The Journal of Toxicological Sciences
Online ISSN : 1880-3989
Print ISSN : 0388-1350
ISSN-L : 0388-1350
Original Article
Dose- and time-dependent changes in blood and adrenal levels of multiple steroids in rats after administration of ketoconazole with or without ACTH
Tomoaki TochitaniAkihito YamashitaIzumi MatsumotoMami KouchiYuta FujiiIzuru Miyawaki
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2019 Volume 44 Issue 9 Pages 601-610

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Abstract

To verify simultaneous measurement of blood levels of adrenal steroids as a tool to evaluate drug effects on adrenal steroidogenesis, dose- and time-dependent changes in blood levels of corticosterone and its precursors (pregnenolone, progesterone and deoxycorticosterone), as well as their relationship with the pathological changes in the adrenal gland, were examined in rats dosed with ketoconazole (KET). Also examined were whether effects on adrenal steroidogenesis that were not obvious in the blood steroid levels after sole administration of KET could be revealed by post-administration of ACTH, and the correlation between the blood and adrenal steroid levels. Male rats were dosed with 15, 50, or 150 mg/kg of KET for 1 or 7 days with or without ACTH, and the blood and adrenal concentrations of the steroids were measured. KET increased the blood deoxycorticosterone level even at a dose level and time point at which histopathological changes were not obvious. KET-induced changes in blood levels of other steroids were revealed by ACTH, and the blood and adrenal levels were generally correlated especially after ACTH post-administration. Thus, blood levels of adrenal steroids, including precursors, can be a sensitive and early marker of drug effects on the adrenal steroidogenesis that reflect adrenal levels of steroids. The usefulness of the multiple steroid measurement as a method for mechanism investigation of drug effects on the adrenal gland can be further enhanced by ACTH.

INTRODUCTION

The adrenal gland is the most common toxicological target within the endocrine system (Ribelin, 1984; Briggs et al., 2015), and drug-induced inhibition of adrenal steroidogenesis can be fatal in humans (Harvey and Everett, 2003). However, methods to evaluate drug effects on adrenal steroidogenesis are scarce, and novel methods are needed (Harvey and Everett, 2003; Hinson and Raven, 2006).

In the adrenal gland of rats, corticosterone is synthesized as the major glucocorticoid from cholesterol (Hanukoglu, 1992; Hinson and Raven, 2006). Cholesterol is first metabolized to pregnenolone by cytochrome P450 (CYP)-11A, next to progesterone by hydroxysteroid dehydrogenase (HSD)-3B, then to deoxycorticosterone by CYP21, and finally to corticosterone by CYP11B1 (Rosol et al., 2001; Harvey and Sutcliffe, 2010). The glucocorticoid synthesis is under the control of adrenocorticotropic hormone (ACTH) from the pituitary gland, which is in turn regulated by hypothalamic corticotrophin-releasing hormone and arginine vasopressin (Hinson and Raven, 2006). ACTH upregulates glucocorticoid synthesis by promoting expression of the CYP enzymes (especially the rate-limiting enzyme CYP11A), and by increasing availability of cholesterol to CYP11A (Rosol et al., 2001; Miller and Bose, 2011; Inomata and Sasano, 2015).

In our previous studies using rats, we showed that simultaneous measurement of blood levels of multiple adrenal steroids, including corticosterone and its precursors, can be a sensitive method to detect effects of various drugs on adrenal steroidogenesis, and also can be useful to investigate the underlying mechanisms (Tochitani et al., 2016, 2017). However, since the drug effects were evaluated only at a single dose level and single time point, the dose- and time-dependency of the changes in the blood steroid levels were unknown. Also, changes in steroid levels could not be detected for some mechanisms.

The aim of the present study was to further verify the simultaneous measurement of blood levels of multiple adrenal steroids as a tool to evaluate drug effects on adrenal steroidogenesis. With this aim, the dose- and time-dependent changes in blood levels of corticosterone and its precursors (pregnenolone, progesterone and deoxycorticosterone), as well as their relationship with the pathological changes in the adrenal gland, were examined in rats dosed with ketoconazole (KET). Also, it was examined whether effects on adrenal steroidogenesis that were not obvious in the blood steroid levels after sole administration of KET could be revealed by post-administration of ACTH. Furthermore, the adrenal steroid levels and their correlation with the blood levels were examined. KET was chosen as the tool compound, because it inhibits several CYP enzymes involved in the adrenal steroidogenesis, and in the previous study in rats, it caused marked increase in the blood deoxycorticosterone level through CYP11B1 inhibition, while its effect on other CYP enzymes were not obvious in the blood steroid levels (Tochitani et al., 2016, 2017).

MATERIALS AND METHODS

Compounds

KET was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). ACTH (adrenocorticotropic hormone) (1-24) was purchased from Bachem AG (Bubendorf, Switzerland).

Animals and husbandry

Animal usage was approved by the Committee for the Ethical Usage of Experimental Animals of Sumitomo Dainippon Pharma Co., Ltd.

Male Sprague-Dawley (Crl:CD) rats purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan) were used. The rats were fed commercial pellet diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum. In Experiment 1, the animals were housed individually or 2 animals per cage in a room with a 12-hr light (8 a.m. to 8 p.m.)/dark cycle. In Experiment 2, the animals were housed 5 animals per cage in a room in which the light was turned on at 8 a.m. and off at 6 p.m. Females were not used considering possible influence of the estrous cycle.

Animal experiment

Experiment 1

Animals were randomly assigned to KET and 0.5% methyl cellulose solution (0.5% MC: vehicle) groups at 8 weeks of age. The dose levels of KET were 15, 50, and 150 mg/kg. The high dose was chosen based on previous reports (Tochitani et al., 2016, 2017), and the middle and low doses were chosen using a common ratio of approximately 3. The group composition is shown in Table 1. The animals in the KET group were dosed with KET by oral gavage once daily at about 10 a.m. for 1 (single-dose group) or 7 days (repeated-dose group). The dosing period of 7 days was chosen based on previous reports (Tochitani et al., 2016, 2017). KET was suspended in 0.5% MC, and the dosing volume was 5 mL/kg. The animals in the vehicle control group were dosed with the same volume of 0.5% MC in the same manner as in the KET group.

Table 1. Group composition in Experiment 1.
Group Dose level
(mg/kg)
Number of animals
Single dose Repeated dose
0.5% MC 0 5 5
KET 15 0 5
50 0 5
150 5 5

During the dosing period, daily observation for clinical signs and mortality was performed before dosing. Body weights were measured before and 2 days after the initiation of dosing, and on the day of necropsy.

1, 3 and 6 hr after the single or 7-day repeated dosing of KET, up to 0.5 mL of blood was sampled from the tail vein without anesthesia, using a syringe with a needle coated with EDTA. Plasma was prepared by centrifuging the blood samples, and stored at -80°C until use.

About 24 hr after the final dosing, the rats were euthanized by exsanguination under isoflurane anesthesia and were necropsied. At necropsy, the bilateral adrenal glands and thymus were rapidly removed, grossly examined, and weighed. The organ weight relative to body weight was calculated using the body weight recorded on the day of necropsy, and after the organ weight measurement, the adrenal gland was fixed in 10% neutral-buffered formalin (NBF).

Experiment 2

Animals were randomly assigned to KET and 0.5% MC groups at 6 weeks of age. The group composition is shown in Table 2. The animals in the KET group were given a single dose of 150 mg/kg of KET by oral gavage between 9 and 10 a.m. KET was suspended in 0.5% MC, and the dosing volume was 5 mL/kg. 5 hr after the KET dosing, the rats were subcutaneously dosed with saline or 0.4 mg/kg of ACTH. ACTH was dissolved in saline, and the dosing volume was 1 mL/kg. The animals in the 0.5% MC group were given a single dose of 0.5% MC, followed by saline or ACTH dosing in the same manner as in the KET group.

Table 2. Group composition in Experiment 2.
Group Dose level
(mg/kg)
Number of animals
Saline ACTH
0.5% MC 0 5 5
KET 150 5 5

Body weights were measured before the dosing of KET, and observation for clinical sings and mortality was performed before blood sampling 6 hr after the dosing of KET.

Six hr after the KET dosing (1 hr after the ACTH dosing), about 250 μL of blood was sampled from the tail vein without anesthesia, using a syringe with a needle coated with EDTA. Plasma was prepared by centrifuging the blood samples, and stored at -80°C until use.

Immediately after the blood sampling, the rats were euthanized by exsanguination under isoflurane anesthesia and were necropsied. At necropsy, the bilateral adrenal glands were rapidly removed, grossly examined and weighed. Then, the adrenal gland was frozen in liquid nitrogen, and stored at -80°C until use.

Histopathology and image analysis

In Experiment 1, the adrenal glands fixed in 10% NBF were embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and examined with a light microscope by a certified pathologist of the Japanese Society of Toxicologic Pathology.

Whole images of the HE-stained adrenal sections were captured using the Aperio ScanScope AT2. Image analysis of cortical vacuolation was conducted using ImageScope software (Leica Biosystems Imaging Inc., Vista, CA, USA) as described previously (Tochitani et al., 2017), with slight modification to improve the sensitivity to detect vacuoles and specificity to exclude non-vacuole areas such as vascular lumen. In the zona fasciculata/reticularis, the total vacuole area (V) was quantified using the Nuclear Algorithm (version 9.1). The tissue area excluding vacuoles (T) was quantified using the Positive Pixel Count Algorithm (version 9.1). The ratio of the total vacuole area to the total tissue area including vacuoles (100 (%) × V/(V+T)) was used as the vacuolation index.

Measurement of blood and adrenal concentrations of steroids

Both in Experiments 1 and 2, plasma concentrations of corticosterone and its precursors (deoxycorticosterone, progesterone, and pregnenolone) were simultaneously measured, using a liquid chromatograph (Nexera, Shimadzu, Kyoto, Japan) coupled with a tandem mass spectrometer (LC-MS/MS) (Triple Quad 6500 or 6500+, AB Sciex, Massachusetts, USA) as described previously (Tochitani et al., 2017).

In Experiment 2, adrenal concentrations of corticosterone, deoxycorticosterone, and progesterone were measured as described previously (Maeda et al., 2013).

Statistical analysis

Statistical analysis was performed using SAS v9.4 software (SAS Institute Inc., Cary, NC, USA). Group differences in the body weight, organ weight, adrenal vacuolation index, and blood and adrenal steroid level were analyzed as follows.

In Experiment 1, for comparison of the 0.5% MC and KET single-dose groups, homogeneity of variance between the two groups was first tested using an F-test (significance level of 25%). When the variance was homogeneous, Student’s t-test was used, and when the variance was not homogeneous, Welch’s t-test was used. For comparison of the 0.5% MC group and KET repeated-dose groups, Dunnett’s test was used.

In Experiment 2, for comparison of the 0.5% MC and KET with or without ACTH dose groups, Tukey’s test was used. Also, Pearson’s correlation coefficient between the blood and adrenal steroid levels were calculated each in the saline and ACTH groups.

Unless otherwise specified, two-tailed test was performed with significance level of 5%. Before the statistical analysis, blood and adrenal steroid concentration values were log-transformed. Also, blood steroid concentration values below the LLOQ were treated as LLOQ/2.

RESULTS

Experiment 1: Dose- and time-dependent changes in organ weights, histopathology, and blood steroid levels after KET administration

Necropsy and organ weights

Throughout the dosing period, no animal died or showed clinical signs or body weight changes. At necropsy after repeated doses, the adrenal glands were pale and large in the KET 150 mg/kg group.

Table 3 shows the organ weights. After repeated doses, the adrenal weight was significantly high in the KET 150 mg/kg group. Also after a single dose, the adrenal weight tended to be high in the KET 150 mg/kg group, though without statistical significance. No significant change was seen in the thymus weight in any group.

Table 3. Organ weights.
Group Final body
weight (g)
Adrenal glands Thymus
AB (mg) RE (%) AB (g) RE (%)
Single dose
0.5% MC 344 ± 16 55 ± 12 16.1 ± 3.3 0.59 ± 0.10 0.17 ± 0.02
KET 150 mg/kg 339 ± 13 69 ± 7 20.3 ± 2.4 0.57 ± 0.11 0.17 ± 0.03
Repeated dose
0.5% MC 383 ± 26 54 ± 10 13.9 ± 1.7 0.56 ± 0.09 0.15 ± 0.03
KET 15 mg/kg 375 ± 21 60 ± 5 16.1 ± 1.5 0.53 ± 0.10 0.14 ± 0.03
50 mg/kg 380 ± 25 63 ± 11 16.5 ± 2.7 0.51 ± 0.06 0.14 ± 0.01
150 mg/kg 363 ± 21 126* ± 25 34.8* ± 2.7 0.63 ± 0.07 0.17 ± 0.02

N = 5. The values are shown as the group mean ± standard deviation.

AB = absolute weight; RE = relative weight to body weight.

*p < 0.05.

Histopathology and image analysis

Table 4 shows the histopathological changes in the adrenal gland. After repeated doses, increased vacuolation of the zona fasciculata/reticularis was observed in the KET 50 mg/kg and above groups (Fig. 1). The vacuoles were thought to be lipid droplets based on their morphology. In addition, hypertrophy of the zona fasciculata/reticularis was observed in the KET 150 mg/kg repeated-dose group. Minimal hypertrophy was also observed in the KET 150 mg/kg single-dose group.

Table 4. Histopathological changes in the adrenal gland.
Findings Group 0.5% MC KET
Dose level (mg/kg) 0 15 50 150
Single dose Number of animals 5 0 0 5
Hypertrophy, ZF/ZR - 5 4
± 1
Repeated dose Number of animals 5 5 5 5
Hypertrophy, ZF/ZR - 5 5 5
± 1
+ 3
2+ 1
Vacuolation, increased, ZF/ZR - 5 5 2
±
+ 3
2+ 3
3+ 2

ZF = zona fasciculata; ZR = zona reticularis.

- = not remarkable; ± = minimal; + = mild; 2+ = moderate; 3+ = severe.

Fig. 1

Histopathological images of the adrenal cortex of a rat from the 0.5% MC (A) and KET 15 mg/kg (B), 50 mg/kg (C) and 150 mg/kg (D) repeated-dose groups (HE sections). Insets show higher magnification of affected cells. While there are no remarkable changes in the 0.5% MC and KET 15 mg/kg groups, vacuolation of the zona fasciculata/reticularis can be seen in the KET 50 mg/kg and 150 mg/kg repeated-dose groups, as well as hypertrophy of the same area in the KET 150 mg/kg repeated-dose group.

Image analysis confirmed statistically significant increase in vacuolation of zona fasciculata/reticularis in the KET 150 mg/kg repeated-dose group (Fig. 2). Also, the vacuolation index tended to be high in the KET 50 mg/kg repeated-dose group. Changes were not observed in the KET 15 mg/kg repeated-dose group or KET 150 mg/kg single-dose group.

Fig. 2

Image analysis of the vacuolation of the zona fasciculata/reticularis. Each dot shows an individual animal value (N = 5/group). The double-asterisk indicates statistically significant difference in the group mean from that in the corresponding 0.5% MC group (**p < 0.01).

Blood levels of steroids

Figure 3 shows blood levels of corticosterone and its precursors. After repeated doses, the deoxycorticosterone level was significantly high in the all KET groups including 15 mg/kg group, generally with dose-dependency. The change was greater at later time points in the KET 150 mg/kg group. Also after a single dose, the deoxycorticosterone level was significantly high in the KET 150 mg/kg at every time point, and the absolute values were higher than those after repeated doses. In addition, while the corticosterone level increased with time after a single dose of 0.5% MC, similar tendency was not observed after a single dose of KET 150 mg/kg, though there was no significant difference between the 0.5% MC and KET groups.

Fig. 3

Blood levels of corticosterone and its precursors. Each dot shows the group mean at each time point and the error bar shows the standard deviation (N = 5/group). Asterisks indicate statistically significant difference from the corresponding 0.5% MC group (*p < 0.05; **p < 0.01).

Other than the above, high pregnenolone level was observed 1 hr after repeated doses of KET 150 mg/kg, and high progesterone level was observed 1 hr after a single dose of KET 150 mg/kg; however, they were not considered to be treatment-related, since they were transient and the values were within the physiological range.

Experiment 2: Blood and adrenal levels of steroids after KET administration with or without ACTH, and their correlation

Blood levels of steroids

Figure 4 shows blood levels of corticosterone and its precursors after administration of KET with or without ACTH. Consistent with Experiment 1, KET significantly increased the deoxycorticosterone level in the saline group. In contrast, ACTH significantly increased all 4 steroid levels in the 0.5% MC group, and administration of KET before ACTH significantly suppressed these increases except that in the deoxycorticosterone level.

Fig. 4

Blood levels of corticosterone and its precursors. Each dot shows an individual animal value (N = 5/group). The hash symbols (#) indicate statistically significant difference from the 0.5% MC with saline group (#p < 0.05; ##p < 0.01). Daggers (†) indicate statistically significant difference from the 0.5% MC with ACTH group (†p < 0.05; ††p < 0.01).

Adrenal levels of steroids

Figure 5 shows adrenal levels of steroids after administration of KET with or without ACTH. Similarly as in the blood, KET significantly increased the deoxycorticosterone level in the adrenal gland in the saline group. Also, KET significantly increased the pregnenolone level, and decreased the progesterone and corticosterone levels in the saline group. In contrast, ACTH only significantly decreased the corticosterone level in the 0.5% MC group, and the difference in the corticosterone level between the 0.5% MC and KET groups were smaller in the ACTH group than in the saline group.

Fig. 5

Adrenal levels of corticosterone and its precursors. Each dot shows an individual animal value (N = 5/group). The hash symbols (#) indicate statistically significant difference from the 0.5% MC with saline group (#p < 0.05; ##p < 0.01). Daggers (†) indicate statistically significant difference from the 0.5% MC with ACTH group (†p < 0.05; ††p < 0.01).

Figure 6 shows the correlation between the blood and adrenal levels of steroids. In the saline group, the correlation was weak or not observed, except in the deoxycorticosterone level. On the other hand in the ACTH group, the correlation was moderate to strong except in the pregnenolone level.

Fig. 6

Correlation between the blood and adrenal levels of steroids. Each dot shows an individual animal value (N = 5/group).

DISCUSSION

First in Experiment 1, the dose- and time-dependent changes in blood levels of corticosterone and its precursors after KET administration were examined, as well as their relationship with the pathological changes in the adrenal gland. Consistent with previous studies (Tochitani et al., 2016, 2017), repeated doses of KET 150 mg/kg caused hypertrophy and vacuolation (lipidosis) of zona fasciculata/reticularis of the adrenal gland, and increased the blood deoxycorticosterone level. These changes were not accompanied by changes in the general condition or non-specific stress responses such as decrease in thymus weight, which is typically induced by stress (Elmore, 2012; Harvey and Sutcliffe, 2010). Therefore, it was thought to be unlikely that the adrenal and steroid changes were due to stress responses. The adrenal lipidosis was thought to be caused by accumulation of cholesterol and/or cholesterol ester, via inhibition of CYP enzymes such as CYP11A (Tochitani et al., 2017). Also, the increased deoxycorticosterone level was considered to be caused via CYP11B1 inhibition. The adrenal hypertrophy was thought to be a compensatory change to the disrupted steroidogenesis.

The extent of the above KET-induced changes was generally dose-dependent, and after repeated doses, the organ weight and histopathological changes were not present or only mild at 15 and 50 mg/kg. On the other hand, the blood deoxycorticosterone level was significantly increased even at 15 mg/kg. Also, after a single dose of KET 150 mg/kg, though the organ weight and histopathological changes were not yet obvious, the blood deoxycorticosterone level was significantly increased. Thus, it was shown that the blood deoxycorticosterone level can be a sensitive and early marker of the KET effect on the adrenal gland.

After repeated doses, the changes in the deoxycorticosterone level were greater at later time points in the KET 150 mg/kg group, while similar tendency was not observed in the KET 15 or 50 mg/kg group. This may be related to longer half-life and Tmax (time to maximum concentration) of KET at higher dose levels (Hamdy and Brocks, 2009). Also, the absolute levels of deoxycorticosterone were higher after a single dose than those after repeated doses. This may be related to adaptive changes that occurred after repeated dosing (Stanislaus et al., 2012).

In the 0.5% MC group in Experiment 1, the corticosterone level tended to be higher at later time points, especially after a single dose. This is probably due to the diurnal variation of the corticosterone level, which peaks in the evening in rats (Atkinson and Waddell, 1997). On the other hand, similar tendency was not observed in the KET 150 mg/kg group, suggesting impaired corticosterone secretion. However, there was no significant difference between the 0.5% MC and KET groups, further supporting low sensitivity of the basal blood corticosterone level as a marker for impaired adrenal steroidogenesis (Yarrington and Reindel, 1996).

Next, in Experiment 2, it was examined whether effects on adrenal steroidogenesis that were not obvious in the blood steroid levels after sole administration of KET could be revealed by ACTH. Considering that KET inhibits not only CYP11B1, but also other CYP enzymes involved in the adrenal steroidogenesis such as CYP11A (Johansson et al., 2002), changes were expected in levels of steroids other than deoxycorticosterone.

Consistent with Experiment 1, sole administration of KET only increased the deoxycorticosterone level. Though the animals used in Experiment 2 were slightly younger than those used in Experiment 1, there were no apparent differences in the basal steroid levels and responses to KET, and it was judged that the difference in the animal age did not affect the study results. On the other hand, ACTH alone increased blood levels of all the 4 steroids. This pattern of steroid changes is different from that caused by KET and other inhibitors of steroidogenesis (Tochitani et al., 2017). Considering that stress responses are also caused via increased ACTH (Harvey and Sutcliffe, 2010), it was further evidenced that the steroid changes caused by the compounds were not related to stress responses.

Administration of ACTH after KET revealed changes in blood levels of steroids other than deoxycorticosterone. The decreases in the pregnenolone and progesterone levels were thought to be caused via inhibition of CYP11A. Thus, ACTH enhanced the sensitivity of this method to detect KET-induced changes in the blood steroid levels, and this would be especially beneficial for investigation of underlying mechanisms of effects on the adrenal steroidogenesis.

Moreover, to investigate whether the changes in the blood levels of steroids were reflecting the adrenal changes, we examined the adrenal levels of steroids and their correlation with the blood levels. Similarly as in the blood, KET alone increased the deoxycorticosterone level in the adrenal gland, and the blood and adrenal levels of deoxycorticosterone were strongly correlated. Unlike in the blood, however, KET alone also increased the pregnenolone level and decreased the progesterone and corticosterone levels in the adrenal gland, and the correlation between the blood and adrenal levels was weak or not observed. On the other hand, after administration of ACTH, the correlation was moderate to strong except in the pregnenolone level. Thus, it was shown that the changes in the blood levels of steroids were reflecting the adrenal changes. It was uncertain how KET increased the adrenal pregnenolone level, but this may be a compensatory change, considering that pregnenolone synthesis is a rate-limiting step in the adrenal steroidogenesis that is rapidly upregulated by ACTH (Rosol et al., 2001; Miller and Bose, 2011), Tmax of KET after oral administration to rats is about 1 to 2 hours and earlier than the time point at which the steroid level was evaluated in the present study (Hamdy and Brocks, 2009), and KET inhibits CYP enzymes only reversibly (Yan et al., 2002). Also, sole administration of ACTH unexpectedly decreased the adrenal level of corticosterone, and this may be due to rapid release of preformed corticosterone in the adrenal gland (Mohn et al., 2005).

In conclusion, it was shown in this study that blood levels of adrenal steroids, including precursors, can be a sensitive and early marker of drug effects on the adrenal steroidogenesis that reflect adrenal levels of these steroids. The usefulness of the multiple steroid measurement as a method to investigate the underlying mechanism of drug-effects on the adrenal gland can be further enhanced by ACTH.

ACKNOWLEDGMENTS

We wish to thank members of Preclinical Research Unit of Sumitomo Dainippon Pharma Co., Ltd., especially Mr. Yasuhiro Sasaki and Mr. Kentaro Abe for conducting the animal experiments, Ms. Yumi Tateishi, Ms. Kaori Kunito, and Ms. Izuru Mise for their excellent histotechnical work, and Ms. Chikako Horike for her contract management work. Also, we wish to thank members of Sumika Chemical Analysis Service, Ltd. (Osaka, Japan) and Safety Research Institute for Chemical Compounds Co., Ltd (Hokkaido, Japan) for the measurement of blood and adrenal levels of the steroids, respectively.

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES
 
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