• View in gallery
    Figure 1—

    Mean ± SD serum cortisol concentration during the mitotane administration period. Notice the changes in serum cortisol concentrations during the induction and maintenance periods (closed circles) in the mitotane treatment group dogs and changes in the serum cortisol concentration after ACTH stimulation testing performed at the completion of the induction period and the maintenance period (open circles).

  • View in gallery
    Figure 2—

    Mean ± SD serum cortisol concentrations (before and after ACTH stimulation) and sodium and potassium concentrations (during the maintenance period) in control group dogs (closed bar), premitotane treatment group dogs (hatched bar), and postmitotane treatment group dogs (open bar). Significant (*P < 0.05, †P < 0.01, ‡P < 0.001) difference among groups.

  • View in gallery
    Figure 3—

    Mean baseline plasma ACTH concentrations before CRH stimulation in control group dogs (closed bar), premitotane treatment group dogs (hatched bar), and postmitotane treatment group dogs (open bar). See Figure 2 for remainder of key.

  • View in gallery
    Figure 4—

    Mean ± SD plasma ACTH concentrations after CRH stimulation in control group dogs (closed bar), premitotane treatment group dogs (hatched bar), and postmitotane treatment group dogs (open bar). See Figure 2 for remainder of key.

  • View in gallery
    Figure 5—

    Magnetic resonance image of the pituitary gland (transverse plane, T1-weighted images) of 3 dogs before (A, B, and C, respectively) and after (D, E, and F, respectively) mitotane administration. In all images, pituitary glands were enhanced in a nonuniform manner with gadolinium. In postmitotane treatment group dogs, thickness of the adenohypophysis and height of the entire pituitary gland (arrows) increased visually with treatment. Bars = 10 mm.

  • View in gallery
    Figure 6—

    Comparison of mean ± SD organ weights between control group dogs (closed bar) and mitotane treatment group dogs (open bar). See Figure 2 for remainder of key.

  • View in gallery
    Figure 7—

    Photomicrographs of sections of the adrenal cortex of a control group dog (A) and a treatment group dog after mitotane administration (B). Notice that the zonae fasciculata and reticularis were markedly atrophied in the mitotane treatment group dog, compared with the control group dog. H&E stain; bars = 200 μm.

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    Figure 8—

    Photomicrograph of immunohistochemically stained ACTH-positive cells of control group dogs (A, B) and mitotane treatment group dogs (C, D). Notice that the size of ACTH-positive cells in mitotane treatment group dogs was visually greater than in control group dogs. Bar = 5 μm.

  • View in gallery
    Figure 9—

    Mean ± SD comparison of the percentage of ACTH-, Pit-1–, and T–pit-positive cells between control group dogs (closed bar) and mitotane treatment group dogs (open bar). See Figure 2 for remainder of key.

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Effect of mitotane on pituitary corticotrophs in clinically normal dogs

Takahiro TaodaDivision of Veterinary Surgery, Department of Veterinary Science, Faculty of Veterinary Medicine, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musasino-shi, Tokyo 180-8602, Japan.

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Yasushi HaraDivision of Veterinary Surgery, Department of Veterinary Science, Faculty of Veterinary Medicine, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musasino-shi, Tokyo 180-8602, Japan.

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Susumu TakekoshiDepartment of Pathology, School of Medicine, Tokai University, Bohseidai, Isehara-shi, Kanagawa 259-1193, Japan

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Johbu ItohCell Science, Teaching and Research Support Center, School of Medicine, Tokai University, Bohseidai, Isehara-shi, Kanagawa 259-1193, Japan

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Akira TeramotoDepartment of Neurosurgery, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan

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Robert Y. OsamuraDepartment of Pathology, School of Medicine, Tokai University, Bohseidai, Isehara-shi, Kanagawa 259-1193, Japan

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Masahiro TagawaDivision of Veterinary Surgery, Department of Veterinary Science, Faculty of Veterinary Medicine, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musasino-shi, Tokyo 180-8602, Japan.

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Abstract

Objective—To evaluate the effects of mitotane administration on the function and morphology of pituitary corticotrophs in clinically normal dogs.

Animals—12 clinically normal adult Beagles.

Procedures—Dogs were randomly assigned to the control group or the mitotane treatment group. In mitotane treatment group dogs, mitotane was administered for 1 month. In both groups, ACTH stimulation testing and corticotrophin-releasing hormone (CRH) stimulation testing were performed. Magnetic resonance imaging (MRI) of the pituitary gland and brain was performed in mitotane treatment group dogs before and after administration of mitotane. After CRH stimulation testing and MRI, dogs were euthanatized and the pituitary gland and adrenal glands were excised for gross and histologic examination.

Results—ACTH concentrations in mitotane treatment group dogs were significantly higher than in the control group dogs following CRH stimulation. Magnetic resonance imaging revealed that pituitary glands were significantly larger in treatment group dogs after administration of mitotane, compared with before administration. On gross and histologic examinations, the adrenal cortex was markedly atrophied. Immunohistochemistry revealed hypertrophy of corticotrophs in pituitary glands of mitotane treatment group dogs.

Conclusions and Clinical Relevance—These findings indicate that inhibition of the adrenal cortex by continuous administration of mitotane leads to functional amplification and morphologic enhancement of corticotrophs in clinically normal dogs. In instances of corticotroph adenoma, hypertrophy of individual corticotrophs induced by mitotane may greatly facilitate enlargement of the pituitary gland and increases in ACTH secretion.

Abstract

Objective—To evaluate the effects of mitotane administration on the function and morphology of pituitary corticotrophs in clinically normal dogs.

Animals—12 clinically normal adult Beagles.

Procedures—Dogs were randomly assigned to the control group or the mitotane treatment group. In mitotane treatment group dogs, mitotane was administered for 1 month. In both groups, ACTH stimulation testing and corticotrophin-releasing hormone (CRH) stimulation testing were performed. Magnetic resonance imaging (MRI) of the pituitary gland and brain was performed in mitotane treatment group dogs before and after administration of mitotane. After CRH stimulation testing and MRI, dogs were euthanatized and the pituitary gland and adrenal glands were excised for gross and histologic examination.

Results—ACTH concentrations in mitotane treatment group dogs were significantly higher than in the control group dogs following CRH stimulation. Magnetic resonance imaging revealed that pituitary glands were significantly larger in treatment group dogs after administration of mitotane, compared with before administration. On gross and histologic examinations, the adrenal cortex was markedly atrophied. Immunohistochemistry revealed hypertrophy of corticotrophs in pituitary glands of mitotane treatment group dogs.

Conclusions and Clinical Relevance—These findings indicate that inhibition of the adrenal cortex by continuous administration of mitotane leads to functional amplification and morphologic enhancement of corticotrophs in clinically normal dogs. In instances of corticotroph adenoma, hypertrophy of individual corticotrophs induced by mitotane may greatly facilitate enlargement of the pituitary gland and increases in ACTH secretion.

Amajor endocrine disorder in dogs, hyperadrenocorticism, is the result of chronic excess secretion of cortisol from the adrenal cortex. Major clinical signs of hyperadrenocorticism are polydipsia-polyuria, polyphagia, abdominal distension, muscle wasting in extremities, signs of depression, thinning and calcification of skin, and depilation.1–4 It has been reported that 80% to 90% of dogs with spontaneous hyperadrenocorticism had PDH, and 10% to 20% were caused by an adrenal tumor. Furthermore, 85% of dogs with PDH had adenomas of the pituitary corticotrophs (ie, ACTH-producing adenomas).5–7

Current medical treatment of PDH in dogs includes administration of mitotane (formerly o,p'-DDD;[1,1-dichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl) ethane]) that destroys the adrenal cortex and administration of corticosteroid synthesis inhibitors such as trilostane, ketoconazole, and metyrapone. For surgical treatment, bilateral adrenalectomy and hypophysectomy are performed. Transsphenoidal hypophysectomy is reported as an especially effective treatment.6,8,9 Radiation therapy with a high-energy beam is also performed for treatment of PDH in dogs. However, medical treatment with mitotane is more commonly used than surgical treatment because of difficulties in securing adequate equipment and personnel and postoperative treatment.7,10,11

Mitotane is a useful drug that suppresses excess secretion of cortisol by selective destruction of the cortisol-producing zonae fasciculata and reticularis of the adrenal cortex, resulting in resolution of various signs of hyperadrenocorticism.7,10,12,13 However, development of neurologic signs in dogs with PDH treated with mitotane for a long period has been reported.14,15 This suggests that with mitotane treatment, organic and functional adverse effects on the pituitary gland or cerebral tissues may develop.

Activity of the HPA axis is regulated by CRH and vasopressin secreted from the hypothalamus. These hormones activate secretion of ACTH from the pituitary gland, and ACTH induces cortisol secretion from the adrenal cortex. Cortisol regulates functions of almost all tissues in the body by interaction with receptors in various target tissues. At the same time, cortisol binds to receptors of the HPA axis and exerts negative feedback on ACTH secretion from the pituitary gland and CRH secretion from the hypothalamus.16,17 When the bilateral adrenal glands are excised, negative feed back by cortisol to the pituitary gland and hypothalamus is lost and secretion of CRH and ACTH increases. Exposure of pituitary corticotrophs to these stimuli is believed to induce adenoma formation in humans.18,19

Various effects of the lack of negative feedback to the hypothalamus and pituitary gland have been reported,10,11,14 but to our knowledge, no studies exist on the effects of mitotane-induced inhibition of the adrenal gland on function and morphology of corticotrophs in the anterior pituitary gland. The objective of the study reported here was to evaluate the effect of mitotane on pituitary corticotrophs in clinically normal dogs.

Materials and Methods

Animals—The Bioethics Committee at Nippon Veterinary and Animal Science University approved the experimental protocol. Twelve clinically and neurologically normal adult Beagles were used. Their health status was confirmed on the basis of clinical history, physical examination findings that included a neurologic examination, CBC determination, and serum biochemical analysis, for which all results were within reference ranges. The group characteristics were as follows: sex, 5 males and 7 females; age, 1 to 8 years old (mean, 2.9 years old); and body weight, 9 to 17 kg (mean, 12.4 kg). Dogs were randomly assigned to a control group (n = 6; mean age, 2.5 years; mean weight, 12.2 kg) and a mitotane treatment group (n = 6; mean age, 3.3 years; mean weight, 12.7 kg).

ACTH stimulation testing—In all dogs in the control and mitotane treatment groups, ACTH stimulation testing was performed according to a method previously reported.20–22 First, a blood sample was collected from the right cephalic vein before stimulation testing. Second, ACTHa (0.25 mg [tetracosactide acetate {0.28 mg}]) was injected into the left cephalic vein with 2 mL of physiologic saline (0.9% NaCl) solution as an injection vehicle. At 1 hour after ACTH injection, a blood sample was collected from the right cephalic vein. Serum was immediately separated from the blood samples by use of a cold centrifuge (at 4°C and 1,470 X g). The serum cortisol concentrations were measured by use of an assay kit.23,24,b Detection limit of this assay was calculated as 0.20 μg/dL. In mitotane treatment group dogs, ACTH stimulation testing was performed before mitotane administration, during the mitotane administration dose escalation period, and after the mitotane fixed dose period.

CRH stimulation testing—The CRH stimulation testing was performed in all dogs in the control and mitotane treatment groups according to a method previously reported.25–27 First, a blood sample was collected from the right cephalic vein before stimulation testing. Second, ovine CRHc (1.5 μg/kg) was injected into the left cephalic vein with 1 mL of physiologic saline solution as an injection vehicle. At 30 minutes after the CRH injection, a blood sample was collected from the right cephalic vein. Blood samples were immediately treated with EDTA, and the plasma was separated by use of a cold centrifuge (at 4°C and 1,470 X g). Plasma ACTH concentrations were measured by use of an assay kit.28,d Detection limit of this assay was calculated as 6.6 pg/mL. In mitotane treatment group dogs, CRH stimulation testing was performed before mitotane administration and after completion of the administration period.

MRI of the pituitary gland and brain—Magnetic resonance imaging was performed in the mitotane treatment group by use of a superconducting magnetic instrumente before and after mitotane administration. All dogs underwent general anesthesia for MRI. After pretreatment with droperidol,f anesthesia was induced by propofol,g and maintained by use of oxygen and isoflurane.h In MRI, T1-weighted images were made before and after the injection of contrast medium at a spin echo that obtained 380 to 400 milliseconds repetition time and 15 to 18 milliseconds echo time. Seven contiguous slices each of the sagittal view and transverse plane locating the pituitary gland in the center were imaged at 2.0-mm slice thickness and 0.0-mm slice gap.29,30 After location of the slice, T1-weighted images of the sagittal plane and the transverse plane were made for 7 minutes. Contrastenhanced sagittal and transverse T1-weighted images were made for 7 minutes, following IV drip infusion of gadoliniumi (0.2 mL/kg) as contrast medium.29–31

PBR measurement—The PBR was measured according to the method used in a study on computed tomography.32 The image with the largest view of the pituitary gland was selected from the T1-weighted transverse plane images acquired by MRI of the pituitary gland and brain for each dog, and the height of the pituitary gland and area of the whole brain were measured in the image. In each dog, the height of the pituitary gland was divided by the area of the whole brain and multiplied by 102 to calculate PBR (ie, PBR = height of the pituitary gland/cross sectional area of the whole brain X 102).

Mitotane administration—Following CRH stimulation testing and MRI of the pituitary gland and brain, adrenocortical function was inhibited for 30 days by administration of mitotanej to dogs in the mitotane treatment group. Mitotane was dissolved in sesame oil and administered orally twice a day with meals as previously reported.33 Mitotane administration began at a dosage of 10 mg/kg/d, and the dosage was increased by 5 mg/kg/d up to 50 mg/kg/d during the induction period. During the induction period, blood samples were collected every 3 days to measure the serum cortisol concentration. When the serum cortisol concentration in induction period decreased to the minimum detection limit (ie, ≤ 0.2 μg/dL), ACTH stimulation testing was performed. If the serum cortisol concentration after ACTH stimulation was ≤ 1.0 μg/dL, the mitotane dosage was changed to the maintenance dosage. If serum cortisol concentration after ACTH stimulation in the induction period did not decrease to ≤ 1.0 μg/dL, the induction period was continued at 50 mg/kg/d. If the serum cortisol concentration did not decrease to ≤ 0.2 μg/dL for 30 days in the induction period, the dog began the maintenance period. In the maintenance period, the drug was administered at a dosage of 50 mg/kg/d for 30 days. During this maintenance period, the serum cortisol concentrations were measured every 5 days. These dosages were established from several studies.7,10,11 In the mitotane administration period, glucocorticoid supplementation was not performed. During the period, the general condition of dogs was observed and the serum electrolyte concentrations (ie, sodium and potassium) were measured to confirm secretion of aldosterone from zona glomerulosa.

Necropsy examination—Dogs were euthanatized by excess anesthesia with pentobarbitalk following CRH stimulation testing and MRI of the pituitary gland and brain in the control group and after administration of mitotane in the mitotane treatment group. Dogs immediately underwent necropsy examination, and the pituitary gland and bilateral adrenal glands were excised. These organs were weighed and fixed with 4% formaldehyde at 4°C for 24 hours. The fixed tissues were embedded in paraffin, and tissue sections were prepared. Pituitary gland and adrenal gland tissue sections were stained with H&E. Sections of the median pituitary gland were subjected to immunohistochemistry by use of a standard enzyme antibody technique. For immunohisto chemistry, antibodies against human ACTHl and human pituitary gland transcription factors (Pit-1m and T-pitn) were used as primary antibodies, and 3, 3-diaminobenzidine for demonstration of peroxidase activity was used. The number of cells expressing Pit-1 and T-pit suggests the number of growth hormone, prolactin, and thyroid-stimulating hormone-positive cells and ACTH-positive cells, respectively.34,35 Specificities of the human antibodies for use with tissues from Beagles were confirmed by western blot analysis. With pituitary gland tissue excised from healthy Beagles, western blot analysis was performed, and single bands were detected at the positions corresponding to the molecular weights of the proteins recognized by the antibodies. Following immunohistochemical staining of the pituitary gland tissue, the area of ACTH-positive cells in the anterior pituitary gland was measured. Numbers of ACTH-positive cell were determined by microscopico examination of 5 fields at 400X magnification with an imaging analysis system.p For microscopic examination, the ventral region of the pituitary gland was equally divided into 5 regions between the dorsal end and the ventral end of the pituitary gland in the median section, and 1 field each was randomly selected from the 5 regions. Similarly, the total number of cells and the numbers of ACTH-positive, Pit-1–positive and T-pit–positive cells per field of the anterior pituitary gland were measured in 5 fields at 400X magnification for each dog, and the ratios of ACTH-positive, Pit-1–positive, and T-pit–positive cells were calculated.

Statistical analysis—Plasma ACTH concentrations were measured before and after CRH stimulation. These values were compared among control group dogs, treatment group dogs before mitotane administration, and treatment group dogs after mitotane administration (ie, control, premitotane treatment, and postmitotane treatment group dogs, respectively). The Mann-Whitney U test was used to make comparisons among control, premitotane treatment, and postmitotane treatment group dogs, and the Wilcoxon signed rank test was used to make comparisons between treatment group dogs before and after mitotane administration. The PBRs calculated from MRI images were compared between treatment group dogs before and after mitotane administration by use of the Wilcoxon signed rank test. With respect to the excised tissues, weights of the pituitary gland and left and right adrenal glands were compared between control and mitotane treatment groups by use of the Mann-Whitney U test. The ACTH-positive cell area measured in immunohistochemically stained tissues was compared between the control and mitotane treatment groups by use of the Mann-Whitney U test. The ratios of ACTH-positive, Pit-1–positive, and T-pit–positive cells per field measured in immunohistochemically stained tissues were compared between control and postmitotane treatment group dogs by use of the Mann-Whitney U test. For all tests, a value of P < 0.05 was considered significant. Unless otherwise stated, results are reported as mean ± SD.

Results

Suppression of the adrenal cortex by mitotane— Before initiation of mitotane administration and at completion of the induction period and maintenance period, serum cortisol concentrations after ACTH stimulation in mitotane treatment group dogs were 19.17 ± 4.11μg/dL, 0.26 ± 0.07μg/dL, and 0.54 ± 0.59 μg/dL, respectively (Figure 1). Serum cortisol concentrations before and after ACTH stimulation in control group dogs were 2.77 ± 2.30 μg/dL and 16.07 ± 4.88 μg/dL, respectively. Serum cortisol concentrations after ACTH stimulation were significantly (P < 0.001) lower in the postmitotane treatment group dogs, compared with control and premitotane group dogs (Figure 2).

Figure 1—
Figure 1—

Mean ± SD serum cortisol concentration during the mitotane administration period. Notice the changes in serum cortisol concentrations during the induction and maintenance periods (closed circles) in the mitotane treatment group dogs and changes in the serum cortisol concentration after ACTH stimulation testing performed at the completion of the induction period and the maintenance period (open circles).

Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1385

The duration of the mitotane induction period was 18.8 ± 3.4 days. Serum cortisol concentrations during the induction periodin mitotane treatment group dogs were 3.27 ± 2.15, 2.03 ± 2.22, 1.49 ± 1.47μg/dL, 0.79 ± 1.18 μg/dL, 0.80 ± 0.86 μg/dL, 0.45 ± 0.32 μg/dL, and 0.27 ± 0.09 μg/dL on days 0, 3, 6, 9, 12, 15, and 18, respectiveley (Figure 1). During the subsequent maintenance period, serum cortisol concentrations were 0.38 ± 0.19, 0.29 ± 0.17 μg/dL, 0.29 ± 0.06 μg/dL, 0.30 ± 0.14 μg/dL, 0.69 ± 0.79 μg/dL, and 0.28 ± 0.11 μg/dL on days 5, 10, 15, 20, 25 and 30, respectively. For 1 dog in which the serum cortisol concentration did not decrease to ≤ 0.2 μg/dL during the 30-day induction period, the dog began the maintenance dosage of mitotane on day 31. For all dogs in which the serum cortisol concentration decreased to ≤ 0.2 μg/dL within the induction period, serum cortisol concentrations after ACTH stimulation were ≤ 1.0 μg/dL, and the dogs began the maintenance dosage of mitotane. Comparison of the serum sodium and potassium concentrations among control, premitotane treatment, and postmitotane treatment group dogs revealed no significance differences (Figure 2). Clinical signs of adrenocortical insufficiency were not observed in any treatment dogs during the mitotane administration period.

Figure 2—
Figure 2—

Mean ± SD serum cortisol concentrations (before and after ACTH stimulation) and sodium and potassium concentrations (during the maintenance period) in control group dogs (closed bar), premitotane treatment group dogs (hatched bar), and postmitotane treatment group dogs (open bar). Significant (*P < 0.05, †P < 0.01, ‡P < 0.001) difference among groups.

Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1385

Plasma ACTH concentrations following CRH stimulation testing—Plasma ACTH concentrations before and after CRH stimulation were 24.83 ± 12.94 pg/mL and 195.67 ± 52.95 pg/mL, respectively, in control group dogs. In premitotane treatment group dogs, plasma ACTH concentrations before and after CRH stimulation were 21.83 ± 15.83 pg/mL and 205.50 ± 75.18 pg/mL, respectively. In postmitotane treatment group dogs, plasma ACTH concentrations before and after CRH stimulation were 470.17 ± 245.39 pg/mL and 766.00 ± 231.57 pg/mL, respectively.

The ACTH concentration before CRH stimulation was significantly higher in postmitotane treatment group dogs than in control and premitotane treatment group dogs (P = 0.004 and P = 0.028, respectively; Figure 3). Similarly, plasma ACTH concentrations after CRH stimulation were significantly higher in postmitotane treatment group dogs than in control and premitotane treatment group dogs (P = 0.004 and P = 0.028, respectively; Figure 4).

Figure 3—
Figure 3—

Mean baseline plasma ACTH concentrations before CRH stimulation in control group dogs (closed bar), premitotane treatment group dogs (hatched bar), and postmitotane treatment group dogs (open bar). See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1385

Figure 4—
Figure 4—

Mean ± SD plasma ACTH concentrations after CRH stimulation in control group dogs (closed bar), premitotane treatment group dogs (hatched bar), and postmitotane treatment group dogs (open bar). See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1385

PBR obtained from MRI—Magnetic resonance imaging revealed that pituitary glands had nonuniform enhancement of the adenohypophysis surrounding central enhancement of the neurohypophysis. The nonuniform enhancement of the adenohypophysis was different from the uniform enhancement of the pituitary gland obtained on delayed contrast-enhanced T1-weighted images in a study.31 In postmitotane treatment group dogs, thickness of the adenohypophysis increased visually, compared with the adenohypophysis in premitotane treatment group dogs (Figure 5). The height of the pituitary gland was 4.65 ± 0.40 mm in premitotane treatment group dogs and 5.68 ± 0.53 mm in postmitotane treatment group dogs. The PBR was 0.263 ± 0.028 in premitotane treatment group dogs and 0.321 ± 0.024 in postmitotane treatment group dogs. In mitotane treatment group dogs, the PBR was significantly (P = 0.028) increased after mitotane administration, compared with before mitotane administration.

Tissue weights—Weights of the pituitary glands that were excised after the dogs were euthanatized were 0.061 ± 0.016g and 0.090 ± 0.021 g in the control and mitotane treatment group dogs, respectively. Weights of the right adrenal gland were 0.629 ± 0.163 g and 0.288 ± 0.101 g in control and mitotane treatment group dogs, respectively, and weights of the left adrenal gland were 0.599 ± 0.118 g and 0.256 ± 0.081 g in control and mitotane treatment group dogs, respectively. Pituitary gland weight was significantly (P = 0.031) higher in mitotane treatment group dogs than in control group dogs, and weights of both adrenal glands were significantly (left adrenal gland, P = 0.0103; right adrenal gland, P = 0.0103) lower in mitotane treatment group dogs than in the control group dogs (Figure 6). In adrenal gland tissue sections stained with H&E, the zonae fasciculata and reticularis were markedly atrophied in mitotane treatment group dogs, compared with control group dogs (Figure 7).

Figure 5—
Figure 5—

Magnetic resonance image of the pituitary gland (transverse plane, T1-weighted images) of 3 dogs before (A, B, and C, respectively) and after (D, E, and F, respectively) mitotane administration. In all images, pituitary glands were enhanced in a nonuniform manner with gadolinium. In postmitotane treatment group dogs, thickness of the adenohypophysis and height of the entire pituitary gland (arrows) increased visually with treatment. Bars = 10 mm.

Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1385

Figure 6—
Figure 6—

Comparison of mean ± SD organ weights between control group dogs (closed bar) and mitotane treatment group dogs (open bar). See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1385

Figure 7—
Figure 7—

Photomicrographs of sections of the adrenal cortex of a control group dog (A) and a treatment group dog after mitotane administration (B). Notice that the zonae fasciculata and reticularis were markedly atrophied in the mitotane treatment group dog, compared with the control group dog. H&E stain; bars = 200 μm.

Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1385

ACTH-positive cell area in pituitary gland tissue—The ACTH-positive cell areas in control and mitotane treatment group dogs were 40.30 ± 15.58 μm2 and 67.99 ± 27.02 μm2, respectively. The ACTH-positive cell area was significantly (P < 0.001) greater in mitotane treatment group dogs than in control group dogs (Figure 8). The total numbers of areas measured for ACTH-positive cells per dog in the control and mitotane treatment group dogs were 461.5 ± 116.0 and 379.7 ± 112.2, respectively; no significant difference was found between the 2 groups.

Figure 8—
Figure 8—

Photomicrograph of immunohistochemically stained ACTH-positive cells of control group dogs (A, B) and mitotane treatment group dogs (C, D). Notice that the size of ACTH-positive cells in mitotane treatment group dogs was visually greater than in control group dogs. Bar = 5 μm.

Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1385

ACTH-positive cell-to-total cell ratio—For control and mitotane treatment group dogs, ACTH-positive cell ratios were 21.05 ± 7.23% and 16.00 ± 6.82%, Pit-1–positive cell ratios were 47.65 ± 9.35% and 51.29 ± 7.74%, and T-pit–positive cell ratios were 17.32 ± 4.30% and 10.14 ± 3.41%, respectively. The ACTH-positive cell and T-pit–positive cell ratios were significantly (ACTH, P = 0.008; T-pit, P < 0.001) lower in mitotane treatment group dogs than in control group dogs. No significant difference was found in Pit-1–positive cell ratios between control and mitotane treatment group dogs (Figure 9).

Figure 9—
Figure 9—

Mean ± SD comparison of the percentage of ACTH-, Pit-1–, and T–pit-positive cells between control group dogs (closed bar) and mitotane treatment group dogs (open bar). See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 67, 8; 10.2460/ajvr.67.8.1385

Discussion

Hyperadrenocorticism caused by an ACTH-producing pituitary gland tumor is a major endocrine disorder often found in dogs. Morphologically, pituitary gland tumors are classified into microadenoma and macroadenoma. In macroadenoma, various neurologic signs caused by compression of the surrounding cerebral tissues by the tumor have been reported.5,36,37 However, the mechanism of development of these pituitary gland tumors, and the differences between microadenoma and macroadenoma, have not been clarified.

In the field of small animal internal medicine, mitotane, trilostane, and the monoamine oxidase inhibitor L-deprenyl are used for treatment of PDH in dogs.7 Notably, mitotane, which selectively destroys the zonae fasciculata and reticularis in the adrenal cortex, is most commonly used.11,38 Treatment of dogs with PDH with mitotane has achieved good outcomes, although careful monitoring is necessary because of the difficulty in establishing the dose resulting from individual differences in sensitivity. It has been reported that when 200 dogs with PDH were surveyed, good to excellent reactions were observed in > 80% of dogs that received treatment for ≥ 3 months. Serum cortisol concentrations of dogs in that study before and after ACTH stimulation were within or lower than range values.10

However, there have been many reports of adverse effects with mitotane treatment. It has been reported that adverse effects were found in 25% and 31% of dogs during the mitotane induction period and maintenance period, respectively.10,39 The main adverse effects that have been reported include anorexia, vomiting, diarrhea, lethargy, and collapse.10,40,41 Furthermore, mitotane has been reported to directly affect the CNS and induce neurologic signs such as ataxia, blindness, and head pressing.11 In dogs with PDH that are treated with mitotane, iatrogenic adrenocortical insufficiency with hyperkalemia and hyponatremia has also been reported.10 Moreover, in other studies42,43 on mitotane, decreases in serum cortisol concentrations as a result of a reduction in negative feedback to the pituitary gland and an increase in ACTH secretion have been found. Tumor growth rate that may have been increased in several dogs was suggested by the findings of another study.14 This phenomenon may be similar to Nelson syndrome in human medicine.44

Promotion of pituitary gland hypertrophy caused by the loss of negative feedback to the pituitary gland has been reported, which strongly suggests that suppression of adrenal glands in dogs with pituitary corticotroph adenomas may result in enlargement of the pituitary gland. However, in other studies, macroadenomas of the pituitary gland grew slowly and independently of years of mitotane treatment in dogs with PDH.11,37 Thus, in the treatment of PDH, the effect of a decrease in negative feedback to the pituitary gland caused by suppression of the adrenal cortex is still controversial, and the details of the direct effect on the pituitary gland and other adverse effects have not been clarified.

In our study, we induced suppression of adrenocortical function for 1 month in healthy Beagles by continuous administration of mitotane. We performed CRH stimulation testing, MRI of the pituitary gland and brain, and immunohistochemistry to investigate mitotane-induced functional and morphologic changes in pituitary corticotrophs. From our results, we evaluated the effect of mitotane on the pituitary gland.

In mitotane treatment group dogs, on the basis of serum cortisol concentrations measured in the maintenance period and results of ACTH stimulation testing performed before mitotane administration and at completions of the induction period and the maintenance period, continuous administration of mitotane at a dosage of 50 mg/kg/d was considered to be sufficient for functional inhibition of the adrenal glands. This dosage of mitotane used in treatment group dogs was higher than that typically used to treat dogs with PDH. Weights of bilateral adrenal glands were significantly lower in the mitotane treatment group, compared to control group dogs. In the H&E-stained adrenal gland tissue sections, severe atrophy of the zonae fasciculata and reticularis was found in mitotane treatment group dogs, as previously reported.45,46 These findings confirmed that mitotane induced sufficient destruction and functional suppression of the adrenal gland.

To investigate the effect of mitotane on the pituitary gland, MRI of the pituitary gland and brain was performed in the pre-and postmitotane treatment groups. Reference range values for the height of the canine pituitary gland measured by computed tomography (3.2 to 5.1 mm) and PBR values (PBR < 0.31) have been reported.9,32,47 In our study, the height of the pituitary gland exceeded the reference range and the PBR exceeded 0.31 in postmitotane treatment group dogs, indicating that mitotane administration did indeed result in enlargement of the pituitary gland. Weights of the excised pituitary glands were also significantly higher in postmitotane treatment group dogs than in control group dogs, confirming that the pituitary gland was actually enlarged. In the immunohistochemical measurement of ACTH-positive cells, the cell area was significantly higher in postmitotane treatment group dogs than in control group dogs. These results indicate that mitotane induces hypertrophy of corticotrophs, and this hypertrophy may have induced hypertrophy of the entire pituitary gland. However, the number of ACTH-positive cells per field on microscopic evaluation was significantly lower in postmitotane treatment group dogs than in control group dogs. We also immunohistochemically investigated T-pit, a cellspecific factor for expression of pituitary gland proopiomelanocortin gene, and Pit-l specifically expressed in differentiation to somatotrophs, mammotrophs, and thyrotrophs.34,35 The numbers of T-pit– and Pit-1–positive cells reflect the numbers of corticotrophs and cell group of somatotrophs, mammotrophs, and thyrotrophs, respectively. The number of T-pit–positive cells was also significantly lower in mitotane treatment group dogs, compared with control group dogs, and this result was consistent with the ACTH-positive cell counts. Because cell counts were made per field in pituitary gland tissue sections, the probability that fewer cells may have been counted as a result of distribution of positive cells is a consideration. No significant difference was found in the number of Pit-1–positive cells between mitotane treatment and control group dogs. Because Pit-1–positive cells accounted for most of the cells in the anterior pituitary gland, distribution of positive cells may not have markedly affected the number of Pit-1–positive cells.

To investigate the effect of persistent suppression of the adrenal cortex by mitotane on ACTH secretory function, plasma ACTH concentrations were measured following CRH stimulation testing before and after 1 month of continuous administration of mitotane. Baseline plasma ACTH concentrations were significantly increased in postmitotane treatment group dogs, compared with control and premitotane treatment group dogs, and the concentration greatly exceeded reference range values of healthy dogs (13 to 46 pg/mL).48 This increase in ACTH production and secretion may have been induced by decreased negative feedback by cortisol and increased CRH secretion from the hypothalamic paraventricular nucleus that sensed the decreased blood cortisol concentrations resulting from suppression of the adrenal cortex. Plasma ACTH concentrations after CRH stimulation were also significantly higher in postmitotane treatment group dogs, compared with control and premitotane treatment group dogs. These findings indicate that ACTH storage increased in corticotrophs.

In humans with PDH, occurrence of rapidly growing, ACTH-producing pituitary gland tumors, called Nelson syndrome, after bilateral adrenalectomy has been reported.44,49,50 The loss of negative feedback to the pituitary gland resulting from excision of the adrenal glands may increase ACTH secretion and promote tumor growth. As a similar example, hypertrophy of thyrotrophs in experimental thyroidectomized rats has been reported.51–53 Thyroid hormone secreted from the thyroid gland negatively controls secretion of thyrotropin-releasing hormone from the hypothalamus and thyroid-stimulating hormone from the pituitary gland in the hypothalamic-pituitary-thyroid axis. Sudden loss of negative feedback by thyroid hormone after thyroidectomy subsequently may induce excess TRH secretion and result in enlarged thyrotrophs.

With regards to the outcomes of long-term mitotane treatment for PDH, various reports10,14,15,39 discuss the adverse effects of this drug. In 1 study,15 mitotane caused enlargement of the pituitary gland and negatively affected brain tissues that resulted in development or aggravation of neurologic signs. In our study, we investigated the pituitary gland in dogs treated with mitotane and found corticotroph hypertrophy with subsequent enlargement of the entire pituitary gland caused by suppression of cortisol production. Hyperfunction of pituitary corticotrophs in the dogs of our study was confirmed as increases in ACTH secretion and storage in the pituitary gland. The ratio of corticotrophs to all cells in the anterior pituitary gland has been reported to be 2% to 15.5% in clinically normal rats54,55 and 15% to 20% in clinically normal humans.56 But the ratio may be several times higher in instances of pituitary corticotroph adenoma.57 Several groups have reported that dogs with pituitary gland macroadenoma are less likely to respond to dexamethasone suppression testing58,59; however, clinically high-dose dexamethasone suppression tests are used in dogs with PDH in general, and approximately 60% of dogs with PDH have plasma cortisol concentrations < 50% of baseline values.60 Results of several studies29,30,61 reveal partial but not total correlation between the size of a pituitary gland tumor and plasma cortisol response to dexamethasone. So it is possible that dogs with pituitary gland adenoma that contain a macroadenoma have negative feedback inhibition. Dogs with PDH in which ACTH concentrations increased after mitotane treatment have been reported,62 indicating a release of suppressive effects of cortisol by mitotane on pituitary corticotrophs. Thus, proliferated corticotrophs in microadenoma and macroadenoma may swell rapidly as a result of negative feedback inhibition of cortisol by mitotane.

ABBREVIATIONS

PDH

Pituitary-dependent hyperadrenocorticism

HPA

Hypothalamic-pituitary-adrenal

CRH

Corticotropin-releasing hormone

MRI

Magnetic resonance imaging

PBR

Pituitary gland brain ratio

a.

Cortrosyn, Daiichi Pharmaceutical Co, Tokyo, Japan.

b.

Spotchem Bidas cortisol assay kit, ARKRAY Inc, Kyoto, Japan.

c.

Ovine CRH, Peptide Institute Inc, Osaka, Japan.

d.

Allegro ACTH assay kit, Kyowa Medex Co, Tokyo, Japan.

e.

Magnetic type 1.5T-MRI instrument, Toshiba Medical Systems Co, Tochigi, Japan.

f.

Droperidol, Sankyo Co, Tokyo, Japan.

g.

Propofol, Takeda Schering-Plough Animal Health K. K., Osaka, Japan.

h.

Isoflurane, Dainippon Pharmaceutical Co, Tokyo, Japan.

i.

Gadolinium, Daiichi Pharmaceutical Co, Tokyo, Japan.

j.

Op'-DDD, Yakult Honsha Co, Tokyo, Japan.

k.

Pentobarbital, Dainippon Pharmaceutical Co, Tokyo, Japan.

l.

Monoclonal mouse anti-adrenocorticotropin, Dako Japan Co, Kyoto, Japan.

m.

Pit-1 rabbit polyclonal IgG, Santa Cruz Biotechnology Inc, Santa Cruz, Calif.

n.

T-pit rabbit polyclonal IgG, Invitrogen Co, Carlsbad, Calif.

o.

Carl Zeiss Axiophot microscope, Carl Zeiss AG, Yena, Germany.

p.

KS400 imaging analysis system, Carl Zeiss AG, Yena, Germany.

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Contributor Notes

This study was supported by grants from the Grant-in-Aid for Scientific Research (C) in Japan and the Japan Society for the Promotion of Science.

Address correspondence to Dr. Taoda.