Journal of Alzheimer’s Disease 62 (2018) 1663–1681 DOI 10

Journal of Alzheimer’s Disease 62 (2018) 1663–1681DOI 10.3233/JAD-170875IOS Press

1663

Induction of Amyloid-42 Productionby Fipronil and Other Pyrazole Insecticides

Morgane Cama,1, Emilie Durieua,1, Marion Bodina, Antigoni Manousopouloub, Svenja Koslowskia,c,Natalia Vasylievad, Bogdan Barnychd, Bruce D. Hammockd, Bettina Bohle, Philipp Koche,f ,Chiori Omorig,h, Kazuo Yamamotoh, Saori Hatag, Toshiharu Suzukig, Frank Kargi, Patrick Gizzij,Vesna Erakovic Haberk, Vlatka Bencetic Mihaljevick, Branka Tavcark, Erik Porteliusl,Josef Panneel,m, Kaj Blennowl,m, Henrik Zetterbergl,m,n,o, Spiros D. Garbisb, Pierrick Auvrayc,Hermeto Gerberp,q,r, Jeremy Fraeringp,q, Patrick C. Fraeringp,q and Laurent Meijera,∗aManRos Therapeutics, Centre de Perharidy, Roscoff, Bretagne, FrancebFaculty of Medicine, Cancer Sciences and Clinical and Experimental Medicine,University of Southampton, Southampton, UKcC.RIS Pharma, Parc Technopolitain, Atalante Saint Malo, Saint Malo, FrancedDepartment of Entomology and Nematology and UCD Comprehensive Cancer Center,University of California, Davis, CA, USAeInstitute of Reconstructive Neurobiology, University of Bonn, Bonn, Germanyf Central Institute of Mental Health, University of Heidelberg/Medical, Faculty Mannheim and HectorInstitut for Translational Brain Research (HITBR gGmbH), Mannheim, GermanygLaboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University,Sapporo, JapanhDepartment of Integrated Bioscience, Graduate School of Frontier Sciences, University of Tokyo,Kashiwa, JapaniHPC INTERNATIONAL SAS and Atlantis Developpement SAS, Noyal-Chatillon sur Seiche, Saint-Erblon, FrancejPlate-forme TechMedILL, UMR 7242, ESBS - Pole API, Illkirch cedex, FrancekFidelta d.o.o., Zagreb, CroatialClinical Neurochemical Laboratory, Sahlgrenska University Hospital, Molndal, SwedenmDepartment of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology,University of Gothenburg, Molndal, SwedennDepartment of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, UKoUK Dementia Research Institute, London, UKpFoundation Eclosion, SwitzerlandqCampus Biotech Innovation Park, Geneva, SwitzerlandrDepartment of Biology, University of Fribourg, Fribourg, Switzerland

Accepted 8 January 2018

Abstract. Generation of amyloid- peptides (As) by proteolytic cleavage of the amyloid- protein precursor (APP),especially increased production of A42/A43 over A40, and their aggregation as oligomers and plaques, represent a charac-teristic feature of Alzheimer’s disease (AD). In familial AD (FAD), altered A production originates from specific mutationsof APP or presenilins 1/2 (PS1/PS2), the catalytic subunits of -secretase. In sporadic AD, the origin of altered production ofAs remains unknown. We hypothesize that the ‘human chemical exposome’ contains products able to favor the production of

1These authors contributed equally to this work.∗Correspondence to: Laurent Meijer, ManRos Therapeutics,

29680 Roscoff, France. Tel.: +33 6 08 60 58 34; E-mail: [email protected].

ISSN 1387-2877/18/$35.00 © 2018 – IOS Press and the authors. All rights reserved

mailto:meijer@penalty -@M manros-therapeutics.com

1664 M. Cam et al. / Aβ42 Inducers in the Human Chemical Exposome

A42/A43 over A40 and shorter As. To detect such products, we screened a library of 3500 + compounds in a cell-basedassay for enhanced A42/A43 production. Nine pyrazole insecticides were found to induce a - and -secretase-dependent,3-10-fold increase in the production of extracellular A42 in various cell lines and neurons differentiated from inducedpluripotent stem cells derived from healthy and FAD patients. Immunoprecipitation/mass spectrometry analyses showedincreased production of As cleaved at positions 42/43, and reduced production of peptides cleaved at positions 38 andshorter. Strongly supporting a direct effect on -secretase activity, pyrazoles shifted the cleavage pattern of another -secretase substrate, alcadein, and shifted the cleavage of APP by highly purified -secretase toward A42/A43. Focusingon fipronil, we showed that some of its metabolites, in particular the persistent fipronil sulfone, also favor the production ofA42/A43 in both cell-based and cell-free systems. Fipronil administered orally to mice and rats is known to be metabolizedrapidly, mostly to fipronil sulfone, which stably accumulates in adipose tissue and brain. In conclusion, several widelyused pyrazole insecticides enhance the production of toxic, aggregation prone A42/A43 peptides, suggesting the possibleexistence of environmental “Alzheimerogens” which may contribute to the initiation and propagation of the amyloidogenicprocess in sporadic AD.

Keywords: A38, A40, A42, A43, A42/A40 ratio, aftins, Alzheimer’s disease, alzheimerogen, amyloid-, amyloid- protein precursor, fipronil, -secretase, human chemical exposome, pesticides, phenylpyrazoles, prevention, pyrazoles,triazines

INTRODUCTION

Alzheimer’s disease (AD) is a major disease incountries with aging populations. Despite unresolvedquestions on the initial causes of AD and numer-ous clinical trial failures in recent years, the lackof satisfactory treatments and the extremely highprevalence of AD calls for fundamental researchto determine its underlying molecular and cellularcauses and mechanisms and for applied research toidentify options for prevention, therapeutic targetsand disease-modifying drug candidates (reviews in[1–4]).

Although the aggregation of specific forms ofamyloid- peptides (As) into soluble oligomers isundoubtedly associated with the onset of AD, theupstream etiological events underlying this patholog-ical process remain unclear, including whether thereis an absolute, or relative, increase in the produc-tion of longer, aggregation-prone A species. Asare derived from the successive action of two pro-teases, -secretase and -secretase, on one of theirnumerous substrates, the transmembrane amyloid-protein precursor (APP). According to its cleav-age site, -secretase liberates As of different sizes.While A40 is considered as the main physiologi-cal product, the appearance of A42 and A43, orincrease of the A42-A43/A40 ratio is clearly asso-ciated with the onset of AD [5–7]. These longerAs show an increased propensity to form oligomers,which can assemble into large extracellular deposits,the amyloid plaques, a characteristic feature of

AD. A42/A43 oligomers are now considered asthe toxic, AD initiating elements rather than themore prominent plaques initially discovered by AloısAlzheimer. This ‘amyloid cascade theory’ is furthersupported by the identification of the genetic causesof early onset familial AD (FAD). These rare formsof AD (less than 1% of all AD cases) are indeed allassociated with specific mutations of either AβPP orPS1 or PS2 (Presenilins) genes (the latter encode thecatalytic subunits of the -secretase complex) (reviewin [8, 9]). In addition, a specific, protective mutationof APP was recently found to be associated withreduced risk for developing AD [10]. Furthermore,animal models that express mutated forms of APPand/or PS1, or human A42/A43, develop molec-ular, cellular and cognitive deficits reminiscent ofAD ([6], review in [11, 12]). Yet, the level of thedifferent forms of As is not well correlated withcognitive deficits in mice, suggesting that As mayrepresent markers rather than inducers of AD in thesemodels [13]. An altered processing of -secretasesubstrates other than APP might also be importantin the pathogenesis of AD. Although FAD is clearlya consequence of genetic mutations of genes encod-ing the proteins responsible for the production ofAs, the initial trigger(s) of late-onset, sporadic AD(>99% of AD cases) remain unknown, despite exten-sive genome-wide association studies (GWAS) andthe identification of various risk factors (review in[14]).

Environmental neurotoxic agents including pesti-cides, organic solvents, metals and some natural tox-

M. Cam et al. / Aβ42 Inducers in the Human Chemical Exposome 1665

ins (cyanobacteria) are likely sources of AD-inducingfactors (reviews in [15–25]). According to the USEnvironmental Protection Agency (EPA) Toxic Sub-stances Control Act, over 84,000 chemicals aremanufactured or imported at levels >10 tons per year,not including pesticides, cosmetics, food stuffs, andfood additives which are covered by other legisla-tions (http://www.epa.gov). It is estimated that we areexposed to over 85,000 substances which, along withall natural substances to which we are exposed fromconception to death, constitute the ‘human chemicalexposome’ (HCE) [26–34]. In search for potential“Alzheimerogens” (named by analogy with carcino-gens), and encouraged by the discovery of Aftins(Amyloid Forty-Two Inducers) [35–37], a classof synthetic compounds which specifically inducethe production of A42, we have started to assem-ble and screen a library of compounds, belonging tothe HCE, for A42-inducing products. Our first studyidentified some triazine herbicides as products stimu-lating the production of A42/A43 in numerous celllines [38].

In this new study, we show that several mem-bers of the pyrazole class of insecticides, exemplifiedby fipronil, also increase the -secretase-dependentproduction of A42/A43 peptides in various celllines and in a cell-free system with highly purified-secretase. Interestingly fipronil is metabolized tofipronil sulfone, a very persistent product that accu-mulates in adipose tissue and brain. These resultsshow that the HCE contains various brain per-meable products able to induce the production ofpathogenic As. If long exposures occur in real lifeand if accumulation of A42/A43 is sufficient totrigger neurodegeneration/neuroinflammation, suchproducts may contribute to the initiation, develop-ment and acceleration of sporadic AD and might thusbe collectively qualified as potential “Alzheimero-gens”. Some of them might also be used todevelop useful chemically-induced animal modelsof AD.

MATERIAL AND METHODS

Material and methods are described in full in theSupplementary Material They include: 1) Pyrazolesand other reagents; 2) Cell cultures: cell lines, iPSCs-derived neurons and HEK293-alcadein cells; 3)Purified -secretase preparation; 4) A cell-basedand cell-free assays; 5) Cell viability assay; 6) Phar-macokinetics studies; 7) Proteomics study.

RESULTS

Screening HCE compounds reveals pyrazoleinsecticides as Aβ42 inducers

We first screened a library of over 3,500 low molec-ular weight compounds representative of the HCE fortheir ability to induce the production and secretion ofA42 by N2a cells stably expressing APP695 (N2a-APP695). An MTS-based cell viability assay wasrun in parallel to assess cell survival. Although thevast majority of compounds were unable to induceA42 production, a few active products including sev-eral triazine herbicides [38] and pyrazole insecticides(this article) were identified. We next assembled asmall library of 18 pyrazoles (1–18, SupplementaryTables 1 and 2) that we further tested, along withaftin-5 (19) as a positive control, for their ability totrigger A42 production at 5, 10, 25, or 50 M, inboth N2a and CHO cells stably expressing APP695and APP751, respectively (Supplementary Table 2and Fig. 1). Nine pyrazoles were found to inducemore than a 3-fold change in A42 levels, showingthat A42 induction is an intrinsic property of some,but not all, pyrazoles: fenpyroximate (2), tebufen-pyrad (3), bixafen (4), fluxapyroxad (5), isopyrazam(6), penthiopyrad (8), chlorantraniliprole (14), tolfen-pyrad (16) and fipronil (18) (Figs. 1 and 2). We nextfocused on fipronil (18) 2, a widely produced insecti-cide used to treat crops and pet parasites (reviews in[39–44]), and we assembled a small library of fipronilanalogues and metabolites (Fig. 3) which were testedfor their A42 production ability in N2a-APP695cells (Supplementary Table 3). The two fipronil enan-tiomers (18a, 18b) were separated by SFC chiralchromatography (Regis Whelk O1 SS column) andfound to be roughly equipotent. Interestingly themain metabolites of fipronil (18) [45], fipronil sulfone

2Although fipronil is not the most potent A42 inducer, it wasselected for more detailed studies, among other pyrazoles, for sev-eral reasons: 1) its very wide use all over the world (despite itsban in Europe in America for food plant cultures), 2) its wide useagainst fleas and ticks in domestic animals, 3) its wide presencein urban and rural environment, 4) its chemical stability and envi-ronmental persistence (especially that of its metabolite, fipronilsulfone), 5) the biological activity of the main metabolite, 6) thefact fipronil sulfone accumulates in adipose tissue, crosses theblood-brain barrier and accumulates in the brain, and 8) its avail-ability in large quantities for very cheap prices (in preparationof our current long term exposure experiments the supply issueneeded to be solved). Incidentally, fipronil was selected before thepublic health scandal of summer 2017 in Europe (contaminationof eggs by fipronil, presumably even more by fipronil sulfone)!

http://www.epa.gov


Fig. 1. Some pyrazoles trigger the production of extracellular amyloid A42. Effect of 18 pyrazoles on extracellular amyloid A42 productionby N2a-APP695 and CHO-7PA2-APPsw cells. Cells were treated with 100 M of each compound for 18 h and cell supernatants were collectedfor extracellular A42 levels measurement by an ELISA assay. Aftin-5 was used as a positive control and the corresponding volume of vehicle(DMSO) was used as a negative control. Levels are expressed as fold change, ± SE, of A42 levels over the A42 level of control, vehicle-treated cells. Average of five experiments performed in triplicate. Horizontal dotted lines indicate levels for 1- and 3- fold increases in A42concentration.

(20), fipronil desulfinyl (21) and fipronil sulfide (22)displayed A42 inducing activity equivalent or closeto that of the parent compound. In contrast, a fewother environmental metabolites such as fipronil chlo-ramine (24) [46] and hydroxy-fipronil (25) [47] werefound to be inactive (Supplementary Table 3).

A42 production triggered by pyrazoles in N2a-APP695 cells requires - and -secretase activity asdemonstrated by the fact that it was strongly inhib-ited by - (inhibitor IV) and -secretases (BMS299897, DAPT) inhibitors and by a -secretasemodulator (‘Torrey Pines’ compound, inducing anincrease in A38 and a decrease in A42 and A40)[48, 49] (Fig. 4). A38 production was also stronglyreduced following pyrazole treatment, while A40levels were only modestly affected, as measured byELISA (data not shown) and by mass spectrome-try (Fig. 5). Increased A42 production triggered bypyrazoles was further confirmed in HEK293 cells sta-bly expressing APPsw (data not shown) and neuronsderived from human iPSCs (see below).

Mass spectrometric quantification and profileanalysis of pyrazole-induced Aβs

A38, A40, and A42 were quantified in N2a-APP695 cell culture supernatants using LC-MS/MS

[50, 51]. Like aftins [35, 36] and triazines [38], pyra-zoles induced a reduction in A38 levels, a slightincrease in A40 levels and a strong increase in A42levels (Fig. 5).

We next analyzed, by IP-MS [52], the profile ofAs produced by N2a-APP695 exposed to fipronil(18), fipronil sulfone (20) and fipronil desulfinyl(21) (Fig. 6). Cell supernatants were collected andAs were immunoprecipitated and analyzed usingMALDI-TOF/TOF [52] (Fig. 6). Exposure to pyra-zoles decreased the production of A1-18, A1-19,A1-33, A1-37, and A1-38. A1-40 levels showeda modest increase. In contrast, the two highly neu-rotoxic [6, 7, 53–55] amyloid peptides A1-42 andA1-43, which were undetectable in supernatants ofcontrol cells, were strongly induced in cells treatedwith fipronil (18) and its two metabolites.

Neurons differentiated from human iPSCs of ADpatient and healthy control

We next tested the effects of the active pyrazolesand aftin-5 on neurons differentiated for 4 weeksfrom human iPSCs derived from a healthy individual(APP WT, wild-type) or from a patient with familialAD (APP K724N mutation) [56–58] (Fig. 7). APPK724N neurons produced more A42 versus A40


Fig. 2. Molecular structure of the nine active pyrazoles.

compared to APP WT neurons. As observed withtriazines [38], addition of any of the active pyrazolesor aftin-5 resulted in further increase in A42 produc-tion, in both APP WT and APP K724N neurons(Fig. 7).

Pyrazoles affect the specificity of APP-C99cleavage and Aβ production in a cell-freeγ-secretase activity assay

In order to assess whether the pyrazole-based com-pounds modulate A production through a directeffect on the specific processing of APP-C99 bythe -secretase complex, we tested fipronil (18),fipronil sulfone (20), and fipronil desulfinyl (21) ina previously described cell-free activity assay per-formed with highly purified -secretase proteaseand APP-C99 substrate (a recombinant APP C-terminal fragment expressed in Escherichia coli asa fusion protein consisting of a N-terminal Met

for translation initiation, amino acids 597–695 ofthe 695-amino acid human isoform of APP, and aC-terminal Flag tag sequence) [59, 60]. More specif-ically, As generated by purified -secretase in thepresence of 100 M of the pyrazole compoundswere analyzed by immunoprecipitation and MALDI-TOF (IP/MS), as previously described [60]. Wefound that all three pyrazole compounds increasedthe A42/A40 ratio when compared to the DMSOcontrol (Fig. 8). Importantly, our analysis furtherrevealed that fipronil sulfone (20), but not fipronil(18) or fipronil desulfinyl (21), drastically increasedthe A43/A40 ratio by ∼2-fold when compared tothe DMSO control (Fig. 8). These results stronglysupport the increased A43 production seen exclu-sively with fipronil sulfone (20) in N2a-APP695cells (Fig. 6). Altogether, we found that pyrazolesfavor the production of the toxic A42 and/or A43peptides, both of which contribute to the formationof amyloid plaques and the development of AD.


Fig. 3. Molecular structure of fipronil and some of its metabolites and derivatives.

Fig. 4. Extracellular A42 production induced by pyrazoles is inhibited by -secretase inhibitor IV, -secretase inhibitors DAPT and BMS299897, and -secretase modulator ‘Torrey Pines’. N2a-APP695 cells were exposed to 10 M -secretase inhibitor IV, 2 M BMS 299897,2 M DAPT or 10 M ‘Torrey Pines’ compound. 1.5 h later cells were exposed to 50 M (2, 5, 8, 14, 16, 18) or 25 M (3, 4, 6) pyrazoles or50 M aftin-5. Extracellular A42 levels were measured after 18 h and are expressed as fold change, ± SE, of A42 level in pyrazole-treatedcells over the A42 level of control, vehicle-treated cells. Representative of two independent experiments performed in triplicates. Errorsbars represent SE of all six values.


Pyrazoles shift the cleavage pattern of anotherγ-secretase substrate, alcadeinα

Like APP, alcadeins (Alcs) are sequentiallycleaved by secretases, first by -secretase, leading

Fig. 5. Absolute quantification of A38, A40, and A42 usingLC-MS/MS. Levels of the three As were determined by massspectrometry in supernatants of N2a-APP695 cells following 18 htreatment with DMSO, 100 M of pyrazoles 18, 20, or 21. Amyloidlevels are expressed as percentage of levels in vehicle-treated cells(average ± SE of quadriplicate values; absolute values in controlcell supernatants are indicated) and A42/A40 ratios are indicatedin parentheses. Horizontal dotted line indicates basal A levelsversus the values in DMSO-treated cells.

to N- and C-terminal fragments, the latter beingthen cleaved by -secretase to an intracellulardomain and the p3-Alcs peptide, in a way similar toAPP [61, 62] (see Fig. 6A in [38]). HEK293 cellsstably expressing full length alcadein were used toinvestigate the effects of pyrazoles on alcadein cleav-age. Alcadein is first cleaved on the N-terminalside (two possible sites) followed by cleavage by-secretase leading to p3-Alc35 and p3-Alc2N+35, the latter representing the major peptidein cultured cells. HEK293-alcadein cells weregrown till 70% confluence and treated with 100 Mpyrazoles for 48 h. The secreted p3-Alc peptideswere recovered by immunoprecipitation and ana-lyzed by MALDI TOF/MS (Fig. 9A). Quantificationof p3-Alc peptides showed that, compared tothe p3-Alc peptide profile in vehicle treatedcells, concentrations of the main alcadein pep-tide (p3-Alc2N+35) and p3-Alc2N+37 peptideremained stable. In contrast, both p3-Alc2N+34and p3-Alc2N+36 concentrations dropped signif-icantly while the level of p p3-Alc2N+38 peptideconcentration strongly increased (Fig. 9B). Theseresults show that, like for APP, pyrazoles inducea shift in the cleavage pattern of Alcadein, another-secretase substrate. Similar effects were seenpreviously with aftin-5 and triazines [38]. Theseobservations further suggest that pyrazoles are morelikely to interact with -secretase than with theirsubstrates.

Fig. 6. Pattern of As produced by N2a-APP695 cells exposed to pyrazoles 18, 20, or 21. Cells were treated for 18 h with DMSO or 20 Mof each pyrazole. Cell supernatants were collected and analyzed as described. Quantification of all As in N2a-APP695 cells supernatants arepresented as percentage of total amyloids. Note the decrease in peptides A1-19, A1-37, A1-38, the increase in A1-40 and the appearanceof A1-42 and A1-43 in the supernatants of pyrazole-treated cells (these two peptides are undetectable in the supernatant of DMSO-treatedcells).


Fig. 7. Pyrazoles trigger enhanced production of A42 versusA40 in neurons differentiated from human induced pluripotentstem cells. Neurons were derived from iPSCs obtained fromhealthy donor (APP WT, white bars) or from an AD patient withAPP K724N mutation (grey bars). They were exposed for 24 hto DMSO (control), 100 M aftin-5 or the nine pyrazoles. Cellsupernatants were collected for extracellular A40 and A42 lev-els measurement by an ELISA assay. Levels are expressed asA42/A40 ratios ± SE of triplicate values. Horizontal dotted lineindicates level for basal A42/A40 ratios.

Proteomics study of fipronil’s effects

In order to understand the molecular mechanismsof action of pyrazoles, we decided to analyze proteinexpression of cells exposed to fipronil sulfone (20)and dipropetryn, two structurally unrelated productswhich, despite their unidentified targets, share theproperty of inducing A42 production. N2a-APPcells were exposed for 18 h to 20 M fipronil sul-fone (20), 100 M dipropetryn or 0.1% DMSO(control) and were processed for proteomics anal-ysis. Cell supernatant A42 levels were increasedas expected (data not shown). A total of 8,027 pro-teins were identified in the multiplex experiment (datanot shown). The expression of 1,634 and 1,638 pro-teins was modified, respectively, in fipronil sulfone(20)- and dipropetryn-treated cells compared to con-trol, DMSO-treated cells (Fig. 10A). Among these,261 proteins were shared by cells treated with bothproducts, out of which 178 were upregulated or down-regulated in the same direction for both products(Supplementary Table 4). DAVID analysis of these178 proteins showed that they gather in mitochondrialpathways, cell cycle control, AD (Fig. 10B).

Fipronil is metabolized to fipronil sulfone,a stable metabolite which accumulatesin adipose tissue and brain

We next investigated the pharmacokinetics andbiodistribution of fipronil (18) and its metabolites in

mice (Fig. 11) and rats (Supplementary Figure 2).Mice first received a single oral dose of fipronil (18)and blood, brain and adipose tissue were collected atvarious times during 72 h (Fig. 11). Plasma pharma-cokinetics showed rapid elimination of fipronil (18)and parallel appearance of fipronil sulfone (20) whichremained stable during the 72 h time-course (Supple-mentary Figure 1). No fipronil desulfinyl (21) wasdetected. Similarly, fipronil (18) transiently appearedin brain and adipose tissues, while fipronil sulfone(20) accumulated to a stable level in both tissues(Fig. 11A, B). Fipronil sulfone (20) reached a 5-6-fold higher concentration in adipose tissue comparedto brain. Given the metabolic stability of fipronil sul-fone (20), we next ran a two months duration studyfollowing a single oral administration of fipronil (18)(Fig. 12A). Fipronil sulfone (20) levels were deter-mined at various times up to 56 days after the singleadministration. The half-life of fipronil sulfone (20)was found to be 14 ± 3 days, 17 ± 2 days, and 26 ±3 days in plasma, brain, and adipose tissue, respec-tively (Fig. 11A). We next administered fipronil (18)daily, 5 days/week, for 3 weeks and measured fipronilsulfone (20) in plasma, brain and adipose tissue(Fig. 12B). This repeated oral dosing allowed themaintenance of a stable level of fipronil sulfone (20)in plasma, brain and adipose tissue (Fig. 12B). Phar-macokinetics and biodistribution of fipronil (18) andfipronil sulfone (20) were next studied in rats duringa 14 days period, following a single oral administra-tion. Results confirmed rapid metabolism of fipronil(18) to fipronil sulfone (20) (Supplementary Fig-ure 2A). Fipronil sulfone (20) was much more stableand showed extensive accumulation in adipose tis-sue (peak concentration 48 h after oral administrationof fipronil (18)) followed by slow release and/ormetabolism (Supplementary Figure 2B).

DISCUSSION

This study reports on the induction of A42/A43production, and the increased ratio of long versusshort As, in various cell cultures and by highlypurified -secretase following exposure to severalpyrazole insecticides. These results support previousresults obtained with various drugs (fenofibrate, cele-coxib, indomethacin, isoprenoids) [63], DAPT undercertain conditions [64, 65], steroids [66], ceramideanalogs [67], the peroxynitrite donor SIN-1 [68],Zinc [60], aftins [35–37] and several triazine herbi-cides [38]. Altogether these results have two mainimplications:


Fig. 8. Mass spectrometric analysis of the As generated in cell-free -secretase assays, in the presence of DMSO or fipronils 18, 20, or 21.A) The As generated by highly purified -secretase in the presence of 100 M of fipronil (18), fipronil sulfone (20) or fipronil desulfinyl(21) or DMSO (vehicle) were pooled from triplicates of the activity assays, immunoprecipitated overnight with the anti-A antibody 4G8and protein G and analyzed by MALDI-TOF in a reflectron mode. The As generated from the recombinant APP-C99 contain an N-terminalmethionine, which results in a mass shift of +149m/z when compared to endogenous A peptides. B) Note the increased A42/A40 ratiofor fipronils 18, 20, and 21, and the increased A43/A40 ratio for fipronil sulfone (20).

1) They support the idea that the HCE containsseveral compounds able to shift the cleavageof APP toward the production of long, toxic,aggregation-prone As, such as A42/A43,those which are classically associated with ADin humans and numerous animal models ofAD. These compounds, if proven to modifythis balance in animals (as suggested by thefirst preliminary results obtained with aftin-4[28] and celecoxib or FT-1 [63]) and in human,may contribute to the onset, development and/oracceleration of AD. We suggest that they be

collectively named “Alzheimerogens”, by anal-ogy with cancer-inducing carcinogens. Identi-fication of such compounds in our environmentappears to be a priority to develop a preventionstrategy.

2) Some of these compounds, particularly themetabolically stable, brain-permeable mole-cules, constitute new pharmacological researchtools to investigate the role of -secretase,its substrates and products, in the onset ofAD, in cell lines and animal models. In thiscontext, one of our objectives is, by analogy


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Fig. 9. Pyrazoles alter the cleavage pattern of Alcadein, leading to increased p3-Alc38 production. A) Immunoprecipitation/mass spec-trometry spectra of p3-Alc peptides produced by HEK cells expressing full length Alcadein exposed to various pyrazoles or DMSO(control). Cells were treated for 48 h with 100 M of each reagent and p3-Alc peptides were analyzed by MALDI-TOF/MS. A) Represen-tative profile for each product showing the the p3-Alc2N+34, p3-Alc2N+35, p3-Alc2N+36, p3-Alc2N+37 and p3-Alc2N+38 peaks.B) Relative quantification of p3-Alc peptides produced by cells exposed to DMSO (control), fipronil (18), fipronil sulfone (20) or fipronildesulfinyl (21). Levels of each peptide are presented as ratios of p3-Alc2N+38 versus p3-Alc2N+35 (average ± SE of triplicate values).

with MPTP (1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine)-induced Parkinsonism, to de-velop a pesticide-induced animal model of AD,which would allow the study of AD onset inwild-type (WT) animals. This is exemplifiedby the orally available fipronil (18) which israpidly metabolized to fipronil sulfone (20), avery stable compound which accumulates inadipose tissue, crosses the blood-brain barrierand reaches stable levels in the brain (Figs. 11and 12; Supplementary Figure 2). We are cur-rently running long-term exposure experimentswith fipronil (18) administered orally in WTmice and Tg2576 mice (overexpressing isoform695 of human APP with the Swedish mutation(KM670/671NL)).

Mechanism of action of fipronil and otherpyrazoles

As also observed with aftins [36] and triazines [38],pyrazoles insecticides, including fipronil derivatives,show a structure/activity relationship (Supplemen-tary Table 1) in their effects on A production – some

are active, others are inactive-, showing that pyra-zole insecticides devoid of A42 induction propertiescan be developed. The limited number of pyrazolesand their wide structural diversity precludes a clearidentification of key structural features interactingwith the unknown target leading to A42 produc-tion. Unspecific, detergent, hydrophobic, membrane,or protein structure disrupting actions are unlikely toaccount for the effects of pyrazoles. Several argu-ments support an effect on -secretase and/or itsmicro-environment: 1) induction of A productionby pyrazoles is inhibited by -secretase inhibitors ormodulators (Fig. 4); 2) pyrazoles also affect the cleav-age of another substrate of -secretase, alcadeins(Fig. 9); and 3) pyrazoles modify the cleavage patternof APP by highly purified -secretase.

Our cell-free -secretase activity assays per-formed with highly purified enzyme and substrate(Fig. 8) show that fipronil (18), Fipronil sulfone(20), and fipronil desulfinyl (21) clearly increased theA42/A40 ratio. Remarkably, Fipronil sulfone (20)additionally increased by ∼2-fold the A43/A40ratio. At the molecular level, the -secretase complexcleaves the APP-C99 substrate within the transmem-


Fig. 10. Proteomics and phosphoproteomics analysis of N2a-APP695 cells exposed to fipronil sulfone (20) and dipropetryn. A) N2a-APP695 cells were exposed for 18 h to 20 M fipronil sulfone (20), 100 M dipropetryn or DMSO. This led to increased extracellular A42expression (3.58 ± 0.05 and 7.87 ± 0.81 fold change for fipronil sulfone (20) and dipropetryn, respectively, compared to DMSO-treatedcells). Up/downregulated proteins in fipronil sulfone- (1634) and dipropetryn- (1638) treated cells versus DMSO-treated cells were identifiedand compared. Among the 261 common proteins, 178 proteins (listed in Supplementary Table 1) were either upregulated by both treatmentsor downregulated by both treatments. B) DAVID analysis of the 178 proteins common to fipronil sulfone (20) and dipropetryn treatments.Data are selected with p-value < 0.01 and FDR < 0.05. FDR, False Discovery Rate.

brane domain (TMD), first at the -site located atthe intracellular interface, to generate two differentC-terminal APP intracellular domains (AICDs) of50 or 51 aa length [69]. To generate As of differ-ent lengths, -secretase next cleaves the N-terminusof the TMD every -helical turn (correspondingto 3-4 amino acids), starting from -sites, follow-ing two production lines: the AICD51/A48 pathway(A48⇒A45⇒A42⇒A38) and the AICD50/A49 pathway (A49⇒A46⇒A43⇒A40) [69].Consistent with a direct effect of pyrazoles onAPP-C99 processing, our results support an alteredA48 pathway leading to an increased A42 pro-duction. Surprisingly, our study further revealed thatfipronil sulfone (20) unambiguously and drasticallyincreases the A43/A40 ratio (Fig. 8). This observa-tion is important since A43 is known to be highlyamyloidogenic and a main species involved in theformation of senile plaques [6, 7, 70]. As of today,A43 is known to be a direct precursor of A40 [69].Therefore, the increased A43 production in the cell-free assay suggests that fipronil sulfone (20) blocksthe processing of APP-C99 at position 43 in the A49pathway, an event which is likely associated withreduced A40 production.

Further quantitative experiments are required toexplain the increased A42/A40 or A43/A40

ratios, caused either by increased A42 or A43production, a reduced A40 production or both. Inany case, an altered A profile in the presence ofpyrazole insecticides strongly suggests that the com-pounds directly affect the positioning of the APPsubstrate in the lipid-bilayer, as previously observedfor compounds known as inverse -secretasemodulators (iGSM) [64]. Similarly to what weobserved with fipronil (18) and fipronil desulfinyl(21), the iGSM nonsteroidal anti-inflammatory drugs(NSAIDs) Fenofibrate and Celecoxib have indeedbeen reported to be A42-raising and A38-loweringcompounds [63]. Interestingly, Celecoxib is a pyra-zole. The pyrazole scaffold has in fact been usedextensively in therapeutic drug development (reviewin [71]). In contrast to iGSMs, some NSAID-based-secretase modulators (GSM), including sulindac-sulfide, lower A42 and increase A38 productions[63]. GSMs are of great pharmaceutical interest andare currently under clinical evaluation for their abilityto reduce the production of the amyloidogenic A42and A43 peptides. At the same time the potentialrisk from A42 and A43 producing iGSMs needscareful evaluation since some of these compoundsare FDA-approved and widely used as NSAIDs. Atthe molecular level, it has further been shown thatresidues 29–36 (GAIIGLMV) of the A motif in


Fig. 11. Short-term (72 hrs) time-course of fipronil (18) and fipronil sulfone (20) bioaccumulation in brain (A) and epididymal adiposetissue (B) following a single oral administration of fipronil (18) to mice. Fipronil (18) (10 mg/kg) was administrated by oral gavage at time 0.Animals were sacrificed at various times and plasma, epididymal adipose tissue and brain were collected. Fipronil (18) and fipronil sulfone(20) levels were quantified by LC-MS/MS. Concentrations are expressed as g/brain or g/epididymal adipose tissue.

Fig. 12. Long-term (56 days) time-course of fipronil sulfone (20) production and accumulation following single (A) or repeated (B) oraladministrations of fipronil (18) to mice. A) Fipronil (18) (10 mg/kg) was administrated by oral gavage on day 0. Animals were sacrificed atvarious times and plasma, epididymal adipose tissue and brain were collected. Fipronil sulfone (20) levels were quantified by LC-MS/MS.B) Fipronil (18) (10 mg/kg) was administrated by oral gavage 5 days /week for 3 weeks (times of administration are indicated by black dots).Animals were sacrificed at various times and plasma, epididymal adipose tissue and brain were collected. Fipronil sulfone (20) levels werequantified by LC-MS/MS. Concentrations are expressed as g/brain, g/epididymal adipose tissue or g/mL plasma.

APP-C99 located at the TMD N-terminus representthe binding site of both A42-lowering GSMs andA42-raising iGSMs [63]. This raises the questionwhether the same binding site could be involved in thepyrazole-induced A42/A40 and A43/A40 ratioincrease. Interestingly, we recently found that zinccan drastically raise the A43/A40 ratio by influ-

encing the positioning of the APP-C99 TMD throughbinding to Lysine 28, which is located at the sur-face of the membrane and is involved in anchoringAPP-C99 in the lipid bilayer [69]. This observationfurther supports the possibility that Lys28 and moreglobally residues 29–36 in APP-C99 are involved inthe pyrazole-induced A43/A40 increase. Finally,


novel GSMs have recently been reported that directlybind on PS1 [72]. Binding of these compounds toPS1 induced a conformational change in the enzyme-substrate interaction that modulated the proteaseactivity and consequently the A profile. Thus, onecannot exclude that fipronil (18), fipronil sulfone(20), or fipronil desulfinyl (21) also modulate APP-processing by directly binding to the -secretasecomplex. Further experiments are needed to deci-pher the mode of action of these compounds (seeSupplementary Figure 3 for working model).

Fipronil exposure and consequences

Fipronil has been developed a selective inhibitorof insect GABA-gated chloride channels (review in[44]). It is one of the most widely used pyrazoleinsecticides in agriculture (seed, culture, and croptreatment), in urban pest control (coackroaches, ter-mites, wasps, flies, and ants management in andaround buildings), and in veterinary applications(fleas and ticks control for pets and cattle). Fiproniland its metabolites are therefore widely found [73]in urban soil, dust, wastewater, and residential runoff[74], in rural soil, rivers, and atmosphere [46, 73,75, 76], but also in various tissues of pet, farmland,and wild-life animals [75, 77, 78] (only a very smallnumber of references are cited). Although fipronil(18) is relatively resistant to degradation, it is mostlyoxidized to fipronil sulfone (20), reduced to fipronilsulfide (22), photodegradated to fipronil desulfinyl(21), hydrolyzed to fipronil amide (23, 28). Manyother metabolites have been described ([45–47, 73],review in [39]). Although fipronil shows low affinityfor mammalian GABA-gated chloride channels com-pared to those of insects, toxicity studies carried outwith fipronil and its metabolites in various vertebrates[39, 40, 43, 44, 77] as well as studies performed withmammalian cells studies [79–83] cast some doubtsabout the safety of fipronil to humans (see Supple-mentary Table 5). This concern is amplified by 1) theexistence of numerous metabolites which are poorlycharacterized in terms of toxicity, 2) their long persis-tence in the environment (soil, water, sediment, andatmosphere), 3) their accumulation in tissues, espe-cially adipose tissue (Figs. 11 and 12), 4) the potencyof fipronil sulfone on mammalian GABA-receptor[84], and 5) the fact that fipronil and its metabolitesessentially meet all druggability criteria of Lipinski’srule of Five (Supplementary Table 6). In addi-tion, fipronil (along with neonicotinoids) is a majorworldwide concern for the survival of pollinators

[85–89]. In mammals, fipronil shows oxidative stress,neurotoxic, thyroid, endocrine, lung inflammation,blood pressure, and reproductive effects (review in[44]) at high doses. In humans, fipronil triggers mildnervous troubles as seen in acute intoxication cases[90, 91] and apparently impacted thyroid functionsof workers following occupational exposure [92].

As shown in Figs. 11 and 12, fipronil (18) is rapidlymetabolized to a very stable product, fipronil sulfone(20), which readily accumulates in fat tissue. Table 1reviews plasma and tissue levels of fipronil sulfone(20) reached experimentally in mice and rat or acci-dentally in humans (our data + literature data). Thehighest plasma levels reached experimentally dur-ing these relatively short exposures (3.7 g/mL, i.e.,8.2 M) are not very far from those which induceA42 and A43 production. Adipose tissue and brainlevels reached experimentally are probably higher.72 h after a single oral administration of 14C-fipronilto rats, about 50% and 2% of the radioactivity (essen-tially fipronil sulfone) were found in adipose tissueand brain, respectively [93]. Fipronil sulfone is alsothe primary metabolite produced in human via hep-atocyte cytochrome P-450 oxydation [90, 92–95].Fipronil sulfone was detected in about 25% of 96human serum samples (0.1–39 ng/mL which corre-sponds to 0.22–86 nM) [45]. These samples wereobtained from individuals with no known fipronilexposure. However, fipronil applied to the fur of petsis easily and rapidly transferred to humans [96, 97].Fipronil and fipronil sulfone levels were measured inurine and blood samples of 159 workers in a factorymanufacturing fipronil-containing veterinary prod-ucts [92]: 33 and 155 workers had detectable serumfipronil and fipronil sulfone (0.37–42.45 ng/mL, i.e.,0.82–9.32 nM), respectively. Incidentally, fipronilhas recently been shown to promote adipogenesis[98] and adiposity/obesity has been suggested as arisk factor for AD [99, 100]. Adipose tissue maythus act as a trap and storage tissue for lipophilic,A42/A43 inducers like fipronil, other pyrazoles,other pesticides. The persistence of fipronil sulfone,its accumulation in adipose tissue, and its abilityto enter the brain raises concerns on its possiblelong-term effects on the central nervous system, inparticular on its possible effects on the productionof toxic, aggregation-prone amyloidogenic A42 andA43, as shown in this article.

In conclusion, we have added some pyrazole insec-ticides, especially the main metabolite of fipronil, inthe growing list of HCE products which are able toalter the specificity of A production. Our hypothesis


Table 1Concentrations of fipronil sulfone reached in mammals following fipronil administration (oral, ip, iv, general exposure). Fipronil sulfone(20) levels are expressed either in g/mL or in g/g. A 4.53 g/mL concentration is equivalent to a concentration of 10 M. bw, body weight

Species/tissue Exposure conditions Fipronil sulfone (20) levels reached Refs.

Mouseplasma 10 mg/kg bw, oral, single

administration1–1.1 g/mL Fig. 11, 12A

plasma 10 mg/kg bw, oral, 5 days/week/3 weeks

3.7 g/mL Fig. 12B

plasma 2.5 or 10 mg/kg bw, oral,1 day/week/2 weeks

0.029 or 0.210 g/mL SK, unpubl.

plasma 2.5 or 10 mg/kg bw, oral,3 days/week/2 weeks

0.211 or 0.662 g/mL SK, unpubl.

adipose tissue 2 mg/kg bw, ip, daily/6 days 22–24 ppm [102]adipose tissue 10 mg/kg bw, oral, single

administration4.3–4.9 g/epididymal adipose pad Fig. 11, 12A

adipose tissue 10 mg/kg bw, oral, 5 days/ week/3 weeks

13.5 g/epididymal adipose pad Fig. 12B

adipose tissue 2.5 or 10 mg/kg bw, oral, 1day/week/2 weeks

0.730 or 5.276 g/g SK, unpubl.

adipose tissue 2.5 or 10 mg/kg bw, oral,3 days/week/2 weeks


brain 40 mg/kg bw, ip, 6 or 20 min, singleadministration

19 or 32 ppm (6 and 20 min,respectively)

[103]

brain 10 mg/kg bw, oral, singleadministration

0.71–0.75 g/brain Fig. 11, 12A

brain 10 mg/kg bw, oral, 5 days/week/3 weeks

2.7 g/brain Fig. 12B

brain 2.5 or 10 mg/kg bw, oral,1 day/week/2 weeks


brain 2.5 or 10 mg/kg bw, oral,3 days/week/2 weeks


Ratplasma 10 mg/kg bw, oral, single

administration1.4 g/mL Suppl. Fig. 1

plasma 5 or 10 mg/kg bw, oral, daily/2 weeks 2.4 or 3.6 g/mL [104]plasma 30 mg Regent ®/kg bw, oral,

daily/15 days0.46 g/mL [105]

plasma 4 g/kg bw, oral, 3X/ week/90 days 0.008–0.39 ng/mL [106]plasma 3.4 (po) or 6.9 (iv) mole/kg bw,

single administration0.2 (po) or 0.25 (iv) g/mL [107]

plasma 3.4 mole/kg bw, oral, daily/14 days 1.5 g/mL [107]plasma 3 mg/kg bw, oral, daily/13 days 0.043 – 2.8 g/mL [108]adipose tissue 10 mg/kg bw, oral, single

administration152.3 g/g Suppl. Fig. 3

adipose tissue 10 mg/kg bw, oral, 72 h,administration

47.87 g fipronil equivalent (>90%fipronil sulfone)/g wet weight

[93]

brain 10 mg/kg bw, oral, single dose 3.74 g/g Suppl. Fig. 2urine 5 or 10 mg/kg bw, oral, daily/2 weeks

or single dose0.023–0.026 g/mL [47]

urine 5 or 10 mg/kg bw, oral, daily/2 weeks 0.024 or 0.032 g/mL [104]urine 4 g/kg bw, oral, 3X/week/90 days 0.009–4.27 ng/mL [106]hair 4 g/kg bw, oral, 3X/week/90 days 4.58–306 pg/mg [106]

Humanplasma no known fipronil exposure

(96 plasma samples)0.1–3.9 ng/mL; detected in 25% of

the samples.[104]

plasma conducted on 159 workers froma factory manufacturingfipronil-containing veterinaryproducts in France

mean: 7.79 ng/mL (range:0.37–42.45 ng/mL) detected in 155out of 159 workers.

[92]

plasma 6 cases of self-poisoning withRegent ®

maximum fipronil + fipronil sulfonelevel measured was 3.74 g/mL

[109]

urine no known fipronil exposure (84 urinesamples)

undetectable [104]


is that these products may, on a long-term, cumu-lative and additive basis, possibly in parallel withmicroorganisms [101] and microorganisms-derivedproducts, alter the A production pattern sufficientlyto lead to AD pathogenesis, and thus qualify as poten-tial “Alzheimerogens”. Gradual accumulation in andchronic release from adipose tissue and transfer to thebrain may contribute to the onset, development, andacceleration of sporadic AD.

ACKNOWLEDGMENTS

We are grateful to Laetitia Bailly (LaboratoireCobra, University of Rouen) for purifying thetwo fipronil enantiomers. We are grateful to JasnaPadovan and Zeljko Javorscak (Fidelta) for the ratPK studies, to Rebecca L. McMahen, Mark J. Stry-nar for providing fipronil sulfone chloramine. Thiswork was supported by ‘Fonds Unique Intermin-isteriel’ PHARMASEA/TRIAD projects, « Agencenationale de securite sanitaire, de l’alimentation, del’environnement et du travail » (ANSES), Fonda-tion “Jerome Lejeune” and CRITT-Sante Bretagne/FEDER (LM). This research was also partly sup-ported by an FP7-KBBE-2012 grant (BlueGenics)to LM. MC is CIFRE/ManRos Therapeutics PhDfellowship recipient. KB is a Torsten Soderberg Pro-fessor. HZ is a Wallenberg Academy Fellow. Thiswork was supported in part by Grant-in-aid for Sci-entific Research 26293010 (TS) and 24790062 (SH)from the Ministry of Education, Culture, Sports, Sci-ence and Technology in Japan. HG, JF, and PCF weresupported by the Fondation Eclosion. Partial sup-port was provided by NIEH S/ROI ES002710, NIEHS Superfund Research Program P42 ES004699 andNIH/454 NS079202.

Authors’ disclosures available online (https://www.j-alz.com/manuscript-disclosures/17-0875r1).

SUPPLEMENTARY MATERIAL

The supplementary material is available in theelectronic version of this article: http://dx.doi.org/10.3233/JAD-170875.

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Journal of Alzheimer’s Disease 62 (2018) 1663–1681 DOI 10