Carbofuran accelerates the cellular senescence and declines the life span of spns1 mutant zebrafish

Carbofuran is a carbamate pesticide, widely used in agricultural practices to increase crop productivity. In mammals, carbofuran is known to cause several untoward effects, such as apoptosis in the hippocampal neuron, oxidative stress, loss of memory and chromosomal anomalies. Most of these effects are implicated with cellular senescence. Therefore, the present study aimed to determine the effect of carbofuran on cellular senescence and biological ageing. Spinster homolog 1 (Spns1) is a transmembrane transporter, regulates autolysosomal biogenesis and plays a role in cellular senescence and survival. Using senescence‐associated β‐galactosidase staining, we found that carbofuran accelerates the cellular senescence in spns1 mutant zebrafish. The yolk opaqueness, a premature ageing phenotype in zebrafish embryos, was accelerated by carbofuran treatment. In the survival study, carbofuran shortened the life span of spns1 mutant zebrafish. Autophagy is the cellular lysosomal degradation, usually up‐regulated in the senescent cells. To know the impact of carbofuran exposure on autophagy progress, we established a double‐transgenic zebrafish line, harbouring EGFP‐tagged LC3‐II and mCherry‐tagged Lamp1 on spns1 mutant background, whereas we found, carbofuran exposure synergistically accelerates autolysosome formation with insufficient lysosome‐mediated degradation. Our data collectively suggest that carbofuran exposure synergistically accelerates the cellular senescence and affects biological ageing in spns1 defective animals.

medium to strong acidic pH, the least activity that can be found at pH 6, which typically uses to examine the senescent and ageing cells. 6 On the other hand, autophagy is a cellular lysosomal degradation process, essential to regulate energy homeostasis through recycling unnecessary or dysfunctional cytoplasmic constituents. 7 In normal cells, autophagy occurs at low baseline altitudes, which is elevated in the senescent cells. 8 Autophagy is initiated with the formation of the phagophore from a variety of sources, such as ER-mitochondrial junctions, ER-plasma membrane contact sites and ER-Golgi intermediate compartment. Then, phagophore wraps unnecessary or dysfunctional cytoplasmic constituents to build-up autophagosomes, which successively fuse with lysosomes to form autolysosomes. 9,10 Finally, through the utilization of lysosomal hydrolases, the contents of autophagosomes are degraded and recycled. The vacuolar H + -ATPase (V-ATPase) is a proton pump, which establishes and sustains an acidic condition within the lysosome. The V-ATPase promotes autophagosome-lysosome fusion and lysosome-mediated degradation of the contents of autophagosomes. 11 Lysosomal degradation yields are released into the cytosol through the lysosomal efflux transporter system. Spinster homolog 1 (Spns1) in vertebrates is known as Spinster (Spin) in flies, encodes a putative lysosomal efflux permease, whose amino acid sequence has resembled the amino acid sequence of lysosomal sugar carrier. 12 The spin mutation is associated with neurodegeneration, rejection behaviour against the opposite sex and shortened life span in flies. 13 In zebrafish, the loss of spns1 gene leads to the accumulation of opaque substances in the yolk, and yolk extension and reduces biological ageing. 12,14 Recently, we found that the concurrent disruption of the V-ATPase subunit gene, ATPase, H + transporting, lysosomal, V0 subunit Ca (atp6v0ca) in spns1 mutant fish synergistically induced cellular senescence and shorten the life span. 15 Carbofuran is a toxic carbamate pesticide, whose chemical name is 2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate. 16 It is widely used to control pests in agricultural fields, particularly in developing countries. 17,18 It has insecticidal, nematicidal and acaricidal activities. 19,20 In Bangladesh, 37% of the total sold pesticides during 2007 was carbofuran. 21 Humans, particularly in agricultural dependent countries, are often exposed to pesticides including carbofuran through contaminated water, air and vegetables. 22,23 The World Health Organization (WHO) classified carbofuran as a highly hazardous pesticide (category 1b). 24 Its toxicity to mammals leads to sperm abnormalities and chromosomal aberrations. 23 Carbofuran suppresses anti-oxidative enzymes in the body, such as glutathione-S-transferase and superoxide dismutase, and induces oxidative stress through the generation of reactive oxygen species (ROS). 25,26 It causes apoptosis of hippocampal neurons through inducing DNA fragmentation, 27 also causes Alzheimer's syndrome type pathology in the central nervous system. 28 Furthermore, carbofuran reduces ATP generation in neurons through the inhibition of glycolysis and Kreb's cycle. 29,30 The detrimental effects of carbofuran on the anti-oxidative defence system as well as on the nervous system suggest its implication in cellular senescence and biological ageing. However, the role of carbofuran on cellular senescence and/or biological ageing still remains obscure. In the present study, we found that carbofuran synergistically accelerates the cellular senescence and shortens the life span of spns1 mutant zebrafish. In addition, using a transgenic zebrafish line [Tg(EGFP-LC3); spns1 +/− ], expressing EGFP-tagged microtubule-associated protein 1 light chain 3-II (LC3-II) on autophagosome, it was revealed that carbofuran affects the autophagy process in spns1 mutant fish.

| Chemicals and reagents
Carbofuran of analytical grade was procured from Merck, Germany (SKU 32056). Its stock solution was prepared by solubilizing in DMSO, which was diluted in egg water to prepare the final solution for treatment. The solution to be used for the control group was prepared likely by diluting an equal volume of DMSO in egg water.

| Zebrafish husbandry
Adult zebrafish of both wild and mutant types were harboured under a 14-hour light/10-hour dark cycle at 25°C. Zebrafish embryos were maintained and raised at 28.5°C under the same light-dark cycle. After three weeks of embryonic development, all embryos were maintained under adult fish condition. Zebrafish were fed with both brine shrimp and flake food, each once daily. Using a circulation system, water of zebrafish tanks were replaced at each 10-15 minutes interval. Hours of post-fertilization (hpf) of Kimmel et al 31 was considered to determine the developmental stages of embryos.

| Generation of transgenic zebrafish
PT2-EGFP-LC3 and PT2-mCherry-Lamp1 plasmids were kindly donated by Dr Shuji Kishi Lab of the Scripps Research Institute, Florida, USA. Tol2 transposase mRNA was synthesized using SP6 RNA polymerase kit (Ambion, AM1340). Collected plasmids concurrently with Tol2 transposase mRNA were microinjected into embryos of the one-cell stage. Embryos having significant green and/or red fluorescent expression under the fluorescence condition of the microscope were sorted, raised to adulthood and mated with wild fishes to find germline-transmitted embryos. Embryos having a green or red fluorescent expression of nearly similar intensity were founder zebrafish (F0) embryos, which were raised to adulthood and incrossed to give F1 generation as described. 32 Adult F1 fishes were mated, and resulting embryos were used in experiments.

| Senescence-associated β-galactosidase activity assay
Embryonic senescence caused by chemical-induced oxidative stress and gene mutation can be detected using the SAβ-gal assay. 33

| Lysosomal staining
At the beginning of the autophagy process, microtubule-associated protein 1 light chain 3-II (LC3II, hereafter mentioned as LC3) is formed at the inner membrane of the autophagosome. 15

| Microscopy and imaging
Imaging was carried out using both macrofluorescence microscope (Nikon AZ100) and confocal microscope (Olympus; FluoView 1000). Before live imaging, embryos were anaesthetized with tricaine solution (0.16 mg/ml). The whole body of SA-β-gal stained fish was photographed using the reflected bright light of the Nikon AZ100 microscope. In addition, the cellular SA-β-gal staining signal was observed under the confocal microscope. In each experiment, all images were taken under the same condition of the microscope.

| RT-PCR analysis
Regulations of mRNA levels of pai1, p21 and smp30 genes were estimated by RT-PCR analysis. Total RNA was isolated using TRIzol reagent (Invitrogen, 15596026), and double-strand cDNA was synthesized using M-MLV reverse transcriptase (Promega, M1705). RT-PCR primers sequences were presented in Table 1 including their annealing temperatures and amplification cycle.

| Quantitative analysis
Intensities of fluorescence of captured images were quantified using Adobe Photoshop CS software. PCR bands were quantified by ImageJ software (Java-based image processing program developed by NIH). Data analysis was carried out using SPSS statistics software (version 15.0). Data are presented as mean ± SEM. A comparison among different groups was made by Student's t test.

| Carbofuran exacerbates premature ageing phenotype and lifespan in spns1 −/− mutant zebrafish
Embryonic senescence has been implicated in the regulation of the ageing process. Accelerated senescence can trigger ageing symptoms and declines organismal longevity. 5,14 A premature ageing phenotype in spns1 −/− mutant zebrafish is yolk opaqueness, which typically begins from the posterior end of yolk extension and gradually progresses towards other parts of yolk extension and yolk. 14,15 If a small part of the yolk and/or yolk extension of the embryo becomes opaque, then it is denoted as a 'partially opaque.' On the other hand, when the major part of yolk and yolk extension become opaque, then it is denoted as 'mostly opaque' (Figure 2A). 15 Figure 2C).

| Carbofuran accelerates autophagy formation in spns1 −/− mutant zebrafish
Cellular autophagy is essential to maintain cellular homeostasis. 11 It has an advantageous effect on organismal longevity. 12

| Carbofuran affects ageing-associated genes regulation in spns1 −/− mutant zebrafish
The impact of carbofuran exposure on ageing-related genes regulations was examined by RT-PCR analysis. Plasminogen activator inhibitor-1 (pai1) is encoded in humans by the SERPINE1 gene, whose up-regulation is a risk factor for atherosclerosis. 38 In elderly individuals, its level is elevated, which may lead to multiple pathologies such as vascular sclerosis, emotional stress, insulin resistance and obesity. 39 Another gene, p21, is a cyclin-dependent kinase inhibitor, promotes cellular senescence. 40 In both prematurely aged mice and normal aged mice, the p21 level was found elevated. 41 In our investigation, the expression levels of both pai1 and p21mRNA were significantly up-regulated by carbofuran exposure to spns1 mutant fish ( Figure 5). On the other hand, senescence marker protein 30 (Smp30) is a calcium-binding protein, was first recognized as a downregulated protein in elderly individuals. 42 The expression of smp30 was down-regulated by the carbofuran treatment to spns1 mutant fish ( Figure 5).

F I G U R E 2
Carbofuran exacerbated the spns1 deficiency phenotype and shortened the survival of spns1 mutant zebrafish. A, Sorts of yolk opaqueness phenotypes in spsn1 −/− (homozygous) zebrafish based on the extent of opacity in the yolk and/or yolk extension: partially opaque (yellow colour) and mostly opaque (red colour). Wild (spns1 +/+ ) and heterozygous (spns1 +/− ) zebrafish do not show such opaqueness (dark blue colour). B, Effect of carbofuran on the opaqueness phenotype of spns1 mutant fish and death record. The opaqueness phenotype and death records were not affected by 10 μmol/L carbofuran exposure. However, the mostly opaque phenotype and death of embryos of 100 μmol/L carbofuran treated group were found at earlier times than fishes of the control group (egg water treatment). C, Survival curves of spns1 mutant fishes for carbofuran treatment at 10 and 100 μmol/L concentrations. By 100 μmol/L carbofuran exposure, the survival of spsn1 mutant fishes was significantly declined (P = .0003) A B C D

| D ISCUSS I ON
Carbofuran is a broad-spectrum pesticide, which is non-specific in its action. 43 It also exerts its toxic effects on non-target animals, including fishes, beneficial arthropods and humans. 43−45 In mammals, carbofuran exposure leads to oxidative stress, which in turn induces neuronal injury and neuronal cell death through the inhibition of acetylcholinesterase (AChE) activity. 17,27 It was demonstrated that the inhibition of AChE by carbofuran is mediated by the carbamylation of the serine molecule of AChE. 17 Although the toxicity of carbofuran is dose-dependent, it can induce oxidative stress in the brain at sub-lethal doses. 17 The major ROS species in the body are generated via a disorganized electron transfer process of the mitochondrial respiratory chain. 46 At a tolerable extent, ROS that generated in mitochondria may activate an adaptive defence mechanism to prevent unexpected outcome. 47 ROS by acting as nucleophilic compounds might cause epigenetic modification of DNA (such as DNA methylation, histone modification) that control genes transcription and expression. 48 The epigenetic machinery copes with the advanced homeostasis impairment, which reshapes the nervous, cardiovascular and respiratory systems in the elder individuals. 49 When the ROS level exceeds the tolerable extent, the developed  age-associated reduction of DNA methylation was manifested. 48 In senescent cells of the spns1 defective fish, carbofuran-induced oxidative stress might affect the ageing process through DNA hypomethylation.
Senescence is elicited by autophagic commencement. 12 At the beginning of autophagy, phagophore is evolved in autophagosome through the elongation of the membrane of the phagophore. During the elongation process, an ubiquitin-like molecule LC3 is conjugated to phosphatidylethanolamine at the membrane of the autophagosome, leading to the formation of autophagosome-associated LC3. 53,54 LC3 exists with autophagosomes until autolysosomal fusion and then delipidated and recycled. 53 In our in vivo real-time monitoring of EGFP-tagged LC3 protein on spns1 mutant context, carbofuran accelerated LC3 puncta accumulation at the same pattern as we detected the acceleration of senescence induction using SA-β gal staining, suggesting carbofuran-induced autophagic abnormality is associated with accelerated senescence. It was demonstrated that autophagy is usually up-regulated by oxidative stress including cellular senescence, 8 which is consistent with our findings.
Cellular intralysosomal waste materials are collectively known as lipofuscin, whose principal constituents are protein and lipid, and other constituents include carbohydrate, autofluorescent pigment and transition metals. 55 The lipofuscin accumulates in several cell types (such as neurons and cardiac cells) of aged individuals. It was demonstrated that lipofuscin accumulation is associated with lysosomal dysfunction. 56 The accumulation further increases in an ageing allied neurodegenerative disorder such as Alzheimer's disease. 56 Under oxidative stress conditions, increased autophagosome formation leads to the delivery of undegradable oxidized protein to the lysosome that accumulates as lipofuscin. 56 Using LysoTracker red staining and mCherry-tagged Lamp1 expression in spns1 deficit fish, we found excessive lysosomal expression, which was further increased by carbofuran exposure. This lysosomal overexpression upon carbofuran exposure might be because of lipofuscin accumulation under stress conditions, which need further studies to explore the mode of lipofuscinogenesis implicated with carbofuran exposure.
In addition, lysosome was significantly co-localized with autophagosome, suggesting successful autolysosomal fusion with insufficient lysosome facilitated degradation of the content of autophagosome.
Such lysosomal dysfunction also validated under the spns1 deficit stress condition. 15 Some compounds such as chloroquine through oxidative stress and lipofuscin accumulation inhibit lysosomal proteases. 56,57 A decline in lysosomal degradation in carbofuran treated zebrafish might also be because of the inhibition of the lysosomal proteases, but it needs further studies to be confirmed. Overall, carbofuran exacerbated lysosomal dysfunction in spns1 deficit fish.
The effects of carbofuran on developmental senescence and biological ageing are consistent with its effects on ageing allied genes regulation in spns1 −/− mutant fish. It was demonstrated that the inhibition of the pai1 gene down-regulates SA-β-gal activity, 58 whereas, in our investigation, SA-β-gal activity was concurrently increased with the pai1 mRNA level. An increment in the pai1 expression is associated with ageing and age-related multimorbidity such as cognitive dysfunction, hypertension, atherosclerosis and glucose intolerance, 58,59 whereas we consistently found that carbofuran exposure shortened the life span of spns1 mutant fish. A potent cyclin-dependent kinase inhibitor, p21, whose up-regulation is maintained in senescent cells. The intensity of senescence cells was decreased with the loss of the p21 gene in mice. 40 The up-regulation of p21 arrest cellular proliferation and promote age-associated pathologies. 60 It was also reported that the level of p21 in the neuronal cells of rats was up-regulated by carbofuran treatment. 29 Constantly, an elevation of the p21 mRNA level in spns1 mutant zebrafish was observed in our investigation. On the other hand, smp30 is a calcium homeostasis gene, protects apoptosis and prevents oxidative stress by reducing ROS generation. 60 Carbofuran exposure to spns1 mutant fish down-regulated the expression of smp30, which might be because of additional oxidative stress in spns1 mutant fish. Altogether, our data suggest that carbofuran accelerated cellular senescence and shortened biological ageing in spns1 mutant zebrafish through autophagic dysregulation.

ACK N OWLED G EM ENTS
The works of this manuscript mainly supported by The World

CO N FLI C T O F I NTE R E S T
The authors of this manuscript have no conflicts of interest.

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