Chemotherapy Drugs Act Differently in the Expression and Somatic Mobilization of the mariner Transposable Element in Drosophila simulans

Transposable elements (TEs) are abundant in genomes. Their mobilization can lead to genetic variability that is useful for evolution, but can also have deleterious biological effects. Somatic mobilization (SM) has been linked to degenerative diseases, such as Alzheimer’s disease and cancer. We used a Drosophila simulans strain, in which SM can be measured by counting red spots in the eyes, to investigate how chemotherapeutic agents affect expression and SM of the mariner TE. Flies were treated with Cisplatin, Dacarbazine, and Daunorubicin. After acute exposure, relative expression of mariner was quantified by RT-qPCR and oxidative stress was measured by biochemical assays. Exposure to 50 and 100 µg/mL Cisplatin increased mariner expression and ROS levels; catalase activity increased at 100 µg/mL. With chronic exposure, the number of spots also increased, indicating higher mariner SM. Dacarbazine (50 and 100 µg/mL) did not significantly alter mariner expression or mobilization or ROS levels, but decreased catalase activity (100 µg/mL). Daunorubicin (25 and 50 µM) increased mariner expression, but decreased mariner SM. ROS and catalase activity were also reduced. Our data suggest that stress factors may differentially affect the expression and SM of TEs. The increase in mariner transposase gene expression is necessary, but not sufficient for mariner SM.


Introduction
Transposable elements (TEs) are DNA sequences that can change their position in the genome [1]. They are ubiquitous, i.e., they occur in almost all living organisms; they are abundant, having long been an important component of several genomes, such as in Drosophila (20%) [2,3], maize (80%) [4,5], and humans (48%) [2,6]; and they are extremely variable in sequence, transposition mechanisms, and evolutionary history. Because of this, more than a hundred families are known [7,8]. TEs were originally considered "junk DNA", but numerous studies now show that TEs drive evolution by offering new gene-splicing patterns and providing protein domains for coding genes that confer new cellular functions. Moreover, the presence of cis-regulatory sequences in TEs can rewire gene expression [9,10]. However, mobilization of TEs is generally deleterious, and several mechanisms have evolved to control transposition [11,12].
Somatic mobilization (SM), i.e., transposition of TEs in somatic tissues, was, until recently, a little-noticed phenomenon because it was considered rare and had no evolutionary or biological significance [13]. However, recent evidence has shown that SM is common, at least for some TEs, and may be associated with some age-related human diseases, such as Alzheimer's disease and cancer [14,15]. For this reason, it is important to understand the SM process, and its regulation or deregulation. Stress is considered a regulator of the activation of TEs. Indeed, several studies have shown that TEs are activated by stress [5,[16][17][18][19][20][21]. However, at the transcriptional level, this phenomenon is complex, as different stressors can elicit different responses in different TEs, with some being upregulated and others downregulated [22].
Chemotherapy drugs deserve special attention as potential TEs activators due to stress, as TEs are thought to play a role in generating genetic variability that induces resistance to cancer treatments. Cisplatin, daunorubicin, and dacarbazine are chemotherapy drugs largely used for cancer treatment. Cisplatin and dacarbazine are alkylating agents, with cisplatin binding DNA and promoting single-strand breaks, while dacarbazine inhibits DNA synthesis by acting as a purine analogue [23,24]. Daunorubicin has a different mechanism of action. It has a DNA-intercalating molecule that inhibits the enzyme topoisomerase II and stops the DNA replication process [25]. These drugs also promote oxidative stress [24,26].
Drosophila is one of the leading models for the study of TEs, as it has a well-annotated and -characterized genome and mobilome, in addition to an arsenal of research tools [27]. It is also important to note that 20% of Drosophila's TEs are active [28]. Using the Drosophila model and a transcriptomic approach, we have previously shown that chemotherapy drugs, such as cisplatin and cyclophosphamide, cause changes in the expression of some TEs, activating some and suppressing others [29,30]. However, there are few studies investigating how chemotherapy can affect the expression and SM of TEs at the molecular and phenotypic levels. Here, we used the Drosophila simulans white-peach (w pch ) strain to exploit its striking ability to somatically mobilize TEs. The w pch strain is a mutant strain with a non-autonomous mariner-peach in the white gene promoter, which produces peacheyed flies due to the insertion of the mariner. This strain has the autonomous mariner-Mos1 in the genome which produces a transposase responsible for excision of mariner-peach. This leads to mosaicism in the eyes, where some eye-forming cells revert to the red color of wild-type flies [31][32][33]. We tested whether the chemotherapy drugs cisplatin, dacarbazine, and daunorubicin alter the relative expression of mariner and the SM of mariner-peach in the D. simulans w pch strain. We also investigated whether these drugs cause oxidative stress upon acute exposure, and looked for the presence of regulatory regions for oxidative stress in the mariner sequence. Our data suggest that chemotherapy drugs may affect in expression and SM of TEs differently.

Fly Stocks Maintenance
Fly stocks were maintained at 20 • C and fed a medium of cornmeal, sucrose, and yeast. Female flies of the strain D. simulans white-peach (w pch ), which were less variable in PCR assays [13], were used for all experiments. This strain has the autonomous TE mariner-Mos1 in the genome and the non-autonomous mariner-peach TE copy in the white gene promoter, which produces peach-eyed flies due to the insertion of mariner [13].

Acute Exposure and Molecular Analysis
Young flies (1-4 days) were subjected to a 7-h starvation period [29] (Figure 1). Subsequently, the flies were transferred to culture media containing food coloring dye and one of the chemotherapy drugs (or only the dye, in the control treatment) for 24 h. After the exposure period, 20 females were selected for RNA extraction. All samples were taken in triplicate. Selection was done by visual analysis of the color of the abdomen (due to the presence of the red food dye) to standardize the samples. Thus, only flies that had ingested the culture medium and had similar abdomen color intensity were used.
The concentrations of each chemotherapy drug used were based on previously published studies. Cisplatin [34] and dacarbazine [35] were mixed to the culture media at 50 and 100 µg/mL, respectively. Daunorubicin was mixed to the media at a concentration of 25 and 50 µM [36].
to the presence of the red food dye) to standardize the samples. Thus, only flies that had ingested the culture medium and had similar abdomen color intensity were used. Figure 1. Experimental design. Acute exposure: 1-to-4-day-old D. simulans w pch flies were subjected to a 7-hour starvation and then transferred to culture media containing cisplatin, dacarbazine, or daunorubicin plus food coloring dye for further analysis. No dye was added to the culture media of the flies used for the biochemical analyses. Chronic exposure: flies were oviposited for 24 h in culture media containing one of the active compounds, and the new generation was collected and analyzed. Created with BioRender.com October 25th, 2022, Licence AD24KGDIHQ.
The concentrations of each chemotherapy drug used were based on previously published studies. Cisplatin [34] and dacarbazine [35] were mixed to the culture media at 50 and 100 µg/mL, respectively. Daunorubicin was mixed to the media at a concentration of 25 and 50 µM [36].
RNA extraction was performed according to the TRIzol™ Reagent protocol (Invitrogen). RNA concentration was analyzed using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA). Samples were then treated with DNaseI (Promega, Madison, USA) and synthesis of complementary DNA (cDNA) was performed using random primers according to the instructions of the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific).
The RT-qPCR amplification was performed as follows: 95 °C for 7 min, followed by 40 cycles of 94 °C for 15 s, 60 °C for 15 s, and 72 °C for 20 s. Relative expression was calculated according to the 2 −ΔΔCT method [39]. Statistical analysis was performed by one- Figure 1. Experimental design. Acute exposure: 1-to-4-day-old D. simulans w pch flies were subjected to a 7-h starvation and then transferred to culture media containing cisplatin, dacarbazine, or daunorubicin plus food coloring dye for further analysis. No dye was added to the culture media of the flies used for the biochemical analyses. Chronic exposure: flies were oviposited for 24 h in culture media containing one of the active compounds, and the new generation was collected and analyzed. Created with BioRender.com, accessed on 25 October 2022, Licence AD24KGDIHQ.
RNA extraction was performed according to the TRIzol™ Reagent protocol (Invitrogen). RNA concentration was analyzed using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Samples were then treated with DNaseI (Promega, Madison, WI, USA) and synthesis of complementary DNA (cDNA) was performed using random primers according to the instructions of the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific).
The RT-qPCR amplification was performed as follows: 95 • C for 7 min, followed by 40 cycles of 94 • C for 15 s, 60 • C for 15 s, and 72 • C for 20 s. Relative expression was calculated according to the 2 −∆∆CT method [39]. Statistical analysis was performed by one-way Analysis of Variance (ANOVA), followed by Tukey's multiple comparison test. Differences were considered significant when p < 0.05.

Chronic Exposure and Phenotypic Analysis
To determine the mariner-peach somatic mobilization rate (SM), chronic exposure to the drugs was performed at the lowest concentration used for acute exposure: 50 µg/mL cisplatin and dacarbazine, and 25 µM daunorubicin. For oviposition, adult flies were transferred to culture media containing one of the chemotherapy drugs ( Figure 1). After 24 h, the adult flies were removed and the eggs were kept in the vials containing the respective drug throughout their life cycle. The same procedure was followed for the control group, but no drug was added to the medium. For phenotypic analysis, 300 female flies were collected for each group and visually examined for the presence or absence of red spots in the eyes. The collected data were compiled in a contingency table. For statistical analysis, Fisher's exact test was performed and differences were considered significant when p < 0.05.

Biochemical Assays
The biochemical assays were adopted from Ogunsuyi and co-workers [40]. Acute exposure was performed as described in Section 2.2, except that no food coloring dye was added to the culture medium in order not to interfere with spectrophotometric measurements. After 24 h of exposure to one of the drugs, 10 female flies were selected from each vial and macerated in 0.1 M potassium phosphate buffer, and pH 7.4. Samples were centrifuged at 10.000 rpm for 10 min (Figure 1). The supernatant was recovered and the protein content was quantified using NanoDrop 2000. Samples were adjusted to a final concentration of 0.4 mg/mL of protein for all biochemical assays. All samples were taken in triplicate. The data were analyzed by two-way ANOVA followed by a post hoc test. Differences were considered significant when p < 0.05.

Search for Oxidative Stress Regulatory Regions
We searched for an Antioxidant Responsible Element (ARE) binding sequence in the regulatory region of mariner using RSAT http://rsat.sb-roscoff.fr/matrix-scan_form.cgi (accessed on 13 December 2022) [41]. The position-specific scoring matrix (PSSM) for the Nrf2-binding site in promoter was obtained in Malhotra and collaborators [42]. In addition, we manually searched for sequences similar to the ARE consensus sequence "TGACXXXGC" [42][43][44] in the 5 UTR region of the mariner element.

Cisplatin
Flies exposed to 50 and 100 µg/mL cisplatin showed a significant increase in expression at the transcriptional level of the mariner gene compared to the control group (p = 0.0036). There was no significant difference between the concentrations of 50 and 100 µg/mL (Figure 2A).
Since exposure to cisplatin increased mariner expression, we investigated whether the drug also altered mariner-peach SM, by performing phenotypic analysis to identify the presence of red spots in the eyes of the flies. We found that the group chronically exposed throughout their life cycle to 50 µg/mL cisplatin had more flies with spots than the control group (p < 0.0001, Figure 2B).
Catalase activity was significantly higher in protein extracts from flies treated with 100 µg/mL of cisplatin (p < 0.0001). On the other hand, there was no significant difference in catalase activity in the flies treated with 50 µg/mL cisplatin ( Figure 2C). The overall ROS content in protein extracts of the flies exposed to both cisplatin concentrations increased sig-nificantly (p < 0.0001) compared to the non-treated group ( Figure 2D). A dose dependence was observed within the two concentrations tested (p < 0.0001).

Cisplatin
Flies exposed to 50 and 100 µg/mL cisplatin showed a significant increase in expression at the transcriptional level of the mariner gene compared to the control group (p = 0.0036). There was no significant difference between the concentrations of 50 and 100 µg/mL (Figure 2A). Since exposure to cisplatin increased mariner expression, we investigated whether the drug also altered mariner-peach SM, by performing phenotypic analysis to identify the presence of red spots in the eyes of the flies. We found that the group chronically exposed throughout their life cycle to 50 µg/mL cisplatin had more flies with spots than the control group (p < 0.0001, Figure 2B).
Catalase activity was significantly higher in protein extracts from flies treated with 100 µg/mL of cisplatin (p < 0.0001). On the other hand, there was no significant difference in catalase activity in the flies treated with 50 µg/mL cisplatin ( Figure 2C). The overall ROS content in protein extracts of the flies exposed to both cisplatin concentrations increased significantly (p < 0.0001) compared to the non-treated group ( Figure 2D). A dose dependence was observed within the two concentrations tested (p < 0.0001).

Dacarbazine
Acute exposure to 50 and 100 µg/mL dacarbazine did not significantly alter the relative expression levels of mariner in any comparison (p = 0.0712, Figure 3A). The levels of SM were also unaffected (p = 0.1176), as there was no significant difference in the number of flies with red eye spots after chronic exposure ( Figure 3B).

Dacarbazine
Acute exposure to 50 and 100 µg/mL dacarbazine did not significantly alter the relative expression levels of mariner in any comparison (p = 0.0712, Figure 3A). The levels of SM were also unaffected (p = 0.1176), as there was no significant difference in the number of flies with red eye spots after chronic exposure ( Figure 3B). There was a significant (p = 0.0133) decrease in catalase activity between the flies exposed to 100 µg/mL dacarbazine and the control group ( Figure 3C). There was no difference in catalase activity or between the two concentrations in the flies treated with 50 µg/mL dacarbazine. Moreover, the flies treated with both concentrations of dacarbazine showed no significant change in fluorescence values for the DCFDA assay (p > 0.05) compared to the control group ( Figure 3D). There was a significant (p = 0.0133) decrease in catalase activity between the flies exposed to 100 µg/mL dacarbazine and the control group ( Figure 3C). There was no difference in catalase activity or between the two concentrations in the flies treated with 50 µg/mL dacarbazine. Moreover, the flies treated with both concentrations of dacarbazine showed no significant change in fluorescence values for the DCFDA assay (p > 0.05) compared to the control group ( Figure 3D).

Daunorubicin
Exposure to daunorubicin for 24 h significantly (p = 0.0004) increased the relative expression of the mariner gene at both concentrations compared with the control group ( Figure 4A). In contrast, chronic exposure significantly decreased mariner-peach SM expression despite the increase in relative expression of mariner (p = 0.0232, Figure 4B).  Flies exposed to Daunorubicin presented a significant decrease (p < 0.05) in the overall ROS content intracellularly for both concentrations tested, as seen by the depletion of the fluorescence values ( Figure 4D). Similarly, there was a decrease in catalase activity at the 50 µM exposure (p < 0.0001, Figure 4C).

ARE Binding Sequences
Analysis using RSAT server and the scoring matrix for Nrf2 described in [42] revealed only one binding site (p = 4.4 × 10 −5 ). However, this site is located within the coding region and in the reverse strand and, based on these properties, should probably not be involved in transposase regulation. On the other hand, we found three ARE sites with some similarity to the consensus sequence of the Nrf2 site in the 5'UTR region of mariner ( Figure 5C). We create a matrix with these sequences and reanalyze the mariner sequence using this matrix. One of these sites (ARE1) was indicated by RSAT as a putative binding site (p = 6.4 × 10 −5 ). The promoter region of the mariner element with the putative ARE binding site is shown in Figure 5. Flies exposed to Daunorubicin presented a significant decrease (p < 0.05) in the overall ROS content intracellularly for both concentrations tested, as seen by the depletion of the fluorescence values ( Figure 4D). Similarly, there was a decrease in catalase activity at the 50 µM exposure (p < 0.0001, Figure 4C).

ARE Binding Sequences
Analysis using RSAT server and the scoring matrix for Nrf2 described in [42] revealed only one binding site (p = 4.4 × 10 −5 ). However, this site is located within the coding region and in the reverse strand and, based on these properties, should probably not be involved in transposase regulation. On the other hand, we found three ARE sites with some similarity to the consensus sequence of the Nrf2 site in the 5 UTR region of mariner ( Figure 5C). We create a matrix with these sequences and reanalyze the mariner sequence using this matrix. One of these sites (ARE1) was indicated by RSAT as a putative binding site (p = 6.4 × 10 −5 ). The promoter region of the mariner element with the putative ARE binding site is shown in Figure 5. The transcription factor CncC binds to the putative ARE binding sequence of mariner when exposed to oxidative stress, which increases the expression of the mariner transposase gene. (C) The logo of the consensus sequence ARE [41] and the three similar sequences to "ARE" found in the 5' UTR region of mariner. Only "ARE 1" was statistically significant (p = 6.4 × 10 −5 ).

Discussion
Nrf2 (nuclear factor E2 p45-related factor 2) is a transcription factor (TF) that mediates one of the major defense mechanisms against oxidative stress. The fly homolog of Nrf2 is the Cap'n'collar (CncC) protein. This TF binds to the cis-regulatory sequence ARE and stimulates the expression of detoxification enzymes, such as catalase, glutathione S-transferase, peroxiredoxin, heme oxygenase-1, and others [42][43][44][45]. Nrf2 also promotes the expression of enzymes involved in the degradation of xenobiotics [43]. The chemotherapy drugs cisplatin, dacarbazine, and daunorubicin are xenobiotics known to induce oxidative stress. Therefore, the presence of a putative ARE sequence in the promoter region of the mariner transposase gene is expected to lead to activation of this gene in the presence of these agents ( Figure 5B). It is worth noting that the position of the ARE sequence in the promoter region of the mariner transposase gene is similar to that of artificial promoters, which have been shown to be efficient in activating reporter genes under oxidative stress conditions [44]. It should be noted, however, that the ARE site that we suspect is present in mariner is only a guess and experimental tests are needed to verify if it is functional.
We had expected that increased activation of mariner would lead to an increase in flies with red spots in the eyes. However, this was not the case with all drugs. Cisplatin increased the general ROS level and catalase activity, indicating oxidative stress. As expected, an upregulation of the transposase gene and an increase in mariner SM was observed, as indicated by the number of flies with red spots in the eyes. For dacarbazine, no increased oxidative stress was detected, since there was no difference in the general ROS levels and catalase activity decreased for the highest concentration tested. There was also no change in mariner expression or SM in dacarbazine exposure. It can be suggested that the drug concentration used was below that capable of inducing oxidative stress in Drosophila, in contrast to mammals in which it was previously tested [35].
Daunorubicin, at the concentrations used, induced a decrease in catalase activity and ROS levels. There was also higher mariner expression, but lower SM. Doxorubicin, a drug that is similar to daunorubicin, also presented a decrease in ROS damage on breast cancer cell lineages [46]. However, increased levels of H2O2 and superoxide dismutase expression have been found [46]. We suggest that a significant decrease in ROS levels improved the cell's ability to handle the drug and control the activity of TEs.
We found that the mariner transposase gene can be upregulated even if no oxidative stress was detected. The mariner element has other regulatory elements, such as HSE, which is activated by heat shock TF. However, this TF may be sensitive not only to heat [47]. The upregulation of mariner could be induced by regulatory elements other than The transcription factor CncC binds to the putative ARE binding sequence of mariner when exposed to oxidative stress, which increases the expression of the mariner transposase gene. (C) The logo of the consensus sequence ARE [41] and the three similar sequences to "ARE" found in the 5 UTR region of mariner. Only "ARE 1" was statistically significant (p = 6.4 × 10 −5 ).

Discussion
Nrf2 (nuclear factor E2 p45-related factor 2) is a transcription factor (TF) that mediates one of the major defense mechanisms against oxidative stress. The fly homolog of Nrf2 is the Cap'n'collar (CncC) protein. This TF binds to the cis-regulatory sequence ARE and stimulates the expression of detoxification enzymes, such as catalase, glutathione S-transferase, peroxiredoxin, heme oxygenase-1, and others [42][43][44][45]. Nrf2 also promotes the expression of enzymes involved in the degradation of xenobiotics [43]. The chemotherapy drugs cisplatin, dacarbazine, and daunorubicin are xenobiotics known to induce oxidative stress. Therefore, the presence of a putative ARE sequence in the promoter region of the mariner transposase gene is expected to lead to activation of this gene in the presence of these agents ( Figure 5B). It is worth noting that the position of the ARE sequence in the promoter region of the mariner transposase gene is similar to that of artificial promoters, which have been shown to be efficient in activating reporter genes under oxidative stress conditions [44]. It should be noted, however, that the ARE site that we suspect is present in mariner is only a guess and experimental tests are needed to verify if it is functional.
We had expected that increased activation of mariner would lead to an increase in flies with red spots in the eyes. However, this was not the case with all drugs. Cisplatin increased the general ROS level and catalase activity, indicating oxidative stress. As expected, an upregulation of the transposase gene and an increase in mariner SM was observed, as indicated by the number of flies with red spots in the eyes. For dacarbazine, no increased oxidative stress was detected, since there was no difference in the general ROS levels and catalase activity decreased for the highest concentration tested. There was also no change in mariner expression or SM in dacarbazine exposure. It can be suggested that the drug concentration used was below that capable of inducing oxidative stress in Drosophila, in contrast to mammals in which it was previously tested [35].
Daunorubicin, at the concentrations used, induced a decrease in catalase activity and ROS levels. There was also higher mariner expression, but lower SM. Doxorubicin, a drug that is similar to daunorubicin, also presented a decrease in ROS damage on breast cancer cell lineages [46]. However, increased levels of H 2 O 2 and superoxide dismutase expression have been found [46]. We suggest that a significant decrease in ROS levels improved the cell's ability to handle the drug and control the activity of TEs.
We found that the mariner transposase gene can be upregulated even if no oxidative stress was detected. The mariner element has other regulatory elements, such as HSE, which is activated by heat shock TF. However, this TF may be sensitive not only to heat [47]. The upregulation of mariner could be induced by regulatory elements other than ARE, or maybe even by ARE, but through the action of daunorubicin as a xenobiotic instead of oxidative stress. A remarkable observation is that an increase in transposase transcription was followed by a decrease in mariner mobilization, as measured by the number of flies with red eye spots. TE mobilization is a process involving several steps, and the transcription of genes encoding the enzymes for transposition is among the first ones. A possible explanation for the reduction in mariner mobilization could be the fact that daunorubicin is a DNA intercalator. Intercalating substances can distort the DNA helix and, in this way, reduce the binding of the enzyme to the DNA. An alternative hypothesis is that daunorubicin is a topoisomerase II inhibitor, and normally, transposition requires DNA replication [48]. Alterations caused by the drug in DNA replication may affect transposition. Another explanation could be an inhibitory effect of transposase over expression [49]. Therefore, it is possible that mariner transposase expression is greatly increased by daunorubicin upon chronic exposure, but mobilization does not occur and is still reduced because little or no transposase activity occurs.
The literature repeatedly emphasizes that stress activates the mobilization of TE, and this fact is supported by several lines of evidence. However, the abundance of transcriptomes recently obtained in organisms under different stress conditions has revealed a more complex scenario: a given stress activates one subset of TEs and represses others. Different stress factors promote different responses [22]. Similarly, the data shown here also suggest that the stressors used elicited different responses for the mariner transposon. The most important finding, however, is that even if a drug can increase transcription of transposon genes, other steps of transposition may be affected and the end result may be a reduction in mobilization.

Conclusions and Perspectives
In summary, different chemotherapeutic agents may have different effects on the same TE. Moreover, transposase gene expression is necessary but not sufficient for TE mobilization. These results contribute to understanding how TEs respond to different stresses and that different methods beyond mere expression are required to conclude that a TE is indeed mobilized. Questions remain about TEs activated by stress: is there a pattern to the activation of TEs? Which TEs are active during which stress? Does stress result in different responses at different developmental stages? Can the accumulation of different levels of stress alter the response of TEs? Future work could focus on exploiting this variability in stress response to target treatment of cancer and other diseases. Moreover, the choice of chemotherapeutic agent used in cancer treatment could take into account the activity of TEs to reduce side effects and/or improve treatment efficacy, as appropriate.