Pre-imaginal exposure to mancozeb induces morphological and behavioral deficits and oxidative damage in Drosophila melanogaster

Abstract Mancozeb (MZ), a manganese/zinc containing ethylene-bis-dithiocarbamate, is a broad-spectrum fungicide. Chronic exposure to MZ has been related to several organisms’ neurological, hormonal, and developmental disorders. However, little is known about the post-natal effects of developmental exposure to MZ. In this study, Drosophila melanogaster was subjected to a pre-imaginal (eggs-larvae-pupae stage) model of exposure to MZ at 0.1 and 0.5 mg/mL. The emergence rate, body size, locomotor performance, sleep patterns, and molecular and biochemical parameters were evaluated in post-emerged flies. Results demonstrate that pre-imaginal exposure to MZ significantly impacted early emerged flies. Additionally, reduced progeny viability, smaller body size and delaying in emergence period, locomotor impairment, and prolonged sleep time were observed. Content of glucose, proteins, and triglycerides were altered, and the bioenergetics efficiency and oxidative phosphorylation at complex I were inhibited. mRNA stade state levels of genes responsive to stress, metabolism, and regulation of circadian cycle (Nrf2, p38, Hsp83, Akt1, GPDH, tor, per, tim, dILP2, and dILP6) were augmented, pointing out to stimulation of antioxidant defenses, insulin-dependent signaling pathway activation, and disruption of sleep regulation. These data were followed by increased lipid peroxidation and lower glutathione levels. In addition, the activity of catalase and glutathione-S-transferase were induced, whereas superoxide dismutase was inhibited. Together, these results demonstrate that developmental exposure to MZ formulation led to phenotype and behavioral alterations in young flies, possibly related to disruption of energetic metabolism, oxidative stress, and deregulation of genes implied in growth, sleep, and metabolism.


Introduction
Mancozeb is a broad-spectrum fungicide; it belongs to the ethylene-bis-dithiocarbamate (EBDC) class of compounds containing manganese and zinc and a multi-site mode of action. This compound plays a significant role in the global agrochemical market, representing one of the most used fungicides (Fitsanakis et al. 2002, Gullino et al. 2010, and is recommended for a sort of crops like fruits, vegetables, and cereals (Goldoni and da Silva 2012).
MZ is described as a mildly toxic compound for vertebrates; however, toxicological studies indicated pro-oxidative effects causing DNA damage and cell death in human and rat cells (Calviello et al. 2006, Srivastava et al. 2012. Moreover, our group reported alterations in oxidative stress markers in Cyprinus carpio (Costa-Silva et al. 2018) and adult Drosophila melanogaster (Saraiva et al. 2018). Besides, exposure to similar dithiocarbamates caused neurotoxic effects and Parkinson-like symptoms in humans and Caenorhabditis elegans model (Zhou et al. 2004, Negga et al. 2011, Harrison Brody et al. 2013, and hormonal disruption in humans (Rom an 2007, Goldner et al. 2010. MZ contamination was also associated with human neural tube defects (Nordby et al. 2005), and prenatal MZ exposure retarded rat's physical development (Castro et al. 1999). Recent studies have associated farmers' exposure to MZ with sleep problems (Fuhriman et al. 2022). In this aspect, there is no information about the implication of embryonic exposure to MZ on oxidative stress parameters, metabolism indicators, and sleep quality.
The toxicological effects of MZ are attributed to the presence of metals manganese and zinc in its formulation, which could lead to metal accumulation in tissues like the brain (Costa-Silva et al. 2018). Together with the presence of metal, the main MZ degradation byproduct ethylene thiourea (ETU), which demonstrated genotoxic potential (Deardfield 1994) has been implied in MZ toxicity. The mechanisms of action underlying MZ toxicity are not entirely known; however, oxidative stress (OS) is recognized as a primary factor in MZ. Oxidative stress (OS) results from an imbalance between the levels of reactive oxygen species (ROS) and antioxidant defense acting in the neutralization of those species (Davies 2000). In addition, a cellular pro-oxidant environment may damage biomolecules like proteins, lipids, and nucleic acids and has been implied in the etiology of pathologies like tumors and neurodegenerative disorders (Valko et al. 2006, Uttara et al. 2009, Phom et al. 2014. In this context, inhibition of mitochondrial complexes, NADPH oxidase, and xanthine oxidase induction and modulation in the activity of the antioxidant enzymes were reported to MZ (Zhang et al. 2003, Hogarth 2012, Todt et al. 2016, Costa-Silva et al. 2018.
D. melanogaster has contributed to describing biochemical and molecular mechanisms implied in xenobiotics toxicity. Furthermore, the fast developmental cycle of this species permits investigating the outcomes of xenobiotic exposure on developmental stages and over the generations (Ternes et al. 2014, Algarve et al. 2018. Besides, behavioral studies such as sleep alterations can be conducted in D. melanogaster. Like mammals, flies' sleep comprises periods of sustained quiescence with an increased arousal threshold and is modulated by stimulants, hypnotics, and heavy metals (Bushey et al. 2010, Algarve et al. 2018.
The present study aims to investigate the effects of developmental exposure to MZ on behavioral, phenotypic, and biochemical parameters, thus contributing to the knowledge about the toxicity of this compound.

Materials and methods
2.1. Drosophila stock and culture D. melanogaster flies (Harwich strain) was obtained from our breeding. The flies were maintained at 25 ± 1 C on 12h light/dark cycle in glass bottles (50 mm Â 85 mm) containing 20 mL of the standard medium mixture, composed of cereal flour, cornflour, water, antifungal agent (NipaginV R ), and supplemented with dried yeast as previously described (Paula et al. 2014). All experiments were performed with the same strain.

Mancozeb formulation exposure
For exposure of D. melanogaster to commercial Mancozeb (80% purity, EmzebV R 800 WP, Sabero Organics America S.A., Minas Gerais, Brazil), during the pre-imaginal phase (eggmaggot-pupa), adult flies of both genders were placed for 24 h in glass tubes with standard culture medium and MZ formulation in two different concentrations for oviposition. After this period, parents were removed from the bottles, and eggs were maintained until the emergence of new flies (cycle of 11-14 days). The treatment was composed of three experimental groups: control (standard culture medium without treatment), MZ formulation 0.1 mg/mL of medium, and MZ formulation 0.5 mg/mL of medium. The concentrations used in this study were 10 and 50 times lower than those of previous studies (Saraiva et al. 2018) where 50% of mortality was reported at 5 mg/mL and lower than concentrations recommended by the manufacturer to the field application (200 g of commercial product/100 L of water) (Sabero Organics America SA, Brazil) and by the US Environmental Protection Agency (2005).

Time of pre-imaginal development and emergence rate
For pre-imaginal development time and emergence rate evaluation, 50 eggs (in triplicates totaling 150 eggs) per experimental group were collected and placed in plastic vials containing standard medium with or without MZ formulation. The time taken to complete pre-imaginal development stages (egg, maggot, pupa, and emergence) was recorded, together with the number of emerged flies.

Body size measure
The newly emerged flies (1 day old) were visualized and photographed using a Carl ZeissStemiV R 2000 C stereo microscope linked to Moticam 2000V R digital camera, 1.6 Â zoom. The pictures were analyzed in the ImageJV R software. Body size was evaluated by measuring the length from the top of the head to the abdomen's end ( Figure S1 for illustration). It was considered 60 individuals per treatment group; males and females were evaluated separately (30 per group).

Locomotor activity
Flies locomotor activity was determined through a negative geotaxis behavioral test (climbing ability) in individual flies, according to Bland et al. (2009), with some alterations. After the treatment, 30 newly emerged flies per group were anesthetized in ice and placed individually in vertical glasses tubes (25 Â 1.5 cm), closed with cotton. After 30 min of recovery, flies were gently tapped to the bottom of the tube, and the time taken by each fly to climb 5 cm into the glass column was recorded on video and analyzed in slow motion. The test was repeated three times per fly (with 20 s between the repetitions).

Sleep analysis
It was used the Drosophila Activity MonitorV R (DAM) (DAMSystem 308-TriKinetics In, Waltham, MA, USA) for the sleep analysis. In this apparatus, individual flies are allocated in glass tubes (5 Â 65 mm) containing standard culture medium at one end and arranged horizontally on specific monitors supporting up to 32 tubes. The monitors were maintained in a DigiThermV R CircKinetics TM incubator (Tritech Research Inc, Los Angeles, CA, USA) set at 25 C, with 12 h day/night cycles during the experiments. The activity is registered when the fly passes through the monitor's infrared beams. Herein, newly emerged flies (1-4 days old) previously exposed to MZ formulation were maintained in the incubator taking access to the standard culture medium without MZ formulation for eight days (period of analysis). The first day in the apparatus is considered an adaptive period and was not included in the sleep analysis. Sleep patterns were analyzed on pySoloV R software, which converts the DAM results into diagrams of activity, sleep cycles, sleep amount, and sleep fragmentation (Gilestro and Cirelli 2009

Determination of glucose, triglycerides, and protein levels
Indicators of energetic metabolism were evaluated, including glucose, triglycerides, and protein levels in the whole homogenate by colorimetric kits, according to the method described by Macedo et al. (2017) and Saraiva et al. (2021). Twenty newly flies of both sexes per group were homogenized in 500 mL of 200 mM HEPES buffer, pH 7.0, and centrifuged at 22 000 Â g for 30 min at 4 C. The supernatant was used for the measurement of glucose and triglycerides levels according to the manufacturer's recommendation (LabtestV R , Minas Gerais, Brazil) with some modifications. The results were adjusted per weight (in mg) of each group of flies and were expressed as mg/mL of sample (homogenate of flies) in relation to the control group.
The protein concentration of all groups was determined using Bovine Serum Albumin (BSA) as standard according to the Bradford method (Bradford 1976), which is based on Coomassie's adsorption Brilliant Blue G-250 reagent.

High-resolution respirometry (HRR)
Alterations in mitochondrial bioenergetics patterns were measured by high-resolution respirometry using Oxygraph-2kV R (O2k, Oroboros Instruments, Innsbruck, Austria). The Drosophilas mitochondria preparations were obtained according to Gnaiger (2009). For each mitochondrial preparation, 50 newly emerged flies of every experimental group were anesthetized on ice and homogenized in 2 mL ice-cold mitochondrial isolation buffer containing 250 mM sucrose, 0.1% BSA, 2 mM EGTA and 5 mM Tris-HCl (pH 7.4). The resulting homogenate was filtered in order to remove the tissue particles with a nylon filter membrane (10 mm pore size) and centrifuged at 200 Â g for 3 min. The supernatant was collected, and further centrifugation at 9000 Â g for 10 min was performed. Therefore, the pellet was re-suspended carefully in 2 mL of ice-cold albumin-free isolation buffer, followed by new centrifugation at 9000 Â g for 10 min. This final pellet was re-suspended in 100 mL of albumin-free isolation buffer and then used in respiration assay.
D. melanogaster isolated mitochondria (0.05 mg/mL) were transferred to 2 mL of MiR05 solution (110 mM sucrose, 60 mM K-lactobionate, containing 0.5 mM EGTA, 3 mM MgCl2, 20 mM taurine, 10 mM KH 2 PO 4 , 20 mM HEPES and 0.1% essentially fatty acid-free BSA, pH 7.1) (Gnaiger 2009). All experiments were performed at 24 C using DatLab 4.0V R software (Oroboros Inc., Austria), with continuous stirring at 759 rpm. Titration protocols evaluated the abilities of several substrates and inhibitors to change the mitochondrial function as a reflex of difference in respiratory states. L-Proline þ pyruvate þ malate þ succinate þ ascorbate and tetramethyl-p-phenylenediamine (TMPD) were used as oxidizable substrates in all experiments (Carvalho et al. 2017). Respiratory rates, changes in mitochondrial respiratory chain complexes, mitochondrial membrane integrity, and production of oxidative oxygen species were determined according to Rodrigues et al. (2018).
After signal stabilization, the basal respiration supported by endogenous substrates was evaluated. The Complex I (CI)mediated leak (LEAK) respiration was determined using 5 mM pyruvate, 5 mM L-proline, and 1 mM malate. CI-mediated OXPHOS (OXPHOS) was determined using ADP (2.5 mM). The convergent electron flow during the maximal OXPHOS respiration (CIc þ CII OXPHOS ) was evaluated with substrates of CIc and CII (10 mM succinate). The ETS respiration represents the not coupled respiration using FCCP (optimum concentration reached between 0.5 and 1.5 mM); CIc þ CII-mediated ETS respiration (CI c þ CII ets ) was determined using FCCP (optimum concentration reached between 0.5 and 1.5 mM). CII-mediated E.T.S. respiration (CII ETS ) was determined with 0.5 mM rotenone. The addition of 2.5 mM antimycin A inhibited Complex III, resulting in non-mitochondrial respiration, the residual oxygen consumption (ROX) with minor contributions from electron leak in the uncoupled state. Respiration of CIV was determined by adding ascorbate 0.5 mM and TMPD 1 mM. For HRR, three independent mitochondrial preparations from each experimental group were used (n ¼ 3). All HRR experiments were performed in duplicates.

Oxidative stress markers analysis
For measurements of antioxidant enzymatic activity and lipid peroxidation, 20 newly emerged flies per group were homogenized in 20 mM HEPES buffer (pH 7.0). The homogenate was centrifuged at 20 000 Â g for 30 min (4 C) (Eppendorf 5427 RV R , rotor FA-45-30-11). The supernatant was isolated and used for measuring the activity of antioxidant enzymes (superoxide dismutase, catalase, and glutathione-S-transferase) and lipid peroxidation, based on protocols described below.

Antioxidant enzymes activity
Superoxide dismutase (SOD) activity consists of the inhibition of superoxide-induced oxidation of quercetin by SOD at 406 nm. The complete system of reaction is constituted by 25 mM phosphate buffer at pH 10, 0.25 mM EDTA, 0.8 mM TEMED, and 0.05 mM quercetin, according to Kostyuk and Potapovich protocols (Kostyuk and Potapovich 1989). Catalase (CAT) activity was determined after H 2 O 2 release in 240 nm in reaction medium containing 0.05 M phosphate buffer, pH 7.0, 0.5 mM EDTA, 10 mM H 2 O 2 , 0.012% TRITON X100 according to Aebi (1984). Glutathione-S-transferase (GST) activity was determined following the procedure of Habig and Jakoby (1981) using 1-chloro 2,4-dinitrobenzene (CDNB) as substrate. The assay was based on a conjugated complex formation of CDNB and GSH at 340 nm. Reaction was conducted in a mix with 0.1 M phosphate buffer pH 7.0, 1 mM EDTA, 1 mM GSH, and 2.5 mM CDNB. All spectrophotometric assays were carried out in an Agilent Cary 60 UV/VISV R spectrophotometer with an 18-cell holder accessory coupled to a Peltier Water System V R temperature controller. The values were normalized by protein concentration and expressed as mU/mg protein in relation to the control group.

Lipid peroxidation assay
The final lipid peroxidation products were quantified as thiobarbituric acid reactive substances (TBARS) according to Ohkawa et al. (1979), with some alterations. The mitochondrial-enriched supernatant was incubated in acetic acid 0.45 M/HCl pH 3.4, thiobarbituric acid 0.28%, SDS 1.2%, at 95 C for 60 min, and the absorbance was then measured at 532 nm. The TBARS values were normalized by protein concentration. Results were expressed as nmol/mg of protein in relation to the control group.

ROS production and resazurin reduction assay
Finished the treatment period, 20 flies per group were homogenized in 1000 mL of mitochondrial isolation buffer (220 mM mannitol, sucrose 60 mM, KCl 10 mM, 10 mM HEPES, 1% BSA) following centrifugation at 1000 Â g for 10 min (4 C). The mitochondrial-enriched supernatant was used for the determination of reactive oxygen species (ROS) formation (DCF-DA assay) and cell metabolic viability (resazurin assay). We quantified the oxidation of 2 0 ,7 0 -dichlorofluorescein diacetate (DCF-DA) as a general ROS production index (P erez-Severiano et al. 2004). The DCF fluorescence resulting from oxidation of DCF-DA was monitored in regular breaks at 485 nm ex / 530 nm em . The values were expressed as fluorescence concerning the control group.
The resazurin reduction assay was conducted as indicative of cellular metabolic functions; this assay is based on mitochondrial ability to convert resazurin into a final fluorescent product (resofurin) due to the presence of dehydrogenases. Impaired cells do not generate the fluorescent signal (O'Brien et al. 2000). The fluorescence was monitored in regular breaks of 1 h using the fluorescence plate reader (PerkinElmer Enspire 2300V R ) at 544 nm ex /590 nm em . The experiment was conducted six times independently in triplicates. The values were expressed as fluorescence concerning the control group.

Quantitative real-time qRT-PCR and gene expression analysis
Expression of Nrf2, NFkB, p38b, Hsp83, Akt1, Gpdh, dIlP2, dIlP6, FOXO, tor, per, and tim genes of newly emerged Drosophila were analyzed (Table 1). Trizol Reagent (Invitrogen TM ) was used to extract about 1 mg of total RNA from 20 flies, according to the manufacturer's protocol, and then treated with DNAse I (DNAse I Amplification Grade -Invitrogen TM , NY). cDNA was synthesized with iScript cDNA Synthesis Kit and random primers again according to the manufacturer's protocol (Biorad# CA, USA). The quantitative real-time polymerase chain reaction was performed according to the protocol previously described by Rodrigues et al. (2018), with the exception of the tubulin gene, used as an endogenous reference gene in this study, presenting no alterations in response to the treatment.

Statistical analysis
The statistical analysis was performed using One-way Analysis of Variance (ANOVA) followed by Tukey's post hoc test. With the exception of the emergence rate evaluation, the two-way ANOVA was performed, followed by the Bonferroni test when considering two variables (concentration and time). The results were deemed to be significant when p 0.05.

Fly emergence
MZ formulation exposure during the pre-imaginal development period caused a delay in approximately 24 h in the emergence of flies. It reduced the total emergence rate by 50% at a higher MZ formulation concentration (Figure 1).

Body size analysis
Newly emerged flies (1 day old) exposed to MZ formulation during the pre-imaginal development were smaller than controls, as observed in Figure 3. The male's length was reduced significantly at 0.1 and 0.5 mg/mL of MZ formulation in about 7 and 15%, respectively (Figure 2(a)). Female body size was also reduced by 20% but only at the highest concentration of MZ formulation (Figure 2(b)). The reduced body of flies can be visualized in Figure 2(c-f). Figure 2(c) represents a male fly developed in a standard medium without MZ formulation, and Figure 2(d) is a male grown in medium with MZ formulation 0.5 mg/mL. Figure 2(e) shows a female fly developed in a standard medium without MZ formulation, and Table 1. Genes tested by qRT-PCR analysis and forward and reverse primers.

Gene
Primer sequences Forward 5

Locomotor performance
Individual negative geotaxis assay demonstrated that flies exposed to MZ formulation during the pre-imaginal development presented locomotor deficits, taking an average of 7.1 s to reach the 5 cm mark on the tube, while control flies took an average of 2.7 s (Figure 3).

Sleep/wake cycle analysis
The total sleep, expressed as the total time of sleep along the day in minutes, was increased in the first 48 h at 0.5 mg/ mL, then returning to control levels (Figure 4(a)). Treatments did not alter the number of daytime sleep fragmentation (Figure 4(b)); however, the number of nocturnal sleep fragmentation was significantly decreased at 0.5 mg/mL, at the first 24 h of analysis, this effect returned gradually to control levels over the days of study (Figure 4(c)). A significant reduction in the duration of daytime sleep bout was observed on the first day of analysis at 0.1 mg/mL. It remained lower than control up to the end of the analysis period. The higher concentration gradually reduced this sleep parameter, which was significant only at five days of treatment (Figure 4(d)). The nocturnal sleep bout duration was increased up to the third day of analysis, then gradually returned to control levels at 0.5 mg/mL (Figure 4(e)).

Levels of glucose, triglycerides, and protein
Some energetic metabolism parameters of newly emerged flies were evaluated, such as triglycerides, glucose, and protein levels. The glucose levels ( Figure 5(a)) decreased significantly at higher concentrations (p 0.05) in comparison to control while triglycerides ( Figure 5(b)) decreased only at MZ 0.1 mg/mL. The total protein levels dropped at both MZ concentrations (p 0.05 and p 0.0001 in comparison to the control group), as visualized in the Figure 5(c).

High-resolution respirometry
The oxygen flux through mitochondrial complexes in response to MZ formulation was evaluated to determine the mitochondrial bioenergetics function, using high-resolution respirometry (HRR) (Figure 6(a)). Basal respiration was unchanged on MZ formulation treatments. After L-proline þ pyruvate þ malate substrates (CI LEAK ) addition, a significant decrease (p 0.05 and p 0.01) on CI activity was induced by MZ formulation 0.1 mg/mL and MZ formulation 0.5 mg/mL, respectively. Likewise, the addition of saturating ADP to induce OXPHOS indicated that MZ formulation exposure in the highest concentration decreased (p 0.05) CI OXPHOS respiration when compared to control. The same result was observed after the addition of cytochrome C (CIc OXPHOS ). The convergent electron flow during the maximal oxidative phosphorylation (CIc þ CII OXPHOS ), induced by the addition of succinate, remained unchanged. The addition of FCCP to uncoupling respiration (CIc þ CII ETS ) shows no significant alterations between control and treated groups, as well as CII ETS respiration, analyzed after inhibition of CI by rotenone. Furthermore, the addition of antimycin A used to evaluate the residual oxygen consumption (ROX) and decreased the O 2 flux to the basal levels without any significant differences between treatment groups. Lastly, the addition of TMPD plus ascorbate indicated that MZ formulation 0.5 mg/mL was able to decrease (p 0.05) CIV respiration (Figure 6(a)). The mitochondrial bioenergetics capacity was determined by subtracting the ADP-induced CI OXPHOS values from the CI LEAK values, showing that pre-imaginal exposure of flies to MZ formulation 0.5 mg/mL was able to reduce (p 0.05) the mitochondrial bioenergetics efficiency, as compared to control ( Figure 6(b)).

Oxidative stress markers and resazurin
MZ formulation exposure during pre-imaginal period inhibited SOD activity in both concentrations (p 0.05 and p 0.01 in comparison to control group, at 0.1 and 0.5 mg/ mL, respectively). CAT and GST activities were both augmented by MZ formulation exposure in the higher concentration (p 0.01 in comparison to control group). Lipid peroxidation measured through TBARS assay was significantly increased (p 0.05 and p 0.01 in comparison to control group, respectively, at 0.1 and 0.5%) ( Table 2). Both MZ formulation concentrations induced the levels of ROS in the flies concerning control, with a more noticeable increase at 0.1 mg/mL (p 0.001) ( Table 2). In parallel, the resazurin reduction to fluorescent resofurin was diminished by treatments (p 0.001 for 0.1 mg/mL and p 0.0001 for 0.5 mg/mL in comparison to control group), indicating reduction in cell metabolic viability (Table 2).

mRNA steady-state levels analysis
The pre-imaginal exposure of flies to MZ formulation altered the expression of many genes involved in cellular processes such as homeostasis, oxidative stress, growth, metabolism, Figure 2. Body size of male and female flies exposed to MZ (0, 0.1, and 0.5 mg/mL). Males (a) and females (b) flies were exposed to MZ during the developmental period. After emergence, the body length of flies was evaluated. Data are expressed as mean ± SEM; Ã p 0.05; ÃÃ p 0.01; ÃÃÃ p 0.001 compared to the control group and ###p 0.001 compared to the other treated group. and sleep regulation ( Table 1). Levels of Nrf2 increased in a concentration-dependent manner (p 0.01 in MZ 0.1 mg/mL and p 0.001 in MZ 0.5 mg/mL in comparison to control group) (Figure 7(a)). MZ exposure did not alter the NFkB mRNA levels (Figure 7(b)). Similarly, p38, Hsp83, Gpdh, dILP6, tor, per, and tim mRNA levels were increased mainly at the higher concentration of MZ (p 0.05) (Figure 7(c,d,f,h,. qRT-PCR analysis revealed significant inhibition of mRNA steady-state levels of FOXO at MZ 0.1 mg/mL (p 0.05 about control group) (Figure 7(i)). Levels of Akt1 and dILP2 were increased in both concentrations (p 0.05) (Figure 7(e,g)).

Discussion
Outcomes from developmental exposure to agrochemicals are scarcely explored. Generally, developmental studies are complex and provide inconclusive data, taking a long period to be conducted and being susceptible to many variables. In this regard, D. melanogaster has the advantage of presenting a short life cycle, permitting different models of exposure in all developmental stages.
In the present study, flies grown in MZ contaminated medium presented reduced body length, triglycerides, protein, glucose levels, and locomotor and sleep alterations. The  insulin/insulin-like growth factor signaling regulates metazoans' growth, development, and metabolism (Broughton et al. 2005). Thus, this study analyzed the mRNA steady-state levels Akt1, tor, and insulin-like peptides dILPs 2 and 6, demonstrating augmented levels concerning the control flies. One of the critical signaling enzymes responsive to insulin is phosphoinositide 3 kinase (PI3K) that phosphorylate phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) to phosphatidylinositol-3,4, 5-trisphosphate (PIP3). Subsequently, the recruitment of pleckstrin homology domain-containing signaling proteins like Akt1 kinase to the plasma membranes occurs. Downstream PI3k effectors like Akt1 are major cellular, metabolism, growth, and proliferation regulators. Tor is another critical regulator of metabolism and growth downstream of PI3k; it stimulates the biosynthesis of proteins, lipids, and nucleic acids. Additionally, tor can promote energy (ATP) production, inhibit the breakdown of lipids via lipolysis and b-oxidation, and promote anabolic activity, which possibly underlies the effects on organismal growth (Dibble and Cantley 2015). If the target of insulin pathway mRNA was stimulated for one side, lower growth body, glucose, and protein were observed on the other side. Although unexpected, similar results showed weight loss and lower glucose levels in flies chronically exposed to MZ (Saraiva et al. 2021). Both cases might result from a lower uptake or absorption of nutrients due to the gastrointestinal tract's potential injuries impacting the availability of energy substrates and compromising mitochondrial respiration (Yasugi et al. 2017). mRNA steady-state levels analysis of Nrf-2 transcriptional factor was increased by MZ treatment suggesting the stimulation of Nrf-2 pathway. Various Nrf-2 activators were described, including synthetic and natural quinones, heavy metals, ROS, or substances that promote their generation. A common feature of these compounds comprises their electrophilic character, leading to the reduction of a disulfide bond in Keap 1 protein, impairing the interaction with Nrf2, which is translocated to the nucleus, subsequently binding to ARE (antioxidant responsive element) of the target genes. Additionally, Nrf-2 pathway is intimately associated with the PI3k/Akt1 signaling pathway, as demonstrated by Sha et al. (2019), when the compound Maltol (aromatic compound) activated Nrf/ARE and PI3k/Akt1 leading to a protective action against oxidative stress. Nfr2/ARE positively regulates tor, a possible mechanism was proposed by Bendavit et al. (2016), demonstrating the existence of an ARE sequence in the promoter of the tor gene, leading to the direct regulation by Nrf2.
Transcription factors of the forkhead box, class O (FOXO), act in the antioxidant cell response to stress by stimulating the transcription of genes coding for antioxidant proteins such as superoxide dismutase-2, peroxiredoxins 3 and 5, catalase, selenoprotein P, and ceruloplasmin. The insulin-dependent signaling and stressful stimuli like ROS results in activation of Akt1 and ERK, culminating with modulation of FOXO activity. Akt1 leads to the inactivation of FOXO by phosphorylation, whereas the activation of p38b and ERK in a ROS-dependent manner induces FOXO phosphorylation leading to the nuclear accumulation and activation of this factor (Klotz et al. 2015). In summary, different kinases can phosphorylate FOXO in response to elevated ROS levels. Herein, MZ inhibited FOXO transcriptional factor mRNA levels and inhibited SOD activity, probably due to a diminished expression of this enzyme.
Environmental factors like air pollutant toxic metals, including manganese and persistent organic pollutant, activates epigenetic changes that consist of acquirable phenotypic variations in chromatin structure, which can interfere in the expression of genes involved in several life processes (Ijomone et al. 2020). Evidence has shown that oxidative stress causes DNA aberrations altering the capacity of DNA methyltransferases to use DNA as a base, disrupting gene methylation (Donkena et al. 2010). In this sense, prenatal exposure to Mn caused temporary hypermethylation and downregulation in different genes in mouse blood (Wang et al. 2013). Another epigenetic change observed following Mn exposure was histone modification. It is supposed that increased histone acetylation has a role in increasing ROS levels and decreasing GSH in neurons. This is probably attributed to the involvement of histone acetylation in enhancing Figure 6. Effects of MZ exposure (0, 0.1, and 0.5 mg/mL) on mitochondria respiration (HRR). O 2 flux was measured on isolated mitochondria. Mitochondrial respiration is represented by abbreviation(s) of the complex(es) involved followed by the state of respiration measured in the presence of L-proline þ pyruvate þ malate (CI LEAK ), þ ADP (CI OXPHOS ),þcytochrome c (Cic OXPHOS ),þ succinate (CIc þ CII OXPHOS ), þ FCCP (CIc þ CII ETS ), þ rotenone (CII ETS ), þ antimycin A (ROX) þ TMPD/ascorbate (CIV) (a). Analysis of bioenergetics capacity is demonstrated in (b). Results are presented as means ± S.E.M of three independent experiments (n ¼ 3); Ã p 0.05, ÃÃ p 0.01 compared to control. Data are expressed as a mean ± S.D. of six independent experiments. Ã p 0.05; ÃÃ p 0.01; ÃÃÃ p 0.001 compared to control group.
Nrf2-HO1 expression pathway (Zhang et al. 2017). In this study, MZ has modified the mRNA steady-state levels of different genes. A possible mechanism implied in this effect could involve activating epigenetic changes by MZ exposure; however, further studies are necessary to validate this hypothesis. Mitochondria is the center of energy generation in the cell. In previous studies, MZ disrupted mitochondrial Complex I, decreasing the ATP levels and oxygen consumption in cells of C. elegans (Zhang et al. 2003, Iorio et al. 2015, Todt et al. 2016, our group reported similar results in adult flies (Saraiva et al. 2021). Herein, the respiratory bioenergetic efficiency was impaired by MZ formulation at 0.5 mg/mL (CIoxphos-CILeak); this process is related to a disruption in the electron transport system and phosphorylation capacity and might culminate in lower production of ATP. Also, the HRR data indicates that MZ impairs mitochondrial respiration supported by Complex I and IV substrates. This effect might be attributed to impairment in OXPHOS system, including ADP and ATP carrier and ATP synthase, or even conformational changes in the complex structure. In addition, respiratory deficits associated with dithiocarbamates exposure have been related to these compounds' chelating properties toward transition elements  (e), Gpdh (f), dIlP2 (g), dILP6 (h), FOXO (i), tor (j), per (k), and tim (l). Results are represented as fold increases/decreases compared to control (considered as 1) (mean ± SEM); a total of six independent experiments was performed (n ¼ 6); Ã p 0.05, ÃÃ p 0.01, ÃÃÃ p 0.001 compared to control. ###p 0.001 compared to 0.5 mg/mL. present in proteins, forming complexes inhibiting their activity, as demonstrated for enzyme superoxide dismutase (Hogarth 2012).
Inhibition of mitochondrial Complex I, as reported in this study, causes superoxide anion formation (O2 -), which potentially react with nitric oxide (NO), producing peroxinitrite (ONOO-), that in its turn gives rise to highly oxidizing molecules causing oxidative damage to the cell. The antioxidant enzyme SOD acts in the dismutation of superoxide anion to hydrogen peroxide. The activity of this enzyme was inhibited by MZ exposure, an effect possibly attributed to the chelating ability of Dithiocarbamates toward metals present in the active center of SOD (Biagini et al. 1995). An inhibition of SOD activity could contribute to higher production of peroxinitrite contributing to cellular oxidative damage. Corroborating this hypothesis, we have demonstrated that MZ exposure decreases nitrite levels in flies (Saraiva et al. 2018). Additionally, MZ induced GST activity; this effect was also described in rat hepatic tissue and carp brain and flies (Hissin and Hilf 1976, Deardfield 1994, Saraiva et al. 2018. Important roles displayed by the GST family in the cells are protecting against oxidative stress and potentially toxic compounds via conjugation of glutathione (GSH) to molecules and products of oxidative metabolism (P erez- Severiano et al. 2004), contributing to their excretion. The present study reports an augmented GST activity, drawing attention to the role displayed by GST in MZ detoxification in flies. In parallel, it was demonstrated induction of CAT activity, hydroperoxide production and lipid peroxidation. These findings corroborate with the mRNA steady-state levels of Hsp83, p38b, Gpdh, and Nrf2. These genes display essential functions providing correct assemble of newly synthesized proteins (Hsp83), acting on survival pathways (p38b), providing proton reducing equivalent for scavenging reactions (Gpdh), and expression of antioxidant enzymes (Nrf2), including GST, SOD, and CAT (Navarro-Yepes et al. 2014) and might represent an adaptive response to the continuous presence of MZ in the medium.
The behavioral outcomes of MZ exposure during the developmental period are not entirely known; however, it was reported that prenatal exposure to MZ retarded rat's physical development (Castro et al. 1999). Thus, the present study will shed light on this question, demonstrating the occurrence of locomotor performance and sleep quality impairment in flies exposed to MZ during the developmental period. Sleep is a well-conserved behavior displaying cognitive, housekeeping, and restorative roles, conserving energy and warranting survival in starving conditions (Sehgal andMignot 2011, Frank andHeller 2019). Previous studies demonstrated an association between exposure to MZ and other carbamates with poor sleep quality in farmers (O' Baumert et al. 2018, Fuhriman et al. 2022. Mn, which is present in MZ structure, has been associated with sleep disturbances. Rats treated chronically with Mn presented hypersomnia, characterized by lengthening sleep phases (Roussel and Renaud 1977). In this regard, several types of sleep disorders were reported in Manganism, a neurological condition resultant from chronic exposure of humans to Mn (Sehgal et al. 2007).
Moreover, our group demonstrated that exposure to MZ can increase the levels of Mn, in fishes and flies (Costa-Silva et al. 2018, Saraiva et al. 2018. The biochemical mechanism underlying sleep disorders are not entirely known. Nevertheless, monoamines have been involved in the regulation of states of sleep, and it is well documented that Mn exposure disrupts the dopaminergic system in rats and flies (Bouabid et al. 2014). As long as the locomotor behavior of flies is under the control of dopaminergic neurons (Sun et al. 2018), we can infer that the dopaminergic system was targeted by MZ treatment in flies, once they presented impaired locomotor performance, compared to control flies, and this fact would induce adverse effects on the sleep-wake cycle.
A critical molecular component of sleep is proteins period (per) and Timeless (tim), which are cycling clock proteins whose expression is rhythmically controlled by their mRNAs. During the day, per and tim are accumulated and migrate to the nucleus about the middle of the night, inhibiting the transcriptional factors Clock (CLK) and cycle (CYC), thus blocking tim and per transcription. Posteriorly, the degradation of tim and per proteins relieves CLK and CYC repression, directing to a new cycle (Sehgal et al. 2007). In this study, tim and per mRNA levels were increased concerning the control; this fact points out a disturbance in the tightly regulated mechanisms for controlling the circadian cycle.
In conclusion, this study reports for the first time the vulnerability to MZ using the invertebrate model of D. melanogaster during developmental stage.Additionally, it reinforces the importance of evaluating the effects of xenobiotics on development, promoting new strategies to prevent and minimize their impact on pregnant women and children exposed to MZ.