Effects of cadmium on oxidative stress and cell apoptosis in Drosophila melanogaster larvae

With the increase of human activities, cadmium (Cd) pollution has become a global environmental problem affecting biological metabolism in ecosystem. Cd has a very long half-life in humans and is excreted slowly in organs, which poses a serious threat to human health. In order to better understand the toxicity effects of cadmium, third instar larvae of Drosophila melanogaster (Canton-S strain) were exposed to different concentrations (1.125 mg/kg, 2.25 mg/kg, 4.5 mg/kg, and 9 mg/kg) of cadmium. Trypan blue staining showed that intestinal cell damage of Drosophila larvae increased and the comet assay indicated significantly more DNA damage in larvae exposed to high Cd concentrations. The nitroblue tetrazolium (NBT) experiments proved that content of reactive oxygen species (ROS) increased, which indicated Cd exposure could induce oxidative stress. In addition, the expression of mitochondrial adenine nucleotide transferase coding gene (sesB and Ant2) and apoptosis related genes (Debcl, hid, rpr, p53, Sce and Diap1) changed, which may lead to increased apoptosis. These findings confirmed the toxicity effects on oxidative stress and cell apoptosis in Drosophila larvae after early cadmium exposure, providing insights into understanding the effects of heavy metal stress in animal development.

Toxicity determination. Based on the results of LC 50 experiment, four cadmium concentrations (1.125 mg/ kg, 2.25 mg/kg, 4.5 mg/kg, and 9 mg/kg) were set up for subsequent experiments. Similar to the LC 50 experiment, male and female Drosophila were collected within 8 h after eclosion and cultured separately for three days. They were then crossed in fresh control medium (without Cd) or Cd mediums for 12 h. Parental adult flies were then removed, and third instar larvae developed from different medium were selected for the experiments.
NBT reduction test was used to detect the ROS content in Drosophila larvae cells 16 . Larvae haemolymph cells (extracted from fifty larvae per sample) were incubated with NBT for 1 h in the incubation medium. The insoluble formazan crystals formed were solubilized by adding 150 µL 50% acetic acid followed by 5 min vortexing. The absorbance of the final solution was measured at 595 nm using a spectrophotometer.
Trypan blue staining was used to detect cellular damage in the midgut of larvae 17 . Seventy third instar larvae were randomly selected from each treatment group, cleaned with PBS, put in 0.02% trypan blue staining solution, and incubated for 2 h with vibration. After cleaned with PBS, the samples were checked and photographed under a stereomicroscope.
Crawling experiment was used to identify the neuronal damage in the early stage of larval development. Three instar larvae were randomly selected from Cd medium with different concentrations and washed in PBS solution, then placed in the center of Petri dish containing 0.8% agarose gel. A layer of grid paper was put under the Petri dish. After the larvae adapted for 10 min, the crawling track, turning angle and number of peristalsis contractions (full anterior to posterior movement = 1 contraction) of larvae within 2 min were recorded 18,19 .
Comet assay can effectively detect and analyze the degree of DNA damage in cells 20 . Fifty third instar larvae were randomly selected from each group. The midgut was dissected under an anatomical microscope, digested in collagenase solution for fifteen minutes, and the cells were collected by centrifugation, washed with PBS and centrifuged to make cell suspension. Then the experiment was performed according to the instructions of the DNA Damage Detection Kit (Jiangsu KeyGEN BioTECH Corp., Ltd, China). Finally, the samples were checked under the fluorescence microscope (Carl Zeiss Jena CO., LTD, Germany) (515-560 nm wavelength).
Quantitative real-time PCR. Total RNA from six third instar larvae was isolated using the RNAiso Plus Kit (TAKARA, Beijing, China), according to the manu-facturer's instructions. The coding sequence (CDS) sequences of two mitochondrial adenine nucleotide transferase (ANT) coding genes (sesB and Ant2) and six apoptosis-related genes (Debcl, hid, rpr, p53, Sce, and Diap1) were queried using NCBI (https:// www. ncbi. nlm. nih. gov/). Primer design and synthesis were carried out at the Shenggong Bioengineering Co. Ltd., Shanghai. cDNA was obtained via RNA reverse transcription, following the instructions of the PrimeScript ®RT Master Mix reverse transcription kit. The reaction system was prepared according to the instructions of the SYBR Green kit to detect the mRNA expression of the aforementioned genes.
The qPCR protocol used was as follows: following initial denaturation at 95 ℃ for 30 s, the template cDNA was amplified for 40 cycles of denaturation (95 ℃, 5 s), annealing (60 ℃, 34 s), and extension (75 ℃, 45 s). The experiments were performed in triplicate and the results were averaged. The amplification primers are shown in Table 1.

Statistical analysis.
All the experimental data were analyzed by SPSS 20.0 statistical software (IBM Company, New York, America) using one-way ANOVA and Tukey's significance testing (P < 0.05). GraphPad Prism 7.0 (GraphPad Software Company, California, America) was used to create all graphs. www.nature.com/scientificreports/ The images obtained from the comet assay were analyzed by Comet Assay Software Project (Beijing Biolaunching Technologies Co., Ltd., Beijing, China), and the tail length (TL, µm), tail moment (TM), olive tail moment (OTM), and tail DNA % (TD) were determined. Ethics declarations. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Results
LC 50 value of Cd to Drosophila larvae. The eclosion rate of Drosophila from larvae under different Cd concentrations was presented in Fig. 1. These results showed that the eclosion rate decreased with increasing Cd concentration, that is, the mortality increased gradually. The Cd concentration corresponding to 50% mortality was 4.449 mg/kg at the 95% confidence limit. LC 50 was used as the basis for setting the cadmium concentration in the follow-up study.
Toxicity effects of Cd on Drosophila larvae. ROS content. The ROS content of blood lymphocytes of third instar larvae was shown in Fig. 2. Compared with the control group, the low Cd concentration group of 1.125 mg/kg has no significant difference. The absorbance values of treatment groups of 2.25 mg/kg, 4.5 mg/ kg and 9 mg/kg show significant differences compared with the control group (P < 0.01), and the ROS level increased gradually with the increase of Cd concentration. These results showed that higher concentration Cd stress could induce a significant increase in ROS content in Drosophila larvae. These results showed that the content of ROS in Drosophila larvae increased significantly under higher concentration of cadmium stress.
Midgut cell damage. The third instar larvae after trypan blue staining were showed in Fig. 3. Midguts of third instar larvae picked randomly in each treatment group appeared stained of varying degrees. The larger the staining area, the darker the color, indicated that the more serious the cell damage. Moreover, the results also showed Table 1. Primers for real-time quantitative PCR amplification.

Gene
Sense primer 5′-3′ Anti-sense primer 5′-3′ Drosophila larvae were exposed to a range of Cd concentrations (Mean ± SD, n = 3, 175 eggs per sample). Cd concentration corresponding to 50% mortality was 4.449 mg/kg at the 95% confidence limit. www.nature.com/scientificreports/ that the proportion of stained Drosophila larvae was higher with increasing concentrations of cadmium (data not shown). These results indicated that the midgut cells of Drosophila larvae showed cell damage under the cadmium exposure, and the degree of damage increased dose-dependently.

Rp49
Crawling behavior. As shown in Fig. 4, the crawling trajectory of larvae in the control group was smooth and simple. As Cd concentration increased, the larvae crawling trajectory became increasingly irregular, with many sharp or circular turns (Fig. 4A). Crawling speeds of larvae in the 4.5 mg/kg (P < 0.05) and 9 mg/kg (P < 0.01) treatment groups were significantly lower than that of the control group (Fig. 4B). Additionally, larvae exposed to 9 mg/kg had a significantly lower number of body wall contractions compared with the control group (P < 0.01) (Fig. 4C). These results showed that high concentrations of cadmium induced abnormal behavior of Drosophila larvae, including abnormal turns and twists, crawling speed reduction and abnormal body wall contraction.
DNA damage. The images of comet assay of midgut cells of the third instar larvae were shown in Fig. 5A. Tail DNA% of comet and tail length of comet were shown in Fig. 5B, C respectively. Tail DNA% and tail length are used to assess the degree of DNA damage in comet assay. According to the grade of DNA damage, tail DNA% in the control group was not damaged. Treatment groups of 2.25 mg/kg and 4.5 mg/kg belonged to mild damage, group of 9 mg/kg was moderately damaged. Tail DNA% of comet was significantly increased in the three treatment groups compared with the control group (P < 0.05 or P < 0.01). The tail length of comet of the treatment groups (2.25 mg/kg, 4.5 mg/kg and 9 mg/kg) increased significantly compared with the control group (P < 0.01). These results indicated that the DNA "smearing" phenomenon became more severe, and there was a higher degree of DNA damage in Drosophila larvae with the increase of cadmium concentration.     Fig. 6. The expression of sesB in treatment groups of 2.25 mg/kg, 4.5 mg/kg and 9 mg/kg decreased significantly compared with the control group (P < 0.01). The expression level of Ant2 appeared to decline first, then increase and then decrease, but there was no significant difference compared with the control group (P > 0.05). These results showed that Cd exposure inhibited the expression of sesB gene in Drosophila larvae. Increased ROS induced by Cd stress will lead to oxidative stress and apoptosis. Therefore, we also detected the expression changes of pro-apoptotic genes (Debcl, hid, rpr, p53) and anti-apoptotic genes (Sce, Diap1) in Drosophila. As shown in Fig. 7, the expression of pro-apoptotic and anti-apoptotic genes changed in third instar larvae under different Cd concentrations. The expression of pro-apoptotic genes, Debcl, hid, rpr, and p53 ( Fig. 7A-D), increased with increasing Cd concentrations. Compared with the control group, the expression of pro-apoptotic genes in the two highest concentration treatment groups (4.5 mg/kg and 9 mg/kg) increased significantly (P < 0.01). The expression of the anti-apoptotic gene Sce was significantly downregulated in the 4.5 mg/kg and 9 mg/kg treatment groups (P < 0.01) (Fig. 7E). The expression of Diap1 showed a downward trend and decreased significantly in 2.25 mg/kg, 4.5 mg/kg and 9 mg/kg treatment groups compared with the control group (P < 0.01) (Fig. 7F).

Discussion
In this study, trypan blue staining showed that Drosophila intestinal cell damage increased with increasing exposure to Cd. The NBT experiments proved that ROS increased after Cd treatment. As excessive ROS has been shown to induce DNA damage 22 , it is unsurprising that the comet assay analysis indicated significantly more DNA damage in Drosophila exposed to high Cd concentrations. Cd could interact with ROS regulators, such as glutathione, reducing the ability to eliminate ROS, indirectly increasing ROS levels and inducing oxidative stress 23 . Previous studies have found that heavy metal exposure caused a decrease in catalase (CAT) and superoxide dismutase (SOD) activity and an increase in MDA content in Drosophila larvae 24 . This leads to the accumulation of ROS in cells, which can cause oxidative stress and subsequent DNA damage.
Our results indicated that larvae in the high Cd concentration groups exhibited abnormal crawling behavior compared with the control group. Similarly, other studies have found that Drosophila eggs treated with 250 and 300 mg graphene/L resulted in a significantly reduced crawling speed 25 , and those treated with high concentrations of NaF showed significantly reduced crawling speed and body wall contractions 19 . The abnormal crawling behavior of larvae exposed to high Cd concentrations indicates that mechanical motor neurons are affected by toxic stress, which may be caused by brain coordination defects and damage to the subesophageal ganglion. The slower crawling speed may be caused by damage to the cholinergic neurons, which can result in abnormal muscle contraction.
This study also identified that the relative expression of sesB in the third instar larvae of Drosophila decreased with increasing Cd concentration. sesB primarily encodes the mitochondrial ANT protein: mitochondria are the only source of adenylate triphosphate (ATP) within the synapses, therefore, ANT is essential for the transport of ATP from the mitochondrial membrane to the cytoplasmic matrix 26 . DeVorkin et al. 27 found that Drosophila caspase1 (Dcp-1) is located upstream of sesB, and can promote autophagy in the salivary glands, midgut, and other parts of larvae by inhibiting sesB activity. Thus, sesB was shown to be a new negative regulator of autophagic flux in the mid-stage of Drosophila development. With a decrease in sesB expression, the mitochondrial morphology changed from elongated organelles to spherical particles, causing changes in their metabolic state and possibly inducing cell apoptosis 28 . The remaining mitochondria maintain tissue viability through increased activity, which can lead to an increase in ROS. When sesB was inhibited, the H 2 O 2 content increased sharply, and O 2 − was www.nature.com/scientificreports/ produced by the G3PDH active enzyme and ubiquinone on the cytoplasmic side of the inner mitochondrial membrane 29 . This indicates that O 2 − /H 2 O 2 are created by a phosphoglyceride shuttle in the body of Drosophila, making sesB-knockout cells more sensitive to oxidative stress. sesB also regulates calcium levels within cells and mitochondria; therefore, when sesB was knocked out, calcium signaling in mitochondria was blocked 30 . Changes in sesB expression generally impair mitochondrial function, leading to reduce toxic metabolism, oxidative damage, and even cell death 31 .
Debcl has been shown to affect a wide range of other genes and functions. Debcl inhibits the expression of Buffy 32 , a gene involved in stress-and DNA damage-induced apoptosis that is not required for normal development in Drosophila. Additionally, the tumor suppressor gene, Rbf1, interacts with Debcl to increase the amount of ROS produced by mitochondria, leading to the activation of the JNK pathway and, eventually, to apoptosis in Drosophila 33 . Debcl may also increase mitochondrial ROS content through glycerophosphate oxidase 1 34 . www.nature.com/scientificreports/ Overexpression of Debcl in the eyes of Drosophila also leads to eye-cell apoptosis 35 . Based on these previous findings and the results of this study, it can be presumed that the upregulation of Debcl expression may ultimately induce apoptosis by increasing the activity of glycerophosphate oxidase 1 34 and the mitochondrial fission protein Drp1 36 . ROS content may be further increased by the increase in Gpo-1 expression, caused by the upregulation of Debcl genes in the Bcl-2 family. p53 can promote DNA repair; however, when DNA damage repair is incomplete or fails, it can promote apoptosis instead 37 . The upregulation of p53 may additionally stimulate the upregulation of other pro-apoptotic genes, rpr and hid in the RHG family, and inhibit the expression of the anti-apoptotic gene, Diap1 38 . Accordingly, in addition to the upregulation of p53, the expression of rpr and hid were also upregulated after Cd stress in this study, whereas the Diap1 expression was significantly downregulated. Diap1 is a key enzyme that controls cell fate, and regulates apoptosis by selectively degrading pro-apoptotic proteins or itself 36 , helping to prevent apoptosis caused by abnormal caspase activation. However, the protein produced by rpr and hid inhibits the expression of Diap1 and induces the release of Dronc (a caspase-9 homolog). After Dronc is released, it binds to the scaffold protein Ark (Apaf-1) and hydrolysis activates Drice, Dcp-1 (a caspase-3 homolog) 37 . These caspases induce cell DNA fragmentation and the formation of apoptotic bodies 39 . This study indicated that Cd stress induced the upregulation of pro-apoptotic genes (Debcl, hid, rpr, and p53) and the downregulation of anti-apoptotic genes (Sce and Diap1) at the mRNA level in Drosophila larvae. Changes in the expression of these genes may also change the membrane potential in the mitochondria and lead to a harmful increase in ROS content and further DNA damage.

Conclusions
Heavy metal cadmium induced damage of midgut cells of third instar larvae in Drosophila melanogaster. The DNA 'smearing' phenomenon indicated DNA damage induced by cadmium exposure. Moreover, the content of reactive oxygen species increased, suggesting oxidative stress. Cadmium could also lead to behavioral changes of Drosophila larvae, such as excessive distortion, increased turning times and slower crawling speed. In addition, Cd induced changes in the expression of adenine nucleotide transferase coding gene sesB in Drosophila larvae, suggesting that Cd stress may produce more ROS by affecting the functional changes of mitochondria. The relative expression of pro-apoptotic genes Debcl, hid, rpr and p53 increased, meanwhile, anti-apoptotic genes Sce and Diap1 decreased. In conclusion, cadmium exposure may induce oxidative stress and apoptosis in Drosophila larvae by increasing the content of reactive oxygen species. Consistent with previous studies 40,41 , this study provided further evidence about the toxicity effects of early cadmium exposure on Drosophila larvae. The present study suggests that cadmium exposure induces oxidative stress and cell apoptosis, which could be one of the main mechanisms for induction of genotoxicity.

Data availability
All data included in this study are available upon request by contact with the corresponding author (zhang-min451@snnu.edu.cn).

Code availability
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