RIP1 and RIP3 contribute to Tributyltin-induced toxicity in vitro and in vivo

Highlights

• TBT-induced cell death was significantly downregulated by RIP1 inhibition or knockdown, or RIP3 deficiency.

• The mortality induced by acute TBT exposure was markedly reduced by RIP1 inhibitor pretreatment or RIP3 deficiency.

• The immunotoxic effects induced by TBT were attenuated in RIP3^−/− mice or WT mice treated with RIP1 inhibitor.

Abstract

Tributyltin (TBT), a widely distributed environmental pollutant, is toxic to animals and human beings. Although its toxicity, especially the immunosuppressive effect, has been reported a lot, the underlying molecular mechanisms are still unclear. In this study, we investigated the mechanisms of TBT-induced cytotoxicity both in vitro and in vivo. TBT induced cell death in both J774A.1 macrophages and mouse bone marrow-derived macrophages (BMDMs) as measured by the LDH and Annexin V-FITC/PI dual staining assays. Pretreatment with RIP1 inhibitor Necrostatin-1 (Nec-1) or transfection with Rip1 siRNA significantly suppressed TBT-induced cytotoxicity in J774A.1 macrophages or human embryonic kidney cell line (HEK293 cells). TBT-induced cell death was also markedly inhibited in RIP3^−/− BMDMs. In agreement with in vitro results, TBT-induced in vivo immunotoxic effects including leukocyte depletion and thymus atrophy were significantly attenuated in RIP3^−/− mice or WT mice treated with Nec-1. Notably, the mortality rate induced by TBT was remarkably reduced in RIP3^−/− mice (100% vs. 12.5% lethality) or Nec-1-treated mice (100% vs. 59.2% lethality) respectively. These results reveal a critical role of RIP1 and RIP3 in TBT-induced toxicity both in vitro and in vivo.

Introduction

Tributyltin (TBT), which is an organotin compound, has been reported to cause neurotoxicity, reproductive/developmental toxicity and immunotoxicity and hence the use of TBT is currently restricted in some fields (Boyer, 1989; Pagliarani et al., 2013). However, due to its previous widespread use in agriculture and industry (Boyer, 1989; Fent, 1996), the risk of human exposure to TBT through drinking water and contaminated fish products is still high (Sousa et al., 2017). TBT has been detected in human blood and liver with concentration up to 8.18 ng mL⁻¹ (Kannan and Giesy, 1999). Therefore, the toxic effects of TBT and the underlying mechanisms should be comprehensively investigated.

High dose of TBT treatment causes the acute toxicity, and results in high mortality rate in animals (Takagi et al., 1992; Ohtaki et al., 2007). Additionally, the toxicity of TBT on immune system and its immunosuppressive effects have been extensively reported. TBT exposure can cause thymus atrophy, depletion of T-lymphocytes in spleen and lymph nodes, decreased macrophage and NK cell activity, and diminished T-helper (Th) 2 cell polarization (Snoeij et al., 1987; Boyer, 1989; Kergosien and Rice, 1998; Whalen et al., 2002; Kato et al., 2006). However, the molecular mechanisms by which TBT exerts these immunotoxic effects are still not clear. Previous studies proposed that TBT induced thymus atrophy through inhibiting cell proliferation or inducing apoptosis in immune cells (Raffray and Cohen, 1993; Gennari et al., 1997; Tomiyama et al., 2009). Regarding the underlying mechanism of TBT-induced apoptosis, although different mechanisms including intracellular Ca²⁺ overload, inhibition of ATP synthesis, caspase activation and release of cytochrome c have been detected and reported (Viviani et al., 1995; Stridh et al., 2001; Tomiyama et al., 2009), however, there is no direct genetic evidence confirming the mechanisms of cytotoxicity and accordingly the molecular targets by TBT have not been identified yet. Moreover, some studies have reported that high concentration of TBT also induced necrosis (Stridh et al., 1999b; Botelho et al., 2015; Khondee et al., 2016), suggesting that TBT-induced cytotoxicity is not limited to apoptosis, and other mode of cell death might also contribute to the immunotoxicity of TBT. And the studies which investigate the mechanism of TBT-induced necrosis are still lacking.

Necrosis, which has gained intensive investigations in recent years, represents another type of programmed cell death in addition to apoptosis. Programmed necrosis can be tightly regulated by two Receptor-interacting protein (RIP) family members, RIP1 and RIP3. Upon exposure to death stimuli, RIP1 interacts with RIP3, which leads to the assembly of necrosome complex and the induction of necrosis via downstream executor MLKL (mixed-lineage kinase domain-like) (Sun et al., 2012; Wu et al., 2013; Christofferson et al., 2014; Chan et al., 2015). Necrosis can be induced by various stimuli, including death cytokines, intracellular ATP depletion, mitochondrial depolarization, poly-(ADP-ribose) polymerase (PARP) activation, reactive oxygen species (ROS) production and certain viruses, etc (Vanlangenakker et al., 2012). To date, at least to our knowledge, there is still no report that a certain environmental pollutant can induce necrosis via RIP1 and RIP3.

In this study, we have investigated the cytotoxic effects of TBT and explored the underlying molecular mechanisms both in vitro and in vivo. Our results indicated that TBT induced the occurrence of necrotic cell death in macrophages through RIP1-RIP3 pathway. Inhibition or genetic deletion of RIP1-RIP3 pathway was able to rescue the cell death induced by TBT. Moreover, pretreatment with RIP1 inhibitor or RIP3 deficiency significantly protected the mice from TBT-induced mortality and alleviated the tissue damage as well as immunosuppressive effect induced by TBT, suggesting that RIP1 and RIP3 also contribute to the toxicity of TBT in vivo.