National Chiayi University National Chiayi University Dep. Of Microbiology, Immunology & Biopharmaceuticals. Dep. Of Microbiology, Immunology & Biopharmaceuticals.

Arsenic Induced Overexpression of Arsenic Induced Overexpression of Inflammatory Cytokines Based on the Inflammatory Cytokines Based on the Human Urothelial Cell Model in Vitro Human Urothelial Cell Model in Vitro and Urinary Secretion of Individuals and Urinary Secretion of Individuals

Chronically Exposed to ArsenicChronically Exposed to ArsenicChemical Research in Toxicology (2014) : 27, 1934-1942Chemical Research in Toxicology (2014) : 27, 1934-1942

TOXICOLOGY 12/87TOXICOLOGY 12/87Impact Factor- 4.19Impact Factor- 4.19

Advisor:Advisor:Yi-Wen Liu, PhD.Yi-Wen Liu, PhD.

Speaker: Speaker: Mezbahul HaqueMezbahul Haque

Date: 2015.01.24Date: 2015.01.24

Inflammation and cancer Inflammation and cancer

Numerous studies suggest that inflammation is likely to have an important role in bladder carcinogenesis. Urologic Oncology: Seminars and Original Investigations (2007) ;25: 260–268

It is now well-established that the induction of inflammation by bacterial and viral infections increases cancer risk. Crit Rev Oncol Hematol (2009);70:183–194.

Inflammation impacts every single step of tumorigenesis, from initiation through tumor promotion, all the way to metastatic progression. Cell. (2010) ; 140: 883–899

Various types of immune and inflammatory cells are frequently present within tumors. Chronic inflammation increases cancer risk. Cell. (2010) ; 140: 883–899

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Role of inflammation in tumor Role of inflammation in tumor initiation and promotion initiation and promotion

2Cell. (2010) ; 140: 883–899

Inflammatory cytokine TGF-α Inflammatory cytokine TGF-α

Arsenic induces inflammation and/or cell proliferation, which may ultimately lead to tumorigenesis, by modulation of keratinocyte-derived

inflammatory or growth promoting cytokines, respectively. Toxicology and applied pharmacology(1996) 141, 308–318

TGF-α overexpression has the unique ability to complement both initiation

and promotion by serving as a tumor enhancer. Toxicology and applied pharmacology(1996) 141, 308–318

TGF-a appears to be a useful complementary tumor marker. CANCERRESEARCH (1987) ;47, 896-901

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TGF- α expression correlates recurrences, agressive phenotype,invasiveness, and death in bladder cancer. Urol.Res. (1997) 25, 9−17

Inflammatory cytokines TGF-βInflammatory cytokines TGF-β

TGF-beta can act as both tumor suppressor and as a significant stimulator of tumor progression, invasion and metastasis. Trends Cell Biol (2001) 11, S44-51

As TGF-beta induces extra-cellular matrix accumulation, angiogenesis and immuno-suppression, it is reported to facilitate progression of tumors under certain conditions. Science (1996) 271, 350-353 Oncogene (1999) 18, 3098-3103

TGF-beta exhibits pro-oncogenic effects that directly target the tumor itself in addition to indirect effects on the stromal compartme.

Front Biosci (2003) 8, 740-749

The TGF-beta receptor signal transduction pathway plays critical roles in many biological processes, including the development and progression of human tumors. Front. Biosci. (2005) 10, 1135−1145.

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TNF-α and IL-8 TNF-α and IL-8

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Tumor necrosis factor-α (TNF-α) is a cytokine involved in variousphysiological processes, such as inflammation, cell proliferation,and apoptosis. Am. J. Physiol. Gastrointest. Liver Physiol.(2004) 287, G497−G502.

IL-8 induces the expression of genes related to cell growth and production of angiogenic factors as well as genes or proteins involved in tissue remodeling. Clin. Cancer Res. (2008) 4, 6735−6741.

Significantly increased levels of TNF-α by arsenic exposure were found consistently both in vitro and in vivo. Toxicol. Appl. Pharmacol.(2005) 204, 18−26 Carcinogenesis (2002) 23, 867−876

It has been indicated that arsenic exposure can markedly increase theexpression of IL-8 in cultured endothelial cells30,31 and the urothelial cell line. Pharmacol. (2012) 258, 10−18.

Aims Aims

Want to observe urinary secretion of inflammatory cytokines in an occupational arsenic exposed population,

workers in a nonferrous metal plant.

Also want observe the TGF-β1, TGF-α, TNF-α, and IL-8 expression in human urothelial cells.

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Materials and Methods Materials and Methods

Cell Culture and Treatment.

METHODS

RT-PCR and Quantitative Real-Time PCR.

Western Blots

Study Population

Determination of Urinary Arsenic Species.

ELISA Assays

The SV-40 immortalized human uroepithelial cell line (SV-HUC-1)

NaAsO2 were 1, 2,4, 8, and 10 μM, a 24 hr exposure

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Flow charts Flow charts

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Arsenical Metabolite Levels and Methylation ProfileArsenical Metabolite Levels and Methylation Profilein the Urine of Workers in the Urine of Workers

Variables Group 1 (TAs < 50 μg/g Cr) Group 2 (TAs ≥ 50 μg/g Cr)

Number of subjects 26 46iAs (μg/g Cr) 4.14 ± 2.48 16.36 ± 12.69b

MMA (μg/g Cr) 3.88 ± 3.17 23.09 ± 25.81b

DMA (μg/g Cr) 24.32 ± 8.56 135.77 ± 106.11b

TAs (μg/g Cr) 32.33 ± 10.26 175.22 ± 133.84b

iAs% 12.76 ± 8.07 10.58 ± 8.14MMA% 11.32 ± 8.28 12.02 ± 7.04DMA% 75.92 ± 13.20 77.40 ± 10.50PMIa 0.872 ± 0.081 0.894 ± 0.081SMIa 0.866 ± 0.111 0.865 ± 0.079

Urinary Concentrations of Various Arsenic Species (X ± SD)

aPMI = (MMA + DMA)/TAs and SMI = DMA/(MMA + DMA). bp < 0.01 compared with Group 1. 9

Summary Summary

The concentrations of all arsenical metabolites (iAs, MMA, DMA, and TAs) in urine of Group 2 were

significantly higher than those of Group 1.

The arsenic species with the highest concentration in urine was DMA.

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Flow charts Flow charts

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Urinary Levels of Inflammatory Cytokines in ArsenicUrinary Levels of Inflammatory Cytokines in ArsenicExposure Workers. Exposure Workers.

(*) p < 0.05 and (**) p < 0.01 compared to the Group 1.

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Summary Summary

The concentrations of IL-8, TNF-α, and TGF-α present in urine were significantly elevated in the high urinary

arsenic, Group 2 compared with the low urinary arsenic, Group 1.

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Flow charts Flow charts

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Correlations between Urinary IL-8 and Urinary Correlations between Urinary IL-8 and Urinary Arsenical Metabolites Arsenical Metabolites

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Correlations between Urinary TNF-α, and log TGF-αCorrelations between Urinary TNF-α, and log TGF-αand Urinary Arsenical Metabolites and Urinary Arsenical Metabolites

Summary Summary

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1. Urinary IL-8 level was significantly positively associated with the concentrations of urinary iAs, and negatively associated with MMA.2. TNF-α level was positively associated with the concentrations of urinary iAs and SMI. 3. TGF-α level was significantly positively associated with the concentrations ofurinary iAs and SMI, and negatively associated with age.

There has significant correlations between the urinary levels of IL-8, TNF-α, and TGF-α and urinary arsenical metabolites.

Flow charts Flow charts

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mRNA expressions of inflammatory factors in the mRNA expressions of inflammatory factors in the human uroepithelial cell line. human uroepithelial cell line.

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Protein expressions of inflammatory factors in human Protein expressions of inflammatory factors in human uroepithelial cell line. uroepithelial cell line.

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Summary Summary

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The level of mRNA expression of IL-8, TNF-α and TGF-α was significantly elevated in SV-HUC-1 cells after exposure to 4 μM arsenite for 24 h as compared to the control group.

Consistent with mRNA expressions, IL-8 and TGF-α, TGF-β1 protein expressions increased in SV-HUC-1

cells after exposed to arsenite.

Conclusion Conclusion

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Our results support that IL-8, TNF-α, and TGF-α expression may be a useful biomarker of effects of arsenic

exposure in workers of nonferrous plants.

Discussion Discussion

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low concentrations of arsenite increased the expressions of IL-8, TNF-α, TGF-α, and TGF-β1 in human uroepithelial cells, and the highest expression was in 2−8 μM arsenite.

The deficiency of the secondary methylation step resulting in the higher MMAV and lower DMAV percentages in urine has been shown to be related to an increased risk of urothelial carcinoma.

Arsenic injure uroepithelial cells, which may increase secretion of theinflammatory cytokines (IL-8, TGF-α) in arsenic exposed individuals. Arsenic also injures the hepatic cells and increases the secretion of the inflammatory cytokines to serum, then filtering into urine.

Atomic absorption spectrophotometer

Jaffe reaction

Pearson or Spearman correlation