Article pubs.acs.org/crt
Arsenic Induced Overexpression of Inflammatory Cytokines Based on the Human Urothelial Cell Model in Vitro and Urinary Secretion of Individuals Chronically Exposed to Arsenic Shengnan Liu, Qingshan Sun, Fei Wang, Lin Zhang, Yingli Song, Shuhua Xi,* and Guifan Sun Department of Environmental and Occupational Health, Liaoning Provincial Key Laboratory of Arsenic Biological Effect and Poisoning, School of Public Health, China Medical University, District of Heping, North Er Road, No. 92, Shenyang City, China, 110001 ABSTRACT: Chronic persistent inflammation could play an important role in the pathogenesis of some malignancies, and inflammation is a critical factor for bladder cancer development. In this study, we measured urine levels of transforming growth factor-α (TGF-α), tumor necrosis factor-α (TNF-α), and IL-8 in arsenic exposure workers and expressions of inflammatory cytokines in human urothelial cells in vivo and in vitro. We found the concentrations of IL-8, TNF-α, and TGFα presented in urine were significantly elevated in the high urinary arsenic workers compared with the low urinary arsenic workers. Multiple regression analysis showed that the urinary IL-8 level was significantly positively associated with urinary iAs concentration after adjusting for the confounding effects of age, employed years, body mass index (BMI), smoking, alcohol, and seafood consumption in recent 3 days. Urinary TNF-α and TGF-α levels were also significantly positively associated with urinary iAs concentration, and SMI. TGF-α level was negatively associated with age after adjusting for the confounding effects. Consistent with the results in vivo, mRNA expressions of TNF-α, TGF-α, and IL-8 and protein expressions of TGF-α, TGF-β1, and IL-8 were significantly elevated in SV-HUC-1 cells after exposure to lower concentrations of arsenite for 24h as compared to the control group. These data indicated that arsenic increased the secretion of inflammatory factors and IL-8, TNF-α, and TGF-α expression may be a useful biomarker of the effect of arsenic exposure.
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and the prevalence of plasma TGF-α overexpression.9 These results suggest that TGF-α may serve as a useful biomarker of adverse health effects of arsenic. TGF-α is a known potent mitogenic polypeptide and is present in the extracellular environment, and it is readily detectable in a variety of biological fluids, such as urine, serum, or plasma. TGF-α overexpression has the unique ability to complement both initiation and promotion by serving as a tumor enhancer.10 Elevated TGF-α levels have been detected in patients with various malignancies.11 Transforming growth factor-β (TGF-β) is a family of multifunctional regulatory peptides involved in a range of processes, including growth, differentiation, angiogenesis, and carcinogenesis.12 In normal cells, TGF-β typically exerts tumor suppressing activities through its ability to induce cell cycle arrest and apoptotic reactions, and in human malignancies, TGF-β is utilized as an oncogenic factor that promotes carcinoma growth, invasion, and metastasis.13 It has three mammalian isoforms, TGF-β1, TGF-β2, and TGF-β3. TGF-β1 is released from parenchymal cells and inflammatory cells, and TGF-β1 protein and its
INTRODUCTION Inorganic arsenic (iAs) is a potent human carcinogen and widely distributed in water, air, and soil. For most people, drinking water is the major source of As. In addition, there is significant occupational exposure to arsenic in nonferrous smelting.1 Epidemiologic studies have identified arsenic exposure as a cause of human cancers, such as of the skin, lung, and bladder.2 Some reports indicated that chronic persistent inflammation played an important role in the pathogenesis of some malignancies,3 and inflammation was a critical factor for bladder cancer development.4 Our previous study found that low concentrations of arsenic induced the expression of cyclooxygenase-2 (COX-2) in human uroepithelial cells (SV-HUC-1).5 COX-2 is an inducible proinflammatory enzyme and is overexpressed in patients with bladder cancer.6 At present, it is especially interesting that exposure to arsenic induces an increase in the inflammatory response. In a population exposed to arsenic in drinking water, transforming growth factor-α (TGF-α) levels both in urine and in exfoliated bladder urothelial cells separated from urine were correlated with urinary total As (TAs), particularly in those individuals with arsenic-associated skin lesions.7,8 There was also a significant linear trend between cumulative arsenic exposure © 2014 American Chemical Society
Received: July 10, 2014 Published: September 25, 2014 1934
dx.doi.org/10.1021/tx5002783 | Chem. Res. Toxicol. 2014, 27, 1934−1942
Chemical Research in Toxicology
Article
Table 1. Gene-Specific Primer Sets and PCR Parameters Gene name
Accession number of genes
IL-8
NM_000584.3
TNF-α
NM_000594.3
TGF-α
NM_003236.3
TGF-β1
NM_000660.5
β-actin
NM_001101.3
Primer sequences Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense
5′-AAGCTGGCCGTGGCTCTCTTG-3′ 5′-AGCCCTCTTCAAAAACTTCTC-3′ 5′-CTCGAACCCCGAGTGACAAG-3′ 5′-TGAGGTACAGGCCCTCTGAT-3′ 5′-TGGAGAACAGCACGTCCC-3′ 5′-TGATGGCCTGCTTCTTCTGG-3′ 5′-GGAAATTGAGGGCTTTCGCC-3′ 5′-CCGGTAGTGAACCCGTTGAT-3′ 5′-GTCCACCTTCCAGCAGATGTG-3′ 5′-GCATTTGCGGTGGACGAT-3′
Product (bp)
30
279
30
159
30
234
30
90
30
76
supplemented with 10% FBS and antibiotics at 37 °C in a 5% CO2 humified cell culture incubator. Two ×105 cells at logarithmic growth phase were treated with NaAsO2. Our previous study found the peak expression of COX-2, a pro-inflammatory inducible enzyme, was in 4 μM arsenite-treated SV-HUC-1 cells for 24 h.5 Therefore, a 24 h exposure was selected. The final concentrations for NaAsO2 were 1, 2, 4, 8, and 10 μM. All experiments were done three times to assess reproducibility. RT-PCR and Quantitative Real-Time PCR. Total cellular RNA was isolated using the Trizol reagent according to the supplier’s recommendations, and RT-PCR was performed as described previously.22 Amplification was conducted in an ABI Prism 2720 sequence detection system. PCR conditions were as follows: initial denaturation at 94 °C for 15 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 °C for 30 s, followed by a final extension at 72 °C for 5 min. The PCR-amplified products were analyzed on a 2.0% agarose gel and stained with ethidium bromide. mRNA expressions of IL-8, TNF-α, TGF-α, and TGF-β1 were measured and normalized to the corresponding β-actin (an internal control) expression levels. Quantitative real-time PCR was performed in the ABI 7500 real-time detection system (Applied Biosystems, Foster City, CA). Fold changes for each gene expression were calculated using the Delta Ct method normalizing to β-actin expression for each sample. All of the primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China), and primer sequences in RT-PCR and quantitative real-time PCR tests were the same and were summarized in Table 1. Each experiment was repeated at least three times. Western Blots. SV-HUC-1 cell lysates were prepared with a lysis buffer as described previously.22 Protein concentrations were measured using the Bio-Rad microprotein assay reagent. The protein extracts (30 μg) were separated on SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad) at 15 V for 40 min. The membranes were blocked in Odyssey blocking buffer (diluted 1:1 with PBS) for 1 h. This was followed by incubating with primary antibody at a dilution of 1:1000 anti-IL-8, anti-TNF-α, anti-TGF-α, anti-TGF-β1, and 1:3000 polyclonal antibodies to β-actin, overnight at 4 °C. After four washes with PBST, the blots were incubated with secondary antibodies at a dilution of 1:5000 for 1 h at room temperature; it is important to protect the secondary antibody incubation from light to avoid loss of signal. Following incubation with the secondary antibody, the membrane was washed four times for 5 min each with PBST. The membrane should be rinsed with PBS to remove Tween-20 prior to imaging, and then the infrared signal was visualized with an LI-COR Odyssey Infrared Imaging system. Study Population. This study was carried out with 72 male workers from nonferrous plants located in the northeastern part of China. At enrollment, there was no other malignant disease, inflammatory disease, and/or dysfunction of the heart, liver, or kidney in any participants. All participants provided informed consent before the questionnaire interview and then donated a biospecimen. The study was approved by the China Medical University Ethics Committee. An interview was performed using a structured questionnaire that elicited information about age, work history, diet (including consumption of seafood, meat, vegetable, and fruit in the 3
receptor I were overexpressed in urine samples in the bladder carcinoma group compared with chronic cystitis.14 Tumor necrosis factor-α (TNF-α) is a cytokine involved in various physiological processes, such as inflammation, cell proliferation, and apoptosis.15 IL-8 is an important mediator in the amplification of immunological and inflammatory reactions and has been reported to be related to certain kinds of tumors.16 Numbers of studies have indicated that arsenic increased the expression of TNF-α, IL-6, and IL-8.17−19 Based on the literature, more studies on arsenic-induced inflammation were in vitro performed in keratinocytes,10 intestinal epithelial cells,20 and bronchial and lung epithelial cells.19 In experimental animals, chronic administration of iAs in mice caused inflammation in the liver.17 Few studies reported the association of arsenic with inflammatory factors in human urothelial cells models. There was no study on the urinary secretion of inflammatory cytokines in an occupational arsenic exposed population. It has been reported that monomethylarsonic acid (MMA), a metabolite of iAs, increased the expression of TNF-α, IL-6, and IL-8 and is associated with tumor progression in human urothelial cells.18,21 Therefore, we measured urinary levels of TGF-α, TNF-α, and IL-8 in workers in a nonferrous metal plant, and TGF-β1, TGF-α, TNF-α, and IL-8 expression in human urothelial cells in vivo and in vitro.
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Cycles
MATERIALS AND METHODS
Chemicals and Reagents. Sodium arsenite (NaAsO2, ≥99.0%) was procured from Sigma Chemical Company (St. Louis, MO, USA) and prepared in sterile PBS, pH 7.4, immediately before each experiment. Fetal bovine serum (FBS), Ham’s F-12 medium, Trizol, antibiotics, trypsin, phosphate-buffered saline (PBS), and other analytical laboratory chemicals and reagents were obtained from Sigma, Invitrogen (Carlsbad, CA, USA), Hyclone (Logan, UT, USA), and Biochrom AG (Berlin,Germany). Antibodies against IL-8 were purchased from Abcam (Cambridge, MA, USA), TNF-α was purchased from Cell Signaling Technology (Boston, MA, USA), and Anti TGF-α and TGF-β1 were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Secondary antibody was from LI-COR Biosciences (Lincoln, NE, USA). The easy RT-PCR kit was obtained from TransGen Biotech Co., Ltd. (Beijing, China). The quantitative real-time PCR kit was obtained from Takara Biotech Co., Ltd. (Dalian, China).The immunological analyses of IL-8 and TNF-α were performed by the kits purchased from Dakewe Biotech Co., Ltd. (Shenzhen, China), and the TGF-α kit was purchased from Boster Biotech Co., Ltd. (Wuhan, China). All other chemicals used in the experimental studies were the highest analytical grade commercially available. Water used in studies was distilled and deionized. Cell Culture and Treatment. The SV-40 immortalized human uroepithelial cell line (SV-HUC-1), which was derived from transformation of normal uroepithelium, was provided by the American Type Culture Collection (Wiltshire, USA). The SV-HUC1 cells were routinely maintained in Ham’s F-12 medium 1935
dx.doi.org/10.1021/tx5002783 | Chem. Res. Toxicol. 2014, 27, 1934−1942
Chemical Research in Toxicology
Article
ELISA Assays. Levels of IL-8, TNF-α, and TGF-α secreted in urine were measured using human TGF-α ELISA kits, human IL-8 ELISA kits, and human TNF-α ELISA kits according to the manufacturer’s instructions. Each urine sample was centrifuged at 2000 rpm and 4 °C for 5 min. Urine samples were added to the ELISA plate, together with a standard curve. The chemiluminescence was measured using a plate reader (Shanghai, China). IL-8, TNF-α, and TGF-α levels were calculated by using a standard curve. Each measurement was repeated in triplicate, and all samples were run at one time. The average value of IL-8, TNF-α, and TGF-α is expressed in ng/g Cr. Statistical Analysis. The data were analyzed using the SPSS 13.0 software. The Kolmogorov−Smirnov test was used to evaluate the normality of the data. Normally distributed and logarithmic transformation normalized distribution variables were analyzed with parametric tests. Differences between two groups were analyzed by Student’s unpaired t test. One-way analysis of variance (ANOVA) followed by least significant difference (LSD) or Dunnett T3 test was used to determine differences among groups of arsenic-treated SVHUC-1 cells. No normally distribution variables were analyzed with a nonparametric test (Mann−Whitney test). The positivity rates were compared by chi-square test. Dose−response relationships between urinary arsenicals concentrations and the secretion of IL-8, TNF-α, and TGF-α were explored using Pearson or Spearman correlation. Furthermore, multiple linear regression models were used to evaluate the effects of variables on levels of IL-8, TNF-α, and TGF-α. In these models IL-8, TNF-α, and TGF-α levels were evaluated in relation to arsenic metabolites and confounder/covariates. Potential confounding factors included the following variables of age, employment years, smoking habits, BMI, taking Chinese medicine of bezoar, and seafood and alcohol consumption in recent 3 days. The results are expressed as means ± standard deviation (SD). A p-value
