Ptaquiloside Reduces NK Cell Activities by Enhancing Metallothionein Expression
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Toxicology 304 (2013) 100–108 Contents lists available at SciVerse ScienceDirect Toxicology jou rnal homepage: www.elsevier.com/locate/toxicol Ptaquiloside reduces NK cell activities by enhancing metallothionein expression, which is prevented by selenium a,∗ a b c d Andreia O. Latorre , Beatriz D. Caniceiro , Heidge Fukumasu , Dale R. Gardner , Fabricio M. Lopes , e a e b a Harry L. Wysochi Jr. , Tereza C. da Silva , Mitsue Haraguchi , Fabiana F. Bressan , Silvana L. Górniak a Department of Pathology, School of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, SP 05508-270, Brazil b Department of Basic Sciences, School of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, SP 13635-900, Brazil c Poisonous Plant Research Lab, N. Logan, UT 84341, USA d Federal University of Technology, Paraná, Brazil e Biological Institute of São Paulo, São Paulo, SP 04014-002, Brazil a r t i c l e i n f o a b s t r a c t Article history: Pteridium aquilinum, one of the most important poisonous plants in the world, is known to be carcinogenic Received 29 September 2012 to animals and humans. Moreover, our previous studies showed that the immunosuppressive effects Received in revised form of ptaquiloside, its main toxic agent, were prevented by selenium in mouse natural killer (NK) cells. 11 December 2012 We also verified that this immunosuppression facilitated development of cancer. Here, we performed Accepted 17 December 2012 gene expression microarray analysis in splenic NK cells from mice treated for 14 days with ptaquiloside Available online 26 December 2012 (5.3 mg/kg) and/or selenium (1.3 mg/kg) to identify gene transcripts altered by ptaquiloside that could be linked to the immunosuppression and that would be prevented by selenium. Transcriptome analy- Keywords: sis of ptaquiloside samples revealed that 872 transcripts were expressed differentially (fold change > 2 Bracken fern and p < 0.05), including 77 up-regulated and 795 down-regulated transcripts. Gene ontology analysis Pteridium aquilinum Immunotoxicity mapped these up-regulated transcripts to three main biological processes (cellular ion homeostasis, Toxicogenomics negative regulation of apoptosis and regulation of transcription). Considering the immunosuppressive DNA microarray effect of ptaquiloside, we hypothesized that two genes involved in cellular ion homeostasis, metallothi- onein 1 (Mt1) and metallothionein 2 (Mt2), could be implicated because Mt1 and Mt2 are responsible for zinc homeostasis, and a reduction of free intracellular zinc impairs NK functions. We confirm these hypotheses and show increased expression of metallothionein in splenic NK cells and reduction in free intracellular zinc following treatment with ptaquiloside that were completely prevented by selenium co-treatment. These findings could help avoid the higher susceptibility to cancer that is induced by P. aquilinum-mediated immunosuppressive effects. © 2012 Elsevier Ireland Ltd. Open access under the Elsevier OA license. 1. Introduction Shahin et al., 1999; Ulian et al., 2010), and this feeding pattern has already been associated with a greater prevalence of certain types Bracken fern (genus Pteridium) is a ubiquitous plant known to of cancers (e.g., esophageal, gastric) (Abnet, 2007; Sugimura, 2000). cause toxicity syndromes in herbivores and cancer in animals and Similarly, concerns exist regarding the indirect consumption of humans (Gil da Costa et al., 2012a; Vetter, 2009). Nevertheless, bracken’s toxins through consumption of milk from livestock that humans have used its crosiers and rhizomes as food in some regions have fed on the plant (Alonso-Amelot and Avendano, 2002; Shahin of the world such as Brazil and Japan (Kamiyama et al., 1986; et al., 1999). Environmental contamination could also be a prob- lem, as has already been demonstrated for ptaquiloside in soil and water (Rasmussen et al., 2003). In fact, epidemiologic studies have ∗ attributed high rates of stomach cancer to people living in areas Corresponding author at: Department of Pathology, Faculty of Veterinary infested by bracken fern, for example, in the highlands of western Medicine and Animal Sciences, University of Sao Paulo, Ave. Prof. Orlando Marques Venezuela (Alonso-Amelot and Avendano, 2001) and in Gwynedd, de Paiva, 87, Sao Paulo 05508-270, Brazil. Tel.: +55 11 99391 4865; fax: +55 11 3091 7829. North Wales (Galpin et al., 1990). More recently, meat was identi- E-mail addresses: [email protected], [email protected] fied as another potential source of intoxication, as bracken toxins (A.O. Latorre), [email protected] (B.D. Caniceiro), [email protected] were detected in the skeletal muscle and liver of cattle fifteen days (H. Fukumasu), [email protected] (D.R. Gardner), [email protected] after bracken consumption had ended (Fletcher et al., 2011). (F.M. Lopes), [email protected] (H.L. Wysochi Jr.), [email protected] (T.C. da Silva), The main toxic agent found in P. aquilinum is ptaquiloside, [email protected] (M. Haraguchi), [email protected] (F.F. Bressan), [email protected] (S.L. Górniak). which has been proven to be responsible for carcinogenic effects 0300-483X © 2012 Elsevier Ireland Ltd. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.tox.2012.12.010 A.O. Latorre et al. / Toxicology 304 (2013) 100–108 101 and a number of well-recognized toxicity syndromes in herbivores 0.1 mM for the in vitro studies. This concentration was determined by preliminary tests that demonstrated an increase in NK cell cytotoxicity in vitro. (Yamada et al., 2007). These toxic effects consist of a thiamine defi- ciency in horses and pigs, an acute hemorrhagic syndrome in cattle, 2.4. In vivo experimental design retinal degeneration in sheep and a urinary bladder neoplasm of epithelial and mesenchymal origin and an epithelial tumor of the Mice were separated into four groups, with five mice per group, as follows: con- upper gastrointestinal tracts of ruminants. The complete descrip- trol (Co), ptaquiloside (Pt), ptaquiloside and selenium (PtSe), and selenium (Se). In general, experimental mice were treated by daily gavage for 14 days with ptaquilo- tion of the effects of bracken fern has been reviewed recently (Gil side (5.3 mg/kg) and/or selenium (1.3 mg/kg). The Co mice received only water and da Costa et al., 2012a). were treated at the same time as the experimental mice. The body weight of each Our previous studies showed that ptaquiloside is an immuno- mouse was measured every 3 days for dose adjustment. On day 15 of the exper- suppressor that caused a reduction in mouse splenic NK iment, mice from all groups were killed with an overdose of CO2 and splenic cell cell-mediated cytotoxicity and IFN␥ production (Latorre et al., suspensions were then prepared to isolate NK cells (see below). 2009). Moreover, we verified that selenium supplementation 2.5. Isolation of splenic NK cells can prevent this reduction in NK-mediated cytotoxicity (Latorre et al., 2011). A greater incidence of chemical-induced preneopla- Spleens were removed aseptically and made into a single-cell suspension. sic lesions are noted in mice immunosuppressed with bracken fern Briefly, for each mouse, the isolated spleen was gently squeezed by the distal end of a syringe into a plate of cold RPMI medium (Gibco). The erythrocytes present in the (Caniceiro et al., 2011), and our findings may be of great relevance in suspension were then lysed using sterile 0.4% ammonium chloride solution. Spleno- avoiding the increased susceptibility to cancer caused by the plant. ◦ cytes were centrifuged at 1200 rpm (4 C, 8 min), and the pelleted cells were then The molecular mechanism underlying ptaquiloside-induced re-suspended in RPMI-complete medium (supplemented with 10% FBS, Gibco). To immunosuppression and its prevention by selenium are unknown. separate non-adherent from adherent cells, the samples were incubated on 6-well ◦ Thus, the objective of this study was to verify the mechanism of plates for 2 h at 37 C in a humidified atmosphere containing 5% CO2. Next, non- adherent cells were harvested and filtered through a 70 m cell strainer. Untouched action of ptaquiloside-induced immunosuppression in splenic NK NK cells were isolated according to the manufacturer’s protocol using an NK cell cells using gene expression microarray analysis. We performed isolation kit, LS columns and a QuadroMACS cell separator system (Miltenyi Biotec, transcriptome analysis in splenic NK cells from mice treated for Inc.). The viability of the separated cells was verified using the Trypan Blue assay 14 days with ptaquiloside (5.3 mg/kg) and/or selenium (1.3 mg/kg) and was found to be 100%. − + The percentage of splenic NK cell (CD3 NK1.1 ) recovery after the isolation to identify gene transcripts altered by ptaquiloside that could be procedure was evaluated using splenic cells from five mice that were processed linked to the immunosuppression and that would be prevented by with PE-labeled anti-CD3 (clone 17A2) and PerCP-Cy5.5-labeled anti-NK1.1 (clone selenium. TM PK136) antibodies (BD Pharmingen) in a FACSCalibur flow cytometer equipped ® with Cell Quest Pro software (Becton Dickinson [BD] Immunocytometry System) ® and analyzed with FlowJo 7.2.6 software (Tree Star Inc, Ashland, KY) as demon- 2. Materials and methods strated in Fig. 1A and B. The percentages of splenic NK cells presented in Fig. 1A and 2.1. Mice B represent the mean obtained from five mice. Fifty eight sixty-day-old male C57BL/6J mice, bred in the Department of Pathol- 2.6. RNA isolation and whole-genome gene expression profile ogy at the School of Veterinary Medicine and Animal Sciences, were used. The mice ◦ were housed in controlled temperature (22–25 C), relative humidity (50–65%) and Isolated splenic NK cells from Co (n = 5), Pt (n = 5), PtSe (n = 5) and Se (n = 5) lighting (12 h/12 h light/dark cycle) conditions.