Low-dose ionising radiation exerts effects comparable to MPTP through alterations in locomotor activity, oxidant/antioxidant status and mitochondrial homeostasis in zebrafish embryos

Low-dose ionising radiation exerts effects comparable to MPTP through alterations in locomotor activity, oxidant/antioxidant status and mitochondrial homeostasis in zebrafish embryos | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Low-dose ionising radiation exerts effects comparable to MPTP through alterations in locomotor activity, oxidant/antioxidant status and mitochondrial homeostasis in zebrafish embryos Ezgi Cahide Aydas Bayramov, Merih Beler, Derya Cansiz, Ismail Unal, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6594085/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Prenatal exposure to environmental factors including low-dose ionising radiation and neurotoxins may disrupt the oxidant-antioxidant balance. Our aim was to assess the effects of exposure to low-dose ionising radiation (LDIR) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is a neurotoxin used to model Parkinson's disease (PD), on developing zebrafish embryos, focusing on the oxidant-antioxidant system and markers of mitochondrial damage associated with PD. Zebrafish embryos were divided into four groups: control, LDIR, MPTP, and LDIR combined with MPTP (LDIR+MPTP). A dental diagnostic x-ray unit (0.08 seconds, 60 kVp, 7mA) was used for the exposures. The LDIR exposure was measured as 0,065 mSv using optically simulated dosimeters. At the end of 72 hours after fertilization, locomotor activities, acetylcholine esterase (AChE) activity, oxidative stress and antioxidant status were assessed. Expressions of genes associated with in PD as markers of mitochondrial damage ( pink1, parkin, dj1 and lrrk2 ) were determined by RT-PCR. Developmental toxicity was observed in all exposure groups as evidenced by pericardial edema, yolk sac edema and spinal curvature. LDIR exposure in zebrafish embryos affected oxidative and mitochondrial stress markers, as well as locomotor activity and AChE as a marker of cognitive function at levels comparable to the MPTP exposure. Our study is the first to determine the effects of LDIR from a dental x-ray unit on the response to MPTP, and we aim to further elucidate the mechanism of these changes observed particularly in the LDIR+MPTP group. Biological sciences/Biochemistry/Neurochemistry Biological sciences/Chemical biology/Rna Low-dose ionising radiation 1-methyl-4-phenyl-1 2 3 6-tetrahydropyridine oxidative stress antioxidant locomotor activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Dental diagnostic imaging is an integral part of the practice of dentistry and certain types of examinations [ 1 ] . Radiation doses have been reduced with the development of digital techniques [ 1 , 2 ] . Exposure to ionising radiation (IR) is harmful, although radiation-related health risks decrease at lower doses [ 3 ] . In view of the lifetime frequency of exposure to dental diagnostic x-rays, even a small increase in health risk would be of significant public health importance [ 4 ] , and repeated exposure may increase the risk of cancer [ 5 ] . The mechanism of deleterious IR effects is strongly linked to increased oxidative stress in irradiated tissues [ 6 ] . IR is able to penetrate the cells of living organisms, where it induces the ionization of both organic and inorganic compounds [ 7 , 8 ] . Due to the high water content in cells, the radiolysis of water molecules by IR is the main process that contributes to the increased formation of reactive oxygen species (ROS) [ 9 ] . ROS react rapidly with macromolecules, including proteins, nucleic acids and lipids, leading to cell dysfunction, and apoptotic cell death [ 6 ] . As a result of elevated oxidative stress, not only direct negative side effects, but also ROS-related diseases can develop. Therefore, it is particulary important to identify effective and safe prophylactic compounds to protect humans from IR damage [ 10 ] . Parkinson's disease (PD), clinically characterized by tremor, bradykinesia and rigidity, is the second most common neurodegenerative disorder in the world. The disease involves loss of dopaminergic neurons in the substantia nigra pars compacta (SNc), leading to dopamine depletion at nerve terminals [ 11 ] . Multiple environmental and genetic factors play a key role in the etiology of PD [ 12 , 13 ] . Environmental factors influencing PD risk include mitochondrial toxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [ 14 ] and IR [ 13 ] . Oxidative stress has also been linked to PD [ 19 ] , and IR induces oxidative stress causing neurobiological responses [ 16 ] . MPTP is a neurotoxin that induces selective loss of dopaminergic neurons in the mammalian midbrain. In different model organisms, exposure to MPTP leads to the characteristic symptoms of PD [ 17 ] . MPTP exposure in zebrafish embryos has been shown to damage dopaminergic neurons and reduce the number of dopaminergic cells in the diencephalon [ 18 ] . In zebrafish, the dopaminergic system is well characterized both in the embryonic and adult stages. For this reason, zebrafish is a suitable model organism to study the molecular pathways in neurodevelopment and neurotoxicology, as well as to explore potential therapeutic agents [ 19 ] . Environmental exposures during sensitive developmental windows in the embryonic phase can have lasting effects on the health [ 20 ] . Previous investigations have focused on elements such as DNA damage mechanisms in the adult organism, free radical generation, and elevated cancer markers at low doses of IR [21]. Cells with high levels of mitotic activity are known to be hypersensitive to radiation. Although organogenesis and especially neurogenesis in the early fetal period are the most susceptible periods of the fetus to radiation, research on LDIR exposure during the embryonic stage is limited [ 22 ] . Prenatal effects have been studied in animal models [ 23 ] . We hypothesized that LDIR exposure in developing zebrafish embryos would cause oxidative stress and mitochondrial stress, similar to MPTP, and could affect the locomotor functions of the embryo, and that LDIR exposure prior to MPTP could also affect the response to MPTP in the organism. To determine the molecular mechanisms of the effect of LDIR and MPTP, molecules involved in the oxidant-antioxidant system (malondialdehyde, nitric oxide, superoxide dismutase, glutathione S-transferase, glutathionine and catalase) and activity of acetylcholinesterase were evaluated to determine cholinergic effects. In addition, to assess whether MPTP and IR induced changes in mitochondrial stress-related genes associated with PD, the expressions of pink1, parkin, dj1 , and lrrk2 were determined. Methods Chemicals Tested MPTP (purity ≥98%) was purchased from Sigma-Aldrich, St Louis, MO, USA. It was analytical grade with the highest purity available. Maintenance of Zebrafish Wild-type AB/AB strain zebrafish were maintained under apparently disease-free conditions. Animal husbandry and spawning were performed in accordance with the relevant guidelines and regulations and the protocols approved by the University of Marmara Institutional Animal Care and Use Committee. Fish were kept in an aquarium rack system (Zebtec, Tecniplast, Italy) at 27 ± 1 °C under a light/dark cycle of 14/10-hour and they were fed with commercial flake fish food complemented with live Artemia twice a day. The pH of system water ranges from 6.9 to 7.2. Reverse osmosis water that contains 0.018 mg L -1 Instant Ocean TM salt was used for all experiments . Zebrafish embryos (AB/AB) were obtained from the Zebrafish Research Laboratory in Marmara University and maintained in E3 medium. After natural spawnings, fertilized embryos were gathered and staged according to their developmental and morphology as described before [24] . We used zebrafish embryos up to 72 hpf and the European Commission Directive 2010/63/EU, permits experimentation in fish embryos at earliest life stages without beingregulated as animal experiments; zebrafish are considered models in vitro until 120 hpf (http://data.europa.eu/eli/dir/2010/63/2019-06-26; accessed 21 May 2025 EFSA opinion: https://doi.org/10.2903/j.efsa.2005.292; accessed 21 May 2025). Embryo Exposure Zebrafish embryos of the AB/AB strain were selected under the stereomicroscope after fertilisation and divided into four groups including control group, MPTP-exposed group, LDIR- exposed group from a dental x-ray unit (0,08 seconds, 60 kVp, 7 mA) and MPTP-exposed combined with LDIR-exposed group. The dental x-ray unit, Belmont AR-33RK5EU (Osaka, Japan) (60 kVp, 7 mA) with long cone (30 cm) was used for the low-dose ionizing radiation exposure. The exposure time on the dental x-ray unit was set to 0,08 seconds. The MPTP, LDIR, and LDIR+MPTP groups were exposed to 0,08 seconds of x-ray, 800 µM MPTP, and 0,08 seconds of x-ray before 800 µM MPTP, respectively, at 24 h post-fertilization. Developmental parameters were monitored and documented daily under a stereomicroscope (Zeiss Discovery V8, Germany) until 72 hpf. During the experiment, an OSL dosimeter (Epsilon Landauer, Istanbul, Turkey) was used for each exposure group (LDIR and LDIR+MPTP groups). These dosimeters had a sensitivity range of 0,05 mSv to 10,0 Sv with an uncertainty of 8% for deep and shallow dose Hp, which was done to compare the reliability of dose measurements. Each OSL dosimeter was placed in the centre of a petri dish to measure absorbed doses. The same conditions but without embryos were used to expose of two other OSL dosimeters. This procedure was repeated for each x-ray group. One dosimeter in each group was not exposed and served as a control. The exposed OSLs were then processed using an InLight Auto200 reader (Epsilon Landauer, Istanbul, Turkey) and the numbers read by the reader were recorded. Developmental parameters were monitored using a stereomicroscope (ZEISS Discovery V8, Germany) and recorded daily until 72 hpf. Locomotor activity The locomotor activity of the zebrafish embryos at 72 hpf was evaluated as described previously [25] . This was performed by placing a 60 mm Petri dish containing embryo medium on top of the motility wheel which is on the microscope stage. Then, by using an embryo poker tool the zebrafish embryo was positioned in the middle of the motility wheel and the time it took for an embryo to swim a predetermined distance was recorded and the average escape response was calculated. Biochemical Assays Zebrafish embryos were prepared as replicate pools of 72 hpf zebrafish. For each pool 100 embryos were homogenized in 1 ml PBS, followed by centrifuging briefly. The supernatant was used for the determination of biochemical parameters [26] . Total Protein Assay The method of Lowry was used to determine the levels of total proteins in the samples [27] . In this method, proteins first react with copper ions in an alkali medium and then they are reduced by Folin reagent. The absorbances are determined at 500 nm. The total protein levels were calculated and used to present the results per protein. Lipid Peroxidation Assay The method of Yagi was used to determine malondialdehyde (MDA) levels, the end product of lipid peroxidation (LPO), as thiobarbituric acid reactive substances. The extinction coefficient of 1.56x105 M -1 cm -1 was used LPO was expressed in terms of MDA equivalents as nmol MDA/mg protein [28] . Nitric Oxide Assay Nitric oxide (NO) was determined through the reduction of nitrate to nitrite by vanadium (III) chloride. The colored complex was measured at 540 nm with a spectrophotometer and the results were expressed as nmol NO/mg protein [29] . Glutathione-S-Transferase Assay The activity of glutathione-S-transferase (GST) was determined spectrophotometrically at 340 nm and the absorbance of the product formed by GSH and 1-chloro-2,4-dinitro-benzenin (CDNB) conjugation was evaluated [30] . Superoxide Dismutase Assay Superoxide dismutase (SOD) activity was determined using the method based on the ability of SOD to increase the effect of riboflavin-sensitized photo-oxidation of o-dianisidine. Superoxide activity is produced by illuminating the reaction mixture containing O-dianisidine dihydrochloride and riboflavin by the light of a fluorescent lamp. The oxidation of O-dia-nisidine is sensitized by riboflavin and enhanced by SOD and the increase is linearly dependent on the concentration of SOD. Absorbances at 0 and 8th min of the illumination were measured using a spectrophotometer at 460 nm and the net absorbances were calculated. The results were expressed as U/mg protein [31] . Glutathionine (GSH) Assay The amount of glutathione (GSH) in the samples was measured using Beutler’s method (1975), and the results are expressed as percentage GSH [32] . Acetylcholinesterase (AChE) assay The Ellman et al. method was used to measure the activity of acetylcholinesterase in the supernatants [33] . Acetylcholinesterase generates thiocholine in this mechanism, which combines with 5.5′-dithiobis (2-nitrobenzoic acid) to produce a yellow hue. At 412 nm, the yellow color’s intensity is evaluated and directly correlated with the sample’s enzyme activity. Catalase assay Using Aebi’s approach, the catalase activity was assessed in this study. The technique assesses the enzyme’s ability to convert hydrogen peroxide into water [34] . Reverse Transcription (cDNA synthesis) and Quantitative Real-Time PCR RNA was isolated from the embryos. Rneasy Mini Kit and Qiacube (Qiagen, Hilden, Germany) were used according to the instructions of the manufacturer. A single-stranded complementary DNA (cDNA) was obtained from 1 lg of total RNA using RT2 First Strand Kit (Qiagen, Hilden, Germany). DNA Master SYBR Green kit (Qiagen, Hilden, Germany) was used to perform RT-PCRs. The beta-actin is housekeeping gene and was used as a reference gene. Relative levels of transcription were calculated using the DD CT method based on the normalization of the values using the housekeeping gene [35] . The list of the primers used is shown in Table 1. Table 1 . Forward and reverse primers used in the study. Statistical Analysis The effects of low-dose ionizing radiation and MPTP on zebrafish embryos were assessed using one-way analysis of variance (ANOVA) test and Dunn's multiple comparison test was used as a post-hoc test to compare the effects of MPTP on low dose IR exposed embryos. Statistical analysis was performed using GraphPad Prism 10 and p<0.05 was considered significant. Results Results of absorbed dose Individual dose measurements were obtained by averaging the exposures within each LDIR group. The absorbed deep and shallow doses for the LDIR exposed group were both 0,065 mSv. Results of developmental analyses The representative images of the zebrafish embryos are presented in Fig. 1 yolc sac edema, pericardial edema and scoliosis were observed in all exposure groups at 24, 48 and 72 hpf. Results of locomotor activities Average speed, total distance swam and explored areas were determined to evaluate the effects of low dose ionising radiation and MPTP on locomotor activity. Compared to the control group average speed significantly decreased in MPTP, LDIR, and LDIR + MPTP groups (p < 0,0001). Total distance swam was found to be decreased in MPTP, LDIR and LDIR + MPTP groups (p < 0,0001). The explored areas also significantly decreased in MPTP, LDIR, and LDIR + MPTP groups (p < 0,0001; p < 0,01; p < 0,0001 respectively). Furthermore, when compared to the LDIR group; the average speed, total distance swam and explored areas of the LDIR + MPTP group were significantly lower (p < 0,0001). Similarly, compared to the LDIR group; the average speed, total distance swam and explored areas of the MPTP group were significantly low (p < 0,0001) (Fig. 2 ). Results of oxidant-antioxidant analyses Figure 3 A shows the outcomes of lipid peroxidation analyses for the control, MPTP, LDIR, and LDIR + MPTP groups. Compared to the control group malondialdehyde (MDA) concentrations were significantly increased in the MPTP, LDIR, and LDIR + MPTP groups (p < 0,0001; p < 0,05; p < 0,0001 respectively). Malondialdehyde (MDA) concentrations of the LDIR group were significantly lower when compared to the MPTP and MPTP + LDIR groups (p < 0,01, p < 0,0001 respectively). The results of Glutathione S-Transferase (GST) analyses of the control, MPTP, LDIR, and LDIR + MPTP groups are shown in Fig. 3 B. GST activity was significantly increased in the MPTP and the LDIR + MPTP groups compared to the control group (p < 0,0001; p < 0,001 respectively). Compared to the MPTP group, GST activity was significantly lower in the LDIR + MPTP group (p < 0,001). On the other hand, GST activity of the LDIR + MPTP group was significantly higher when compared to the LDIR group (p < 0,05). The results of CAT analyses of the control, MPTP, LDIR and LDIR + MPTP groups are shown in Fig. 3 C. CAT activities were significantly decreased in the MPTP and LDIR groups compared to the control group (p < 0,001; p < 0,05 respectively). In the LDIR + MPTP group, CAT activities were significantly increased compared to the control group (p < 0,01). CAT activities of the LDIR + MPTP group were significantly increased when compared with the MPTP and LDIR groups (p < 0,0001 and p < 0,001, respectively). In our study SOD activity was significantly increased in the MPTP, LDIR and LDIR + MPTP groups compared to the control group (p < 0,0001; p < 0,01; p < 0,0001 respectively). SOD activity was significantly higher in the MPTP group than in the LDIR and LDIR + MPTP groups (p < 0,0001; p < 0,001 respectively). On the other hand, SOD activity in the LDIR + MPTP group was significantly higher than in the LDIR group (p < 0,05) (Fig. 3 D). The results of GSH analyses of the control, MPTP, LDIR and LDIR + MPTP groups are shown in Fig. 3 E. GSH levels were significantly decreased in the MPTP and LDIR groups compared to the control group (p < 0,0001; p < 0,01 respectively). GSH levels of the LDIR group were significantly higher than in the MPTP group, and GSH levels in the LDIR + MPTP group were significantly higher than the in the MPTP group (p < 0,01 and p < 0,001, respectively) (Fig. 3 E). Results of AChE analysis The results of Acetylcholine esterase (AChE) analyses of the control, MPTP, LDIR and LDIR + MPTP groups are shown in Fig. 4 A. AChE activity was significantly decreased in the, MPTP, LDIR and LDIR + MPTP groups compared to the control group (p < 0,0001; p < 0,01; p < 0,0001 respectively). In the LDIR group, AChE activity was significantly higher than in the MPTP and LDIR + MPTP groups (p < 0,01). Results of NO analysis The results of nitric oxide (NO) analyses in the control, MPTP, LDIR and LDIR + MPTP groups are shown in Fig. 4 B. Accordingly, NO levels were significantly increased in the MPTP, LDIR, and LDIR + MPTP groups compared to the control group (p < 0,01; p < 0,05; p < 0,0001 respectively). NO levels in the LDIR + MPTP group were significantly higher than in the MPTP and LDIR groups (p < 0,01; p < 0,0001 respectively). Results of gene expression analysis The mRNA expression levels of pink1 of the control, MPTP, LDIR and LDIR + MPTP groups are shown in Fig. 5 A. pink1 expression was significantly increased in the MPTP, LDIR and LDIR + MPTP groups compared to the control group (p < 0,01; p < 0,0001; p < 0,01 respectively). pink1 expression was significantly higher in the LDIR group when compared to the MPTP and LDIR + MPTP groups compared to the LDIR group (p < 0,01 and p < 0,001 respectively). The results of parkin expression analyses of the control, MPTP, LDIR and LDIR + MPTP groups are shown in Fig. 5 B. parkin expression was significantly increased in the LDIR, MPTP and LDIR + MPTP groups compared to the control (p < 0,05, p < 0,001, and p < 0,05 respectively). In addition, when compared to the LDIR and LDIR + MPTP groups, the parkin expression of the MPTP group was significantly higher (p < 0,05 and p < 0,01 respectively). dj1 expressions was significantly increased in the MPTP, LDIR, and LDIR + MPTP groups compared to the control group (p < 0,05; p < 0,0001, and p < 0,05 respectively). dj1 expression was significantly higher in the LDIR group, compared to the MPTP and LDIR + MPTP groups (p < 0,001) (Fig. 5 C). The results of lrrk2 expression analyses of the control, MPTP, LDIR, and LDIR + MPTP groups are shown in Fig. 5 D. lrrk expressions were significantly increased in the MPTP and LDIR groups compared to the control group (p < 0,0001 and p < 0,05 respectively). In the MPTP group, lrrk2 expression was significantly higher than the LDIR and LDIR + MPTP groups (p < 0,0001). In addition, lrrk2 expression was significantly lower in the LDIR + MPTP group than in the LDIR group (p < 0.001). Discussion Ionising radiation; mainly x-rays, is used in the dental radiodiagnostics. Our study is the first to show that low-dose ionizing radiation can induce comparable effects to MPTP by activating the expression of PD genes associated with mitochondrial damage, in developing zebrafish embryos. The results of our study showed that LDIR exposure in zebrafish embryos altered oxidative and mitochondrial stress markers, as well as locomotor and cognitive function markers at levels comparable to MPTP exposure. Environmental exposures such as LDIR or MPTP can have adverse effects on health during the embryonic phase, a sensitive stage of development [ 20 ] . MPTP is a neurotoxin that has been shown to induce a selective loss of dopaminergic neurons in the mammalian midbrain, leading to characteristic symptoms of PD in various animal models including zebrafish [ 19 ] . Several studies showed that MPTP exposure reduces locomotor activity in zebrafish embryos [ 18 , 36 ] . In our study a significant decrease in locomotor activity was observed in all exposure groups, as evidenced by decreased average speed, total distance and areas explored. It was also noteworthy that the decrease in locomotor activity was accompanied by a decrease in AChE activity which is a cholinergic enzyme. Our results are consistent with those of Cansız et al. (2022) and Kollayan et al. (2024) who showed decreased AChE activity and decreased locomotor activity in zebrafish embryos treated with MPTP and LDIR, respectively [ 37 , 38 ] . The inhibition of locomotor activity in the LDIR group may be related to the deleterious effects of x-rays on cholinergic function [ 39 ] . While no difference was observed between the MPTP and LDIR + MPTP groups, Although the decrease was significant in the LDIR + MPTP group compared with the LDIR group, indicating that LDIR before neurotoxin exposure had no effect on locomotor activity. When MPP + is taken by the dopaminergic neurons, it inhibits mitochondrial oxidative phosphorylation complex 1, leading to mitochondrial stress. Mitophagy is the process by which damaged mitochondria are targeted for lysosomal degradation. pink1 and parkin work coordinately to regulate mitophagy [ 36 ] . Defects in pink1/parkin regulated mitophagy lead to the accumulation of damaged mitochondria which contributes to PD [ 40 ] . pink1 has been shown to protect cells against oxidative stress induced apoptosis [ 41 ] . Increased lrrk and pink1 expressions were found in MPTP exposed zebrafish embryos [ 37 ] . In a previous study, Ustündag et al. (2022) reported that pink1, parkin and lrrk expressions were significantly increased in the MPTP exposed zebrafish embryos [ 36 ] . As mitochondrial impairment activates lrrk2 before the presence of neurodegeneration and pink1 regulates mitophagy to target the damaged mitochondria for lysosomal degradation, increased lrrk and pink1 expressions may indicate mitochondrial stress due to MPTP and LDIR exposures [ 40 , 42 ] . Consistent with these reports MPTP has been show to increase pink1, parkin and lrrk gene expression in zebrafish embryos. dj1 plays a role in antioxidative stress reactions and the loss of its function has been suggested to lead to the onset of PD [ 43 ] . Decreased dj1 gene expressions was found in MPTP exposed zebrafish embryos [ 36 , 37 ] . In contrast to these studies, we found increased expressions of the dj1 gene. In our study, LDIR, MPTP and LDIR combined with MPTP exposure led to changes in the expression of pink1, parkin, dj1 and lrrk genes as indicators of mitochondrial damage. The expression of pink1, parkin and dj1 genes increased in all exposure groups compared to the control group. On the other hand, lrrk gene expression was found to be increased in the LDIR and MPTP groups, whereas a decrease was observed in the LDIR combined with MPTP exposure group. Our study is the first to show that LDIR may act like MPTP by activating the expressions of genes that regulate mitochondrial stress in developing zebrafish embryos. One of the major causes of radiation-induced damage is the generation of ROS and free radicals as a result of radiation exposure. ROS react with tissues, causing LPO, DNA damages, and enzyme inactivation, all of which are radiation damage mediators [ 44 ] . Rats showed an increase in LPO levels following exposure to 3 Gray (Gy) of x-rays [ 45 ] . Consistent with this finding, our group has recently shown increased oxidative stress in low dose x-ray radiation exposed zebrafish embryos as evidenced by increased LPO [ 38 , 44 ] . Similarly, increased LPO levels were observed in LDIR-exposed zebrafish embryos in our study. In the neurodegenerative diseases including PD, the generation of free radicals particularly ROS and RNS, is known to be detrimental, as they affect proteins, lipids, and nucleic acids [ 46 – 48 ] . The increase in ROSs damages the target neuronal cells [ 49 ] and dopaminergic neurons are more susceptible to oxidative stress. MPTP [ 14 ] and IR [ 13 ] are environmental factors that may influence the risk of PD by inducing oxidative stress on the neurobiological responses [ 16 ] . As a neurotoxin MPTP has been shown to induce selective loss of dopaminergic neurons in the mammalian midbrain, leading to characteristic symptoms of PD in various animal models including zebrafish [ 19 , 36 ] . Zebrafish embryos have been shown to be susceptible to the dopaminergic neurotoxin MPTP [ 18 ] . When MPTP is metabolized by monoamine oxidase-B, 1-Methyl-4- phenylpyridinium (ion) (MPP + ) is formed as the ultimate toxic agent [ 36 ] . The conversion of MPTP to MPP + has been shown to destroy the dopaminergic neurons by leading to the generation of hydroxyl radicals and causing LPO in experimental models [ 50 – 55 ] . Similarly in our study, increased LPO levels were observed in MPTP-exposed zebrafish embryos when compared with the control group. Another free radical produced by irradiation is NO, which is a biological mediator in biochemical reactions [ 56 ] . After high-dose whole-body x-ray irradiation, in vivo NO generation was measured in mice, resulting in delayed NO synthase expression and NO formation [ 57 ] . Increased NO levels were found in rabbits after exposure to 550 rads of x-rays [ 58 ] . In previous studies from our zebrafish research laboratory, Karagoz et al. (2023) and Kollayan et al. (2024) found a significant increase in NO levels in LDIR-exposed zebrafish embryos [ 38 , 44 ] . A similar increase in NO levels was observed in the LDIR group. In addition, we observed a dramatic and significant increase in LPO and NO levels in the LDIR combined with MPTP group compared to the LDIR group, indicating that MPTP treatment enhanced LPO and NO levels and increased oxidative stress in the LDIR-exposed zebrafish embryos. In addition to being an endogenous free radical, NO is a potent vasodilator. The edema associated with radiation has been reported to indicate an increase in the permeability of the capillary wall. Vessel dilation is one of the most common early changes observed in the capillaries and pre-arterioles after irradiation [ 38 ] . Accordingly, increased NO levels in the ionizing radiation groups may be associated with the observed pericardial edema findings. Consistent with this, in our study the increase in NO levels was accompanied by a pericardial edema finding. According to the oxidative stress hypothesis of PD, NO plays an important role in creating an environment that can damage the dopaminergic neurons [ 59 ] . The results of our study showed increased NO levels in MPTP-exposed zebrafish embryos. In a previous study, Cansız et al. (2022) observed increased NO levels in MPTP-exposed zebrafish embryos, and our results were in agreement with their findings [ 37 ] . In addition, we observed a significant increase in NO levels in the LDIR + MPTP group compared to the MPTP and LDIR groups, suggesting an enhancement of the NO response by both treatments. SOD can protect cells from the damaging effects of reactive oxygen species through the detoxification of superoxide radicals [ 60 ] . There are several reports on the role of SOD activities in the effect of LDIR [ 62 , 63 ] . Kojima et al. (1999) showed that SOD activity was increased of in LDIR-exposed mice [ 52 ] . Increased SOD activity has also been reported in zebrafish embryos exposed to LDIR [ 44 ] . In the present study, we observed increased SOD activities in zebrafish embryos exposed to MPTP, LDIR and LDIR combined with MPTP. Increased levels of NO or LPO due to LDIR and MPTP exposure may be the reason for the increase in SOD activity. Catalase is also an antioxidant enzyme that prevents hydroxyl radical generation and separates hydrogen peroxide into water and oxygen [ 63 ] . Few studies in the literature show that environmental factors affect the CAT activity [ 52 , 64 ] . Kollayan et al. (2024) reported that dental x-ray exposure decreased CAT activities in zebrafish embryos [ 38 ] . We found decreased CAT activity in LDIR and MPTP exposed zebrafish embryos and when compared to the LDIR and MPTP groups an activation of CAT activity was observed in the LDIR combined with MPTP exposure group. In our study, exposure to MPTP and LDIR caused changes in the activities of elements of the antioxidant defence system, as well as causing oxidative stress. We found decreased levels of GSH, which is the major antioxidant in cells, in MPTP and LDIR exposed zebrafish embryos. In accordance with our results Karagoz et al. (2023) reported decreased GSH levels in standard panoramic x-ray exposed zebrafish embryos, however, in the same study increased GSH levels were found in the pedodontic panoramic x-ray exposed group [ 44 ] . It has been reported that minor oxidative stress can boost intracellular GSH levels as an adaptive response, whereas severe oxidative stress might cause GSH levels to drop [ 65 ] . Exposure to MPTP has also been shown to decrease GSH levels [ 66 ] . The reason for the more dramatic decrease in GSH levels in the MPTP group compared to the LDIR group may be due to the higher LPO and NO, as indicators of oxidative stress in the MPTP group. GST enzymes catalyse the conjugation of reduced glutathione to electrophilic groups on the substrate molecules to make the products of oxidative stress soluble for their easy elimination from the cell [ 37 , 67 ] . Previous studies have shown an increase in GST levels due to oxidative stress and environmental stimuli [ 68 , 69 ] . Transient overexpression of GST has been shown in MPTP-injected mice, suggesting that GST acts as an endogenous regulator of the cellular stress induced by MPTP to protect cells from ROS [ 67 ] . The expression and activity of GST were detected as early as 4 hpf in zebrafish embryos showing that GST enzymes are not only expressed but are also active during early embryogenesis [ 70 ] . In accordance with these reports we found significant increases in the GST activities of the MPTP and LDIR combined with MPTP exposure groups. Increased GST activities in x-ray exposed zebrafish embryos have been reported previously [ 38 , 25 ] . On the other hand, the increase in the GST activity of the LDIR group was not statistically significant. LDIR exposure before MPTP treatment decreased the GST response compared to the MPTP group, but increased it compared to the LDIR group. In the practice of dental radiology, lower radiation doses are required for digital imaging sensors [ 71 ] . Considering our previous research, an exposure time of 0.08 seconds, which is one of the frequently used exposure settings in periapical dental imaging, was chosen as a representative exposure time of LDIR [ 38 ] . Our study is the first to determine the effects of LDIR from a dental diagnostic x-ray unit on the response to MPTP, focusing on the oxidant-antioxidant system and the expression of genes related with in PD as markers of mitochondrial homeostasis. Based on our results, we may suggest that exposure to LDIR during the embryonic period may alter the response to neurotoxin treatment in terms of mitochondrial stress, oxidant-antioxidant status, locomotor and cholinergic functions in zebrafish embryos. On the other hand, in the LDIR + MPTP group, the aggravation of the response to oxidative damage by LDIR exposure before MPTP treatment, and the suppression of the expressions of genes responsible for the regulation of mitochondrial stress are the issues that need to be supported by further research and their mechanism needs to be revealed. Statements and Declarations Acknowledgement The authors would like to thank Dr. Hüseyin Gündüz for providing OSL dosimetries and further guidance. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Consent to participate All the authors have agreed for authorship, read and approved the manuscript, and given consent to participate. Consent for publication All the authors have agreed for authorship, read and approved the manuscript, and given consent for publication. Author contributions statement Conception/Design of study: Ebru Emekli-Alturfan; Sebnem Ercalik Yalcinkaya; Ezgi Cahide Aydas Bayramov. Data Acquisition: Ezgi Cahide Aydas Bayramov; Merih Beler; Derya Cansiz, Ismail Unal; Gizem Egilmezer; Selma Yaltkaya; Atakan Karagoz. Data Analysis/Interpretation: Ebru Emekli-Alturfan; Sebnem Ercalik Yalcinkaya; Ezgi Cahide Aydas Bayramov.Drafting Manuscript: Sebnem Ercalik Yalcinkaya; Ezgi Cahide Aydas Bayramov. Critical Revision of Manuscript: Ebru Emekli-Alturfan, Sebnem Ercalık Yalcinkaya.Supervision: Ebru Emekli-Alturfan; Sebnem Ercalik Yalcinkaya. Disclosure statement The authors report no conflicts of interest. Funding This study was supported by Marmara University Scientific Research Project Unit (BAPKO) number of 10981. Ethical approval Currently, the European Commission Directive 2010/63/EU, permits experimentation in fish embryos at earliest life stages without being regulated as animal experiments; zebrafish are considered models in vitro until 120 hpf (http://data.europa.eu/eli/dir/2010/63/2019-06-26; accessed 21 May 2025 EFSA opinion: https://doi.org/10.2903/j.efsa.2005.292; accessed 21 May 2025). References Hwang, S. Y., Choi, E. S., Kim, Y. S., Gim, B. E., Ha, M. & Kim, H. Y. Health effects from exposure to dental diagnostic x-ray. Environmental health and toxicology. 33, 018017; https://doi.org/10.5620/eht.e2018017 (2018). Bahreyni Toossi, M. T., Akbari, F. & Bayani Roodi, S. Radiation exposure to critical organs in panoramic dental examination. Acta medica Iranica . 50, 809–813 (2012). Brenner, D. J. et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6594085","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":467424271,"identity":"6c2916ef-a6ab-4341-bc6d-9b5c6b5aa1e0","order_by":0,"name":"Ezgi Cahide Aydas Bayramov","email":"","orcid":"","institution":"Marmara University","correspondingAuthor":false,"prefix":"","firstName":"Ezgi","middleName":"Cahide Aydas","lastName":"Bayramov","suffix":""},{"id":467424273,"identity":"bf191ce5-565e-4e5a-a82f-78c39002e6cd","order_by":1,"name":"Merih Beler","email":"","orcid":"","institution":"Marmara University","correspondingAuthor":false,"prefix":"","firstName":"Merih","middleName":"","lastName":"Beler","suffix":""},{"id":467424274,"identity":"784efece-6ce9-402f-ad83-6da734c170d9","order_by":2,"name":"Derya Cansiz","email":"","orcid":"","institution":"Istanbul Medipol University","correspondingAuthor":false,"prefix":"","firstName":"Derya","middleName":"","lastName":"Cansiz","suffix":""},{"id":467424275,"identity":"b0ba516b-6b8b-41fc-9c93-69e09a9e98e6","order_by":3,"name":"Ismail Unal","email":"","orcid":"","institution":"Istanbul Medipol University","correspondingAuthor":false,"prefix":"","firstName":"Ismail","middleName":"","lastName":"Unal","suffix":""},{"id":467424278,"identity":"225316ea-634a-4044-bd28-f2701ead9b0a","order_by":4,"name":"Gizem Egilmezer","email":"","orcid":"","institution":"Marmara University","correspondingAuthor":false,"prefix":"","firstName":"Gizem","middleName":"","lastName":"Egilmezer","suffix":""},{"id":467424280,"identity":"66b82ce4-dcaf-4f0a-a77f-e32a684bcb1a","order_by":5,"name":"Selma Yaltkaya","email":"","orcid":"","institution":"Marmara University","correspondingAuthor":false,"prefix":"","firstName":"Selma","middleName":"","lastName":"Yaltkaya","suffix":""},{"id":467424282,"identity":"fa47d463-9e67-484a-8a5a-dd4318d66e7c","order_by":6,"name":"Atakan Karagoz","email":"","orcid":"","institution":"Fenerbahçe University","correspondingAuthor":false,"prefix":"","firstName":"Atakan","middleName":"","lastName":"Karagoz","suffix":""},{"id":467424284,"identity":"70b5d85e-e844-4a7b-ae7a-e5568b8bf2f5","order_by":7,"name":"Ebru Emekli-Alturfan","email":"","orcid":"","institution":"Marmara University","correspondingAuthor":false,"prefix":"","firstName":"Ebru","middleName":"","lastName":"Emekli-Alturfan","suffix":""},{"id":467424285,"identity":"6da2dc18-5d8d-43b5-a0fc-23c21a454dbe","order_by":8,"name":"Sebnem Ercalik Yalcinkaya","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYFACHgjFD8SMDSAWM3MDcVokG8BaDIBaGInUYnAApoWBgBb59t5jDz7m2OQb3z588OOMij/R/O1ALT8qtuHUYnDmXLrhzG1pltvOpSVLbjhjkDvjMGMDY8+Z27i1SOSYSfNuO2xgdobHjPFhm0FuA1ALM2Mbbi3y89+YSf/d9t/AuAek5Z9B7nxCWhhu8JhJM247YGDAA9SyscEgdwMhLQZn8tIke7clG0icYUuWnHHMOHcjUMtBfH6Rbz97TOLnNjsD/h7mgx97auRy550/fPDBjwo8DsMKDpCofhSMglEwCkYBGgAAr7FYnc7w1UYAAAAASUVORK5CYII=","orcid":"","institution":"Marmara University","correspondingAuthor":true,"prefix":"","firstName":"Sebnem","middleName":"Ercalik","lastName":"Yalcinkaya","suffix":""}],"badges":[],"createdAt":"2025-05-05 11:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6594085/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6594085/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-29382-8","type":"published","date":"2025-11-25T15:58:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84203295,"identity":"5c46acc0-ecd5-42a4-a06f-6349e1e44f0a","added_by":"auto","created_at":"2025-06-09 08:40:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":81352,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of the zebrafish embryos in the groups. C: Control; MPTP: MPTP-exposed group; LDIR: LDIR-exposed group; LDIR+MPTP: MPTP-exposed combined with LDIR-exposed group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6594085/v1/12a0eabdcdea31b34796b838.png"},{"id":84204100,"identity":"7c5c5f07-2f66-4658-9cad-ae437a26b3bb","added_by":"auto","created_at":"2025-06-09 08:48:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":28894,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Average speed (mm/sec); (B) Total distance swam (mm); (C) Explored areas (%) of the groups\u003cstrong\u003e \u003c/strong\u003e*p\u0026lt;0,05; **p\u0026lt;0,01; ***p\u0026lt;0,001; ****p\u0026lt;0,0001 C: Control group; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine exposed group; LDIR: low dose ionising radiation exposed group; LDIR+MPTP: MPTP combined with LDIR exposed group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6594085/v1/d5ea13acff9a5fe47e54ee49.png"},{"id":84203296,"identity":"ccb17a73-90b7-4cc9-8bee-8a41e935d6e9","added_by":"auto","created_at":"2025-06-09 08:40:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34358,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Malondialdehyde (MDA) levels as an index of lipid peroxidation (LPO); (B) Glutathione S-transferase (GST) activities of the groups; (C) Catalase (CAT) activities of the groups; (D) Superoxide dismutase (SOD) activities of the groups; (E) Glutathione (GSH) levels of the groups. *p\u0026lt;0,05; **p \u0026lt;0,01; ***p\u0026lt;0,001; ****p\u0026lt;0,0001 C: Control group; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine exposed group; LDIR: low-dose ionising radiation exposed group; LDIR+MPTP: MPTP combined with LDIR exposed group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6594085/v1/6fe69f0b5f59cefe220c1ea8.png"},{"id":84204101,"identity":"71485c9e-6ec9-4625-9029-0c662ec2aa26","added_by":"auto","created_at":"2025-06-09 08:48:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":21349,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Acetylcholine esterase activities (AChE) of the groups (B) Nitric oxide (NO) levels of the groups. (\u003cem\u003en\u003c/em\u003e=10) *\u0026lt;0,05; **p\u0026lt;0,01; ***p\u0026lt;0,001; ****p\u0026lt;0,0001 C: Control group; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine exposed group; LDIR: low dose ionising radiation exposed group; LDIR+MPTP: MPTP combined with LDIR exposed group.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6594085/v1/8d6cd8f064450b0b736d9054.png"},{"id":84205076,"identity":"4ffa83ea-0c27-45ee-a079-e540f3cfdf6a","added_by":"auto","created_at":"2025-06-09 08:56:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":33203,"visible":true,"origin":"","legend":"\u003cp\u003eBar graph presentation of the fold change of (A) \u003cem\u003epink1\u003c/em\u003e, (B) \u003cem\u003eparkin\u003c/em\u003e, (C) \u003cem\u003edj1\u003c/em\u003eand (D) \u003cem\u003elrrk2\u003c/em\u003e transcripts quantified by RT-PCR. All RT-PCR results are normalized to b-actin, the housekeeping gene and expressed as change from their respective controls. Data presented are mean ± SD. *p\u0026lt;0,05; **p\u0026lt;0,01; ***p\u0026lt;0,001; ****p\u0026lt;0,0001 C: Control group; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine exposed group; LDIR: low dose ionising radiation exposed group; LDIR+MPTP: MPTP combined with LDIR exposed group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6594085/v1/e383e4f00423a1a12b1a930b.png"},{"id":97179254,"identity":"77ecc5a1-3f2f-4457-842b-49738d92ff78","added_by":"auto","created_at":"2025-12-01 16:14:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1252015,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6594085/v1/9bec49a7-91dc-40f2-be3b-1e3eaee62012.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Low-dose ionising radiation exerts effects comparable to MPTP through alterations in locomotor activity, oxidant/antioxidant status and mitochondrial homeostasis in zebrafish embryos","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDental diagnostic imaging is an integral part of the practice of dentistry and certain types of examinations\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Radiation doses have been reduced with the development of digital techniques\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Exposure to ionising radiation (IR) is harmful, although radiation-related health risks decrease at lower doses\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. In view of the lifetime frequency of exposure to dental diagnostic x-rays, even a small increase in health risk would be of significant public health importance\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, and repeated exposure may increase the risk of cancer\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe mechanism of deleterious IR effects is strongly linked to increased oxidative stress in irradiated tissues\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. IR is able to penetrate the cells of living organisms, where it induces the ionization of both organic and inorganic compounds\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Due to the high water content in cells, the radiolysis of water molecules by IR is the main process that contributes to the increased formation of reactive oxygen species (ROS)\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. ROS react rapidly with macromolecules, including proteins, nucleic acids and lipids, leading to cell dysfunction, and apoptotic cell death\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. As a result of elevated oxidative stress, not only direct negative side effects, but also ROS-related diseases can develop. Therefore, it is particulary important to identify effective and safe prophylactic compounds to protect humans from IR damage\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eParkinson's disease (PD), clinically characterized by tremor, bradykinesia and rigidity, is the second most common neurodegenerative disorder in the world. The disease involves loss of dopaminergic neurons in the substantia nigra pars compacta (SNc), leading to dopamine depletion at nerve terminals\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Multiple environmental and genetic factors play a key role in the etiology of PD\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Environmental factors influencing PD risk include mitochondrial toxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e and IR\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Oxidative stress has also been linked to PD\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e, and IR induces oxidative stress causing neurobiological responses\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003eMPTP is a neurotoxin that induces selective loss of dopaminergic neurons in the mammalian midbrain. In different model organisms, exposure to MPTP leads to the characteristic symptoms of PD\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. MPTP exposure in zebrafish embryos has been shown to damage dopaminergic neurons and reduce the number of dopaminergic cells in the diencephalon\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. In zebrafish, the dopaminergic system is well characterized both in the embryonic and adult stages. For this reason, zebrafish is a suitable model organism to study the molecular pathways in neurodevelopment and neurotoxicology, as well as to explore potential therapeutic agents\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEnvironmental exposures during sensitive developmental windows in the embryonic phase can have lasting effects on the health\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Previous investigations have focused on elements such as DNA damage mechanisms in the adult organism, free radical generation, and elevated cancer markers at low doses of IR [21]. Cells with high levels of mitotic activity are known to be hypersensitive to radiation. Although organogenesis and especially neurogenesis in the early fetal period are the most susceptible periods of the fetus to radiation, research on LDIR exposure during the embryonic stage is limited\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Prenatal effects have been studied in animal models\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe hypothesized that LDIR exposure in developing zebrafish embryos would cause oxidative stress and mitochondrial stress, similar to MPTP, and could affect the locomotor functions of the embryo, and that LDIR exposure prior to MPTP could also affect the response to MPTP in the organism. To determine the molecular mechanisms of the effect of LDIR and MPTP, molecules involved in the oxidant-antioxidant system (malondialdehyde, nitric oxide, superoxide dismutase, glutathione S-transferase, glutathionine and catalase) and activity of acetylcholinesterase were evaluated to determine cholinergic effects. In addition, to assess whether MPTP and IR induced changes in mitochondrial stress-related genes associated with PD, the expressions of \u003cem\u003epink1, parkin, dj1\u003c/em\u003e, and \u003cem\u003elrrk2\u003c/em\u003e were determined.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eChemicals Tested\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMPTP (purity \u0026ge;98%) was purchased from Sigma-Aldrich, St Louis, MO, USA. It was analytical grade with the highest purity available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaintenance of Zebrafish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWild-type AB/AB strain zebrafish were maintained under apparently disease-free conditions. Animal husbandry and spawning were performed in accordance with the relevant guidelines and regulations and the protocols approved by the University of Marmara Institutional Animal Care and Use Committee. Fish were kept in an aquarium rack system (Zebtec, Tecniplast, Italy) at 27 \u0026plusmn; 1 \u0026deg;C under a light/dark cycle of 14/10-hour and they were fed with commercial flake fish food complemented with live Artemia twice a day. The pH of system water ranges from 6.9 to 7.2. Reverse osmosis water that contains 0.018 mg L\u003csup\u003e-1\u003c/sup\u003e Instant Ocean\u003csup\u003eTM\u003c/sup\u003e salt was used for all experiments\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eZebrafish embryos (AB/AB) were obtained from the Zebrafish Research Laboratory in Marmara University and maintained in E3 medium. After natural spawnings, fertilized embryos were gathered and staged according to their developmental and morphology as described before\u003csup\u003e[24]\u003c/sup\u003e. We used zebrafish embryos up to 72 hpf and the European Commission Directive 2010/63/EU, permits experimentation in fish embryos at earliest life stages without beingregulated as animal experiments; zebrafish are considered models in vitro until 120 hpf (http://data.europa.eu/eli/dir/2010/63/2019-06-26; accessed 21 May 2025 EFSA opinion: https://doi.org/10.2903/j.efsa.2005.292; accessed 21 May 2025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEmbryo Exposure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZebrafish embryos of the AB/AB strain were selected under the stereomicroscope after fertilisation and divided into four groups including control group, MPTP-exposed group, LDIR- exposed group from a dental x-ray unit (0,08 seconds, 60 kVp, 7 mA) and MPTP-exposed combined with LDIR-exposed group.\u003c/p\u003e\n\u003cp\u003eThe dental x-ray unit, Belmont AR-33RK5EU (Osaka, Japan) (60 kVp, 7 mA) with long cone (30 cm) was used for the low-dose ionizing radiation exposure. The exposure time on the dental x-ray unit was set to 0,08 seconds. The MPTP, LDIR, and LDIR+MPTP groups were exposed to 0,08 seconds of x-ray, 800 \u0026micro;M MPTP, and 0,08 seconds of x-ray before 800 \u0026micro;M MPTP, respectively, at 24 h post-fertilization. Developmental parameters were monitored and documented daily under a stereomicroscope (Zeiss Discovery V8, Germany) until 72 hpf.\u003c/p\u003e\n\u003cp\u003eDuring the experiment, an OSL dosimeter (Epsilon Landauer, Istanbul, Turkey) was used for each exposure group (LDIR and LDIR+MPTP groups). These dosimeters had a sensitivity range of 0,05 mSv to 10,0\u0026thinsp;Sv with an uncertainty of 8% for deep and shallow dose Hp, which was done to compare the reliability of dose measurements. Each OSL dosimeter was placed in the centre of a petri dish to measure absorbed doses. The same conditions but without embryos were used to expose of two other OSL dosimeters. This procedure was repeated for each x-ray group. One dosimeter in each group was not exposed and served as a control. The exposed OSLs were then processed using an InLight Auto200 reader (Epsilon Landauer, Istanbul, Turkey) and the numbers read by the reader were recorded.\u003c/p\u003e\n\u003cp\u003eDevelopmental parameters were monitored using a stereomicroscope (ZEISS Discovery V8, Germany) and recorded daily until 72 hpf.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLocomotor activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe locomotor activity of the zebrafish embryos at 72 hpf was evaluated as described previously\u003csup\u003e[25]\u003c/sup\u003e. This was performed by placing a 60 mm Petri dish containing embryo medium on top of the motility wheel which is on the microscope stage. Then, by using an embryo poker tool the zebrafish embryo was positioned in the middle of the motility wheel and the time it took for an embryo to swim a predetermined distance was recorded and the average escape response was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical Assays\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZebrafish embryos were prepared as replicate pools of 72 hpf zebrafish. For each pool 100 embryos were homogenized in 1 ml PBS, followed by centrifuging briefly. The supernatant was used for the determination of biochemical parameters\u003csup\u003e[26]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal Protein Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe method of Lowry was used to determine the levels of total proteins in the samples\u003csup\u003e[27]\u003c/sup\u003e. In this method, proteins first react with copper ions in an alkali medium and then they are reduced by Folin reagent. The absorbances are determined at 500 nm. The total protein levels were calculated and used to present the results per protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLipid Peroxidation Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe method of Yagi was used to determine malondialdehyde (MDA) levels, the end product of lipid peroxidation (LPO), as thiobarbituric acid reactive substances. The extinction coefficient of 1.56x105 M\u003csup\u003e-1\u003c/sup\u003e cm\u003csup\u003e-1\u003c/sup\u003e was used LPO was expressed in terms of MDA equivalents as nmol MDA/mg protein\u003csup\u003e[28]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNitric Oxide Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNitric oxide (NO) was determined through the reduction of nitrate to nitrite by vanadium (III) chloride. The colored complex was measured at 540 nm with a spectrophotometer and the results were expressed as nmol NO/mg protein\u003csup\u003e[29]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlutathione-S-Transferase Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe activity of glutathione-S-transferase (GST) was determined spectrophotometrically at 340 nm and the absorbance of the product formed by GSH and 1-chloro-2,4-dinitro-benzenin (CDNB) conjugation was evaluated\u003csup\u003e[30]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSuperoxide Dismutase Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSuperoxide dismutase (SOD) activity was determined using the method based on the ability of SOD to increase the effect of riboflavin-sensitized photo-oxidation of o-dianisidine. Superoxide activity is produced by illuminating the reaction mixture containing O-dianisidine dihydrochloride and riboflavin by the light of a fluorescent lamp. The oxidation of O-dia-nisidine is sensitized by riboflavin and enhanced by SOD and the increase is linearly dependent on the concentration of SOD. Absorbances at 0 and 8th min of the illumination were measured using a spectrophotometer at 460 nm and the net absorbances were calculated. The results were expressed as U/mg protein\u003csup\u003e[31]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlutathionine (GSH)\u003c/strong\u003e \u003cstrong\u003eAssay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amount of glutathione (GSH) in the samples was measured using Beutler\u0026rsquo;s method (1975), and the results are expressed as percentage GSH\u003csup\u003e[32]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcetylcholinesterase (AChE) assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Ellman et al. method was used to measure the activity of acetylcholinesterase in the supernatants\u003csup\u003e[33]\u003c/sup\u003e. Acetylcholinesterase generates thiocholine in this mechanism, which combines with 5.5\u0026prime;-dithiobis (2-nitrobenzoic acid) to produce a yellow hue. At 412 nm, the yellow color\u0026rsquo;s intensity is evaluated and directly correlated with the sample\u0026rsquo;s enzyme activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCatalase assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing Aebi\u0026rsquo;s approach, the catalase activity was assessed in this study. The technique assesses the enzyme\u0026rsquo;s ability to convert hydrogen peroxide into water\u003csup\u003e[34]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReverse Transcription (cDNA synthesis) and Quantitative Real-Time PCR\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was isolated from the embryos. Rneasy Mini Kit and Qiacube (Qiagen, Hilden, Germany) were used according to the instructions of the manufacturer. A single-stranded complementary DNA (cDNA) was obtained from 1 lg of total RNA using RT2 First Strand Kit (Qiagen, Hilden, Germany). DNA Master SYBR Green kit (Qiagen, Hilden, Germany) was used to perform RT-PCRs. The beta-actin is housekeeping gene and was used as a reference gene. Relative levels of transcription were calculated using the DD CT method based on the normalization of the values using the housekeeping gene\u003csup\u003e[35]\u003c/sup\u003e. The list of the primers used is shown in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e. Forward and reverse primers used in the study.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of low-dose ionizing radiation and MPTP on zebrafish embryos were assessed using one-way analysis of variance (ANOVA) test and Dunn\u0026apos;s multiple comparison test was used as a post-hoc test to compare the effects of MPTP on low dose IR exposed embryos. Statistical analysis was performed using GraphPad Prism 10 and p\u0026lt;0.05 was considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eResults of absorbed dose\u003c/h2\u003e \u003cp\u003eIndividual dose measurements were obtained by averaging the exposures within each LDIR group. The absorbed deep and shallow doses for the LDIR exposed group were both 0,065 mSv.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eResults of developmental analyses\u003c/h2\u003e \u003cp\u003eThe representative images of the zebrafish embryos are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e yolc sac edema, pericardial edema and scoliosis were observed in all exposure groups at 24, 48 and 72 hpf.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eResults of locomotor activities\u003c/h2\u003e \u003cp\u003eAverage speed, total distance swam and explored areas were determined to evaluate the effects of low dose ionising radiation and MPTP on locomotor activity. Compared to the control group average speed significantly decreased in MPTP, LDIR, and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001). Total distance swam was found to be decreased in MPTP, LDIR and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001). The explored areas also significantly decreased in MPTP, LDIR, and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001; p\u0026thinsp;\u0026lt;\u0026thinsp;0,01; p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001 respectively). Furthermore, when compared to the LDIR group; the average speed, total distance swam and explored areas of the LDIR\u0026thinsp;+\u0026thinsp;MPTP group were significantly lower (p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001). Similarly, compared to the LDIR group; the average speed, total distance swam and explored areas of the MPTP group were significantly low (p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eResults of oxidant-antioxidant analyses\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA shows the outcomes of lipid peroxidation analyses for the control, MPTP, LDIR, and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups. Compared to the control group malondialdehyde (MDA) concentrations were significantly increased in the MPTP, LDIR, and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001; p\u0026thinsp;\u0026lt;\u0026thinsp;0,05; p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001 respectively). Malondialdehyde (MDA) concentrations of the LDIR group were significantly lower when compared to the MPTP and MPTP\u0026thinsp;+\u0026thinsp;LDIR groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0,01, p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001 respectively).\u003c/p\u003e \u003cp\u003eThe results of Glutathione S-Transferase (GST) analyses of the control, MPTP, LDIR, and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB. GST activity was significantly increased in the MPTP and the LDIR\u0026thinsp;+\u0026thinsp;MPTP groups compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001; p\u0026thinsp;\u0026lt;\u0026thinsp;0,001 respectively). Compared to the MPTP group, GST activity was significantly lower in the LDIR\u0026thinsp;+\u0026thinsp;MPTP group (p\u0026thinsp;\u0026lt;\u0026thinsp;0,001). On the other hand, GST activity of the LDIR\u0026thinsp;+\u0026thinsp;MPTP group was significantly higher when compared to the LDIR group (p\u0026thinsp;\u0026lt;\u0026thinsp;0,05).\u003c/p\u003e \u003cp\u003eThe results of CAT analyses of the control, MPTP, LDIR and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC. CAT activities were significantly decreased in the MPTP and LDIR groups compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0,001; p\u0026thinsp;\u0026lt;\u0026thinsp;0,05 respectively). In the LDIR\u0026thinsp;+\u0026thinsp;MPTP group, CAT activities were significantly increased compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0,01). CAT activities of the LDIR\u0026thinsp;+\u0026thinsp;MPTP group were significantly increased when compared with the MPTP and LDIR groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001 and p\u0026thinsp;\u0026lt;\u0026thinsp;0,001, respectively).\u003c/p\u003e \u003cp\u003eIn our study SOD activity was significantly increased in the MPTP, LDIR and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001; p\u0026thinsp;\u0026lt;\u0026thinsp;0,01; p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001 respectively). SOD activity was significantly higher in the MPTP group than in the LDIR and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001; p\u0026thinsp;\u0026lt;\u0026thinsp;0,001 respectively). On the other hand, SOD activity in the LDIR\u0026thinsp;+\u0026thinsp;MPTP group was significantly higher than in the LDIR group (p\u0026thinsp;\u0026lt;\u0026thinsp;0,05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe results of GSH analyses of the control, MPTP, LDIR and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE. GSH levels were significantly decreased in the MPTP and LDIR groups compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001; p\u0026thinsp;\u0026lt;\u0026thinsp;0,01 respectively). GSH levels of the LDIR group were significantly higher than in the MPTP group, and GSH levels in the LDIR\u0026thinsp;+\u0026thinsp;MPTP group were significantly higher than the in the MPTP group (p\u0026thinsp;\u0026lt;\u0026thinsp;0,01 and p\u0026thinsp;\u0026lt;\u0026thinsp;0,001, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eResults of AChE analysis\u003c/h2\u003e \u003cp\u003eThe results of Acetylcholine esterase (AChE) analyses of the control, MPTP, LDIR and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. AChE activity was significantly decreased in the, MPTP, LDIR and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001; p\u0026thinsp;\u0026lt;\u0026thinsp;0,01; p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001 respectively). In the LDIR group, AChE activity was significantly higher than in the MPTP and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0,01).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eResults of NO analysis\u003c/h2\u003e \u003cp\u003eThe results of nitric oxide (NO) analyses in the control, MPTP, LDIR and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. Accordingly, NO levels were significantly increased in the MPTP, LDIR, and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0,01; p\u0026thinsp;\u0026lt;\u0026thinsp;0,05; p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001 respectively). NO levels in the LDIR\u0026thinsp;+\u0026thinsp;MPTP group were significantly higher than in the MPTP and LDIR groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0,01; p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001 respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eResults of gene expression analysis\u003c/h2\u003e \u003cp\u003eThe mRNA expression levels of \u003cem\u003epink1\u003c/em\u003e of the control, MPTP, LDIR and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. \u003cem\u003epink1\u003c/em\u003e expression was significantly increased in the MPTP, LDIR and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0,01; p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001; p\u0026thinsp;\u0026lt;\u0026thinsp;0,01 respectively). \u003cem\u003epink1\u003c/em\u003e expression was significantly higher in the LDIR group when compared to the MPTP and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups compared to the LDIR group (p\u0026thinsp;\u0026lt;\u0026thinsp;0,01 and p\u0026thinsp;\u0026lt;\u0026thinsp;0,001 respectively).\u003c/p\u003e \u003cp\u003eThe results of \u003cem\u003eparkin\u003c/em\u003e expression analyses of the control, MPTP, LDIR and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. \u003cem\u003eparkin\u003c/em\u003e expression was significantly increased in the LDIR, MPTP and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0,05, p\u0026thinsp;\u0026lt;\u0026thinsp;0,001, and p\u0026thinsp;\u0026lt;\u0026thinsp;0,05 respectively).\u003c/p\u003e \u003cp\u003eIn addition, when compared to the LDIR and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups, the \u003cem\u003eparkin\u003c/em\u003e expression of the MPTP group was significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0,05 and p\u0026thinsp;\u0026lt;\u0026thinsp;0,01 respectively).\u003c/p\u003e \u003cp\u003e \u003cem\u003edj1\u003c/em\u003e expressions was significantly increased in the MPTP, LDIR, and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0,05; p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001, and p\u0026thinsp;\u0026lt;\u0026thinsp;0,05 respectively). \u003cem\u003edj1\u003c/em\u003e expression was significantly higher in the LDIR group, compared to the MPTP and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0,001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThe results of \u003cem\u003elrrk2\u003c/em\u003e expression analyses of the control, MPTP, LDIR, and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD. \u003cem\u003elrrk\u003c/em\u003e expressions were significantly increased in the MPTP and LDIR groups compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001 and p\u0026thinsp;\u0026lt;\u0026thinsp;0,05 respectively). In the MPTP group, \u003cem\u003elrrk2\u003c/em\u003e expression was significantly higher than the LDIR and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001). In addition, \u003cem\u003elrrk2\u003c/em\u003e expression was significantly lower in the LDIR\u0026thinsp;+\u0026thinsp;MPTP group than in the LDIR group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIonising radiation; mainly x-rays, is used in the dental radiodiagnostics. Our study is the first to show that low-dose ionizing radiation can induce comparable effects to MPTP by activating the expression of PD genes associated with mitochondrial damage, in developing zebrafish embryos. The results of our study showed that LDIR exposure in zebrafish embryos altered oxidative and mitochondrial stress markers, as well as locomotor and cognitive function markers at levels comparable to MPTP exposure.\u003c/p\u003e \u003cp\u003eEnvironmental exposures such as LDIR or MPTP can have adverse effects on health during the embryonic phase, a sensitive stage of development\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. MPTP is a neurotoxin that has been shown to induce a selective loss of dopaminergic neurons in the mammalian midbrain, leading to characteristic symptoms of PD in various animal models including zebrafish\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Several studies showed that MPTP exposure reduces locomotor activity in zebrafish embryos\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. In our study a significant decrease in locomotor activity was observed in all exposure groups, as evidenced by decreased average speed, total distance and areas explored. It was also noteworthy that the decrease in locomotor activity was accompanied by a decrease in AChE activity which is a cholinergic enzyme. Our results are consistent with those of Cansız et al. (2022) and Kollayan et al. (2024) who showed decreased AChE activity and decreased locomotor activity in zebrafish embryos treated with MPTP and LDIR, respectively\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. The inhibition of locomotor activity in the LDIR group may be related to the deleterious effects of x-rays on cholinergic function\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. While no difference was observed between the MPTP and LDIR\u0026thinsp;+\u0026thinsp;MPTP groups, Although the decrease was significant in the LDIR\u0026thinsp;+\u0026thinsp;MPTP group compared with the LDIR group, indicating that LDIR before neurotoxin exposure had no effect on locomotor activity.\u003c/p\u003e \u003cp\u003eWhen MPP\u003csup\u003e+\u003c/sup\u003e is taken by the dopaminergic neurons, it inhibits mitochondrial oxidative phosphorylation complex 1, leading to mitochondrial stress. Mitophagy is the process by which damaged mitochondria are targeted for lysosomal degradation. \u003cem\u003epink1\u003c/em\u003e and \u003cem\u003eparkin\u003c/em\u003e work coordinately to regulate mitophagy\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Defects in \u003cem\u003epink1/parkin\u003c/em\u003e regulated mitophagy lead to the accumulation of damaged mitochondria which contributes to PD\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003epink1\u003c/em\u003e has been shown to protect cells against oxidative stress induced apoptosis\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Increased \u003cem\u003elrrk\u003c/em\u003e and \u003cem\u003epink1\u003c/em\u003e expressions were found in MPTP exposed zebrafish embryos\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. In a previous study, Ust\u0026uuml;ndag et al. (2022) reported that \u003cem\u003epink1, parkin\u003c/em\u003e and \u003cem\u003elrrk\u003c/em\u003e expressions were significantly increased in the MPTP exposed zebrafish embryos\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. As mitochondrial impairment activates \u003cem\u003elrrk2\u003c/em\u003e before the presence of neurodegeneration and \u003cem\u003epink1\u003c/em\u003e regulates mitophagy to target the damaged mitochondria for lysosomal degradation, increased \u003cem\u003elrrk\u003c/em\u003e and \u003cem\u003epink1\u003c/em\u003e expressions may indicate mitochondrial stress due to MPTP and LDIR exposures\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Consistent with these reports MPTP has been show to increase \u003cem\u003epink1, parkin and lrrk\u003c/em\u003e gene expression in zebrafish embryos.\u003c/p\u003e \u003cp\u003e \u003cem\u003edj1\u003c/em\u003e plays a role in antioxidative stress reactions and the loss of its function has been suggested to lead to the onset of PD\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Decreased \u003cem\u003edj1\u003c/em\u003egene expressions was found in MPTP exposed zebrafish embryos\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. In contrast to these studies, we found increased expressions of the \u003cem\u003edj1\u003c/em\u003egene.\u003c/p\u003e \u003cp\u003eIn our study, LDIR, MPTP and LDIR combined with MPTP exposure led to changes in the expression of \u003cem\u003epink1, parkin, dj1 and lrrk\u003c/em\u003e genes as indicators of mitochondrial damage. The expression of \u003cem\u003epink1, parkin\u003c/em\u003e and \u003cem\u003edj1\u003c/em\u003egenes increased in all exposure groups compared to the control group. On the other hand, \u003cem\u003elrrk\u003c/em\u003e gene expression was found to be increased in the LDIR and MPTP groups, whereas a decrease was observed in the LDIR combined with MPTP exposure group. Our study is the first to show that LDIR may act like MPTP by activating the expressions of genes that regulate mitochondrial stress in developing zebrafish embryos.\u003c/p\u003e \u003cp\u003eOne of the major causes of radiation-induced damage is the generation of ROS and free radicals as a result of radiation exposure. ROS react with tissues, causing LPO, DNA damages, and enzyme inactivation, all of which are radiation damage mediators\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Rats showed an increase in LPO levels following exposure to 3 Gray (Gy) of x-rays\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Consistent with this finding, our group has recently shown increased oxidative stress in low dose x-ray radiation exposed zebrafish embryos as evidenced by increased LPO\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Similarly, increased LPO levels were observed in LDIR-exposed zebrafish embryos in our study.\u003c/p\u003e \u003cp\u003eIn the neurodegenerative diseases including PD, the generation of free radicals particularly ROS and RNS, is known to be detrimental, as they affect proteins, lipids, and nucleic acids\u003csup\u003e[\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. The increase in ROSs damages the target neuronal cells\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e and dopaminergic neurons are more susceptible to oxidative stress. MPTP\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e and IR\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e are environmental factors that may influence the risk of PD by inducing oxidative stress on the neurobiological responses\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. As a neurotoxin MPTP has been shown to induce selective loss of dopaminergic neurons in the mammalian midbrain, leading to characteristic symptoms of PD in various animal models including zebrafish\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Zebrafish embryos have been shown to be susceptible to the dopaminergic neurotoxin MPTP\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. When MPTP is metabolized by monoamine oxidase-B, 1-Methyl-4- phenylpyridinium (ion) (MPP\u003csup\u003e+\u003c/sup\u003e) is formed as the ultimate toxic agent\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. The conversion of MPTP to MPP\u003csup\u003e+\u003c/sup\u003e has been shown to destroy the dopaminergic neurons by leading to the generation of hydroxyl radicals and causing LPO in experimental models\u003csup\u003e[\u003cspan additionalcitationids=\"CR51 CR52 CR53 CR54\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. Similarly in our study, increased LPO levels were observed in MPTP-exposed zebrafish embryos when compared with the control group.\u003c/p\u003e \u003cp\u003eAnother free radical produced by irradiation is NO, which is a biological mediator in biochemical reactions\u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e. After high-dose whole-body x-ray irradiation, in vivo NO generation was measured in mice, resulting in delayed NO synthase expression and NO formation\u003csup\u003e[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. Increased NO levels were found in rabbits after exposure to 550 rads of x-rays\u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e. In previous studies from our zebrafish research laboratory, Karagoz et al. (2023) and Kollayan et al. (2024) found a significant increase in NO levels in LDIR-exposed zebrafish embryos\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. A similar increase in NO levels was observed in the LDIR group. In addition, we observed a dramatic and significant increase in LPO and NO levels in the LDIR combined with MPTP group compared to the LDIR group, indicating that MPTP treatment enhanced LPO and NO levels and increased oxidative stress in the LDIR-exposed zebrafish embryos.\u003c/p\u003e \u003cp\u003eIn addition to being an endogenous free radical, NO is a potent vasodilator. The edema associated with radiation has been reported to indicate an increase in the permeability of the capillary wall. Vessel dilation is one of the most common early changes observed in the capillaries and pre-arterioles after irradiation\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Accordingly, increased NO levels in the ionizing radiation groups may be associated with the observed pericardial edema findings. Consistent with this, in our study the increase in NO levels was accompanied by a pericardial edema finding.\u003c/p\u003e \u003cp\u003eAccording to the oxidative stress hypothesis of PD, NO plays an important role in creating an environment that can damage the dopaminergic neurons\u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e. The results of our study showed increased NO levels in MPTP-exposed zebrafish embryos. In a previous study, Cansız et al. (2022) observed increased NO levels in MPTP-exposed zebrafish embryos, and our results were in agreement with their findings\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. In addition, we observed a significant increase in NO levels in the LDIR\u0026thinsp;+\u0026thinsp;MPTP group compared to the MPTP and LDIR groups, suggesting an enhancement of the NO response by both treatments.\u003c/p\u003e \u003cp\u003eSOD can protect cells from the damaging effects of reactive oxygen species through the detoxification of superoxide radicals\u003csup\u003e[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e. There are several reports on the role of SOD activities in the effect of LDIR\u003csup\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/sup\u003e. Kojima et al. (1999) showed that SOD activity was increased of in LDIR-exposed mice\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. Increased SOD activity has also been reported in zebrafish embryos exposed to LDIR\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. In the present study, we observed increased SOD activities in zebrafish embryos exposed to MPTP, LDIR and LDIR combined with MPTP. Increased levels of NO or LPO due to LDIR and MPTP exposure may be the reason for the increase in SOD activity.\u003c/p\u003e \u003cp\u003eCatalase is also an antioxidant enzyme that prevents hydroxyl radical generation and separates hydrogen peroxide into water and oxygen\u003csup\u003e[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/sup\u003e. Few studies in the literature show that environmental factors affect the CAT activity\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/sup\u003e. Kollayan et al. (2024) reported that dental x-ray exposure decreased CAT activities in zebrafish embryos\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. We found decreased CAT activity in LDIR and MPTP exposed zebrafish embryos and when compared to the LDIR and MPTP groups an activation of CAT activity was observed in the LDIR combined with MPTP exposure group.\u003c/p\u003e \u003cp\u003eIn our study, exposure to MPTP and LDIR caused changes in the activities of elements of the antioxidant defence system, as well as causing oxidative stress. We found decreased levels of GSH, which is the major antioxidant in cells, in MPTP and LDIR exposed zebrafish embryos. In accordance with our results Karagoz et al. (2023) reported decreased GSH levels in standard panoramic x-ray exposed zebrafish embryos, however, in the same study increased GSH levels were found in the pedodontic panoramic x-ray exposed group\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. It has been reported that minor oxidative stress can boost intracellular GSH levels as an adaptive response, whereas severe oxidative stress might cause GSH levels to drop\u003csup\u003e[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]\u003c/sup\u003e. Exposure to MPTP has also been shown to decrease GSH levels\u003csup\u003e[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]\u003c/sup\u003e. The reason for the more dramatic decrease in GSH levels in the MPTP group compared to the LDIR group may be due to the higher LPO and NO, as indicators of oxidative stress in the MPTP group.\u003c/p\u003e \u003cp\u003eGST enzymes catalyse the conjugation of reduced glutathione to electrophilic groups on the substrate molecules to make the products of oxidative stress soluble for their easy elimination from the cell\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/sup\u003e. Previous studies have shown an increase in GST levels due to oxidative stress and environmental stimuli\u003csup\u003e[\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]\u003c/sup\u003e. Transient overexpression of GST has been shown in MPTP-injected mice, suggesting that GST acts as an endogenous regulator of the cellular stress induced by MPTP to protect cells from ROS\u003csup\u003e[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/sup\u003e. The expression and activity of GST were detected as early as 4 hpf in zebrafish embryos showing that GST enzymes are not only expressed but are also active during early embryogenesis\u003csup\u003e[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]\u003c/sup\u003e. In accordance with these reports we found significant increases in the GST activities of the MPTP and LDIR combined with MPTP exposure groups. Increased GST activities in x-ray exposed zebrafish embryos have been reported previously\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. On the other hand, the increase in the GST activity of the LDIR group was not statistically significant. LDIR exposure before MPTP treatment decreased the GST response compared to the MPTP group, but increased it compared to the LDIR group.\u003c/p\u003e \u003cp\u003eIn the practice of dental radiology, lower radiation doses are required for digital imaging sensors\u003csup\u003e[\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]\u003c/sup\u003e. Considering our previous research, an exposure time of 0.08 seconds, which is one of the frequently used exposure settings in periapical dental imaging, was chosen as a representative exposure time of LDIR\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur study is the first to determine the effects of LDIR from a dental diagnostic x-ray unit on the response to MPTP, focusing on the oxidant-antioxidant system and the expression of genes related with in PD as markers of mitochondrial homeostasis. Based on our results, we may suggest that exposure to LDIR during the embryonic period may alter the response to neurotoxin treatment in terms of mitochondrial stress, oxidant-antioxidant status, locomotor and cholinergic functions in zebrafish embryos. On the other hand, in the LDIR\u0026thinsp;+\u0026thinsp;MPTP group, the aggravation of the response to oxidative damage by LDIR exposure before MPTP treatment, and the suppression of the expressions of genes responsible for the regulation of mitochondrial stress are the issues that need to be supported by further research and their mechanism needs to be revealed.\u003c/p\u003e"},{"header":"Statements and Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Dr. H\u0026uuml;seyin G\u0026uuml;nd\u0026uuml;z for providing OSL dosimetries and further guidance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors have agreed for authorship, read and approved the manuscript, and given consent to participate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors have agreed for authorship, read and approved the manuscript, and given consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception/Design of study: Ebru Emekli-Alturfan; Sebnem Ercalik Yalcinkaya; Ezgi Cahide Aydas Bayramov. Data Acquisition: Ezgi Cahide Aydas Bayramov; Merih Beler; Derya Cansiz, Ismail Unal; Gizem Egilmezer; Selma Yaltkaya; Atakan Karagoz. Data Analysis/Interpretation: Ebru Emekli-Alturfan; Sebnem Ercalik Yalcinkaya; Ezgi Cahide Aydas Bayramov.Drafting Manuscript: Sebnem Ercalik Yalcinkaya; Ezgi Cahide Aydas Bayramov. Critical Revision of Manuscript: Ebru Emekli-Alturfan, \u0026nbsp; Sebnem Ercalık Yalcinkaya.Supervision: Ebru Emekli-Alturfan; Sebnem Ercalik Yalcinkaya.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Marmara University Scientific Research Project Unit (BAPKO) number of 10981.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCurrently, the European Commission Directive 2010/63/EU, permits experimentation in fish embryos at earliest life stages without being regulated as animal experiments; zebrafish are considered models in vitro until 120 hpf (http://data.europa.eu/eli/dir/2010/63/2019-06-26; accessed 21 May 2025 \u0026nbsp;EFSA opinion: https://doi.org/10.2903/j.efsa.2005.292; accessed 21 May 2025).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHwang, S. 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Effects of x-ray radiation on oxidation products of nitric oxide in rabbits treated with antioxidant compounds. \u003cem\u003eTurkish Journal of Biochemistry.\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;34,\u003c/strong\u003e 15\u0026ndash;18 (2009).\u003c/li\u003e\n \u003cli\u003eJackson-Lewis, V., \u0026amp; Smeyne, R. J. MPTP and SNpc DA neuronal vulnerability: role of dopamine, superoxide and nitric oxide in neurotoxicity. Minireview. \u003cem\u003eNeurotoxicity research\u003c/em\u003e. \u003cstrong\u003e7,\u003c/strong\u003e 193\u0026ndash;202; https://doi.org/10.1007/BF03036449 (2005).\u003c/li\u003e\n \u003cli\u003ePrzedborski, S., Jackson-Lewis, V., Yokoyama, R., Shibata, T., Dawson, V. L., \u0026amp; Dawson, T. M. 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Oblique lateral radiographs and bitewings; estimation of organ doses in head and neck region with Monte Carlo calculations. \u003cem\u003eDento maxillo facial radiology\u003c/em\u003e. \u003cstrong\u003e43,\u003c/strong\u003e 20130419; https://doi.org/10.1259/dmfr.20130419 (2014).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Low-dose ionising radiation, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, oxidative stress, antioxidant, locomotor activity","lastPublishedDoi":"10.21203/rs.3.rs-6594085/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6594085/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePrenatal exposure to environmental factors including low-dose ionising radiation and neurotoxins may disrupt the oxidant-antioxidant balance. Our aim was to assess the effects of exposure to low-dose ionising radiation (LDIR) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is a neurotoxin used to model Parkinson's disease (PD), on developing zebrafish embryos, focusing on the oxidant-antioxidant system and markers of mitochondrial damage associated with PD. Zebrafish embryos were divided into four groups: control, LDIR, MPTP, and LDIR combined with MPTP (LDIR+MPTP). A dental diagnostic x-ray unit (0.08 seconds, 60 kVp, 7mA) was used for the exposures. The LDIR exposure was measured as 0,065 mSv using optically simulated dosimeters. At the end of 72 hours after fertilization, locomotor activities, acetylcholine esterase (AChE) activity, oxidative stress and antioxidant status were assessed. Expressions of genes associated with in PD as markers of mitochondrial damage (\u003cem\u003epink1, parkin, dj1 \u003c/em\u003eand\u003cem\u003e lrrk2\u003c/em\u003e) were determined by RT-PCR. Developmental toxicity was observed in all exposure groups as evidenced by pericardial edema, yolk sac edema and spinal curvature. LDIR exposure in zebrafish embryos affected oxidative and mitochondrial stress markers, as well as locomotor activity and AChE as a marker of cognitive function at levels comparable to the MPTP exposure. Our study is the first to determine the effects of LDIR from a dental x-ray unit on the response to MPTP, and we aim to further elucidate the mechanism of these changes observed particularly in the LDIR+MPTP group.\u003c/p\u003e","manuscriptTitle":"Low-dose ionising radiation exerts effects comparable to MPTP through alterations in locomotor activity, oxidant/antioxidant status and mitochondrial homeostasis in zebrafish embryos","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-09 08:40:50","doi":"10.21203/rs.3.rs-6594085/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-16T10:45:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-14T17:08:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336885081612656907037251180747649573904","date":"2025-09-11T15:31:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-05T02:11:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"20628388390134081316461271224812120852","date":"2025-08-30T11:31:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"12519393200265887985938466784126153848","date":"2025-06-05T12:45:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157775177314825463518999969366565249630","date":"2025-06-05T11:17:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-05T10:14:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-05T10:07:24+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-23T16:12:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-22T14:28:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-05T11:28:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d383e1d4-0bf6-44ad-bb33-45c4ea943d1c","owner":[],"postedDate":"June 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":49627191,"name":"Biological sciences/Biochemistry/Neurochemistry"},{"id":49627192,"name":"Biological sciences/Chemical biology/Rna"}],"tags":[],"updatedAt":"2025-12-01T16:08:08+00:00","versionOfRecord":{"articleIdentity":"rs-6594085","link":"https://doi.org/10.1038/s41598-025-29382-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-25 15:58:31","publishedOnDateReadable":"November 25th, 2025"},"versionCreatedAt":"2025-06-09 08:40:50","video":"","vorDoi":"10.1038/s41598-025-29382-8","vorDoiUrl":"https://doi.org/10.1038/s41598-025-29382-8","workflowStages":[]},"version":"v1","identity":"rs-6594085","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6594085","identity":"rs-6594085","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}