Carbofuran affects cellular autophagy and developmental senescence through the impairment of Nrf2 signalling

Abstract Carbofuran is a broad‐spectrum synthetic pesticide. Its exposure to non‐target mammals affects the biological system through the induction of oxidative stress. Since oxidative stress is a major contributing factor to cellular autophagy and senescence, our present investigation determined the impacts of carbofuran‐induced oxidative stress on cellular autophagy and senescence. A transmembrane protein, Spinster homolog 1 (Spns1), is involved in autophagic lysosomal metabolism. Its mutation accelerates the cellular senescence and shortens the lifespan. Using a transgenic zebrafish line, expressing fluorescent microtubules‐associated protein 1 light chain 3 (EGFP‐LC3) at the membrane of the autophagosome, we found that carbofuran affects autophagic lysosomal biogenesis in wild‐type zebrafish and exacerbates autophagic defect in spns1‐mutant zebrafish. In real‐time mortality study, carbofuran has shortened the lifespan of wild‐type fish. Nrf2 is a stress‐responsive transcription factor that regulates the expression of antioxidant genes (such as gstp1) in the prevention of oxidative stress‐mediated cellular damage. To assess the effect of carbofuran on Nrf2 signalling, we established a dual‐monitoring transgenic zebrafish line, expressing gstp1 promoter‐driven EGFP and mCherry‐tagged Neh2 domain of Nrf2. Our results suggested that the exposure of carbofuran has down‐regulated both Nrf2 and Gstp1 expressions. Overall, carbofuran affects cellular autophagy and accelerates senescence by enervating the Nrf2 signalling.


| INTRODUC TI ON
Senescence is a cellular response towards various stresses, characterized by the permanent proliferative arrest on cells. 1 Pharmacological and/or genetic ablation of senescence/senescent cells improve health outcomes and extend the lifespan. 2 Autophagy is a lysosome-mediated cellular catabolic process, implicated with cellular senescence. In regulating the cellular homeostasis, autophagy usually suppresses cellular senescence by eliminating damaged cytoplasmic organelles and macromolecules. 3 In contrast, autophagy can also be activated by cellular senescence and up-regulated in senescent cells. 3  and insufficient growth factors. 4 At the beginning of the autophagic process, unnecessary or dysfunctional cytoplasmic constituents form double-membraned vesicles (known as autophagosomes), which subsequently form autolysosomes via fusion to lysosomes.
In autolysosome, the contents of autophagosomes are degraded by lysosomal enzymes and recycled. 5 In vertebrates, Spinster homolog 1 (Spns1), a lysosomal efflux permease, functions at the terminal stage of autophagy. 6,7 Mutation of the spns1 gene in zebrafish (Danio rerio) leads to embryonic senescence and premature ageing. 7,8 Senescenceassociated β-galactosidase (SAβ-gal) is a biomarker to monitor cellular senescence in vitro as well as organismal senescence in vivo. 7 While higher activity of the lysosomal β-galactosidase enzyme can be found at lower pH, certainly consistent activity can be detected at pH 6 after treatment with various stressors in zebrafish. Therefore, appropriately conditioned β-galactosidase activity at pH 6 has been utilized to detect senescent cells as well as to identify genes triggering cellular/embryonic senescence. 9 In senescent cells, it has also been reported that the accumulation of non-degradable cellular organelles and macromolecules leads to an increase in lysosomal content. 9 Carbofuran, chemically known as 2,3-dihydro-2,2-dimethyl-7 -benzofuranyl methyl-carbamate, commonly used in agricultural practices to control both leaf-eating and soil-dwelling insects. 10,11 For example, in Bangladesh, about 37% of the total sold pesticides during 2007 was carbofuran. 12 Carbofuran is non-specific in its activity, exerts a lethal effect towards a wide range of pests. 12 Its exposure through contaminated air, water, foods and vegetables may exert noxious effects on non-target animals including humans. 11 Carbofuran increases the generation of reactive oxygen species (ROS) in the brain through the inhibition of glutathione-Stransferase, catalase and superoxide dismutase. 13,14 It has also been demonstrated that carbofuran arrests the proliferation of neuronal cells through the induction of oxidative stress. 10 Excessive ROS generation in the biological system affects cellular fates including apoptosis, autophagy and senescence. 15 ROS via oxidative stress increase the incidences of age-related diseases such as atherosclerosis, arthritis and Alzheimer's disease as well as strongly influence the biological ageing process. 2,15 Recently, we reported that carbofuran hastens the developmental senescence in spns1-mutant zebrafish and affects their lifespan. 16 The current study extends the report in wild zebrafish, whereas we found carbofuran affects cellular autophagy and accelerates developmental senescence in both wild and spns1-mutant zebrafish.
In the biological system, the oxidative stress condition is due to the imbalance between ROS generation and antioxidative defence mechanisms of the body. 10 The nuclear factor erythroid 2-related factor 2 (Nrf2) is an essential transcription factor, which promotes the transcription of a number of antioxidant elementbearing genes. 17

| Zebrafish husbandry and maintenance
Adult wild-type, mutant and transgenic zebrafish were maintained at 25°C in a 14-h light/10-h dark cycle. They were fed with brine shrimp once daily. Flake food was also given once daily. A continuously circulating system was used to maintain zebrafish, F I G U R E 1 Carbofuran exacerbates cellular senescence and shortens lifespan. (A) Based on the magnitude of opaqueness, the yolk opaqueness was sorted as partially opaque (marked by yellow colour) and mostly opaque (marked by red colour). Fish (wild type; spns1 +/+ or heterozygote; spns1 ± ) did not show opaqueness phenotype was marked by black colour. (B) The effect of carbofuran exposure on senescence phenotype (yolk opacity) of spns1-mutant zebrafish. In the cases of carbofuran-treated spns1-mutant zebrafish, both partially and mostly opaque phenotypes were found at earlier times than mutant zebrafish of the control group (egg water treatment). In addition, carbofuran treatment leads mutant zebrafish to die at quicker times than fish of the control group. (C) Survival (in hours) of spns1-mutant embryos has been shortened (P = .0003) upon exposure to carbofuran. (D) Survival (in months) of wild zebrafish was shortened (P < .0007) upon exposure to carbofuran. (E) Carbofuran accelerates SAβ-gal activity in both wild-type (spns1 +/+ ) and spns1-mutant (spns1 −/− ) zebrafish. Carbofuran did not affect morphological phenotype (MP) of wild fish but affected yolk opaqueness of spns1-mutant fish (black arrowhead in the first row). SAβ-gal-stained fish have shown in the second row. The cellular SAβ-gal staining at dorsal to the eye has shown in the third row. (F) Quantification of dark blue particles of SAβ-gal staining of whole fish. The scale bar is 250 μm (stereo microscopic images) and 10 μm (confocal microscopic images). The number of fish was 10 (n = 10). Data are presented as mean ± SE. *P ≤ .05; ***P ≤ .005 which replace the water of each tank within 10-15 minutes of interval. Ultraviolet light was used to disinfect the water of the circulating system. Embryos were raised at 28.5°C until reaching the appropriate stage. Morphological features of embryos and hours of post-fertilization (hpf) of Kimmel et al (1995) were used to fix the developmental stages of embryos. 22 Where necessary, 0.003% PTU (1-phenyl-2-thiourea) was used to prevent pigment development.

| Chemicals and chemical treatment
Carbofuran was collected from Merck, Germany (SKU 32056).

| Senescence-associated β-gal (SAβ-gal) assay
Senescence-associated beta-galactosidase (SAβ-gal) is a biomarker for senescence induction, commonly used in both cellular and organismal senescence and ageing studies. 23  with senescence-associated beta-galactosidase, which signposted with blue staining. The staining intensity was quantified by pixel analysis, using Adobe Photoshop CS software as described previously. 8 Due to nearly similar intense blue staining at yolk and yolk extension regions of all embryos, these regions were excluded during the quantification.

| Generation of transgenic zebrafish
The pT2-gstp1-EGFP plasmid containing a 3.5-kb gene regulatory region of gstp1 was kindly donated by Dr Kobayashi Lab, Tsukuba, Japan. 24 In pT2-Neh2-mCherry plasmid, mCherry was tagged to founder zebrafish (F0) as previously described. 25 Positive founders (F0) were in-crossed to obtain F1 embryos, which were raised to adulthood. Embryos resulting from F1 and/or later generations were used in experiments.

| Survival analysis
Homozygous spns1 −/− embryos were sorted based on the yolk opaqueness phenotype and exposed to carbofuran (100 µM) from 36 hpf to until the death of all embryos. Embryos were monitored and counted under a stereo-microscope at each 6-hour interval.
Whenever a dead embryo was observed, it has been removed from the treatment preparation. In the case of real-time mortality study, adult zebrafish of wild-type background has been continuously exposed to a low dose of carbofuran (5 µM) through fish water, since 3 months of post-fertilization. Survival data were presented through the Kaplan-Meier survival curve.

| LysoTracker red staining
EGFP-tagged microtubules-associated protein 1 light chain 3 (EGFP-LC3) expresses at the membrane of autophagosomes, which is widely used as a biomarker to estimate the progress in the autophagic process. 30,31 In our estimation, we used a trans-

| RT-PCR analysis
The gstp1 mRNA expression level was estimated using RT-PCR

| Microscopy and imaging
Before imaging, embryos were anaesthetized using 0.16 mg/mL tricaine (Sigma, A5040). Imaging was carried out with FluoView 1000 Fluorescence intensities of captured images were quantified using Adobe Photoshop software.

| Statistical analysis
Statistical analyses were carried out using Statistical Package for the Social Sciences (SPSS) software version 20.0. The software was used to generate each of the graphs. Data were expressed as mean ± SE.
Comparisons between different animal groups were made with Student's t test.

F I G U R E 4 Carbofuran down-regulates
Gstp1 (This is fine, no change!) expression and up-regulates autophagosomal LC3 expression. (A) The gross expressions of gstp1-EGFP and mCherry-LC3 in transgenic zebrafish under macromicroscope. Transgenic zebrafish were exposed to carbofuran for 12 hours (48 to 60 hpf). Carbofuran treatment significantly declined gstp1-EGFP expression and significantly increased autophagosomal mCherry-LC3 expression throughout the fish. Scale bar: 250 μm.

| Carbofuran accelerates developmental senescence and affects biological ageing
Embryos, lacking the spns1 gene, can be morphologically identified by yolk opacity, smaller eye and loss of yolk extension. 7,8 Such embryos cannot survive, but die within 3-6 days of post-fertilization.
We used heterozygous spns1 ± fish of males and females for crossmating. Among resultant embryos, homozygous (spns1 −/− ) mutant embryos were identified by the representative phenotypes such as yolk opacity ( Figure 1A). It was estimated that about 25% of resultant embryos were developed with the yolk-opaque phenotype, due to Spns1 deficiency.
Of note, heterozygous spns1 ± fish, as well as normal fish, do not show the yolk-opaque phenotype, 7 and in almost all cases, yolk opaqueness of homozygous spns1 −/− fish originated at the ventral tip of yolk extension and gradually enlarged towards other parts of yolk extension and yolk ( Figure 1A). Based on the magnitudes of opaqueness, the yolk opacities were classified as partially opaque and mostly opaque ( Figure 1A). Carbofuran exposure to spns1mutant zebrafish induced yolk-opaque phenotypes at earlier times than spns1-mutant fish of the control group. Moreover, carbofurantreated mostly opaque fish have died faster than the fish of the control group ( Figure 1B).
The loss of the spns1 gene induces organismal and cellular senescence, which can be determined by the robust up-regulation of SAβ-gal activity. 8 The strong increment of SAβ-gal activity in spns1-mutant zebrafish has also been confirmed in our current study ( Figure 1E, F). Further, we found that carbofuran exposure to wildtype zebrafish has increased SAβ-gal activity. Although the increment was relatively weak, nevertheless the activity was significantly In survival analysis, we reconfirmed that carbofuran exposure significantly shortened the lifespan of spns1-mutant zebrafish ( Figure 1E). 16 Additionally, using a real-time survival study, we found carbofuran significantly abated the survival lengths of wild zebrafish ( Figure 1F), suggesting carbofuran affects lifespan of both wild and spns1 −/− mutant fishes.

| Carbofuran affects autophagy process in both wild and spns1-mutant fishes
The loss of spns1 function causes the accumulation of autolysosome in the cell, which was recognized through the accumulation of enlarged lysosomal and autophagosomal positive structures as well as their co-expression. 6 Furthermore, in our previous study, it has been demonstrated that Spns1 plays a pivotal role in the regulation of cellular autophagy in zebrafish. 31 Thus, carbofuran

| Carbofuran interferes Nrf2-ARE transcriptional regulatory elements
The stress-or chemical-mediated activation of the Nrf2 pathway displaces Nrf2 component from the Keap1-binding site to the nucleus, where it binds to ARE and regulates the expression of genes of the defence system including gstp1. 33  of Nrf2) and gstp1-driven EGFP expressions ( Figure 3A). The transgenic zebrafish embryos were exposed to carbofuran through egg water since 56 hpf and monitored under a fluorescence microscope.

| Differential effects of carbofuran on Nrf2 signalling are independent of its effect on autophagy progress
Under oxidative stress, ROS generated in mitochondria are essential mediators of cellular autophagy. 34 It has been demonstrated that carbofuran induces oxidative stress in mammals through the up-regulation of ROS generation. 14 Figure 4B), where an accumulation of autophagosomal mCherry-LC3 puncta was observed (as a hallmark of autophagy induction). On the other hand, embryos exposed to carbofuran for a short duration (4 hours; short-term exposure) from 56 hpf (by the endpoint of 60 hpf) showed an opposite pattern of expression for gstp1-EGFP; that is the up-regulation of the potential Gstp1 activity ( Figure 5A,B). However, autophagosomal LC3 expression was still increased after the short duration of carbofuran exposure likewise the long duration of carbofuran exposure. The distinction in Gstp1 activity upon long and short intervals of carbofuran exposure was further confirmed by RT-PCR analysis ( Figure 6A,B). Overall, Gstp1 activity was initially up-regulated by carbofuran exposure, but subsequently down-regulated upon persistent continuous exposure, whereas carbofuran-induced autophagosomal puncta seemed constitutive and were observed under both long and short terms of carbofuran exposure.

| D ISCUSS I ON
Among carbamate pesticides, carbofuran is one of the most widely used pesticides in agriculture and forestry, due to its noxious effects against a large number of insects. 35 Its environmental exposure to humans leads to toxic effects such as spermatozoal DNA damage and retinal degeneration. 36 Studies with rats have suggested that carbofuran induces oxidative stress in the nervous system and skeletal muscle by disrupting pro-oxidant/antioxidant balance. 37 Carbofuran also induces oxidative stress in the liver through increasing lipid peroxidation and decreasing hepatic superoxide dismutase. 38 A basal level of ROS generation is required for cell survival. 39 At low to the moderate extent of oxidative stresses, cellular protection against oxidative stress is carried out by Nrf2-mediated transactivation of ARE-targeted genes as well as using cellular adaptation via epigenetic modification such as DNA methylation and/or histone modification ( Figure 7). 39 Persistent oxidative stress may cause cellular senescence. 40 In the present investigation, we revealed carbofuran  47 It has been reported that carbofuran increases ROS and free-radical generation via the impairment of mitochondrial electron transport chain. 13 The subchronic carbofuran exposure increases around 44% of mitochondrial lipid peroxidation. Carbofuran induces an impairment of the electron transport chain in the mitochondria, 13 which might be a stimulating factor in the autophagy process. Autophagy progress was determined by autophagosomal and lysosomal biogenesis, which were up-regulated by carbofuran exposure to both wild-type and spns1mutant zebrafish. As autophagy can also be triggered by cellular senescence, 3 therefore, acceleration of senescence by carbofuran exposure may additionally contribute to the increment in autophagosomal and lysosomal biogenesis. The size of both autophagosomal and lysosomal puncta was increased by carbofuran exposure, and those puncta were sustainably colocalized, which suggests an autolysosomal accumulation, due to their improper biogenesis.
The activation of Nrf2 signalling is crucial for cellular adaptive and protective responses to oxidative stress. 49  Alzheimer's and Parkinson's disease. 54 In elderly people, circulating insulin-like growth factor-1 (IGF-1) is down-regulated, which leads to age-associated vascular diseases, including atherosclerosis. 55 In mice, it was demonstrated that reduced circulating IGF-1 declines the Nrf2 signalling. 56 In Alzheimer's (AD) and Parkinson's diseases, the translocation of Nrf2 into the nucleus is impaired, which decreases cellular protection under oxidative stress. 57 Cellular senescence and ageing are implicated with each other. A rapamycin-insensitive protein complex, mTORC2, facilitates senescence in vascular endothelial cells by reducing Nrf2 transcription. 58 The impairment may also be associated with the neurodegenerative effect of carbofuran as neuronal degeneration inhibits Nrf2 translocation into the nucleus. 57 Since both short and persistent exposure conditions of carbofuran up-regulated autophagosomal LC3 expression, initial up-regulation is due to carbofuran-induced ROS generation, and subsequent up-regulation might be because of carbofuran-induced cellular senescence and neurodegeneration. All these conditions (ROS generation, cellular senescence and neurodegeneration) are effective to stimulate autophagosome formation. 3,47,59 While we found an initial phase of Nrf2 activation by carbofuran, overall, our data suggested that carbofuran persuades impaired autolysosomal accumulation as well as accelerates senescence via subsequently possible dysfunction of the Nrf2 pathway. However, we still need further investigation on the mechanism underlying the role of carbofuran that affects the Nrf2 pathway by linking to Spns1.

CO N FLI C T O F I NTE R E S T
The authors have no competing interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the finding of this article are available from the corresponding author upon reasonable request.