Pathological features of the recovery phase in drug-induced vacuolar lesions caused by steroidogenesis disruption in the canine adrenal cortex

Abstract

Steroidogenesis disruption caused by compounds causes vacuolar lesions in the adrenal cortex. However, research on the reversibility of compound-induced adrenal lesions remains limited. In a 4-week oral repeated-dose toxicity study with a 4-week recovery period in dogs, administration of S-637880—a compound that off-target inhibits steroid hormone synthesis in vitro—resulted in diffuse microvesicular vacuolation, which was considered to reflect excess lipid accumulation in adrenocortical cells. After the 4-week recovery period, these diffuse vacuolar lesions developed into characteristic multifocal macrovesicular vacuolation in the adrenal cortex, making it difficult to evaluate lesion reversibility. Immunohistochemical evaluation suggested that the histopathological changes observed after the recovery period reflected slow degeneration of lipid-laden adrenocortical cells, culminating in cell death accompanied by macrophage activation and aggregation to process apoptotic or necrotic cells and the liberated lipids. These findings are considered to represent a transitional phase in the recovery process. The reversibility of S-637880-induced vacuolar lesions was confirmed in a subsequent 13-week oral repeated-dose toxicity study with a 13-week recovery period. This case study may be helpful in evaluating the reversibility of vacuolar lesions in the adrenal cortex during nonclinical safety assessments and in determining an appropriate recovery periods for assessing adrenal toxicity.

INTRODUCTION

The adrenal glands—particularly the adrenal cortex—are one of the most sensitive target organs within the endocrine system, especially to compounds that possess high fat solubility or affect steroid hormone metabolism (Inomata and Sasano, 2015; Rosol et al., 2001). This sensitivity is due to the adrenal cortex’s primary role in synthesizing and secreting various steroid hormones using cholesterol as a substrate (Harvey et al., 2007; Hinson and Raven, 2006; Inomata and Sasano, 2015; Rosol et al., 2001). Several compounds and drugs, such as ketoconazole, aminoglutethimide, and metyrapone, are known to induce adrenocortical and steroidogenic toxicity (Harvey et al., 2007; Harvey, 2016). Steroidogenesis in the adrenal cortex involves cytochrome P450 enzymatic reactions acting on a variety of precursors, as well as the intracellular transport of cholesterol to the inner mitochondrial membrane by the steroidogenic acute regulatory protein (Miller and Auchus, 2011). Disruption of steroidogenesis may occur through multiple mechanisms of action involving different molecules associated with the steroidogenic pathway (Harvey, 2016). Assessment of adrenocortical and steroidogenic toxicity caused by compounds is essential for estimating human risk, as steroid hormones derived from the adrenal glands are involved in many critical physiological processes, including stress responses, lipid metabolism, immune regulation, blood pressure control, electrolyte balance, and reproductive functions (Holst et al., 2004). Indeed, adrenocortical insufficiency caused by compounds such as aminoglutethimide and etomidate has been reported to result in cardiovascular collapse and death—a condition known as Addisonian crisis—in humans (Harvey, 2016; Hinson and Raven, 2006; Rosol et al., 2001).

Adrenocortical cells physiologically contain cytoplasmic lipid droplets, as they synthesize steroid hormones from cholesterol derived via hydrolysis of cholesterol esters (Nishizato et al., 2014). Disruption of steroidogenesis by compounds can histopathologically lead to vacuolar lesions in the adrenal cortex, typically presenting as increased accumulation of lipid droplets within adrenocortical cell cytoplasm (Brändli-Baiocco et al., 2018; Colman et al., 2021; Woicke et al., 2021). This condition may alter cellular function and disrupt adrenal gland homeostasis. The adrenal cortex consists of three functional zones—namely, the zona glomerulosa, zona fasciculata, and zona reticularis—from outermost to innermost (Mitani, 2014). Vacuolar lesions in adrenocortical cells can affect one or more of these zones, with the zona fasciculata most frequently involved (Brändli-Baiocco et al., 2018).

In nonclinical toxicity evaluations conducted during drug development, the primary objectives are to identify the target organs affected by compound-induced toxicity and to determine the relationship between dose and exposure. It is also important to assess the potential reversibility of toxicity to support human risk assessment and establish safe usage conditions (Salian-Mehta et al., 2024; Sewell et al., 2014). Scientific evaluation of reversibility considers both the extent and severity of the pathological lesion and the regenerative capacity of the affected organ system (ICH, 2012). Several studies showed the regenerative capacity of the adrenal glands and the turnover of adrenocortical cells. One report described an “undifferentiated cell zone” located between the zona glomerulosa and the zona fasciculata in the rat adrenal cortex, which appears to lack endocrine function but contains stem or progenitor cells—suggesting that the adrenal cortex possesses regenerative capacity (Mitani, 2014). Other studies have demonstrated that undifferentiated progenitor cells in the adrenal capsule or subcapsular region give rise to terminally differentiated zona glomerulosa cells, which subsequently undergo lineage conversion into zona fasciculata cells as they migrate centripetally before undergoing apoptosis at the corticomedullary junction (Freedman et al., 2013; Kim et al., 2009).

When adrenal lesions are induced by compounds in nonclinical toxicity studies, it is critical to evaluate their reversibility to inform risk assessment in humans. However, data on the reversibility of compound-induced adrenal lesions remain limited. Nonclinical toxicity studies, including repeated-dose toxicity studies of S-637880—a small-molecule drug candidate synthesized by Shionogi & Co., Ltd.—were conducted prior to first-in-human trials. In a 4-week oral repeated-dose toxicity study in dogs, diffuse adrenocortical microvesicular vacuolation corresponding to macroscopic adrenal enlargement was observed at the end of the administration period. These changes were considered as off-target effects as the target molecule of S-637880 is expected to be absent in the adrenal cortex and the pharmacological function is anticipated not to be related to steroidogenesis. After a 4-week recovery period, the vacuolar lesions developed into multifocal lesions consisting of aggregates of cells with macrovesicular vacuoles, which causes difficulty in the judgement of the reversibility of the vacuolar lesions.

In this study, adrenal tissues from the previously described 4-week oral repeated-dose toxicity study of S-637880 in dogs were examined pathologically to elucidate the pathogenesis of the characteristic adrenal lesions observed after the 4-week recovery period and to discuss lesion reversibility. This evaluation included histological, immunohistochemical, and electron microscopy examinations. To elucidate the molecular mechanism underlying these adrenal changes, an in vitro steroidogenesis assay was also conducted.

MATERIALS AND METHODS

Compound

S-637880, a low-molecular-weight compound, was designed and synthesized by Shionogi & Co., Ltd. (Osaka, Japan) as a drug candidate.

Pathological examination of the adrenal glands in the 4-week oral repeated-dose toxicity study in dogs

Data on organ weights, gross findings, and specimens for pathological evaluation were obtained from dogs administered S-637880 during a 4-week oral repeated-dose toxicity study, as described below. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Shionogi Pharmaceutical Research Center (approval number S16098C-0000). Beagle dogs (Marshall Biotechnology Co., Ltd.), 6 months old at the initiation of the dosing, were assigned to control (0.5% w/v methylcellulose), 3 (low-), 20 (middle-), or 300 mg/kg (high-) dose groups (n = 3/sex/group) and were dosed once daily by gavage (5 mL/kg/day) for 28 days. Additional animals were included in the control and high-dose groups (n = 1 or 2/sex/group) to assess the reversibility of S-637880-related changes following a 4-week drug withdrawal period. The animals were kept in animal rooms of Shionogi Pharmaceutical Research Center during the quarantine, acclimation, and administration periods. Room temperature and relative humidity were controlled to maintain 20 to 26°C and 30 to 70%, respectively. Room lighting was controlled at a 12-hr light-dark cycle (8:00 to 20:00). The animals were supplied with approximately 200 g/dog/day of expanded food (DS-A, Oriental Yeast Co., Ltd, Japan). Drinking water was constantly provided via an automatic watering system with watering nozzles. The following examinations were conducted: clinical observations, body weight measurement, food consumption measurement, ophthalmology, electrocardiography, urinalysis, hematology, blood chemistry, toxicokinetics, necropsy, organ weight measurement, and histopathology. The parameters examined in urinalysis, hematology, blood chemistry, as well as the organs examined in necropsy, organ weight measurement, and histopathology, are presented in Table S2. At the end of the administration and recovery periods, all animals were necropsied following anesthesia with pentobarbital sodium and euthanasia by exsanguination. The adrenal glands, along with other systemic organs and tissues defined by our standard operating procedures, were excised, weighed, and fixed in 10% neutral-buffered formalin. The fixed adrenal glands were routinely processed and embedded in paraffin. Paraffin-embedded sections (3 μm thick) were stained with hematoxylin and eosin (H&E) and evaluated microscopically based on five severity scores: normal (score 0), minimal (score 1), mild (score 2), moderate (score 3), and marked (score 4) (Mann et al., 2012). Statistical significance of organ weight data was determined using Dunnett’s multiple comparison test (Dunnett, 1955) for comparison among three groups or more or Welch’s t-test (Welch, 1947) for comparison among between two groups using SAS 9.2 software in Provantis system (Instem, United Kingdom), with significance defined at p < 0.01 or p < 0.05.

Immunohistochemistry and electron microscopy

For each immunohistochemical stain, serial 3-μm sections were labeled using a modified peroxidase-conjugated immune-polymer method as previously reported (Asaoka et al., 2013; Kato et al., 2013; Nakagawa et al., 2021). Details of the immunohistochemical conditions are listed in Table S1. These immunohistochemical specimens were qualitatively evaluated.

To characterize the increased vacuoles observed in the adrenal cortex following repeated oral administration of S-637880, immunohistochemical staining was performed on representative cases (n = 1/sex for both control and 300 mg/kg groups) using adipophilin and lysosomal-associated membrane protein 2 (LAMP2), as previously reported (Kato et al., 2013; Moroki et al., 2021). Adipophilin is a membrane-associated protein of cytosolic, non-lysosomal lipid droplets. Immunohistochemical staining with adipophilin and LAMP2 has been reported to be useful for distinguishing lipid accumulation (immunolabelled for adipophilin) from phospholipidosis (immunolabelled for LAMP2) (Asaoka et al., 2013; Bodié and Decker, 2014; Obert et al., 2007).

To identify the cell types within the vacuolated adrenal cortex lesions, immunohistochemical staining was also conducted for CYP21A2 and ionized calcium-binding adapter molecule 1 (Iba1) in representative animals (n = 1/sex for both control and 300 mg/kg groups). CYP21A2 is a steroidogenic enzyme expressed in adrenocortical cells and is involved in steroid hormone production (Sushko et al., 2012). Iba1 is a pan-macrophage marker expressed by a wide range of monocyte/macrophage lineage subpopulations (Imai et al., 1996; Pierezan et al., 2014). To further characterize macrophage functions, immunohistochemical staining was also performed using additional macrophage markers, including LAMP2, CD204, and CD206. LAMP2 is essential for lysosomal function and macrophage phagocytosis, and it also serves as an indicator for macrophage activation (Huynh et al., 2007; Wang et al., 2019). CD204 is known to play a key role in the recognition of exogenous antigens by macrophages (Kelley et al., 2014; Tomokiyo et al., 2002; Yamate et al., 2023). CD206 is a mannose receptor of macrophages that is associated with an anti-inflammatory molecule, contributing to the resolution of excessive inflammation and promote wound healing (Tsuchiya et al., 2019).

Ultrastructure of adrenocortical cells in the zona fasciculata was also examined using transmission electron microscopy in representative cases (n = 1/sex for both control and 300 mg/kg groups). The procedure followed a previously described method using formalin-fixed tissue samples (Obara et al., 2021). Formalin-fixed adrenal glands were excised into small pieces and post-fixed in 3% glutaraldehyde, followed by 2% osmium tetroxide. After embedding in epoxy resin, ultrathin sections were prepared. Electron staining was performed using platinum blue (Nissin EM, Tokyo, Japan) and lead citrate (Nacalai Tesque, Kyoto, Japan). The specimens were observed using a JEM-1400 plus electron microscope (JEOL, Tokyo, Japan).

Thirteen-week oral repeated-dose toxicity study in dogs

The general test system, including animal husbandry, test site, and experimental design (including dosage levels), was the same as that used in the 4-week oral repeated-dose toxicity study. Beagle dogs (Marshall Biotechnology Co., Ltd.), 6 months old at the initiation of the dosing, were assigned to control (0.5% w/v methylcellulose), 3 (low-), 20 (middle-), and 300 mg/kg (high-) dose groups (n = 4/sex/group) and were dosed once daily by gavage (5 mL/kg/day) for 91 days. Additional animals were assigned to the control and high-dose groups (n = 3/sex/group) to evaluate the reversibility of S-637880-related changes following drug withdrawal. To confirm the complete reversibility of adrenal lesions observed in the 4-week repeated-dose toxicity study, a longer recovery period of 13 weeks was established. The following examinations were conducted: clinical observations, body weight measurement, food consumption measurement, ophthalmology, electrocardiography, urinalysis, hematology, blood chemistry, bone marrow analysis, toxicokinetics, necropsy, organ weight measurement, and histopathology. The parameters examined in urinalysis, hematology, blood chemistry, as well as the organs examined in necropsy, organ weight measurement, and histopathology, are presented in Table S3. All experimental procedures were approved by the Institutional Animal Care and Use Committee (approval number S18013D-0000).

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay

To investigate the involvement of cell death and the pathological mechanisms underlying histopathological changes in the adrenal glands of dogs administered S-637880, a TUNEL assay was performed on representative cases (n = 1/sex for both control and 300 mg/kg groups) from the 4-week and 13-week oral repeated-dose toxicity studies. Normal thymus tissue was used as a positive control to confirm assay validity. The assay was conducted using the ApopTag® Peroxidase In situ Apoptosis Detection Kit (Sigma-Aldrich, Inc.) to visualize DNA fragmentation. The TUNEL assay can detect not only apoptosis, in which a cell shows nuclear positivity for it, but also necrosis in which a cell shows both nuclear and cytoplasmic positivity (Jaeschke et al., 2018; Mori et al., 2020). Quantitative analysis was performed by manually counting cells with only nuclear positivity and cells with both nuclear and cytoplasmic positivity in zona fasciculata and zona reticularis of the adrenal cortex at a magnification of x100 (20 number of fields of view examined).

In vitro steroidogenesis assay

To investigate the mode of action (MoA) of adrenal lesions observed in the 4-week and 13-week repeated-dose toxicity studies in dogs, the effect of S-637880 on steroidogenesis after exposure to S-637880 was evaluated using H295R cells (a human adrenocortical carcinoma cell line provided by ATCC) (Gazdar et al., 1990). The assay design was modified based on OECD Test Guideline No. 456 and a previous report (Winther et al., 2013). Cultured H295R cells were exposed to S-637880 at concentrations ranging from 0.195 to 100 μg/mL, solubilized in DMSO, for 48 hours at 37 °C in a 5% CO2 incubator. The dose settings were based on the observed range of maximum plasma concentrations of S-637880 (0.809–15.2 μg/mL) in the 4-week oral repeated-dose toxicity study in dogs. After treatment, the culture medium was collected, and cortisol concentrations were measured using a Cortisol ELISA Kit (Enzo Life Sciences, Inc.). Cell viability was also assessed using PrestoBlue® (Thermo Fisher Scientific, Inc.). The cells were treated with ketoconazole (Tokyo Chemical Industry Co., Ltd.) as a positive control in the same manner as S-637880 to confirm assay validity (Winther et al., 2013).

RESULTS

Pathological features of adrenal lesions in the 4-week oral repeated-dose toxicity study in dogs

In both sexes of the high-dose group, enlarged adrenal glands with significantly increased weights were observed at the end of the administration period (Table 1). Histopathologically, a minimal to mild increase in diffuse microvesicular vacuolation was noted in the zona fasciculata and zona reticularis, accompanied by an increase in the thickness of these zones of the adrenal cortex compared with animals in the control group (Table 1; Figs. 1A–1D).

Table 1. Pathological findings of the adrenal gland observed in the 4-week oral repeated-dose toxicity study of S-637880 in dogs

Fig. 1

Photomicrographs of the adrenal glands from dogs treated with 0.5% MC (control) for 4 weeks (A, B), 300 mg/kg of S-637880 for 4 weeks (C, D), and 300 mg/kg of S-637880 for 4 weeks followed by a 4-week recovery period (E, F). Original magnification: 4× (A, C, E); 20× (B, D, F). Staining: H&E (hematoxylin and eosin). 0.5% MC: 0.5% w/v methylcellulose.

In this study, the histopathological term of the “increase in diffuse microvesicular vacuolation” refers to an increase in foamy cytoplasmic vacuoles accompanied by cytoplasmic enlargement (cellular hypertrophy) in cortical cells of the zona fasciculata and zona reticularis. The use of this terminology is considered appropriate, as cellular enlargement primarily caused by vacuole accumulation in adrenocortical cells is typically diagnosed as vacuolation rather than hypertrophy in the nonclinical study (Brändli-Baiocco et al., 2018; Woicke et al., 2021). Following a 4-week drug withdrawal period, adrenal gland sizes and weights were comparable to those in the control group (Table 1). Histopathologically, cluster or aggregate of cells with macrovesicular vacuoles were observed in the zona fasciculata and reticularis of the adrenal cortex, particularly in regions directly beneath the zona glomerulosa (Figs. 1E and 1F). The lesions were not accompanied by an obvious increase in the thickness of zona fasciculata and zona reticularis. The term “increase in multifocal macrovesicular vacuolation” was used to describe this finding.

At the end of the administration period, increased microvesicular cytoplasmic vacuoles in adrenocortical cells were positive for adipophilin (Fig. 2A) but negative for LAMP2 (Fig. 2B). Additionally, a small population of LAMP2-positive cells showing cytoplasmic staining was scattered throughout the vacuolated lesions of the adrenal cortex and was considered to represent macrophages based on their morphological characteristics. Ultrastructural analysis of adrenocortical cells in the zona fasciculata revealed numerous medium-density lipid droplets and occasional cholesterol crystals in the cytoplasm (Fig. 2C). No markedly dilated lysosomes or lamellar structures derived from lysosomes were observed under electron microscopy. These findings suggest that the increased cytoplasmic vacuoles in adrenocortical cells were lipid droplets and not lysosome-derived.

Fig. 2

Immunohistochemical staining for adipophilin (A) and LAMP2 (B), and ultrastructural morphology (C) of the zona fasciculata in a dog treated with 300 mg/kg of S-637880 for 4 weeks. Original magnification: 20× (A, B); 2000× (C). Scale bar: 5.0 μm (C). Arrowhead: medium-density lipid droplets (C); Arrow: cholesterol crystals (C). LAMP2: lysosomal-associated membrane protein 2.

Immunohistochemistry for CYP21A2 and Iba1 was performed to identify the cell types present in the aggregating cells in the adrenal cortex observed after the 4-week drug withdrawal in the 4-week repeated-dose toxicity study. Adrenocortical cells across all three cortical zones were positive for CYP21A2 (Fig. 3A). Iba1-positive macrophages were sparsely distributed throughout the adrenal cortex (Fig. 3B). Following the 4-week recovery period, the aggregate cells with macrovesicular vacuoles in the zona fasciculata and reticularis were negative for CYP21A2 (Fig. 3F) but positive for Iba1 (Fig. 3G). In contrast, adrenocortical cells without increased cytoplasmic vacuoles were positive for CYP21A2 (Fig. 3F) and negative for Iba1 (Fig. 3G). These results indicate that the aggregating cells with macrovesicular vacuoles in the adrenal cortex are macrophages.

Fig. 3

Immunohistochemical staining for CYP21A2, Iba1, LAMP2, CD204, and CD206 in the adrenal cortex from dogs treated with 300 mg/kg of S-637880 for 4 weeks (A–E) and for 4 weeks followed by a 4-week recovery period (F–J). Original magnification: 20×. Iba1: ionized calcium-binding adapter molecule 1; LAMP2: lysosomal-associated membrane protein 2.

Moreover, macrophages positive for LAMP2-, CD204-, and CD206 were sparsely distributed in the adrenal cortex (Figs. 3C–3E), though they were less numerous compared to Iba1-positive macrophages (Fig. 3B). At the end of the recovery period, macrophages positive for LAMP2 (Fig. 3H) and CD204 (Fig. 3I) were diffusely observed in the adrenocortical lesions, whereas only a small population of macrophages were positive for CD206 in the same region (Fig. 3J).

No S-637880-related changes were observed in clinical signs, body weight, food consumption, ophthalmoscopy, electrocardiography, urinalysis, or blood chemistry data during the administration period. Male dogs of the high-dose group had mild decreases in absolute numbers of red blood cells, hemoglobin and hematocrit levels in hematology compared to the control group, and minimal increases in erythroid hematopoiesis were observed in both the spleen and bone marrow on histopathological examination. No S-637880-related changes were observed in any examinations during the recovery period (Table S2).

Pathological features of adrenal lesions in the 13-week oral repeated-dose toxicity study in dogs

In both sexes of the high-dose group, enlarged adrenal glands with their significantly increased weights were observed at the end of the administration period (Table 2). In both sexes of the high- and middle-dose groups, a minimal to moderate increase in diffuse microvesicular and macrovesicular vacuolation in the zona fasciculata and zona reticularis, accompanied by an increase in the thickness in these zones was noted compared with that in the control group (Table 2; Figs. 4A–4H). Similar lesions were also observed in the high-dose group of the 4-week study; however, in the 13-week study, these lesions were additionally present in the middle-dose group, where no such effects had been observed during the 4-week study.

Table 2. Pathological findings of the adrenal gland observed in the 13-week oral repeated-dose toxicity study of S-637880 in dogs

Fig. 4

Photomicrographs of the adrenal glands from dogs treated with 0.5% MC (control) for 13 weeks (A, B), 20 mg/kg of S-637880 for 13 weeks (C, D), 300 mg/kg of S-637880 for 13 weeks (E, F), and 300 mg/kg of S-637880 for 13 weeks followed by a 13-week recovery period (G, H). Original magnification: 4× (A, C, E, G); 20× (B, D, F, H). Staining: H&E (hematoxylin and eosin). 0.5% MC: 0.5% w/v methylcellulose.

At the end of the 4-week administration period, vacuolar lesions were predominantly microvesicular. In contrast, at the end of the 13-week administration period, macrovesicular vacuoles were admixed within the lesions. Immunohistochemically, cells with macrovesicular vacuoles were comprising both macrophages and cortical cells. Additionally, multifocal mononuclear inflammatory cell infiltrates—absent in the 4-week study—were present in both sexes of the high-dose group and in males of the middle-dose group of the 13-week study (Fig. 4F, inset). Following a 13-week drug withdrawal period, adrenal gland sizes and weights had returned to levels comparable to those of the control group (Table 2). Histopathologically, the lesions were considered reversible, as they were markedly milder and less frequent after the 13-week recovery period compared to with those observed at the end of the administration period (Table 2; Figs. 4E–4F). In addition, an increase in the thickness of zona fasciculata and zona reticularis was not observed after recovery period. Minimal vacuolar lesions in the adrenal cortex were also observed in some females in the control group (Table 2; Figs. 4A–4B), which are generally recognized as common spontaneous findings in experimental beagle dogs (Sato et al., 2012); however, test-substance related vacuolar lesions of the adrenal cortex were observed in both sexes of the high- and middle-dose groups with higher grade in severity and dose-response.

No S-637880-related changes were observed in body weight, food consumption, ophthalmoscopy, electrocardiography, urinalysis, or blood chemistry during the administration period in this study. However, the following S-637880-related findings—excluding adrenal effects—were noted at the end of the administration period: increased salivation in males in the high-dose group; decreases in hemoglobin and hematocrit levels and increases in absolute numbers of reticulocytes and platelets compared to the control group, and other changes in red blood cell-related parameters on hematology and bone marrow analysis in both sexes of the high-dose group and in males of the middle-dose group; congestion in the spleen on histopathological examination in both sexes of the high-dose group and in males of the middle-dose group: increased hemosiderin in the spleen or liver on histopathological examination in females of the high-dose group; and increased erythroid hematopoiesis in the bone marrow on histopathological examination in both sexes of the middle- and high-dose groups. During the recovery period, no S-637880-related changes were observed in any examinations, except for adrenal pathological lesions and a mild decrease in mean corpuscular volume and mean corpuscular hemoglobin in hematology (Table S3).

TUNEL assay

In the TUNEL assay, DNA fragmentation in apoptotic cells is visualized by positive nuclear staining, whereas in necrotic cells it is visualized by both nuclear and cytoplasmic staining. Nuclear TUNEL-positive apoptotic cells and both nuclear and cytoplasmic TUNEL-positive necrotic cells were counted to evaluate the involvement of cell death and the pathological mechanisms underlying histopathological changes in the adrenal glands in the 4-week and 13-week oral repeated-dose toxicity studies. In the 4-week study, the number of apoptotic and necrotic cells in the adrenal cortex at the end of the administration period in high-dose group dogs did not differ from that in control group animals. However, at the end of the 4-week recovery period, the number of apoptotic and necrotic cells was higher in the high-dose group than in the control group (Figs. 5A–5E). In the 13-week study, the number of apoptotic and necrotic cells in the adrenal cortex at the end of the administration period was higher in the high-dose group than in the control group. However, by the end of the 13-week recovery period, the number of apoptotic and necrotic cells was similar to that observed in the control group (Figs. 5F–5J).

Fig. 5

TUNEL-stained adrenal glands from dogs treated with 0.5% MC (control) for 4 weeks (A) and 13 weeks (F); 300 mg/kg of S-637880 for 4 weeks (B) and 13 weeks (G); 0.5% methylcellulose (control) for 4 weeks followed by a 4-week recovery period (C) and 13 weeks followed by a 13-week recovery period (H); and 300 mg/kg of S-637880 for 4 weeks followed by a 4-week recovery period (D) and 13 weeks followed by a 13-week recovery period (I). Panels E and J show the average number of nucleus TUNEL-positive cells and combined nucleus and cytoplasm TUNEL-positive cells in the zona fasciculata and zona reticularis for the 4-week (E) and 13-week (J) oral repeated-dose toxicity studies (n = 1/sex for each control and 300 mg/kg group). TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. 0.5% MC: 0.5% w/v methylcellulose. Arrowhead: nucleus and/or cytoplasm TUNEL-positive cells.

In vitro steroidogenesis assay

To investigate the MoA of adrenal lesions observed in the 4-week and 13-week oral repeated-dose toxicity studies in dogs, the inhibitory effect of S-637880 on steroidogenesis was evaluated in vitro using H295R cells. Ketoconazole, a known steroidogenesis inhibitor, was used as a positive control and its treatment showed inhibitory effects on cortisol synthesis. After 48 hours of treatment with S-637880, cortisol concentrations decreased in a dose-dependent manner, indicating inhibition of cortisol synthesis in H295R cells (Fig. 6). No cytotoxicity was observed in either ketoconazole-treated or the S-637880-treated H295R cells across the tested concentration range.

Fig. 6

Effects of ketoconazole (A) and S-637880 (B) on steroid hormone (cortisol) production in H295R cells after 48 hr of treatment. X-axis: concentrations of ketoconazole and S-637880. Y-axis: relative cortisol concentration and cell viability (%), compared to solvent control.

DISCUSSION

Pathological vacuolation of adrenocortical cells has been observed in toxicity studies using animal species such as rats, dogs, and monkeys, and is known to be induced by various compounds (Floettmann et al., 2013; Robertson et al., 2001; Tochitani et al., 2019). The nature of these vacuoles varies depending on the MoA of the toxicity. Several types of vacuolation have been described: increased number and size of cytoplasmic lipid droplets; increased number and size of lysosomes due to phospholipid accumulation, leading to the formation of lamellar bodies; and swelling of cellular organelles such as mitochondria (Breiden and Sandhoff, 2019; Mutsuga et al., 2017). Among these, many compounds that induce increased lipid droplet formation in adrenocortical cells exert their effects through inhibition of steroid hormone synthesis (Brändli-Baiocco et al., 2018). Adrenocortical cells are responsible for synthesizing various steroid hormones using cholesterol as a substrate. Inhibition of steroidogenesis is considered to result in the pathological accumulation of cholesterol, cholesterol esters, or steroid hormone precursors within the cells. In the present study, S-637880 was shown to inhibit steroid hormone synthesis in vitro using H295R cells. Therefore, inhibition of steroidogenesis is considered the likely MoA responsible for the adrenal vacuolar lesions observed in the oral repeated-dose toxicity study of S-637880 in dogs. Because the adrenal glands play a central role in stress responses, similar vacuolar changes have also been reported to occur in response to stress or other physiological stressors (National Toxicology Program). However, no S-637880-related stress responses—such as immunosuppressive changes or stress-related indicators (e.g., decreased body weight)—were observed in any of the repeated-dose toxicity study examinations, apart from pathological adrenal lesions. Although mild anemia was noted in dogs administered S-637880, it was not accompanied by other findings indicative of physiological stress.

By extending the administration period from 4 weeks to 13 weeks in the oral repeated-dose toxicity studies of S-637880 in dogs, adrenal vacuolar lesions became more severe at the end of the administration period. Furthermore, macrovesicular vacuoles and inflammatory changes in the adrenal glands of dogs administered 300 mg/kg/day were more prominent in the 13-week study compared to the 4-week study. Immunohistochemical profiling revealed that, in the 13-week study, the cells containing macrovesicular vacuoles in the adrenal cortex at the end of the administration period included both steroidogenic cortical cells and macrophages. In contrast, after a 4-week recovery in the 4-week study, these cells were primarily macrophages. Interestingly, macrovesicular vacuoles (mainly macrophages) observed after a 4-week drug withdrawal in the 4-week repeated-dose toxicity study were more prominent in the middle layer of the adrenal cortex, particularly in the region directly below the zona glomerulosa. In contrast, in dogs administered S-637880 for 13 weeks, macrovesicular vacuoles—comprising both macrophages and cortical cells—were more prominently distributed across the middle to lower layers of the adrenal cortex, spanning the zona fasciculata and zona reticularis. Although the precise reason for this distribution is unclear, one possible explanation involves the regeneration or turnover mechanisms of adrenocortical cells or adrenal cortex tissue. Several studies have shown that progenitor cells, which differentiate into various steroidogenic cell types, reside in the outer regions of the adrenal cortex, such as the capsular/subcapsular area and the zona glomerulosa (Grabek et al., 2019; King et al., 2009; Oikonomakos et al., 2021). Physiological turnover of adrenal cortex tissue is thought to occur through the proliferation of these progenitor cells, which then migrate centripetally toward the medulla and ultimately undergo cell death at the corticomedullary junction (Grabek et al., 2019; King et al., 2009; Oikonomakos et al., 2021). Macrovesicular vacuoles were more prominent in the middle to lower layers of the adrenal cortex, spanning the zona fasciculata and zona reticularis, in dogs administered S-637880 at the end of the 13-week administration period. This may be because these cells were exposed to S-637880-induced steroidogenesis disruption for a longer duration than those in the upper layers of the adrenal cortex, which may represent newly formed and younger cells.

Macrophages are tissue-resident immune cells found in various organs, where they eliminate foreign substances and pathogens, regulate host defense and inflammatory responses, and maintain tissue homeostasis (Rodríguez-Morales and Franklin, 2023). Macrophages are known to express different molecules depending on the tissues or organs in which they reside, as well as the functions they perform in specific tissue environments (Kaku et al., 2014; Krenkel and Tacke, 2017; Mass et al., 2023). In the current study, characteristic adrenal cortical lesions observed after a 4-week drug withdrawal in the 4-week repeated-dose toxicity study of S-637880 consisted of clustered and aggregated macrophages. To investigate their function, expression profiles of macrophage-related markers were evaluated immunohistochemically. CD204 is known to play an important role in the recognition of exogenous foreign antigens by macrophages, but it has also been implicated in the processing of degenerated lipids and lipid metabolism (Kelley et al., 2014; Tomokiyo et al., 2002). The diffuse expression of LAMP2 and CD204 observed in this study is considered indicative of macrophage activation involved in processing excess tissue lipids and phagocytosing necrotic or degenerating adrenocortical cells. Focal CD206 expression was also detected in clustered and aggregated macrophages. CD206 is reported to be expressed in macrophages of adipose tissue, where these macrophages buffer lipids released from dying adipocytes (Vogel et al., 2022). However, the population of CD206-positive macrophages observed in this study was small, and their role or relationship to the pathogenesis remains unclear.

In the 4-week repeated-dose toxicity study of S-637880, the number of TUNEL-positive cells in the adrenal cortex—positive in both the nucleus and/or cytoplasm—was not increased at the end of the administration period but was elevated after the 4-week drug withdrawal period. This suggests that lipid-accumulated adrenocortical cells underwent gradual degeneration, leading to cell death during the withdrawal period. The subsequent activation and aggregation of macrophages to process dead cells and liberated lipids likely contributed to the characteristic multifocal macrovesicular vacuolar lesions observed in the adrenal cortex at the end of the 4-week recovery period (Fig. 7). In contrast, in the 13-week repeated-dose toxicity study of S-637880, the number of nucleus and/or cytoplasm TUNEL-positive cells in the adrenal cortex was increased at the end of the administration period but not after the 13-week withdrawal period. Taken together with the decreasing incidence and lower severity of histopathological findings, these results indicate that a 13-week withdrawal period was sufficient to confirm near-complete reversibility of the adrenal lesions induced by S-637880.

Fig. 7

Schematic representation of the proposed mechanism underlying the histopathological changes observed in the adrenal glands of dogs in the 4-week oral repeated-dose toxicity study of S-637880.

General toxicity studies using animals such as dogs are conducted to assess human risk associated with drug candidate compounds. In repeated-dose toxicity studies—a component of general toxicity assessments—the objectives include not only evaluating toxicity in target organs but also determining whether such toxic effects are reversible (Perry et al., 2013). Reversibility is typically assessed based on the extent and severity of pathological lesions, the regenerative capacity of the affected organ system, and existing knowledge from other compounds that produce similar effects (ICH, 2012). Recovery periods are commonly set at 2–4 weeks for compounds with half-lives of one day or less (Pandher et al., 2012). The adrenal cortex is known to possess regenerative capacity, and the turnover cycle of adrenocortical cells in rodents has been reported to range from 3 to 6 months (Grabek et al., 2019). Therefore, to adequately evaluate the reversibility of compound-induced adrenal lesions, a longer recovery period than typically used may be necessary. A trend toward reversibility—such as a decrease in lesion incidence or severity—along with a scientific rationale that this trend will eventually result in full recovery, is generally considered sufficient; complete demonstration of full reversibility is not always required (ICH, 2012). However, as demonstrated in the case of S-637880, shorter recovery periods may fail to confirm such trends, making it difficult to establish safe use conditions in clinical studies (e.g., determining an appropriate monitoring period following drug discontinuation due to adrenal adverse events). An additional challenge in reversibility assessment lies in the limitations imposed by animal welfare considerations, particularly the 3Rs principle, which often restricts the number of animals, groups, or time points used for sacrifice and evaluation (typically one recovery period and two animals per sex for non-rodents) (Pandher et al., 2012; Salian-Mehta et al., 2024). Therefore, accumulating knowledge that enables effective assessment of reversibility is essential and requires a deeper understanding of the underlying pathophysiological mechanisms.

To examine the progression of pathological changes in detail, further studies—such as frequent time-course analyses and studies on regenerative and proliferative activities of adrenocortical cells—are required to better elucidate the precise mechanisms underlying the reversibility of vacuolar lesions in the adrenal cortex caused by steroidogenesis disruption.

In conclusion, steroidogenesis disruption caused by S-637880 induced diffuse microvesicular vacuolation of the adrenal cortex in a 4-week repeated-dose toxicity study in dogs. After the 4-week recovery period, these diffuse vacuolar lesions developed into characteristic multifocal macrovesicular vacuolation in the adrenal cortex, making it difficult to evaluate lesion reversibility. Immunohistochemical evaluation suggested that the histopathological changes observed after the recovery period reflected slow degeneration of lipid-laden adrenocortical cells, culminating in cell death accompanied by macrophage activation and aggregation to process apoptotic or necrotic cells and the liberated lipids. These findings are considered to represent a transitional phase in the recovery process. The reversibility of S-637880-induced vacuolar lesions was confirmed after a 13-week recovery period in a subsequent 13-week oral repeated-dose toxicity study. This case study may be valuable in evaluating reversibility of vacuolar lesions in the adrenal cortex caused by steroidogenesis disruption in nonclinical safety assessments. It may also support the establishment of appropriate recovery periods for assessing adrenal toxicity, as well as the determination of adequate follow-up periods and safe usage conditions for drugs with potential adrenal toxicity in humans.

ACKNOWLEDGMENTS

The authors would like to thank Chieko Yabuuchi, Rumiko Mochizuki, Hirotada Murayama, and Tamiko Fumimoto for their technical support with histopathological specimen preparation and staining. We also acknowledge the members of the Kansai Conference on Toxicologic Pathology for their helpful advice, as well as the Shionogi toxicology team and staff members of Osaka Metropolitan University for their experimental support and guidance.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of interest

The authors declare no conflict of interest.

Data availability

The data in this study are included in the article/supplementary materials. Contact the corresponding author(s) directly to request the underlying data.

Author contribution statement

Conceptualization: Ryo Daniel Obara, Yuki Kato

Formal analysis: Ryo Daniel Obara, Hiroyuki Oka, Yukie Murata

Investigation: Ryo Daniel Obara, Yuki Kato, Yoshiji Asaoka, Yukie Murata

Methodology: Ryo Daniel Obara, Yuki Kato, Yoshiji Asaoka, Hiroyuki Oka, Yukie Murata, Takeshi Izawa, Mitsuru Kuwamura

Supervision: Ryo Daniel Obara, Yuki Kato, Mitsuru Kuwamura

Visualization: Ryo Daniel Obara, Hiroyuki Oka, Yukie Murata

Writing – original draft: Ryo Daniel Obara

Writing – review & editing: Ryo Daniel Obara, Yuki Kato, Yoshiji Asaoka, Hiroyuki Oka, Yukie Murata, Takeshi Izawa, Mitsuru Kuwamura

Approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

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