Effects of o,p’-DDE, a Mitotane Metabolite, in an Adrenocortical Carcinoma Cell Line

In South Brazil, the incidence of pediatric adrenocortical carcinoma (ACC) is higher than in other regions and countries worldwide. The ACC treatment includes therapy with mitotane, the only adrenolytic drug approved by the FDA. The mitotane metabolism occurs via two main reactions: the β-hydroxylation, which yields the final product o,p’-DDA, and the α-hydroxylation, which will give the final product o,p’-DDE. It is speculated that o,p’-DDE may be an active metabolite since it has a cytotoxic effect on adrenocortical carcinoma cells (H295R). No further studies have been conducted to confirm this hypothesis; however, it was found that mitotane and its metabolites are present at significantly different concentrations in the plasma of the patients. Our study aimed to assess the in vitro effects of o,p’-DDE and o,p’-DDD in cell death pathways, oxidative parameters, and interaction with adrenal CYP’s involved in the steroidogenic process in the H295R cell line. It was found that o,p’-DDE had a different effect than the o,p’-DDD on apoptosis, inhibiting this cell death pathway, but it promotes cell necrosis at higher concentrations. In contrast to o,p’-DDD, the o,p’-DDE did not have effects on the different oxidative parameters evaluated, but exhibited stimulatory interactions with steroidogenic CYP’s, at intermediate concentrations. Therefore, we demonstrated important cell effects of o,p’-DDE; its plasma levels during mitotane therapy should be monitored as an important therapeutic parameter.


Introduction
Adrenocortical carcinoma (ACC) is an endocrine tumor that affects the adrenal glands, promoting the overgrowth of this organ and increasing the production of steroid hormones, such as androgens and cortisol. In pediatric patients, this leads to early virilization (acne, pubic hair, hirsutism, increased muscle mass) and Cushing's syndrome. The overall incidence of ACC in adults is 1.7 to 2 cases per million per year, whereas in children is even rarer (0.3 per million per year), being more common in women [1,2]. In contrast, in Southern Brazil, the incidence of ACC in pediatric patients is higher than in other countries and this difference is related to the R337H mutation in the TP53 gene, this genotype being related to aggressive and advanced tumors [1].
The drug 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichlorethane (o,p'-DDD), also known as mitotane, was discovered in 1940, as a derivative of the insecticide dichlorodiphenyltrichloroethane (DDT). The adrenolytic effect was first described in dogs, caused by selective necrosis in the fasciculate and reticular zones in the adrenal cortex [3]. The first clinical evidence was published in 1959, showing mitotane effects in ACC [4] and, therefore, mitotane was approved by the FDA for the treatment of ACC. Although the mechanism of action is still not well understood, mitotane remains the main drug currently used for the treatment of postoperative or inoperable ACC, employed as a monotherapy or combined with chemotherapies [5].

Cell Viability of o,p'-DDD and o,p'-DDE in H295R Cell Line
The H295R cells were incubated with different concentrations of o,p'-DDD. In comparison to untreated cells (control), at concentrations of 100, 300, and 1000 µM, the cell viability was reduced by 51. 37 D). Ctrl was the vehicle control, containing 1% of ethanol P.A in DMEM F12. Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Dunnett's post-test. Data represent the mean ± SEM of three independent experiments (n = 6). * p < 0.05; *** p < 0.0005; **** p < 0.0001, compared to the vehicle group (control group).

Effect of o,p'-DDD and o,p'-DDE on Caspase 3/7 Activity in H295R Cell Line
H295R cells were incubated with different concentrations of o,p'-DDE or 50 μM of o,p'-DDD for 48 h. After this period, the activity of the caspase 3/7 was monitored at different times (i.e., 1, 2, 3, 4, 5, and 18 h). In comparison to the control, the cells treated with o,p'-DDE had a diminished activity of caspase 3/7, after 3 h, with a reduction of 68.79% and 71.40% with 50 and 75 μM of o,p'-DDE, respectively. After 4 h, the activity decreased by 72.17%, 75.75%, and 46.60%; and in 5 h, to 70.93%, 75.54%, and 48.88%, at the concentrations of 50, 75, and 100 μM, respectively. In comparison to the control, the incubation with o,p'-DDD promoted an increase in the caspase 3/7 activity, by 125.7%, 63.13%, and 33.99% at 3, 4, and 5 h, respectively, contrasting with the effects induced by o,p'-DDE. After 18 h post incubation, the overall activity of caspase 3/7 has increased in comparison to other monitoring times. Nevertheless, by comparison with the control, the o,p'-DDE promote a decrease of 26.03%, 30.7%, and 15.37%, with concentrations of 50, 75, and 100 μM of, respectively. At this time, cells incubated with o,p'-DDD (50 μM), exhibited a modest decrease in the activity of caspase 3/7, being 18.44% less than the control ( Figure 2).  D). Ctrl was the vehicle control, containing 1% of ethanol P.A in DMEM F12. Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Dunnett's post-test. Data represent the mean ± SEM of three independent experiments (n = 6). * p < 0.05; *** p < 0.0005; **** p < 0.0001, compared to the vehicle group (control group). In the presence (+) or absence (−) of Necrostatin-1 (Nec-1), a RIP1 inhibitor with the capability to prevent the necroptosis process, H295R cells were incubated with different concentrations of o,p'-DDE, or o,p'-DDD at 50 μM for 48 h. The cell viability was monitored by MTT assay, and each group incubated with Nec-1 (+) was compared with those  10,25,50,75, and 100 µM for 48 h. Control was the vehicle, containing 1% of ethanol P.A in DMEM F12. Statistical analyses were performed by two-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. Data represent the mean ± SEM of three independent experiments (n = 6). * p < 0.05 compared to the vehicle group (control group).

Effect of o,p'-DDD and o,p'-DDE on Necroptosis in H295R Cell Line
In the presence (+) or absence (−) of Necrostatin-1 (Nec-1), a RIP1 inhibitor with the capability to prevent the necroptosis process, H295R cells were incubated with different concentrations of o,p'-DDE, or o,p'-DDD at 50 µM for 48 h. The cell viability was monitored by MTT assay, and each group incubated with Nec-1 (+) was compared with those not incubated [(Nec-1 (−)]. A gain in cell viability in the groups Nec-1(+) would indicate the participation of necroptosis in the cell death process, promoted by o,p'-DDE. However, in the groups Nec-1 (+), a slight decrease in cell viability was observed, being more pronounced at 100 µM of o,p'-DDE, which induces a decrease in cell viability of 18.34%, in comparison to group Nec-1 (−). In contrast to the effects of o,p'-DDE, no difference in cell viability was observed in the groups Nec-1 (+) and Nec-1 (−) incubated with o,p'-DDD at 50 µM (Figure 3). This result indicates that the necroptosis process was not involved in the cell death mechanism promoted by o,p'-DDE.

Effect of o,p'-DDD and o,p'-DDE on Necrosis in H295R Cell Line
In order to understand the cell death mechanism induced by o,p'-DDE, H295R cells were treated with different concentrations of o,p'-DDE, and 50 μM of o,p'-DDD for comparison. These cell cultures were incubated with 7-aminoactinomycin D (7-AAD), a fluorescent nuclear stain, that is impermeant to the normal plasma membrane of live cells. The plasma membrane of necrotic cells, on the other hand, is permeable to 7-AAD, indicative of an active necrotic process. H295R cells treated with o,p'-DDE at 75 μM increased their fluorescence by 39.37%, but at 100 μM cells exhibited a fluorescence 206.98% higher than that observed in control cells. This confirms that, at least in part, necrosis is a mechanism of cell death induced by o,p'-DDE. Nevertheless, the effect of o,p'-DDD in cell necrosis was higher than that observed with o,p'-DDE, since at 50 μM, the fluorescence of 7-AAD was increased by 291.65%, in comparison with the control group ( Figure 4). Statistical analyses were performed by two-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. Data represent the mean ± SEM of three independent experiments (n = 6). * p < 0.05 compared to the vehicle group (control group). # p < 0.05 when comparing (−) Nec-1 and (+) Nec-1.

Effect of o,p'-DDD and o,p'-DDE on Necrosis in H295R Cell Line
In order to understand the cell death mechanism induced by o,p'-DDE, H295R cells were treated with different concentrations of o,p'-DDE, and 50 µM of o,p'-DDD for comparison. These cell cultures were incubated with 7-aminoactinomycin D (7-AAD), a fluorescent nuclear stain, that is impermeant to the normal plasma membrane of live cells. The plasma membrane of necrotic cells, on the other hand, is permeable to 7-AAD, indicative of an active necrotic process. H295R cells treated with o,p'-DDE at 75 µM increased their fluorescence by 39.37%, but at 100 µM cells exhibited a fluorescence 206.98% higher than that observed in control cells. This confirms that, at least in part, necrosis is a mechanism of cell death induced by o,p'-DDE. Nevertheless, the effect of o,p'-DDD in cell necrosis was higher than that observed with o,p'-DDE, since at 50 µM, the fluorescence of 7-AAD was increased by 291.65%, in comparison with the control group ( Figure 4).

Effect of o,p'-DDD and o,p'-DDE on Oxidative Parameters in H295R Cell Line
The o,p'-DDE at concentrations of 10, 25, and 50 µM, as well as the o,p'-DDD at 50 µM, did not affect the GSH levels in H295R cells, in comparison to untreated cells ( Figure 5A). However, in comparison to the control, the o,p'-DDE (25 and 50 µM) induced a decrease in catalase activity, by 40.56% and 30.94%, respectively. By contrast, o,p'-DDD (50 µM) increased catalase activity by 46.05%, in comparison to untreated cells ( Figure 5B). The GST was not affected by incubation with o,p'-DDE ( Figure 5C), compared to untreated cells; nevertheless, o,p'-DDD induced an increase in GST activity, by more than 10 fold, in comparison to untreated control. The GPx activity was increased when the cells were treated with 25 and 50 μM of o,p'-DDE, by 115.55% and 151.11%, respectively ( Figure 5D), whereas o,p'-DDD did not influence significantly the activity of GPx. No differences were observed in the accumulation of LPO or SOD levels among the untreated cells, and those treated with o,p'-DDE. On the other hand, the cells treated with 50 μM o,p'-DDD, compared to untreated cells, exhibited an increase in these parameters (85.55% for LPO and 146.85% for SOD ( Figure  5E and Figure 5F, respectively). The o,p'-DDE, and o,p'-DDD at 50 μM decreased ROS levels by 29.31% and 25.1%, respectively ( Figure 5G).

Effects of o,p'-DDD and o,p'-DDE in Hormone Secretion in H295R Cell Line
At the assayed concentrations, the o,p'-DDE did not affect the cortisol secretion in comparison to the control ( Figure 6A   The Vina scores, cavity sizes, docking centers, and sizes of the predicted cavities from the docking simulation of o,p'-DDE for different CYPs are shown in Table 1. A negative value of the Vina score represents a more stable binding of the compound to the target. High values for cavity size indicate that the cavity size is close to or larger than the compound, resulting in the accuracy of docking [20,21]. All docking sketch maps of the target protein with o,p'-DDE are shown in Figure 7.

Molecular Docking of o,p'-DDE with CYP11A1, CYP11B1, CYP21A2, CYP19A1 an CYP17A1
The Vina scores, cavity sizes, docking centers, and sizes of the predicted caviti the docking simulation of o,p'-DDE for different CYPs are shown in Table 1. A n value of the Vina score represents a more stable binding of the compound to the High values for cavity size indicate that the cavity size is close to or larger than th pound, resulting in the accuracy of docking [20,21]. All docking sketch maps of th protein with o,p'-DDE are shown in Figure 7.

Discussion
The compounds o,p'-DDE, and o,p'-DDA are the major products of mitotane meta olism, raising doubts if metabolic activation is important for the therapeutic effect [1 Despite its lower plasma concentration, it has been discussed whether o,p'-DDE could an active metabolite since it displays cytotoxic effects on the adrenocortical cell l (H295R) [12]. Higher concentrations of o,p'-DDE are found associated with chylomicro and this could explain its plasma-free lower concentration [17]. Moreover, o,p'-DDE is n found in feces or urine, indicating it has a cumulative behavior in patients, over the th apy [18]. As a prognosis tool, a previous study reported the medians of o,p'-DDE lev in plasma, finding 2.87 μM for responders, 2.03 μM for patients with stable disease, a 1.56 μM for non-responders [14]. Herein, we observed that o,p'-DDE displayed simi cytotoxicity as mitotane, in H295R cells, after 48 h of incubation, with close IC50 valu This agrees with a previous study, where o,p'-DDE also had cytotoxic effects in the H29 cell line, after 72 h of incubation [18]. Furthermore a similar compound, MeSO2-DDE, sayed at 5, 10, and 15 μM, also reduced significantly the viability of H295R cells after 7 of incubation [22]. Despite this cytotoxic effect on the adrenal cell line, the mechanism action displayed by the o,p'-DDE was not determined. Therefore, we investigated so cell death pathways, such as apoptosis, necroptosis, ferroptosis, and necrosis, to expl the effects of o,p'-DDE.
It has been found that mitotane induces apoptosis in H295R cells, increasing caspa 3/7 activity [23][24][25][26]. Accordingly, we observed an increase in the caspase 3/7 activity H295R cells incubated with mitotane at 50 μM (in comparison to untreated control), co firming apoptosis as an important pathway of cell death. On the other hand, in compa son to untreated cells, o,p'-DDE significantly inhibited the caspase 3/7 activity at alm all concentrations assayed (Figure 2), indicating a different cell death pathway, other th apoptosis. Necroptosis is a highly regulated type of necrosis [27], that occurs through t activation of the receptor-interacting protein (RIP), RIPK1 and RIPK3, and mixed linea kinase domain-like pseudokinase (MLKL) [28,29]. However, the o,p'-DDE was able to d crease the H295R cell viability, even in the presence of Nec-1, an inhibitor of the necro tosis pathway, as also observed after incubation with mitotane. Therefore, no involveme of necroptosis in the cell death mechanism of mitotane or o,p'-DDE was observed. Inste the combination of o,p'-DDE, and Nec-1, induced higher cytotoxicity in H295R cells, th

Discussion
The compounds o,p'-DDE, and o,p'-DDA are the major products of mitotane metabolism, raising doubts if metabolic activation is important for the therapeutic effect [15]. Despite its lower plasma concentration, it has been discussed whether o,p'-DDE could be an active metabolite since it displays cytotoxic effects on the adrenocortical cell line (H295R) [12]. Higher concentrations of o,p'-DDE are found associated with chylomicrons, and this could explain its plasma-free lower concentration [17]. Moreover, o,p'-DDE is not found in feces or urine, indicating it has a cumulative behavior in patients, over the therapy [18]. As a prognosis tool, a previous study reported the medians of o,p'-DDE levels in plasma, finding 2.87 µM for responders, 2.03 µM for patients with stable disease, and 1.56 µM for non-responders [14]. Herein, we observed that o,p'-DDE displayed similar cytotoxicity as mitotane, in H295R cells, after 48 h of incubation, with close IC 50 values. This agrees with a previous study, where o,p'-DDE also had cytotoxic effects in the H295R cell line, after 72 h of incubation [18]. Furthermore a similar compound, MeSO 2 -DDE, assayed at 5, 10, and 15 µM, also reduced significantly the viability of H295R cells after 72 h of incubation [22]. Despite this cytotoxic effect on the adrenal cell line, the mechanism of action displayed by the o,p'-DDE was not determined. Therefore, we investigated some cell death pathways, such as apoptosis, necroptosis, ferroptosis, and necrosis, to explain the effects of o,p'-DDE.
It has been found that mitotane induces apoptosis in H295R cells, increasing caspase 3/7 activity [23][24][25][26]. Accordingly, we observed an increase in the caspase 3/7 activity in H295R cells incubated with mitotane at 50 µM (in comparison to untreated control), confirming apoptosis as an important pathway of cell death. On the other hand, in comparison to untreated cells, o,p'-DDE significantly inhibited the caspase 3/7 activity at almost all concentrations assayed (Figure 2), indicating a different cell death pathway, other than apoptosis. Necroptosis is a highly regulated type of necrosis [27], that occurs through the activation of the receptor-interacting protein (RIP), RIPK1 and RIPK3, and mixed lineage kinase domain-like pseudokinase (MLKL) [28,29]. However, the o,p'-DDE was able to decrease the H295R cell viability, even in the presence of Nec-1, an inhibitor of the necroptosis pathway, as also observed after incubation with mitotane. Therefore, no involvement of necroptosis in the cell death mechanism of mitotane or o,p'-DDE was observed. Instead, the combination of o,p'-DDE, and Nec-1, induced higher cytotoxicity in H295R cells, than o,p'-DDE.
The treatment with o,p'-DDE increased the GPx activity ( Figure 5D), indicating an inhibition of the ferroptosis pathway. Furthermore, the evolution of ferroptosis is accompanied by increased lipid peroxidation (LPO) and ROS production [30,31], which did not occur in the cells treated with o,p'-DDE ( Figure 5E,G). Mitotane did not induce ferropto-sis [30,32], which might explain why GPx levels did not decrease after exposure to mitotane ( Figure 5D). A depletion in GSH impairs the activity of GPX4, resulting in an accumulation of peroxidized lipids which are involved in the ferroptosis mechanism [33]. Herein, both mitotane and o,p'-DDE had no effects in GSH levels ( Figure 5A). However, mitotane at 50 µM increased the lipid peroxidation in H295R cells, in comparison to untreated cells, which was expected since the drug promotes strong induction of lipid peroxidation in ACC cells [30]. By contrast, the treatment of H295R cells with o,p'-DDE did not lead to an LPO accumulation, also indicating that ferroptosis is not involved in the cell death mechanism. Instead, as in mitotane incubation, a significant increase of 7-AAD staining was observed in cells treated with 100 µM o,p'-DDE, in comparison to the untreated control, revealing that cells treated at high concentrations of o,p'-DDE undergo a necrotic process.
Superoxide dismutase (SOD) and Catalase (CAT) are important enzyme classes in cell oxidative stress. Whereas SOD promotes superoxide dismutation, the CAT reacts with H 2 O 2 to form water and oxygen [34][35][36]. As previously observed [16], in our assays, mitotane stimulated the SOD expression in the H295R cells, but the cells treated with o,p'-DDE displayed only a discrete increase (without statistical significance) in SOD activity. Interestingly, another mitotane metabolite, o,p'-DDA, also did not promote a stimulatory effect in the SOD activity in H295R cells [16]. Mitotane also stimulated the CAT expression, which can protect cells from oxidative damage; notwithstanding, the o,p'-DDE slightly decreased the CAT activity in comparison to the control ( Figure 8B). The increase in CAT and SOD activity exerted by mitotane treatment may explain the diminished levels of ROS observed in H295R cells, in comparison to the untreated cells (control). On the other hand, despite o,p'-DDE depleting CAT expression, a decreased level of ROS was observed in the cells treated with o,p'-DDE in comparison to the control. Therefore, o,p'-DDE was unable to stimulate SOD, as mitotane did, and it promoted a depletion in the CAT expression. Nevertheless, at 50 µM, both mitotane and o,p'-DDE were able to deplete the ROS levels equivalently, suggesting there is no involvement of oxidative stress in the mechanism of cell death of o,p'-DDE.
GST has a pivotal role in cell detoxification against xenobiotics. The expression of GST in H295R cells incubated with mitotane 50 µM was augmented about 12-fold, in comparison to the untreated cells (control), indicating that mitotane can be conjugated with GSH for being eliminated. However, the o,p'-DDE did not stimulate the expression of GST as observed with mitotane, but a slight augmentmentation was observed with 50 µM of o,p'-DDE; in comparison to untreated cells, this was not statistically significant. Therefore, in contrast to mitotane, the o,p'-DDE seems not to be eliminated via the GSH-GST system.
Metabolic activation of mitotane may be dependent on CYP11B1, or others CYPs, and the ability of the tumor to metabolize mitotane may predict the response to treatment [15]. In comparison to untreated cells, mitotane decreased the expression of CYP11A1 and CYP17A1 ( Figure 8A,B), as previously observed [24,37]. Nevertheless, it increased the expression of CYP19A1 which, to our knowledge, was not previously described. Thus, we investigated the potential of o,p'-DDE to induce the activity of CYPs involved in the production of aldosterone and DHEA, which could explain the increased levels of these hormones, in comparison to untreated cells ( Figure 6B,C). Indeed, our in silico analysis, following the Vina score and cavity size, showed a strong interaction between o,p'-DDE, and CYP11A1, CYP11B1, CYP21A2, CYP19A1 and, mainly, with CYP17A1, which had the highest value of cavity size. Furthermore, in comparison to the control, the o,p'-DDE, at 25 µM, showed the higher stimulation of CYP17A1 expression ( Figure 8B), confirming their close relationship, as observed in our in silico assays. CYP17A1 converts the 17hydroxypregnenolone to DHEA, explaining the increased concentrations of DHEA in H295R cells treated with o,p'-DDE ( Figure 6C), compared to untreated control.
A previous investigation showed that MeSO 2 -DDE, at the lower concentration, stimulated steroid production and CYP11B1 expression, but had the opposite effect when assayed at higher concentrations [22], suggesting a biphasic response which is reported with a wide variety of compounds [12,37,38]. This behavior is called hormesis, a dose-response relationship characterized by low-dose stimulation and high-dose inhibition [39]. In H295R cells, 1.25 µM of MeSO 2 -DDE increased the levels of CYP11B1, CYP11B2, and StAR, whereas concentrations of 5 and 10 µM did not [39]. A similar biphasic response was observed in steroid secretion; the lower concentrations of MeSO 2 -DDE (at 1.25 µM) increased cortisol and aldosterone levels, whereas at 10 µM the steroid hormones secretion was reduced [39,40]. Herein, we observed that o,p'-DDE stimulated the expression of CYP17A1 and increased aldosterone and DHEA secretion, at the intermediate concentration assayed (25 µM) whereas the levels of cortisol remained unchanged. The stimulation promoted by o,p'-DDE occurred in the intermediate concentration investigated, whereas the lower and higher concentrations did not affect the hormone secretion nor the CYPs expression, being consistent with a biphasic behavior, as described for MeSO 2 -DDE.  [41]. For the experiments with H295R, the cells were used between the 5 and 10 passages as recommended by ATCC (CLR-2128(CLR- , 2007, to ensure the secretion of steroid hormones.

Cell Viability
The H295R cells were seeded at a concentration of 1 × 10 4 in a 96-well plate and incubated for 24 h at 37 • C and 5% CO 2 . After that, the cells were exposed to o,p'-DDD,

Necroptosis Assessment
The cells were seeded at a concentration of 5 × 10 4 in two 96-well plates. After 24 h, the supernatant was removed and cells were treated with o,p'-DDE at 0,1, 3, 10, 30, 100, 300, and 1000 µM and 50 µM of o,p'-DDD, and incubated for 48 h. Then, 100 µL of MTT solution (0.5 mg/mL) in DMEM F12 was added to each well. In one plate 50 mM of Necrostatin-1 (Nec-1) (Sigma-Aldrich, St. Louis, MO, USA), an inhibitor of receptor-associated kinase 1 (RIPK1), was added. The plates were incubated for 3 h at 37 • C and the same cell viability protocol was performed, as described previously in item 4.3. The plate was read at 595 nm by a microplate reader EPOCH BIOTEK ® (BIOTEK, Winooski, VT, USA).

Necrosis Assessment
The cells were seeded at a concentration of 1 × 10 6 in a 6-well plate. After 24 h, the supernatant was removed and the cells were treated with o,p'-DDD at 50 µM and o,p'-DDE at 0, 10, 25, 50, 75, and 100 µM, and incubated for 48 h. After incubation, the supernatant was collected and cells were washed with 1 mL of PBS and trypsinized. The cells were mixed with the supernatant to maintain dead cells. The cells were centrifuged at 1400 rpm for 10 min and resuspended in PBS. The cells were transferred in cytometer tubes and 400 µL of PBS and 5 µL of 7-AAD (BD Pharmingen, Franklin Lakes, NJ, USA) were added. The samples were incubated for 15 min at room temperature and analyzed in a flow cytometer FACSCanto II (Becton Dickinson, Franklin Lakes, NJ, USA).

Oxidative Parameters
H295R cells were seeded at a concentration of 1 × 10 6 in a 6-well plate until reaching the confluence of 80-90% [43]. After that, the cells were incubated with 0, 10, 25, and

Dosage of DHEA, Cortisol and Aldosterone
The cells were seeded at a concentration of 3 × 10 4 in a 24-well plate. After 24 h, the supernatant was removed and the cells were incubated with 10, 25, and 50 µM of o,p'-DDE, for 48 h. After that, the supernatant was collected and the samples were stored at −80 ºC. Cortisol, aldosterone, and DHEA levels were determined by ELISA kits following producer instructions (Invitrogen, Carlsbad, CA, USA).

Molecular Docking
The molecular docking simulation was conducted using a method based on cavity detection by CB-Dock (http://cao.labshare.cn/cb-dock/; accessed on 15 October 2022) [20]. The crystal structures of the CYP11A1, CYP11B1, CYP21A2, CYP19A1, and CYP17A1 were downloaded from the protein data bank (http://www.rcsb.org; accessed on 15 October 2022). The 3D structure of o,p'-DDE was downloaded from the PubChem compound database (https://pubchem.ncbi.nlm.nih.gov/; accessed on 15 October 2022) and converted to ".mol" extension. Then, the crystal structures of proteins and the 3D structure of o,p'-DDE were inputted to CB-Dock and binding activities were analyzed according to vina score, cavity size, docking center, and size [20].

CYP's Expression
The cells were seeded at a concentration of 1 × 10 6 in a 6-well plate. After 24 h, the supernatant was removed and the cells were incubated with 10, 25, and 50 µM of o,p'-DDE, and 50 µM of o,p'-DDD, in triplicate, for 48 h. Following incubation, total RNA was extracted from the cells using the GenElute™ Total RNA Purification Kit, following the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO, USA). 1 µg of the extracted RNA was reverse-transcribed using High-Capacity cDNA Reverse Transcription Kit (Invitrogen, Carlsbad, CA, USA). The cDNAs were amplified by quantitative PCR using the primers for CYP11A1 (Hs00167984), CYP17A1 (Hs01124136), and CYP19A1 (Hs00903411) (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression levels were calculated relative to their respective control GAPDH and expressed as 2-ddCt.

Statistical Analysis
All data were expressed as means ± standard error of the mean (S.E.M.). Results were subjected to the Kolmogorov-Smirnov normality test and by one or two-way analysis of variance (ANOVA), followed by post hoc of Dunnett's, Tukey's or Holm-Sidak's multiple comparisons test, when applicable. For results that failed on the normality test, the Kruskal-Wallis test with Dunn's Multiple Comparison Test were performed. The results were analyzed using GraphPad Prism (v. 5.0, San Diego, CA, USA). The level of significance was set at 95% (p < 0.05).

Conclusions
Despite the speculation over the o,p'-DDE effects on patients during mitotane therapy, few studies were performed to evaluate the action of o,p'-DDE in the adrenocortical cells. Therefore, in this study, we investigated the cell pathway using the H295R cell line and found that necrosis was the main mechanism of cell death promoted by o,p'-DDE. We also reported, for the first time, a biphasic-like behavior of H295R cells against o,p'-DDE, as observed in the hormone secretion effects and stimulation of CYP17A1, which explained the DHEA secretion profile after incubation with different concentrations of o,p'-DDE. Therefore, due to these important findings, and its cumulative behavior, the levels of o,p'-DDE in plasma patients should also be monitored during the mitotane therapy to ensure efficacy and safety to patients.