 compounds were evaluated for the agonistic effects on sirtuin1 (SIRT1). The results showed that compounds 1, 5 and 6 were strongly cytotoxic in HepG2 cell line. Two dammarane triterpenoid glycosides 3 and 10 exhibited agonistic activities on SIRT1 with IC50 of 10 μM and 20 μM, respectively.

Numerous epidemiological studies suggested that there is a variable cancer risk in patients with Parkinson's disease (PD). However, the underlying mechanisms remain unclear. In the present study, the role of metabotropic glutamate receptor 5 (mGluR5) has been investigated in 6-hydroxydopamine (6-OHDA)-induced PD combined with liver cancer both in vitro and in vivo. We found that PD cellular model from 6-OHDA-lesioned MN9D cells suppressed the growth, migration, and invasion of Hepa1-6 cells via down-regulation of mGluR5-mediated ERK and Akt pathway. The application of 2-methyl-6-(phenylethyl)-pyridine and knockdown of mGluR5 further decreased the effect on Hepa-1-6 cells when co-cultured with conditioned media. The effect was increased by (S)-3,5-dihydroxyphenylglycine and overexpression of mGluR5. Moreover, more release of glutamate from 6-OHDA-lesioned MN9D cells suppressed mGluR5-mediated effect of Hepa1-6 cells. Application of riluzole eliminated the increased glutamate release induced by 6-OHDA in MN9D cells and aggravated the suppressive effect on Hepa-1-6 cells. In addition, the growth of implanted liver cancer was inhibited in 6-OHDA induced PD-like rats, and was associated with increased glutamate release in the serum and down-regulation of mGluR5 in tumor tissue. Collectively, these results indicate that selective antagonism of glutamate and mGluR5 has a potentially beneficial effect in both liver cancer and PD, and thus may provide more understanding for the clinical investigation and further an additional therapeutic target for these two diseases.

Anorectal melanoma is a rare disease that carries a poor prognosis. To date, limited genetic analyses confirmed KIT mutations as a recurrent genetic event similar to other mucosal melanomas, occurring in up to 30% of anorectal melanomas. Importantly, a subset of tumors harboring activating KIT mutations have been found to respond to c-Kit inhibitor-based therapy, with improved patient survival at advanced tumor stages. We performed comprehensive targeted exon sequencing analysis of 467 cancer-related genes in a larger series of 15 anorectal melanomas, focusing on potentially actionable variants based on gain- and loss-of-function mutations. We report the identification of oncogenic driver events in the majority (93%) of anorectal melanomas. These included variants in canonical MAPK pathway effectors rarely observed in cutaneous melanomas (including an HRAS mutation, as well as a BRAF mutation resulting in duplication of threonine 599), and recurrent mutations in the tumor suppressor NF1 in 20% of cases, which represented the second-most frequently mutated gene after KIT in our series. Furthermore, we identify SF3B1 mutations as a recurrent genetic event in mucosal melanomas. Our findings provide an insight into the genetic diversity of anorectal melanomas, and suggest significant potential for alternative targeted therapeutics in addition to c-Kit inhibitors for this melanoma subtype.

Objective To observe the effects of Huannao Yicong Formula (还脑益聪方, HYF) on learning and memory and it’s regulating effect on γ-secretase related anterior pharynx defective 1 (APH-1), presenilin enhancer-2 (PEN-2) signaling pathway, so as to discuss and further clarify the mechanism of HYF on Alzheimer’s disease.

Neuroendocrine carcinomas (NECs) of the colorectum are rare but highly aggressive neoplasms. These tumors show some shared genetic alterations with colorectal adenocarcinomas, and most of them have adjacent glandular adenoma or adenocarcinoma components. However, genetic data on colorectal NECs still are sparse and insufficient for definite conclusions regarding their molecular origin. Based on morphological characterization, panel and whole-exome sequencing, we here present results from an in-depth analysis of a collection of 15 colorectal NECs with glandular components, 10 of which by definition were mixed adenoneuroendocrine carcinomas (MANECs). Among shared genetic alterations of both tumor components, we most frequently found TP53, KRAS and APC mutations that also had highest allele frequencies. Mutations exclusive to glandular or neuroendocrine components outnumbered shared mutations but occurred at lower allele frequencies. Our findings not only provide additional evidence for a common clonal origin of colorectal NECs and adjacent glandular tumor components, but strongly suggest their development through the classical adenoma-carcinoma sequence. Moreover, our data imply early separation of glandular and neuroendocrine components during malignant transformation with subsequent independent mutational evolution.

Immune checkpoint inhibitors such as programmed cell death-1 inhibitor pembrolizumab have been shown to be effective in metastatic malignancies such as advanced melanoma. Immune-related adverse effects on multiple organs have been described, such as colitis, skin rash, and hypothyroidism. We present the case of a 44-year-old man with advanced melanoma and recurrent lung metastases who developed symptoms of dyspepsia and gastroesophageal reflux disease after 1 month of therapy with pembrolizumab. Gastric biopsy showed histologic features consistent with lymphocytic gastritis, which was absent on a biopsy 2 months before initiation of therapy. Lymphocytic infiltrates likely secondary to increased autoimmunity after use of immunotherapy have been observed in the colon; however, such histologic findings in the upper gastrointestinal tract have yet to be described. Here, we present a case of lymphocytic gastritis in a patient treated with pembrolizumab, suggesting a new manifestation of toxicity.

Background Non-alcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver diseases. Cyclocarya paliurus (C. paliurus), an edible and medicinal plant in Chinese folk, has been demonstrated to ameliorate diabetes, obesity and lipid metabolism disorders. However, its effects on NAFLD and its potential molecular mechanism have not been clearly expounded. Purpose The present study was designed to explore the therapeutic potential of triterpenic acids-enriched fraction from C. paliurus (CPT), as well as its underlying mechanism in vivo and in vitro models of NAFLD. Methods The metabolic effects and possible molecular mechanism of CPT were examined using HepG2 cells and primary hepatocytes (isolated from C57BL/6 J mice) models of fatty liver induced by palmitic acid (PA) and a high fat diet mouse model. Results In high fat diet-induced C57BL/6 J mice, CPT significantly reduced liver weight index, serum alanine transaminase (ALT), aspartate transaminase (AST), triacylglycerol (TG), total cholesterol (TC) and hepatic TG, TC levels. Moreover, CPT dramatically decreased the contents of blood glucose, insulin, and insulin resistance (HOMA-IR) index. Meanwhile, CPT significantly increased the tyrosine phosphorylation level of IRS and the uptake of 2-deoxyglucose (2DG) in PA-induced HepG2 cells and primary hepatocytes fatty liver models. Furthermore, in PA-induced HepG2 cells and primary hepatocytes, CPT significantly decreased the number of lipid droplets and intracellular TG content. In addition, mechanism investigation showed that CPT increased the phosphorylation of phosphoinositide 3-kinase (PI3K), protein kinase B (Akt) and glycogen synthase-3β (GSK3β) in vivo and in vitro models, which were abrogated by PI3K inhibitor LY294002 in vitro models. Conclusion These findings indicate that CPT may exert the therapeutic effects on NAFLD via regulating PI3K/Akt/GSK3β pathway.

Although there are numerous strategies to counteract the death of dopaminergic neurons in Parkinson's disease (PD), there are currently no treatments that delay or prevent the disease course, indicating that early protective treatments are needed. Targeting axonal degeneration, a key initiating event in PD, is required to develop novel therapies; however, its underlying molecular mechanisms are not fully understood. Here, we studied axonal degeneration induced by 6-hydroxydopamine (6-OHDA) in vitro and in vivo. We found that metabotropic glutamate receptor 5 (mGluR5) expression increased during 6-OHDA-induced axonal degeneration in primary neurons and that blockade of mGluR5 by its antagonists 2-methyl-6-(phenylethynyl)-pyridine (MPEP) and 3-[(2-methyl-1, 3-thiazol-4-yl) ethynyl]-pyridine (MTEP) almost completely attenuated the degenerative process in vitro. Furthermore, a rapid increase in intra-axonal calcium levels following 6-OHDA treatment was visualized using a calcium-sensitive fluorescence probe and a calcium chelator prevented the axonal degenerative process induced by 6-OHDA in vitro, whereas application of the mGluR5 antagonist MPEP partially attenuated the increase in intra-axonal calcium. The screening of calcium targets revealed that calpain activation and an increase in phosphorylated extracellular signal-regulated kinase (p-ERK) were calcium dependent during 6-OHDA-induced axonal degeneration in vitro. Consistent with these in vitro findings, blockade of mGluR5 with MPEP attenuated the degeneration of dopaminergic axons induced by 6-OHDA injection into the striatum prior to soma death in the early stage of PD in an in vivo animal model. In addition, MPEP inhibited the increase in mGluR5 expression levels, calpain activation and the elevation of p-ERK in the striatum triggered by 6-OHDA injection in vivo. Taken together, these data identify an mGluR5-calcium-dependent cascade that causes axonal degeneration, and suggest that mGluR5 antagonists could provide effective therapy to prevent the disease process of PD.

Background: Atopic dermatitis (AD) is an inflammatory skin disease characterized by chronic recurrent dermatitis with profound itching. Most patients have personal and/or family history of atopic diseases. Several criteria have been proposed for the diagnosis of AD. Although the clinical features of childhood AD have been widely studied, there has been less large-scale study on adult/adolescent AD. The aim of this study was to investigate the clinical features of adult/adolescent patients with chronic symmetrical eczema/AD and to propose Chinese diagnostic criteria for adult/adolescent AD. Methods: A hospital-based study was performed. Forty-two dermatological centers participated in this study. Adult and adolescent patients (12 years and over) with chronic symmetrical eczema or AD were included in this study. Questionnaires were completed by both patients and investigators. The valid questionnaires were analyzed using EpiData 3.1 and SPSS 17.0 software. Results: A total of 2662 valid questionnaires were collected (1369 male and 1293 female). Of all 2662 patients, 2062 (77.5%) patients had the disease after 12 years old, while only 600 (22.5%) patients had the disease before 12 years old, suggesting late-onset eczema/AD is common. Two thousand one hundred and thirty-nine (80.4%) patients had the disease for more than 6 months. One thousand one hundred and forty-four (43.0%) patients had a personal and/or family history of atopic diseases. One thousand five hundred and forty-eight (58.2%) patients had an elevated total serum IgE and/or eosinophilia and/or positive allergen-specific IgE. Based on these clinical and laboratory features, we proposed Chinese criteria for adult/adolescent AD. Of all 2662 patients, 60.3% were satisfied with our criteria, while only 48.2% satisfied with Hanifin Rajka criteria and 32.7% satisfied with Williams criteria, suggesting a good sensitivity of our criteria in adult/adolescent AD patients. Conclusion: Late-onset of eczema or AD is common. The clinical manifestations of AD are heterogeneous. We have proposed Chinese diagnostic criteria for adolescent and adult AD, which are simple and sensitive for diagnosis of adult/adolescent AD.

Background Multiple risk factors contribute to the progression of Parkinson’s disease, including oxidative stress and neuroinflammation. Epidemiological studies have revealed a link between higher urate level and a lower risk of developing PD. However, the mechanistic basis for this association remains unclear. Urate protects dopaminergic neurons from cell death induced by oxidative stress. Here, we investigated a novel role of urate in microglia activation in a lipopolysaccharide (LPS)-induced PD model. Methods We utilized Griess, ELISA, real-time PCR, Western blot, immunohistochemistry, and immunofluorescence to detect the neuroinflammation. For Griess, ELISA, Western blot, and immunofluorescence assay, cells were seeded in 6-well plates pre-coated with poly-l-lysine (PLL) and incubated for 24 h with the indicated drugs. For real-time PCR assay, cells were seeded in 6-well plates pre-coated with PLL and incubated for 6 h with the indicated drugs. For animal experiments, rats were injected with urate or its vehicle twice daily for five consecutive days before and after stereotaxic surgery. Rats were killed and brain tissues were harvested after 4 weeks of LPS injection. Results In cultured BV2 cells and rat primary microglia, urate suppressed proinflammatory cytokine production and inducible cyclooxygenase 2 and nitric oxide synthase expression to protect dopaminergic neurons from the toxic effects of activated microglia. The neuroprotective effects of urate may also be associated with the stimulation of anti-inflammatory factors interleukin 10 and transforming growth factor β1. Intracellular urate level was increased in a dose-dependent manner upon co-treatment with urate and LPS as compared with LPS alone, an effect that was abrogated by pretreatment with probenecid (PBN), an inhibitor of both glucose transporter 9 and urate transporter 1 (URAT1). PBN also abolished the anti-inflammatory effect of urate. Consistent with these in vitro observations, the number of tyrosine hydroxylase-positive neurons was decreased and the loss of motor coordination was reversed by urate administration in an LPS-induced rat model of PD. Additionally, increased plasma urate level abolished the reduction of URAT1 expression, the increase in the expression of interleukin-1β, and the number of ionized calcium-binding adaptor molecule 1-positive microglia along with changes in their morphology. Conclusions Urate protects neurons against cytotoxicity induced by microglia activation via modulating urate transporter-mediated intracellular urate level. Electronic supplementary material The online version of this article (10.1186/s12974-018-1175-8) contains supplementary material, which is available to authorized users.

Thioredoxin 1 (Trx1) is known to play an important role in protecting against cell death. However, the mechanism for control of Trx1 in cell death resulting from DNA damage has not been fully investigated. In this study, we used the DNA-damaging agent methyl methanesulfonate (MMS) to investigate the protective effects of Trx1 against DNA damage and cell death in HEK293 cells. We found that MMS application caused dose-dependent changes in the Trx1 redox state determined by redox western blotting. At lower concentrations, both reduced and oxidized Trx1 were observed, whereas the reduced band was fully oxidized at the higher concentration. Trx1 overexpression and small interfering RNA knockdown in cells revealed that reduced Trx1 after exposure to lower doses of MMS attenuated DNA damage, assessed by comet assay, and level of the DNA-damage marker histone γ-H2AX, possibly through scavenging intracellular ROS and an increase in p21 protein level via enhancing its stability. However, oxidized Trx1 lost its protective ability to DNA damage in response to higher concentration of MMS. Corresponding to the redox state control of Trx1, cell death induced by different dose of MMS was also found, by inhibiting phosphorylations of p38 and 4E-BP1. These results indicate that reduced Trx1 plays important protective roles against MMS-induced DNA damage and cell death, suggesting that cell protection is regulated by the intracellular redox state. Control of the redox state of Trx1 and its regulating proteins may offer a novel therapeutic strategy for the control of cancer.

Context.— In advanced gastric, esophageal, and gastroesophageal junction adenocarcinomas (GE-GEJ-AC) that overexpress ERBB2 (erb-b2 receptor tyrosine kinase 2 or HER2), anti-HER2 monoclonal antibody therapy confers survival benefit. To select patients for treatment, HER2 expression and gene amplification are evaluated by immunohistochemistry (IHC) and in situ hybridization. Objective.— To determine whether GE-GEJ-AC tested for HER2 on biopsy specimens of a primary tumor show different IHC scores and/or HER2 amplification by in situ hybridization in matched resection specimens, potentially changing therapy eligibility. Design.— Immunohistochemistry and silver in situ hybridization were performed in biopsy and/or resection specimens from 100 patients. HER2 testing was performed in matched resection and biopsy specimens of 15 cases to determine whether GE-GEJ-AC with IHC scores of 0, 1+, and 2+ in biopsy and resection specimens had different IHC and silver in situ hybridization results. Results.— The IHC 3+ cases showed HER2 amplification in 4 of 5 cases (80%), and IHC scores of 0, 1+, and 2+ showed 3.5%, 14.3%, and 23.5% HER2 amplification by silver in situ hybridization. Among the 15 paired biopsy and resection specimens, 9 (60%) had at least pT2 stage GE-GEJ-AC with HER2 IHC scores of 0, 1+, or 2+ in the biopsy, and 2 of those 9 cases (22%) had IHC 3+ and HER2 amplification by silver in situ hybridization on the resection specimen. Conclusions.— Our data suggest that HER2 testing should be repeated on resection specimens of GE-GEJ-AC with HER2 IHC scores of negative (0 and 1+) or equivocal (2+) and in situ hybridization amplification negative biopsy specimen results to evaluate for HER2 heterogeneity when patients are being considered for anti-HER2 therapy.

Transcriptional changes accompany induced resistance of plants against insect feeding, and monitoring transcriptional reorganization triggered in response to herbivory is an essential step for deciphering the molecular basis of the resistance. To isolate up-regulated genes in tea leaves under mild infestation of green leafhopper (Empoasca vitis Gothe), a subtractive cDNA library was constructed using the suppression subtractive hybridization strategy. Subsequent differential screening, DNA sequencing analyses, and real-time PCR analyses identified 21 unique genes up-regulated by leafhopper feeding from the library. Among the genes likely to be directly involved in defense, those encoding pathogen-related protein and type 2 ribosome-inactivating protein were found in the subtractive library. In addition, genes involved in the CBL (calcineurin B-like)–CIPK (CBL-interacting protein kinases) pathway, cell wall fortification, secondary metabolite synthesis, water transport, and oxidative stress protection were identified in our experiment. Besides, a number of genes that have not been previously associated with defense responses against insects were isolated.

The diagnosis of sebaceous carcinoma presents an important challenge to both clinicians and pathologists, as many cases are initially misdiagnosed both clinically and histopathologically, potentially leading to adverse medical and legal outcomes. The distinction of sebaceous carcinoma from benign sebaceous proliferations and other tumors is therefore of utmost importance, and immunohistochemistry may be useful in this differential. We studied the expression of D2-40 (podoplanin) by immunohistochemistry to determine if it can aid in this differential diagnosis and to evaluate the possibility of lymphangiogenesis in sebaceous carcinoma. A total of 36 cases of sebaceous lesions, including 16 sebaceous carcinomas, 7 sebaceous adenomas, 6 sebaceomas, and 7 cases of normal glands and sebaceous hyperplasia, and 17 cases of basal cell carcinoma and 10 cases of squamous cell carcinoma, were also examined. We found no significant increase in tumor lymphangiogenesis by semiquantitative scoring of lymphovascular density per square millimeter of tumoral/peritumoral stroma in sebaceous carcinoma versus benign sebaceous proliferations. However, D2-40 staining showed a different pattern in the benign tumors, which were positive only in the basaloid cells (most pronounced in sebaceoma), versus sebaceous carcinoma, which was either negative or focally positive in a haphazard pattern in most cases, although some cases of basaloid sebaceous carcinomas showed strong positivity. We also found D2-40 to be only weakly and focally positive in basal cell carcinoma and weakly to moderately positive in squamous cell carcinoma, which showed increased staining with decreased differentiation. Therefore, overall, D2-40 is, of limited diagnostic utility in sebaceous lesions but may be useful in distinguishing sebaceoma and basaloid sebaceous carcinoma from basal cell carcinoma.

Glutamate excitotoxicity contributes to the development of Parkinson's disease (PD) and pharmacological blockade of metabotropic glutamate receptor 5 (mGluR5) has beneficial anti-akinetic effects in animal models of PD; however, the mechanism by which these antagonists alleviate PD symptoms is largely unknown. In our study, the effects of mGluR5 inhibition on DNA damage were investigated in a rotenone-induced model of PD. We first found that the selective mGluR5 antagonist, 2-methyl-6- (phenylethynyl) pyridine, prevented rotenone-induced DNA damage in MN9D dopaminergic neurons through a mechanism involving the downregulation of intracellular calcium release which was associated with a reduction in endoplasmic reticulum stress and reactive oxygen species (ROS)-related mitochondrial dysfunction. Interestingly, the ROS-related mitochondrial dysfunction was accompanied by an increase in expression of the antioxidant protein, Trx2. Treatment of cells with the calcium chelating agent 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or the ROS scavenger N-acetyl-L-cysteine, also reduced rotenone-induced DNA damage, while transfection of a dominant-negative form of Trx2 increased it. In addition, mGluR5 inhibition altered the expression profiles of proteins involved in DNA repair activity. Specifically, the expression of phosphorylated ERK (p-ERK) and CREB, as well as APE1 and Rad51 were elevated after rotenone stimulation and were subsequently downregulated following blockade of mGluR5. These findings were confirmed in vivo in a rotenone-induced rat model of PD. Inhibition of mGluR5 protected against neurotoxicity by mitigating oxidative stress-related DNA damage associated with 8-hydroxy-2'-deoxyguanosine production and also reduced p-ERK activity and Trx2 expression. These findings provide a novel link between mGluR5 and DNA damage in a model of PD, and reveal a potential mechanism by which mGluR5 mediates DNA damage in neurodegenerative diseases.

In this study, we tested the hypothesis that exposure to diesel exhaust particles (DEP) may increase susceptibility of the host to pulmonary infection. Male Sprague-Dawley rats received a single dose of DEP (5 mg/kg), carbon black (CB, 5 mg/kg), or saline intratracheally. Three days later, the rats were inoculated intratracheally with approximately 5,000 Listeria monocytogenes and sacrificed at 3, 5, and 7 days postinfection, and we determined the number of viable Listeria in the left lobe of lungs. The remaining lungs underwent bronchoalveolar lavage (BAL) and the retrieved BAL cells were identified and counted. Luminol-dependent chemiluminescence, a measure of reactive oxygen species (ROS) formation, generated by BAL cells was monitored and the levels of nitric oxide and tumor necrosis factor (TNF)-[alpha] produced by macrophages in culture were determined. At 7 days postinfection, we excised the lung-draining lymph nodes and phenotyped the lymphocyte subpopulations. Exposure of rats to DEP, but not to CB, decreased the clearance of Listeria from the lungs. Listeria-induced generation of luminol-dependent chemiluminescence by pulmonary phagocytes decreased by exposure to DEP but not CB. Similarly, Listeria-induced production of NO by alveolar macrophages was negated at 3, 5, and 7 days after inoculation in DEP-exposed rats. In contrast, CB exposure had no effect on Listeria-induced NO production at 3 days after infection and had a substantially smaller effect than DEP at later days. Exposure to DEP or CB resulted in enlarged lung-draining lymph nodes and increased the number and percentage of CD4(+) and CD8(+) T cells. These results showed that exposure to DEP decreased the ability of macrophages to produce antimicrobial oxidants in response to Listeria, which may play a role in the increased susceptibility of rats to pulmonary infection. This DEP-induced suppression is caused partially by chemicals adsorbed onto the carbon core of DEP, because impaired macrophage function and decreased Listeria clearance were not observed following exposure to CB.

Dichloroacetic acid (DCA) and chloroform (CHCl 3 ) are both major by-products of drinking water chlorination and DCA increases the hepatotoxicity of CHCl 3 . In this study, we further characterized this effect and investigated DCA-induced alterations of CHCl 3 disposition and metabolism as a possible mechanism for this interaction. Both adult male and female Sprague-Dawley rats were gavaged with three doses (09:00, 16:00 and 09:00 the next morning) of DCA (each 2.45 mmol/kg), then challenged with an i.p. injection of CHCl 3 (3.12 or 9.35 mmol/kg). Hepatic damage was assessed 24 h after CHCl 3 administration as increased alanine aminotransferase (ALT), ornithine carbomyl transferase (OCT) and bilirubin in plasma. In a separate experiment, rats were pretreated with DCA or were given 14 CHCl 3 at the same dosages. The disposition of 14 C in various tissues and covalent binding of 14 CHCl 3 -derivatives to liver proteins and lipids were determined 1 h later. CHCl 3 -induced hepatotoxicity was significantly more severe in DCA-pretreated groups. ALT and OCT were more markedly elevated in DCA+CHCl 3 (3.12 mmol/kg) groups than NaCl+CHCl 3 animals. Plasma bilirubin content was elevated only in DCA+CHCl 3 groups and females were more susceptible to this effect. The responses of rats to DCA treatment were somewhat gender-different. DCA treatment increased total cytochrome P450 in females, but not in males. Hepatic glutathione concentration was elevated in males after DCA treatment, but not in females. In the present study we confirmed that DCA pretreatment potentiates the CHCl 3 -hepatotoxicity of both male and female rats. However, there was little evidence that DCA pretreatment significantly affected CHCl 3 disposition or increased CHCl 3 binding in vivo.

Previously, we showed that the pretreatment of dichloroacetic acid (DCA) increased CHCl3-induced hepatotoxicity in vivo and CHCl3 metabolism in vitro in both male and female rats. The effects of DCA on hepatic cytochrome P450-dependent monooxygenases were studied in this experiment. Male and female Sprague-Dawley rats were treated with DCA (each 2.45 mmol/kg) three times (9 AM, 4 PM, and 9 AM) and hepatic microsomes were prepared 3 hr after the last treatment (the same procedure as previous studies). After DCA treatment, the total content of cytochrome P450 (0.67 nmol/mg protein vs 0.79) and the activity of NADPH-dependent cytochrome P450 reductase (227 nmol/mg protein/min vs 332) were significantly increased in female rats, but they were unchanged in males (0.99 vs 0.98 for P450; 315 vs 311 for reductase). Induction of CYP2E1 was observed in both sexes, evidenced as increased activities of aniline and p-nitrophenol hydroxylases and increased CYP2E1 protein amount determined by immunoblot assay. In contrast, the CYP2B-related activity (dealkylation of pentoxyresorufin) and immunoreactive protein did not increase after DCA treatment in either males or females. The activity of ethoxyresorufin dealkylase was decreased in DCA-treated males compared to their controls (310 pmol/mg protein/min vs 229, p0.05), but it was not significantly affected in females. These data demonstrate that DCA treatment of both male and female rats altered the population of hepatic cytochrome P450. The results support the hypothesis that DCA increases CHCl3 metabolism, and therefore hepatotoxicity, by inducing CYP2E1.

Metabotropic glutamate receptors (mGlus) are G-protein-coupled receptors playing an important role in the central nervous system (CNS). Recently, mGlus have been identified in peripheral tissues, and aberrant expression or inhibition of the receptors functions in the development of certain cancers. However, the correlation of mGlu activity with hepatocellular carcinoma (HCC) remains unknown. In this study, we analyzed the effects of inhibiting mGlu5 activity in hepatocarcinoma cell lines and a xenograft model. Inactivation of mGlu5 with 2-Methyl-6-(phenylethyl)-pyridine (MPEP), a specific antagonist of the receptor, caused inhibition of cell growth, migration, and invasion of HepG2 and Bel-7402 cells, assessed by MTT assay, ATP production, wound healing, and Boyden chamber assay, respectively. Moreover, inhibition of tumor growth and the potential metastasis of hepatocellular carcinoma were also found in nude mice. Furthermore, mGlu5-mediated extracellular signal-regulated kinase (ERK) phosphorylation has been found to be partially involved in cell growth and migration, as detected by stimulation of (S)-3,5-Dihydroxyphenylglycine (DHPG), an agonist of the receptor, and blockage of MPEP and U0126, an inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (MEK). These data indicate that inhibiting the activity of mGlu5 has the molecular potential to suppress oncogenic actions by blocking downstream effector molecules. The study suggests that mGlu5 activity may contribute to understanding the development of HCC.

The activation of group I metabotropic glutamate receptor (group I mGlus) has been shown to produce neuroprotective or neurotoxic effects. In this study, we investigated the effects of N-acetylcysteine (NAC), a precursor of the antioxidant glutathione, on group I mGlus activation in apoptosis of glial C6 and MN9D cell lines, and a rat model of Parkinson's disease (PD). We demonstrated that NAC protected against apoptosis through modulation of group I mGlus activity. In glial C6 cells, NAC promoted phosphorylation of ERK induced by (s)-3,5-dihydroxy-phenylglycine (DHPG), an agonist of group I mGlus. NAC enhanced the group I mGlus-mediated protection from staurosporine (STS)-induced apoptosis following DHPG treatment. Moreover, in rotenone-treated MN9D cells and PD rat model, NAC protected against group I mGlus-induced toxicity by compromising the decrease in phosphorylation of ERK, phosphorylation or expression level of TH. Furthermore, the results showed that NAC prohibited the level of ROS and oxidation of cellular GSH/GSSG (E(h)) accompanied by activated group I mGlus in the experimental models. Our results suggest that NAC might act as a regulator of group I mGlus-mediated activities in both neuroprotection and neurotoxicity via reducing the oxidative stress, eventually to protect cell survival. The study also suggests that NAC might be a potential therapeutics targeting for group I mGlus activation in the treatment of PD.

We investigated the systemic immunotoxic potential of respiratory exposure to diesel exhaust particles (DEP) in this study. Female B6C3F1 mice (approximately 8 weeks old) were exposed to increasing concentrations of DEP intratracheally, 3 times every two weeks, and sacrificed 2 or 4 weeks after the first exposure. The systemic toxicity and immune status in mice were evaluated. Mice exposed to DEP (1 to 15 mg/kg) showed no significant changes in body, spleen, or liver weights. Lung weights were increased in the mice exposed to 15 mg/kg DEP for 2 or 4 weeks. Except for a decreased platelet count, no significant alterations occurred in hematological parameters following DEP exposure. The number of splenic anti-sheep red blood cell (sRBC) IgM antibody-forming cells (AFC) decreased following DEP exposure for 2 weeks. This effect was less severe following 4 weeks of exposure and was only evident in the high dose group. Exposure to DEP also resulted in a significant decrease in the absolute numbers and the percentages of total spleen cells for total, CD4(+), and CD8(+) T cells, while the numbers of B cells and total nucleated cells in spleen were not significantly changed. The proliferative response of splenocytes to the T-cell mitogen, concanavalin A (ConA), as well as their production of IL-2 and IFN-gamma, was decreased dose-dependently following exposure of mice to DEP for 2 weeks, whereas proliferation was not changed in response to anti-CD3 monoclonal antibody. In summary, short-term respiratory exposure of mice to DEP resulted in systemic immunosuppression with evidence of T cell-mediated and possibly macrophage-mediated mechanisms.

Exposure to asphalt fumes is a health concern due to the presence of polycyclic aromatic compounds (PACs) in asphalt. Bioactivation of many PACs requires metabolism by the cytochrome P-450 (P-450) system. The objective of this study was to evaluate the effects of exposure of rats to asphalt fume condensate (AFC), collected at the top of a paving asphalt storage tank, on the pulmonary microsomal P-450 system and to determine the genotoxic effects of such exposure. Male Sprague-Dawley rats were intratracheally instilled with saline or with 0.45, 2.22, or 8.88 mg/kg AFC for 3 consecutive days and sacrificed the following day. Lung microsomes were isolated by differential centrifugation of lung homogenates. Microsomal protein level, NADPH cytochrome c reductase activity, and the activities and protein levels of cytochrome P-450 isozymes CYP1A1 and CYP2B1 were monitored to assess the effects of AFC exposure on pulmonary P-450. The activities of CYP2B1 and CYP1A1 were determined by monitoring xenobiotic metabolism of 7-pentoxyresorufin and 7-ethoxyresorufin, respectively. CYP2B1 and CYP1A1 levels were determined by immunochemical analysis. Micronucleus (MN) formation in bone-marrow polychromatic erythrocytes (PCEs) was determined to assess the genotoxic effects of AFC exposure. The results showed that exposure of rats to AFC did not significantly affect total cytochrome P-450 content or cytochrome c reductase activity in the lung. CYP2B1 levels and enzyme activity were not significantly affected by AFC exposure. In contrast, CYP1A1 levels and activity were significantly increased in microsomes isolated from AFC-exposed lungs. Increased MN formation was observed only in high-dose AFC-exposed bone marrow PCEs. These results demonstrate that AFC exposure induced CYP1A1 activity and increased the enzyme levels of CYP1A1 in lung microsomes, suggesting that AFC exposure may alter metabolism of PACs by the cytochrome P-450 system in the lung. Alteration of cytochrome P-450 metabolism of PACs may contribute to the AFC-induced genotoxic effects demonstrated as MN formation.

Both Listeria monocytogenes infection and silica exposure have been shown to significantly alter immune responses. In this study, we evaluated the effect of preexposure to silica on lung defense mechanisms using a rat pulmonary L. monocytogenes infection model. Male Sprague-Dawley rats were instilled intratracheally with saline (vehicle control) or silica using either an acute treatment regimen (5 mg/kg; 3 days) or a subchronic treatment protocol (80 mg/kg; 35 days). At 3 or 35 days after silica instillation, the rats were inoculated intratracheally with either approximately 5000 or 500,000 L. monocytogenes. At 3, 5, and 7 days postinfection, the left lung was removed, homogenized, and cultured on brain heart infusion agar at 37 degrees C. The numbers of viable L. monocytogenes were counted after an overnight incubation. Bronchoalveolar lavage (BAL) was performed on the right lungs, and BAL cell differentials, acellular lactate dehydrogenase (LDH) activity and albumin content were determined. Alveolar macrophage (AM) chemiluminescence (CL) and phagocytosis were assessed as a measure of macrophage function. Lung-associated lymph nodes were removed, and lymphocytes were recovered and differentiated. Preexposure to silica significantly increased the pulmonary clearance of L. monocytogenes as compared to saline controls. Exposure to silica caused significant increases in BAL neutrophils, LDH and albumin, and lymph-nodal T cells and natural killer (NK) cells in infected and noninfected rats. CL and phagocytosis were also elevated in silica-treated rats. In summary, the results demonstrated that exposure of rats to silica enhanced pulmonary immune responses, as evidenced by increases in neutrophils, NK cells, T lymphocytes, and macrophage activation. These elevations in pulmonary immune response are likely responsible for the increase in pulmonary clearance of L. monocytogenes observed with preexposure to silica.

The role of metabolism in dichloroacetic acid (DCA) potentiation of CHCl3 hepatotoxicity was investigated. Male and female Sprague-Dawley rats were given three doses (09:00, 16:00 and 09:00 the next morning) of DCA (each 2.45 mmol/kg) by gavage. The rats were euthanized 3 h after the last dose, hepatic microsomes were prepared and 14CHCl3 metabolism was measured in vitro. The binding of 14CHCl3-derivatives to microsomal proteins and lipids was increased 65 and 100%, respectively, in DCA-treated rats compared to their respective NaCl-treated controls. The formation of CO2 (nmol 14CO2/incubation) was significantly elevated in DCA-treated rats compared to controls (10.4 vs 6.3 in males; 10.8 vs 6.1 in females). DCA treatment decreased the apparent Michaelis constant (Km app) for conversion of 14CHCl3 to 14CO2 in rats (0.665 vs 0.415 mM in males, P < 0.05; 0.161 vs 0.081 in females). 14CO2 production and 14C binding were observed under N2 atmosphere, indicating that reactive metabolites of 14CHCl3 were formed by oxidation as well as reduction. Male and female rats metabolized CHCl3 differently. The Km app for CO2 production was up to 5-fold higher in the males than in the females, regardless of DCA treatment. Inhibition by SKF 525-A and piperonyl butoxide was gender dependent in both control and DCA-treated groups. The results showed, that increased bioactivation of CHCl3 by DCA treatment is one element in the DCA-CHCl3 interaction.