Differential Cell Sensitivity between OTA and LPS upon Releasing TNF-α

The release of tumor necrosis factor α (TNF-α) by ochratoxin A (OTA) was studied in various macrophage and non-macrophage cell lines and compared with E. coli lipopolysaccharide (LPS) as a standard TNF-α release agent. Cells were exposed either to 0, 2.5 or 12.5 µmol/L OTA, or to 0.1 µg/mL LPS, for up to 24 h. OTA at 2.5 µmol/L and LPS at 0.1 µg/mL were not toxic to the tested cells as indicated by viability markers. TNF-α was detected in the incubated cell medium of rat Kupffer cells, peritoneal rat macrophages, and the mouse monocyte macrophage cell line J774A.1: TNF-α concentrations were 1,000 pg/mL, 1,560 pg/mL, and 650 pg/mL, respectively, for 2.5 µmol/L OTA exposure and 3,000 pg/mL, 2,600 pg/mL, and 2,115 pg/mL, respectively, for LPS exposure. Rat liver sinusoidal endothelial cells, rat hepatocytes, human HepG2 cells, and mouse L929 cells lacked any cytokine response to OTA, but showed a significant release of TNF-α after LPS exposure, with the exception of HepG2 cells. In non-responsive cell lines, OTA lacked both any activation of NF-κB or the translocation of activated NF-κB to the cell nucleus, i.e., in mouse L929 cells. In J774A.1 cells, OTA mediated TNF-α release via the pRaf/MEK 1/2-NF-κB and p38-NF-κB pathways, whereas LPS used pRaf/MEK 1/2–NF-κB, but not p38-NF-κB pathways. In contrast, in L929 cells, LPS used other pathways to activate NF-κB. Our data indicate that only macrophages and macrophage derived cells respond to OTA and are considered as sources for TNF-α release upon OTA exposure

Toxicological Profile of Ultrapure 2,2 ',3,4,4 ',5,5 '-Heptachlorbiphenyl (PCB 180) in Adult Rats

Peer reviewe

Exocrine Pancreatic Carcinogenesis and Autotaxin Expression

<div><p>Exocrine pancreatic cancer is an aggressive disease with an exceptionally high mortality rate. Genetic analysis suggests a causative role for environmental factors, but consistent epidemiological support is scarce and no biomarkers for monitoring the effects of chemical pancreatic carcinogens are available. With the objective to identify common traits for chemicals inducing pancreatic tumors we studied the National Toxicology Program (NTP) bioassay database. We found that male rats were affected more often than female rats and identified eight chemicals that induced exocrine pancreatic tumors in males only. For a hypothesis generating process we used a text mining tool to analyse published literature for suggested mode of actions (MOA). The resulting MOA analysis suggested inflammatory responses as common feature. In cell studies we found that all the chemicals increased protein levels of the inflammatory protein autotaxin (ATX) in Panc-1, MIA PaCa-2 or Capan-2 cells. Induction of MMP-9 and increased invasive migration were also frequent effects, consistent with ATX activation. Testosterone has previously been implicated in pancreatic carcinogenesis and we found that it increased ATX levels. Our data show that ATX is a target for chemicals inducing pancreatic tumors in rats. Several lines of evidence implicate ATX and its product lysophosphatidic acid in human pancreatic cancer. Mechanisms of action may include stimulated invasive growth and metastasis. ATX may interact with hormones or onco- or suppressor-genes often deregulated in exocrine pancreatic cancer. Our data suggest that ATX is a target for chemicals promoting pancreatic tumor development.</p> </div

Testosterone increases CA-induced ATX signaling and prevent CA-induced toxicity in Panc-1 cells.

<p>Panel (<b>A</b>) and (<b>B</b>) show Western blots and densitometric analysis of extracellular and intracellular ATX levels. Cells were incubated with 1 nM testosterone for 24 hours and thereafter with 10 µM CA for additional 24 hours. (<b>C</b>) Cells were stained by trypan blue and counted under light microscope. Cells were preincubated with 1 nM testosterone for 24 hours and thereafter treated with 10 µM CA for additional 24 hours. (<b>D</b>) Cell numbers measured by MTT. Cells were preincubated with 1 nM testosterone for 24 hours and thereafter treated with 100 µM CA for additional 24 hours. Data was obtained from three independent experiments. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043209#s3" target="_blank">Results</a> are presented as mean ± SD. *significantly different from controls (*p<0.05, **p<0.01, ***p<0.001). #significantly different from CA alone (#p<0.05, ##p<0.01). ¤significantly different from testosterone alone (¤¤p<0.01).</p

Primer sequences used for RT²-PCR.

<p>Primer sequences used for RT²-PCR.</p

Male-specific pancreatic carcinogens induce MMP-9 expression in Panc-1 cells.

<p>Cells were treated with 10 µM TCP, ChlA, BA, CA, TDI, AMN, MER or ROX for 24 hours. (<b>A</b>) Densitometric analysis of intracellular MMP-9 levels. Level in untreated cells (control) was set to 100. (<b>B</b>) Representative Western blots of intracellular MMP-9 protein levels. (<b>C</b>) Cells were treated with 10 µM BA, CA, TDI, TCP or ChlA for 24 hours. Samples were analyzed by Real Time RT-PCR using primers shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043209#pone-0043209-t002" target="_blank">Table 2</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043209#s3" target="_blank">Results</a> are presented as mean ± SD, n = 3 and control levels were set to one. *significantly different from controls (*p<0.05, **p<0.01, ***p<0.001).</p

Male-specific pancreatic carcinogens increase Ca²⁺ levels and activate α-calcineurin.

<p>(<b>A</b>) Fura-2-loaded Panc-1 cells were treated with ATP (200 µM), ChlA (100 µM), TCP (1 mM), CA (50 µM) or BA (500 µM). *significantly different from controls (set to 100%) (*p<0.05, **p<0.01). (<b>B</b>) BA+ATP-treated cells were pretreated with BA and thereafter with ATP. #significantly different from ATP, p<0.05. (<b>C</b>) Cells were preincubated with KN62 (100 nM) for 10 min followed by CA (50 µM) treatment. (<b>D</b>) Cells were treated with chemicals (10 µM) for 15 min. Levels of α-calcineurin (45/48 kDa, active form and 61 kDa, inactive form) were analyzed by Western blotting. Three different controls (c) were used. In A, B and C results are presented as mean ± SD, n = 3.</p

Summary of results obtained in Panc-1, Mia-PaCa-2 and Capan-2 cells.

<p>Summary of results obtained in Panc-1, Mia-PaCa-2 and Capan-2 cells.</p

Chemicals classified by NTP to be associated with site-specific tumor induction in pancreas acinar cell (pancreatic acinar cell adenoma or carcinoma) in male rats and their major use.

<p>Total PubMed abstracts and NTP's Salmonella results are shown.</p