Stimulation of eryptosis by broad-spectrum insect repellent N,N-Diethyl-3-methylbenzamide (DEET)

https://doi.org/10.1016/j.taap.2019.03.011Get rights and content

Highlights

  • DEET possesses a weak hemolytic potential in human erythrocytes.

  • DEET induces eryptosis characterized by cell membrane scrambling with phosphatidylserine (PS) externalization

  • DEET-induced premature cell death is accompanied by cell shrinkage and dysregulated calcium influx.

  • DEET-induced PS exposure is not ameliorated by extracellular calcium removal nor by intracellular calcium chelation.

  • Suicidal erythrocyte death caused by DEET is not mediated through oxidative stress or a specific signaling pathway.

Abstract

N,N-Diethyl-3-methylbenzamide (DEET) is the most widely used insect repellent in the world. Adverse effects following DEET exposure are well documented. Moreover, DEET has been shown to possess cytotoxic and apoptotic properties in nucleated cells. Although red blood cells (RBCs) lack intracellular organelles, they nevertheless undergo programmed cell death termed eryptosis. Compromised RBC health contributes to the development of anemia; a condition affecting 25% of the global population. This study investigated the interaction between DEET and human RBCs, and explored accompanying biochemical and molecular alterations. RBCs at 5% hematocrit were incubated in presence and absence of 1–5 mM (0.02%–0.1%) of DEET for 6 h at 37 °C. Hemolysis was spectrophotometrically determined by hemoglobin release, while major eryptotic events were analyzed by flow cytometer. Phosphatidylserine (PS) exposure was detected with Annexin-V-FITC, cell volume by forward scatter (FSC) of light, intracellular calcium with Fluo-3/AM, and reactive oxygen species with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). DEET caused slight hemolysis at 4 and 5 mM, and significantly increased Annexin-V-FITC and Fluo3 fluorescence, with reduced FSC at 5 mM. Removal of extracellular Ca2+ abolished DEET-induced Fluo3 fluorescence but had no effect on Annexin-V binding. Importantly, blockade of eryptotic signaling mediators p38 MAPK, caspases, protein kinase C, casein kinase 1, or necroptotic kinases receptor-interacting protein 1 and mixed lineage kinase domain-like protein, with small molecule inhibitors, did not ameliorate DEET-mediated PS externalization. In conclusion, DEET elicits suicidal erythrocyte death; an event characterized by loss of membrane asymmetry, cell shrinkage, and elevations in intracellular Ca2+ mainly through dysregulated Ca2+ influx.

Introduction

The World Health Organization (WHO) reports that a little less than one-fifth of the global burden of infections is caused by vector-borne disease, which accounts for around 700,000 deaths annually (WHO. Global vector control response 2017–2030. Geneva: World Health Organization; 2017. http://apps.who.int/iris/bitstream/handle/10665/259205/9789241512978-eng.pdf?sequence=1). Among the most infamous vectors are mosquitoes in the Anopheles, Aedes, and Culex genera. These blood-sucking insects are responsible for the transmission of serious diseases and pathogens including malaria, filariasis, yellow fever, dengue fever, chikungunya, Zika virus, West Nile virus, Japanese encephalitis, and St. Louis encephalitis (WHO. Vector-borne diseases: Report of an informal expert consultation. New Delhi: World Health Organization; 2014. http://apps.who.int/iris/bitstream/handle/10665/206531/B5139.pdf?sequence=1; WHO: Yellow fever fact sheet. http://www.who.int/en/news-room/fact-sheets/detail/yellow-fever) (Mackenzie et al., 2004; Mulatier et al., 2018). Given the lack of vaccination, prophylaxis by insect repellents remains one of the most efficient and widely used methods to prevent the spread of vector-borne disease worldwide.

N,N-Diethyl-3-methylbenzamide (DEET) is the most commonly used insect repellent in the world (Fig. 1A) (Chen-Hussey et al., 2014). Since its introduction over half a century ago (Nentwig, 2003), DEET has demonstrated efficacy in reducing the transmission of vector-borne disease. The repellence properties of DEET may be attributed to its activation of olfactory receptor neurons (ORNs) in mosquito antennae, among other possible mechanisms (Leal, 2014). Products containing up to 100% DEET are available to consumers in a variety of formulations including sprays, aerosols, gels, lotions, sticks, and wipes (Diaz, 2016).

Pharmacokinetic studies in animals indicate significant variability in oral and dermal absorption of DEET. In humans, although absorption rates may reach as high as 20%, extensive variations observed in animals may still be mirrored in man (Feldmann and Maibach, 1970; Blomquist and Thorsell, 1977; Reifenrath et al., 1980; Reifenrath et al., 1981; Snodgrass et al., 1982; Moody et al., 1989; Moody and Nadeau, 1993; Taylor et al., 1994; Selim et al., 1995). DEET reaches the systemic circulation, crosses the placenta (McGready et al., 2001; Barr et al., 2010; Diaz, 2016), and is chiefly eliminated in the urine. Major DEET metabolites excreted are N,N-diethyl-mhydroxymethylbenzamide and N-ethyl-m-hydroxymethylbenzamide, representing up to 42% of applied dose (Selim et al., 1995). The unchanged parent compound has also been detected in urine (Smallwood et al., 1992). Nevertheless, based on the inherent diversity of human use and consumption, variations in DEET bioavailability and metabolism are expected to be not uncommon.

Intoxication related to DEET is well documented in animals and humans, with dermal, neurologic, ocular, psychotic, respiratory, gastrointestinal, and cardiovascular manifestations (Heick et al., 1980; Snyder et al., 1986; Heick et al., 1988; McKinlay et al., 1998; Briassoulis et al., 2001; Bell et al., 2002; Jortner, 2006). Cases of accidental and suicidal death have also been reported following oral and dermal exposure to DEET (Veltri et al., 1994; Bell et al., 2002; Wiles et al., 2014). Populations at higher risk of adverse DEET effects include children, workers in parks and manufacturing plants, and individuals with preexisting skin conditions (https://www.atsdr.cdc.gov/toxprofiles/tp185.pdf; https://toxnet.nlm.nih.gov/cgi-bin/sis/search2/r?dbs+hsdb:@term+@DOCNO+1582). To date, there exists a paucity in the literature concerning the cellular and molecular effects of DEET, and the potential toxic effects of the repellent on human red blood cell (RBC; erythrocyte) lifespan have not yet been reported. Responsible for gas exchange and transfer of immune complexes, RBCs have a lifespan of 120 days, after which they are removed from the circulation by the monocyte-macrophage system (Lang and Lang, 2015b). Although they lack intracellular organelles, most notably the nucleus and mitochondria, RBCs nonetheless undergo a specific form of programmed cell death known as eryptosis. Characteristics of eryptotic cells include membrane blebbing and phospholipid scrambling, cell shrinkage, intracellular accumulation of calcium and reactive oxygen species (ROS), ceramide formation, and energy exhaustion (Lang et al., 2012). As in apoptosis of nucleated cells, multiple signaling mediators similarly regulate eryptosis. Although still in their infancy, discovery efforts have discerned the existence of caspases, p38 mitogen-activated protein kinase (MAPK), casein kinase 1 (CK1), protein kinase C (PKC), receptor-interacting protein 1 (RIP1), mixed lineage kinase domain-like protein (MLKL), Janus kinase 3 (JAK3), 5’ AMP-activated protein kinase (AMPK), and cGMP-dependent protein kinase (cGKI), (Lang et al., 2012; LaRocca et al., 2014; Lang and Lang, 2015b).

Various xenobiotics and pathological conditions have been identified as modulators of eryptosis (Pretorius et al., 2016) and the effect of DEET on RBC lifespan remains an uncharted territory. In this report, we sought to investigate the interaction between DEET and human RBCs, which represent an excellent model to assess the toxicity of xenobiotics (Hinderling, 1997; Schrijvers, 2003; Farag and Alagawany, 2018). Here, we show that DEET causes premature RBC death characterized by loss of membrane asymmetry and phosphatidylserine (PS) externalization, profound intracellular calcium accumulation secondary to dysregulated influx, and cell shrinkage. Interestingly, the eryptotic effects of DEET were not mediated through oxidative stress, did not depend on calcium availability, and are not significantly ameliorated by blocking of major cell death signaling pathways.

Section snippets

Erythrocytes, chemicals, and solutions

Fresh, citrate-phosphate-dextrose-adenine (CPDA-1)-anticoagulated RBC samples from consented, healthy adults were obtained from ZenBio (Research Triangle Park, NC, USA). Samples were stored at 4 °C according to standard blood banking procedures (Fernandes da Cunha et al., 2005) and used within 20 days of collection. Cell viability and validity for experimentation was verified by low (≤5%) Annexin-V-FITC binding (Lupescu et al., 2014). Prior to experiments, RBCs were washed in phosphate-buffered

DEET induces weak hemolysis in human RBCs

Cell-free hemoglobin is a marker of hemolysis. To determine the hemolytic potential of DEET, RBCs were treated with 1–5 mM of DEET for 6 h at 37 °C, and hemoglobin leakage into the medium was subsequently assayed. Fig. 1B shows that DEET causes a modest, yet statistically significant increase in hemolysis at both 4 and 5 mM. This indicates that the cell membrane could be a major action site targeted by the repellent.

DEET causes PS externalization

Membrane phospholipid scrambling, the principal feature of eryptotic cells, was

Discussion

DEET is considered the gold standard of insect repellents, and formulations containing DEET are more effective than those without it (Tintinalli and Stapczynski, 2011). Environmental and public health agencies encourage DEET use, and more consumption is indeed on the horizon (Chen-Hussey et al., 2014). Although no adverse effects on RBCs following DEET exposure have been observed in humans, reduced hemoglobin and hematocrit found in small-scale animal studies were either unreliable or not

Acknowledgments

We thank the members of the Lee laboratory for helpful advice and discussion during this work. This work was supported in part by the Brody Brothers Grant (BBE216102) to M-H.L. and the Saudi Government Graduate Scholarship (through King Saud University) to M.A.A.

Competing interests

The authors declare they have no competing interests relevant to this manuscript.

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