- Email: [email protected]
Cantharidin and norcantharidin inhibit caprine luteal cell steroidogenesis in vitro
Experimental and Toxicologic Pathology 64 (2012) 37–44
Contents lists available at ScienceDirect
Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Cantharidin and norcantharidin inhibit caprine luteal cell steroidogenesis in vitro Nae-Fang Twu a , Ramanujam Srinivasan c , Chung-Hsi Chou b , Leang-Shin Wu c , Chih-Hsien Chiu c,∗ a
Department of Obstetrics and Gynecology, Taipei Veterans General Hospital, National Yang-Ming University, Taiwan School of Veterinary Medicine, National Taiwan University, Taipei 10617, Taiwan c Department of Animal Science and Technology, College of Bio-Resources and Agriculture, National Taiwan University, Taipei 10617, Taiwan b
a r t i c l e
i n f o
Article history: Received 3 March 2010 Accepted 2 June 2010 Keywords: Cantharidin Norcantharidin Caprine Luteal cells Steroidogenesis
a b s t r a c t Cantharidin and its analog norcantharidin are active constituents of Mylabris, have been demonstrated to ailments for a variety of cancers. But several reports of cantharidin’s natural or accidental toxicoses in field animals and humans showed a strong connection between cantharidin and its abortifacient and aphrodisiac properties. However, their exact cellular mechanisms in steroidogenesis remains poorly understood. Thus this study was aimed to explore the effects of cantharidin on luteal cell steroidogensis and to compare its effect with that of norcantharidin. For this purpose, luteal cells isolated from corpora lutea of native Taiwan goats were maintained in vitro and treated for 4 and 24 h with cantharidin and norcantharidin (0.1, 1.0, and 10 g ml−1 ) to assess their steroidogenic effects. Progesterone (P4 ) levels and steroidogenic enzyme expression were assessed by enzyme immunoassay and Western blot methods, respectively. In caprine luteal cells, cantharidin and norcantharidin repressed basal P4 production, as well as that mediated by ovine luteinizing hormone (oLH), 8-bromo-cyclic AMP (8-Br-cAMP), 22Rhydroxycholesterol (22R-OHC) and pregnenolone (P5 ). They also inhibited the expression of steroidogenic acute regulatory (StAR) protein, cytochrome P450 cholesterol side-chain cleavage (P450scc) enzyme, and 3-hydroxysteroid dehydrogenase (3-HSD) enzyme. Additionally, the greater inhibitory effect was detected using cantharidin, when it is compared with that of norcantharidin. Our results suggest that ingestion of cantharidin may decrease luteal steroidogenesis, and the decline in luteal P4 levels may disrupt reproductive functions in humans as well as animals. © 2010 Elsevier GmbH. All rights reserved.
1. Introduction Cantharidin (Fig. 1A) and its demethylated analog, norcantharidin (Fig. 1B), are natural toxins produced by Chinese blister beetles (Mylabris phalerata or Mylabris cichorii) and Spanish flies (Cantharis vesicatoria). Blister beetles are most commonly found in southern Europe, Africa and Asia (Oaks et al., 1960; Wang, 1989; Tagwireyi et al., 2000; Moed et al., 2001). It has been reported that the use of dried bodies of the Chinese blister beetle belonging to Mylabris species, as a traditional Chinese medicine since antiquity, and also used as a folk medicine still today (Wang, 1989; Tagwireyi et al., 2000). The preparations from these beetles have been used by herbalists in some parts of South Africa as abortifacients and for their claimed aphrodisiac effects in males (Harrisberg et al., 1984; Tagwireyi et al., 2000).
∗ Corresponding author at: Laboratory of Animal Physiology, Department of Animal Science and Technology, College of Bio-Resources and Agriculture, National Taiwan University, Taipei 10617, Taiwan. Tel.: +886 2 3366 4158; fax: +886 02 2733 7095. E-mail address: [email protected] (C.-H. Chiu). 0940-2993/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2010.06.003
Cantharidin toxicoses, often due to the ingestion of feed containing blister beetles, have been documented in a variety of animals, including mammals, birds, and frogs, and continue to present a problem for ranchers (Schmitz, 1989). The presence of cantharidin has been confirmed in field ruminants, especially in horses and sheep diagnosed by chromatographic identification of their stomach contents (Ray et al., 1980). Experimentally-induced cantharidin toxicosis has been studied in experimental animals (rabbit, rat, goat, sheep, and horse) given them cantharidin or dried preparations of blister beetles, and renal dysfunction in sheep and horse has been reported frequently (Ray et al., 1979). In addition, there are several reports of human poisonings resulting from the ingestion of these toxins (Tagwireyi et al., 2000). Both cantharidin and norcantharidin have potential clinical utility as inhibitors of protein phosphatase 1 and 2A (PP1, PP2A) (McCluskey et al., 2003). The possibility that phosphatases might influence secretory processes has also attracted attention recently, and phosphatase-linked mechanisms have been implicated extensively in several endocrine tissues, including islets of langerhans and chromaffin and pituitary cells (Gagliardino et al., 1991; Green and Orme-Johnson, 1991; Iriuchijima et al., 1992; Polettini et al., 1992; Abayasekara et al., 1996; Murphy and Jones, 1996; Ciereszko et al., 2001). Both cantharidin and norcantharidin have
38
N.-F. Twu et al. / Experimental and Toxicologic Pathology 64 (2012) 37–44
Fig. 1. The chemical structures of cantharidin (A) and norcantharidin (B).
been used to assess the role of phosphatases in steroidogenesis (Abayasekara et al., 1996; Ciereszko et al., 2001). However, the molecular mechanisms through by which cantharidin and its analogue norcantharidin exert their effects on steroidogenesis are not fully understood. We have reported previously that the establishment of a luteal cell line (tsCLC-D) by transformation of a temperature-sensitive A209 (tsA209) mutant of simian virus 40 (SV40), a stable cell line derived from corpus luteum of Taiwan goat. We proposed that these cells retaining steroidogenic capacity of tsA209 could make them useful experimental tool to study the regulation of steroidogenesis (Chiu et al., 2008a). Moreover, these cell lines are thought to be very useful tool for the evaluation of luteal function in the field of reproductive toxicology and asses the pathological conditions in animals, especially under the effects of steroidogenically active drugs. The importance of steroidogenic acute regulatory (StAR) protein, cytochrome P450 cholesterol side-chain cleavage (P450scc) enzyme, and 3-hydroxysteroid dehydrogenase (3-HSD) enzyme in basal and hormone-regulated steroidogenesis are well-known (Stocco, 2001; Stocco et al., 2007). However, the effect of cantharidin and norcantharidin on the expression of these enzymes has yet to be determined. In the present study, primary caprine luteal cells and stable caprine luteal cells (tsCLC-D) (Chiu et al., 2008a) were used to investigate the effects of cantharidin and norcantharidin on basal and hormonally-induced progesterone (P4 ) production. In addition, we determined the effects of these toxins on the expression of StAR protein, P450scc enzyme, and 3-HSD enzyme. 2. Materials and methods 2.1. Reagents and chemicals Cell culture medium 199 (M199) with Earle’s salts and lglutamine (without sodium bicarbonate), trypsin–EDTA (0.25%), penicillin-G, streptomycin sulfate, fetal bovine serum (FBS) and trypan blue stain were purchased from Invitrogen Corporation (Grand Island, NY, USA). Ovine luteinizing hormone (NIDDK-oLH-1-3) was a kind gift from the NIDDK National Hormone Pituitary Program (Bethesda, MD, USA). Collagenase 1A, 8-bromo-cyclic AMP (8-BrcAMP), DNase 1, and other general chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Pregnenolone and 22hydroxycholesterol (22R-OHC) were purchased from Steraloids, Inc. (Newport, RI, USA). Cantharidin was purchased from Sigma Chemical Co., and norcantharidin was a generous gift from Dr. Guang-Sheng Wang (Department of Physiology, National YangMing University, Taiwan).
and Use Committee, College of Medicine, National Taiwan University, and all experimental procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory animals. Estrous cycles were assessed by monitoring serum P4 levels daily as described previously (Chiu et al., 2008a). In brief, blood samples were collected from each animal daily and the serum P4 concentration was measured by enzyme immunoassay. Adult females with regular estrous cycles were checked daily to define the phases of the cycle as detailed subsequently. Day 1 of the estrous cycle was defined as the first day of low serum P4 (<2 ng ml−1 ) following by the midcycle peak P4 . Ovaries (n = 3) were collected surgically from goats in spontaneous estrus on day 9–12 and transported to the laboratory in ice-cold PBS immediately after collection. Corpora lutea were dissected and washed thoroughly with sterile saline. The corpus luteum was digested in 5 ml of Hank’s balanced salt solution containing collagenase 1 (400 U ml−1 ) and DNase I (200 U ml−1 ). Cell viability was determined by the trypan blue dye exclusion method (approximately 85–90%). 2.3. Primary luteal cell and tsCLC-D cell line maintenance As we demonstrated in our earlier study, the tsCLC-D cell line responds to 8-Br-cyclic AMP, 22 R-OHC and pregnenolone (P5 ) treatment with an increase in progesterone (P4 ) biosynthesis. And also can express StAR protein, P450scc enzyme and 3-HSD enzyme as similar to normal luteal cells. Their lost responses to gonadotropin, enforced us to use of primary luteal cells which were derived from mid luteal phase in this study. First, the isolated primary luteal cells were resuspended in M199 medium (105 cells ml−1 ), then seeded in 48-well plates (Coster, Cambridge, MA) and grown for 24 h at 37 ◦ C with 5% CO2 . The temperaturesensitive tsCLC-D (Chiu et al., 2008a) cells were grown in T-25 cell culture flasks (Coster) with M199 medium, supplemented with 5% FBS for 24 h at 34 ◦ C with 5% CO2 . At the time of seeding, the nonadherent cells were removed by aspiration and the healthy cells were collected by trypsinization. The trypsinized cell pellets gently resuspended in M199 medium (105 cells ml−1 ) were seeded in 48-well plates and grown for 24 h at 39 ◦ C with 5% CO2 . 2.4. Treatment of luteal cells The time-course effects of cantharidin and norcantharidin on basal P4 secretion and the influence of cantharidin and norcantharidin on oLH- and cAMP-stimulated P4 production were performed by using primary caprine luteal cells. The effects of cantharidin and norcantharidin on 22R-OHC- and pregnenolonesupported P4 production, Western blot study of StAR protein, P450scc enzyme, and 3-HSD enzyme expression, cellular proliferation and viability and cellular imaging studies were performed by using tsCLC-D cells. All experiments in this study were performed in serum-free M199 media. At the time of experiments, experimental cultures were replaced with serum-free M199 media. Stimulation of cells were performed using a maximal stimulatory doses of oLH (100 ng ml−1 ), the cyclic AMP analog 8-Br-cAMP (100 M). In some experiments the optimal doses of 22R-OHC (1 g ml−1 ) a steroidogenic substrate for P450scc enzyme or pregnenolone (1 g ml−1 ) a steroidogenic substrate for 3-HSD enzyme. After 4 or 24 h, culture media were collected and stored at −20 ◦ C until the enzyme immunoassay (EIA) was performed.
2.2. Animals and cell collection 2.5. P4 assay Native crossbred adult female Taiwanese goats (1–3 years of age) were maintained in good health with normal estrous cycles. Animals were given alfalfa hay and mineral block and water ad libitum. All experimental protocols were approved by the Animal Care
The progesterone assay was modified from a direct EIA using G7, an IgM monoclonal antibody with specific affinity of 1.1 × 1010 M−1 as described previously (Guo et al., 2001; Wu et al., 2002).
N.-F. Twu et al. / Experimental and Toxicologic Pathology 64 (2012) 37–44
It exhibits cross-reactivity of <0.01% with bovine serum albumin and other steroids including pregnenolone, testosterone, estradiol, and estrone. Aliquots (50 l) of diluted medium and horseradish peroxide-linked progesterone conjugate (150 l) were added to microtiter plates coated with 200 l of G7 (representing a 1:40,000 dilution). After incubation at room temperature with gentle shaking for 15 min and three washes with Tween-20 in 0.01 M phosphate buffer at pH 7.0, the color was developed with 200 l of 3.7 mM O-phenylenediamine in 0.03% H2 O2 for 15 min. The reaction was stopped by the addition of 50 l of 8N H2 SO4 . The optical density (OD) of each sample was assessed using a dual wavelength reader (Dynatech, Denkendort, Germany) at a wavelength set between 490 and 630 nm. The concentration of P4 was determined by comparing the sample values with a P4 standard curve. The coefficients of variation within and between assays were 7 and 12%, respectively. The sensitivity of the assay was 0.3 pg ml−1 (Guo et al., 2001; Chun et al., 2002). All the standards and samples were assayed in duplicate. 2.6. Western blot analysis After treatments, the cells were rinsed twice with cold PBS and harvested. Then the cells were resuspended in cold lysis buffer (2% SDS, 50 mM Tris, pH 6.8, 5 mM EDTA, 1% 2-mercaptoethanol, 5% glycerol) and whole cell extracts were prepared as described previously (Tosac et al., 2006). Samples containing 50 g of protein were loaded individually on 12% SDS-polyacrylamide gels (SDS-PAGE) as described previously (Tosac et al., 2006). Following electrophoresis, the separated proteins were electro-transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated overnight at 4 ◦ C in PBS containing 0.05% Tween-20 (PBST) and 5% fat-free milk, followed by incubation with appropriate amounts of StAR polyclonal (the epitope-specific polyclonal antibody was produced in a rabbit immunized against a synthetic 26 amino acid peptide (82AMQRALGILKDQEGWKKESRANGDE107) derived from the coding sequences reported for the bovine StAR gene (Gene Bank Accession No. Q28918)) (Chiu et al., 2008b), P450scc and 3-HSD polyclonal antibodies (polyclonal antibodies against P450scc and 3b-HSD conserved recombinant protein was produced by transformation of E. coli BL21 (DE3) with the recombinant plasmid containing the clone of full-length cDNA for P450scc and 3b-HSD in the prokaryotic expression vector pET29a) (Chiu et al., 2008c) in PBST-fat-free milk for 1 h at room temperature. After four washes with PBST, the membrane was incubated for 1 h with a 1:5000 dilution of corresponding anti-mouse peroxidase-conjugated IgG. The membrane was washed again with PBST, and bound antibodies were visualized by the ECL system (Amersham Pharmacia Inc., NJ Biotech, Piscataway, NJ) using Kodak X-OMAT film (Eastman Kodak Co., Rochester, NY). Monoclonal mouse anti-goat -actin and secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). 2.7. 3 H-thymidine incorporation and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays Cellular proliferation was assessed by using the 3 H-thymidine incorporation assay. For proliferation assays, tsCLC-D cells were seeded (1 × 104 cells well−1 ) onto 96-well plates (Nunc, Wiesbaden, Germany) in 200 l M 199 medium supplemented with 5% FBS at 37 ◦ C with 5% CO2 . After 24 h, the culture media was replaced with 200 l of M 199 medium with or without cantharidin or norcantharidin (0.1, 1.0, or 10 l ml−1 ), after which the cells were cultured for 8 h in the presence of 0.5 Ci well−1 [3H]TdR (Amersham-Buchler, Braunschweig, Germany). The cells were collected on fiberglass filter paper with a 96-well harvester (Phar-
39
macia, Freiburg, Germany), and radioactivity was quantified with a 96-well -plate liquid scintillation counter (Pharmacia). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay were used to determine the number of living cells in the sample. In brief, the cells were seeded on 96-well plates with M199 medium supplemented with 5% FBS for 24 h at 37 ◦ C with 5% CO2 , at a density of 1 × 104 cells ml−1 . That would ensure exponential growth for the duration of the assay. After 24 h, the growth medium was replaced with serum-free M199 medium. Six duplicate wells were set up for each sample, and untreated cells served as controls. Cantharidin or norcantharidin were initially dissolved in dimethylsulfoxide (DMSO), which was present at <1% final concentration during assays. The cells were then treated for 24 h with cantharidin or norcantharidin at the concentrations of 0.1, 1, and 10 g ml−1 , after which 10 l MTT (Sigma) was added to each well and the cells were incubated for 4 h at 37 ◦ C. The medium was removed, and MTT stabilization solution (dimethylsulfoxide:ethanol = 1:1) was added to each well and shaken for 10 min until all crystals were dissolved. The OD was measured at 595 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Wells without cells were used to define zero absorbance. Each assay was performed in triplicate. 2.8. Data analysis All experimental values in the figures were expressed as mean ± SEM of triplicates of three individual experiments, except Western blot studies. Statistical analysis was performed using ANOVA (two-factor with replications) followed by Duncan’s multiple comparison (Duncan, 1995). A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Effect of cantharidin and norcantharidin on basal P4 secretion by primary caprine luteal cells To determine the short-term (4 h) and long-term (24 h) effects of cantharidin and norcantharidin on basal P4 secretion, primary caprine luteal cells were cultured with various doses (0.1, 1.0, and 10 g ml−1 ) of the toxins. In this study, the basal P4 secretion was dose-dependently reduced by cantharidin in both 4 and 24 h (Fig. 2A). Norcantharidin reduced basal P4 secretion after 24 h, but the effect was not dose-dependent (Fig. 2B). 3.2. Effect of cantharidin and norcantharidin on oLH- and 8-Br-cAMP-regulated P4 secretion by primary caprine luteal cells To determine the influence of cantharidin and norcantharidin on oLH- and cAMP-stimulated P4 production by primary caprine luteal cells, the cells were incubated with or without cantharidin or norcantharidin (0.1, 1.0, or 10 g ml−1 ), along with or without oLH (100 ng ml−1 ) or 8-Br-cAMP (100 M). The cAMP analog activates PKA directly and induces maximal P4 production. Under oLH and 8-Br-cAMP-stimulated conditions, both cantharidin (Fig. 2C) and norcantharidin (Fig. 2D) produced dose-dependent inhibition of P4 production after 24 h of incubation. 3.3. Effect of cantharidin and norcantharidin on basal; and 22R-OHC- and pregnenolone-supported P4 secretion by tsCLC-D cells We next tested the effects of cantharidin and norcantharidin on basal level; and 22R-OHC- and pregnenolone-supported P4 production in tsCLC-D cells. Cells were treated for 24 h with cantharidin and norcantharidin (0.01, 0.1, 1.0, or 10 g ml−1 ) alone or in the
40
N.-F. Twu et al. / Experimental and Toxicologic Pathology 64 (2012) 37–44
Fig. 2. Effects of cantharidin and norcantharidin on basal, oLH-, and cAMP-stimulated P4 synthesis. Primary caprine luteal cells were cultured in serum-free medium for 24 h with increasing concentrations (0.1, 1, and 10 g ml−1 ) of cantharidin or norcantharidin in the absence (A and B) or presence (C and D) of oLH (100 ng ml−1 ) or (cAMP 100 M). The media was collected after 4 and 24 h, and P4 concentrations were determined by EIA as described in Section 2. All data are the mean ± SEM of triplicates of three individual experiments; each experiment was performed triplicate. Bars with different letters are significantly different at p < 0.05.
presence or absence of 1 g ml−1 22R-OHC or pregnenolone. 22ROHC bypasses the action of StAR protein and supports the P4 synthesis. A dose-dependent inhibited basal level P4 production in tsCLC-D cells by cantharidin and norcantharidin are shown in Fig. 3. As shown in Fig. 4B, norcantharidin barely observed inhibition of 22R-OHC-supported P4 production, but not cantharidin (Fig. 4A) and both were shown their maximal inhibition evident at 10 g ml−1 doses. Both these inhibitors did not interfere with steroidogenic enzymes directly since they did not prevent P4 production by lueal cells, which were supplied with 22R-OHC as a substrate. Cantharidin and norcantharidin also dose-dependently inhibited pregnenolone-supported P4 production in tsCLC-D cells (Fig. 4C and D). The maximal inhibitory effects of both compounds were found at 10 g ml−1 dose. 3.4. Effects of cantharidin and norcantharidin on StAR, P450scc, and 3ˇ-HSD expression As determined by Western blot analysis (Fig. 5), cantharidin produced dose-dependent (1 and 10 g ml−1 ) inhibition of StAR protein, P450scc enzyme, and 3-HSD enzyme expression. While dose-dependent inhibition of StAR protein and P450scc enzyme expression was seen with norcantharidin (1 and 10 g ml−1 ), but
Fig. 3. Effects of cantharidin and norcantharidin on basal P4 secretion in tsCLC-D cells. The tsCLC-D cells were cultured in serum-free medium with increasing concentrations (0.01, 0.1, 1, and 10 g ml−1 ) of cantharidin or norcantharidin. The media was collected after 24 h, and P4 concentrations were determined by EIA. All data are the mean ± SEM of triplicates of three individual experiments; each experiment was performed triplicate. Bars with different letters are significantly different at p < 0.05.
N.-F. Twu et al. / Experimental and Toxicologic Pathology 64 (2012) 37–44
41
Fig. 4. Effects of cantharidin and norcantharidin on 22R-OHC- and pregnenolone-supported P4 synthesis in tsCLC-D cells. Cells were cultured in serum-free medium for 24 h with increasing concentrations (0.01, 0.1, 1, and 10 g ml−1 ) of cantharidin or norcantharidin and co-incubated with 22R-OHC (1 g ml−1 ; A and B) or pregnenolone (1 g ml−1 ; C and D). The media was collected after 24 h, and P4 concentrations were determined. All data are the mean ± SEM of triplicates of three individual experiments; each experiment was performed in triplicate. Bars with different letters are significantly different at p < 0.05.
the inhibition of 3-HSD enzyme expression was only observed in the lower norcantharidin dose. After coincubation with 22R-OHC (1 g ml−1 ), P450scc enzyme expression was inhibited by both the 1 and 10 g ml−1 concentrations of cantharidin, but only by the higher concentration of norcantharidin (Fig. 6). After coincubation with pregnenolone (1 g ml−1 ), inhibition of 3-HSD enzyme expression was observed in response to 10 g ml−1 concentration of cantharidin, whereas norcantharidin was ineffective at both doses. 3.5. Effect of cantharidin and norcantharidin on tsCLC-D cellular proliferation and viability In order to explore whether cantharidin and norcantharidin effects tsCLC-D cell population with two fundamentally different parameters such as proliferation and viability, we performed 3 Hthymidine assay and MTT assay. The 3 H-thymidine assay detects an increase in the proportion of cells in S phase within the target population, whereas the MTT assay detects an increase in total cell number measured by detection of cellular metabolic activity. First, the 3 H-thymidine uptake assay was subjected to determine whether the inhibition of tsCLC-D cell population reflects a decrease in the rate of DNA synthesis. As depicted in Fig. 7A, both cantharidin and norcantharidin produced significant (p < 0.05), rapid (24 h) dose-dependent inhibition of mitogen-induced cell proliferation. However, the effect of cantharidin was more pronounced than that of norcantharidin. Next we assessed the cell viability by using a metabolic assay, MTT assay. As seen in Fig. 7B, the cytotoxic effects were evident
at the highest (10 g ml−1 ) concentration of cantharidin, but not detected for norcantharidin at any concentration. In contrast, treatment with norcantharidin at any concentrations (0.1–10 g ml−1 ) had no apparent effects on cell viability. Cytotoxicity also was confirmed with morphological and ultra-structural analysis, after incubation of tsCLC-D cells for 24 h with 10 g ml−1 cantharidin and norcantharidin. Typical extracellular membrane-bound apoptotic bodies surrounded by ruptured cells, decreased intracellular volume, ruptured intercellular organelles, and condensed chromatin were detected in the cantharidin-treated cells, but not in the norcantharidin-treated or control cells (data not shown).
4. Discussion The deleterious effects of naturally occurring toxins from variety of sources on female reproductive functions are major serious growing problem for human and wild life. The presence of functionally normal corpus luteum (CL) is essential for the maintenance of gestation in mammals. The major secretory product of the CL is the C21 steroid, progesterone (P4 ). This suggests the normal levels of P4 secretion are essential for the implantation and subsequent development of feto-placental unit. Thus, the perturbation in the secretion and/or action of P4 by exogenous compounds can profoundly affect the steroidogenic capacity of the corpus luteum. In this regard, the abnormal luteal function cause failure of implantation and embryonic wastage by effecting the estrus cycle, pregnancy as well as the developing young ones (Oduma et al., 1995) or early embryonic deaths.
42
N.-F. Twu et al. / Experimental and Toxicologic Pathology 64 (2012) 37–44
Fig. 5. Effects of cantharidin and norcantharidin on expression of StAR protein, P450scc, and 3-HSD enzymes in tsCLC-D cells. The tsCLC-D cells were cultured in serum-free medium for 24 h with increasing concentrations (0, 1, and 10 g ml−1 ) of cantharidin or norcantharidin. After treatment, total cellular protein was extracted and loaded (50 g) in SDS-PAGE and immunobloted with appropriate amounts of StAR, P450scc and 3B-HSD antibodies, as described in Section 2. The blot is representative of three separate experiments with similar experimental patterns.
In the present study, we investigated the effects of cantharidin and norcantharidin on progesterone (P4 ) production and the effects of those toxins on the expression of StAR protein, P450scc enzyme, and 3-HSD enzyme by using primary caprine luteal cells and caprine luteal cell line (tsCLC-D) (Chiu et al., 2008a). We assessed the effects of cantharidin and norcantharidin on oLH- and 8-Br-cAMP-stimulated P4 production. In our results the suppressed oLH-stimulated P4 productions by cantharidin demonstrate that the inhibition may be due to partial inhibition of adenylate cyclase, resulting in the reduction of cAMP formation. Furthermore, these finding corroborates with the inhibition of StAR
Fig. 6. Effects of cantharidin and norcantharidin on 22R-OHC-supported P450scc enzyme (upper panel) and pregnenolone-induced 3-HSD enzyme (lower panel) expression in tsCLC-D cells. The tsCLC-D cells were cultured in serum-free medium for 24 h with increasing concentrations (0.1, 1, and 10 g ml−1 ) of cantharidin or norcantharidin and co-incubated with 22R-OHC or pregnenolone (1 g ml−1 ). After treatment, total cellular protein was extracted and loaded (50 g) in SDS-PAGE and immunobloted with appropriate amounts of P450scc and 3B-HSD antibodies as described in Section 2. The blot is representative of three separate experiments with similar experimental patterns.
protein expression by cantharidin and also supports the previous studies (Jones et al., 2000), thereby showed that both steroidogenesis and StAR protein activities were blocked by the inhibition of protein phosphatase 1 and 2A (PP1, PP2A). Our results of the inhibited 8-Br-cAMP-stimulated P4 production by cantharidin suggest that the blocked cAMP-mediated steroid production by preventing the expression of StAR protein and this also supports the earlier studies (Burns et al., 2000). It has been demonstrated that both cantharidin and norcantharidin are strong inhibitors of PP1 and PP2A (Liu et al., 2008). These studies implicate that the important roles of PP1/2A in stimulus-dependent StAR protein expression, and thus of steroidogenesis. However, the
Fig. 7. Effects of cantharidin and norcantharidin on tsCLC-D cell proliferation and viability. The tsCLC-D cells were exposed to cantharidin or norcantharidin (0.1, 1.0, and 10 g ml−1 ) in a regular medium for 24 h following addition of 3 H-thymidine for 24 h (A). Cell viability was measured by MTT assay (B). Each bar represents the mean ± SEM of triplicates of three individual experiments; each experiment was performed in triplicate. Bars with different letters are significantly different at p < 0.05.
N.-F. Twu et al. / Experimental and Toxicologic Pathology 64 (2012) 37–44
hormone-regulated steroidogenesis is dependent upon serine or threonine phosphorylation (Green and Orme-Johnson, 1991), but the role of phosphatases in the regulation of P4 secretion by luteal cells were unknown. In the present study, the inhibition of P4 secretion by cantharidin and norcantharidin, inhibitors of PP1/PP2A can perhaps best be explained in terms of preventing the dephosphorylation of a protein which is essential for steroidogenesis and which is thought to be involved in a phosphorylation/dephosphorylation cycle. This was also been supported by the presence and activity of PP1 and PP2A in rat luteal cells and the consequences of inhibiting PP activity in terms of P4 secretion (Abayasekara et al., 1996). More over the inhibition of PP1 and PP2A by okadaic acid enhanced FSH-mediated P4 secretion in primary granulosa cells (Gonzalez Reyes et al., 1997) and rat preovulatory follicles (Yu et al., 2001). Our results are consistent with previous studies, which suggest that the regulation of the serine/threonine phosphorylation balance is involved in luteal cell steroidogenesis. These results could also imply that cantharidin had no direct influence on the activities of steroidogenic enzymes (P450scc or 3-HSD) since cantharidin did not prevent P4 synthesis by lueal cells supplied with 22R-OHC, as a substrate. This is because 22R-OHC freely crosses the aqueous space between outer and inner mitochondrial membranes (Abayasekara et al., 1996). StAR protein promotes the transport of cholesterol from the outer mitochondrial membrane to the inner membrane, where the P450scc complex is located. Therefore, the suppression of StAR protein expression resulted in reduced steroid production. Our study was strongly supported by other previous studies have reported that organophosphate compounds (Dimethoate, Roundup, diethylumbelliferyl phosphate) inhibit steroidogenesis by blocking StAR protein expression in MA-10 cells (Walsh et al., 2000a,b; Manna et al., 2003). Comparing the results of 3 H-thymidine assay and MTT assay, the data clearly demonstrate that cantharidin and norcantharidin have inhibitory effect on DNA synthesis rather than apoptotic effects and also these results high correspondence concerning the decrease of luteal cell population through mitotic arrest. In caprine luteal cells, the inhibition of P4 production by cantharidin and norcantharidin was primarily due to the result of inhibition of steroidogenesis, providing evidence for the ability of these toxins to modulate gonadotropin-stimulated regulation of luteal function. Our results also suggest that the ingestion of cantharidin may decrease the ovarian steroidogenesis, and the decline in serum P4 levels may lead to a disruption of reproductive functions in humans, as well as other animals. 5. Conclusions We concluded that, cantharidin and norcantharidin repressed basal as well as oLH, 8-Br-cAMP, and 22R-OHC-induced P4 production in caprine luteal cells. Our evidence indicates the reduction in P4 production is mediated through the inhibition of StAR protein, P450scc enzyme, and 3-HSD enzyme expression. Interestingly, cantharidin more effectively inhibited caprine luteal cell steroidogenesis, as compared to norcantharidin. This study broadens our understanding of the possible intermediate targets influenced by cantharidin and its analog norcantharidin. In the present study, we established an in vitro cell system to investigate the effects of the closely related, naturally occurring toxins cantharidin and norcantharidin on luteal cell steroidogenesis. Both cantharidin and norcantharidin were found to inhibit the production of P4 and steroidogenic protein expression in luteal cells. In contrast to cantharidin, the inhibition of luteal cell steroidogenesis by norcantharidin is low. Steroidogenesis is regulated, at least in part, by the involvement of protein phosphorylation.
43
Acknowledgements The authors thank Dr. Guang-Sheng Wang for providing the norcantharidin. This study was supported by the research grants from the National Science Council, Republic of China (NSC93-2313-B002-113 and NSC95-2313-B-002-060-MY3). References Abayasekara DR, Ford SL, Persaud SJ, Jones PM. Role of phosphoprotein phosphatases in the corpus luteum. II. Control of progesterone secretion by isolated rat luteal cells. J Endocrinol 1996;150:213–21. Burns CJ, Gyles SL, Persaud SJ, Sugden D, Whitehouse BJ, Jones PM. Phosphoprotein phosphatases regulate steroidogenesis by influencing StAR gene transcription. Biochem Biophys Res Commun 2000;273:35–9. Chiu CH, Guo IC, Lin JH, Wu LS. Characterization of a stable steroidogenic caprine luteal cell line transformed by a temperature-sensitive simian virus 40. Chin J Physiol 2008a;51:369–75. Chiu CH, Wu LS, Jong DS. Production and application of a polyclonal peptide antiserum for universal detection of StAR protein. Chin J Physiol 2008b;51: 54–61. Chiu CH, Wei HW, Wu LS. Generation and utilization of P450 cholesterol side-chain cleavage enzyme and 3beta-hydroxysteroid dehydrogenase antibodies for universal detection. J Immunoassay Immunochem 2008c;29:152–60. Chun WB, Cheng WF, Wu LS, Yang PC. The use of plasma progesterone profiles to predict the reproductive status of anestrous gilts and sows. Theriogenology 2002;58:1165–74. ´ Ciereszko R, Opałka M, Kaminska B, Wojtczak M, Okrasa S, Dusza L. Luteotrophic action of prolactin during the early luteal phase in pigs: the involvement of protein kinases and phosphatases. Reprod Biol 2001;1:62–83. Duncan DB. Multiple range and multiple F test. Biometrics 1995;11:1–42. Gagliardino JJ, Krinks MH, Gagliardino EE. Identification of the calmodulin-regulated protein phosphatase, calcineurin, in rat pancreatic islets. Biochim Biophys Acta 1991;1091:370–3. Gonzalez Reyes J, Santana P, Gonzalez Robaina I, Cabrera Oliva J, Estevez F, Hernandez I, et al. Effect of the protein phosphatase inhibitor okadaic acid on FSH-induced granulosa cell steroidogenesis. J Endocrinol 1997;152: 131–9. Green EG, Orme-Johnson NR. Inhibition of steroidogenesis in rat adrenal cortex cells by a threonine analogue. J Steroid Biochem Mol Biol 1991;40:421–9. Guo IC, Wu LS, Lin JH, Chung BC. Differential inhibition of progesterone synthesis in bovine luteal cells by estrogens and androgens. Life Sci 2001;68:1851– 65. Harrisberg J, Deseta JC, Cohen L, Temlett J, Milne FJ. Cantharidine poisoning with neurological complications. S Afr Med J 1984;65:614–5. Iriuchijima T, Michimata T, Ogiwara T, Mizuma H, Yamada M, Murakami M, et al. Inhibitory effects of okadaic acid on thyrotropin and prolactin secretion from rat anterior pituitaries. Neuropeptides 1992;21:207–10. Jones PM, Sayed SB, Persaud SJ, Burns CJ, Gyles S, Whitehouse BJ. Cyclic AMP-induced expression of steroidogenic acute regulatory protein is dependent upon phosphoprotein phosphatase activities. J Mol Endocrinol 2000;24:233–9. Liu FY, Li Y, Peng YM, Ye K, Li J, Liu YH, et al. Norcantharidin ameliorates proteinuria, associated tubulointerstitial inflammation and fibrosis in protein overload nephropathy. Am J Nephrol 2008;28:465–77. Manna PR, Eubank DW, Lalli E, Sassone-Corsi P, Stocco DM. Transcriptional regulation of the mouse steroidogenic acute regulatory protein gene by the cAMP response-element binding protein and steroidogenic factor 1. J Mol Endocrinol 2003;30:381–97. McCluskey A, Ackland SP, Bowyer MC, Baldwin ML, Garner J, Walkom CC, et al. Cantharidin analogues: synthesis and evaluation of growth inhibition in a panel of selected tumour cell lines. Bioorg Chem 2003;31:68–79. Moed L, Shwayder TA, Chang MW. Cantharidin revisited: a blistering defense of an ancient medicine. Arch Dermatol 2001;137:1357–60. Murphy LI, Jones PM. Phospho-serine/threonine phosphatases in rat islets of Langerhans: identification and effect on insulin secretion. Mol Cell Endocrinol 1996;117:195–202. Oaks WW, Ditunno JF, Magnani T, Levy HA, Mills LC. Cantharidin poisoning. Arch Intern Med 1960;105:574–82. Oduma JA, Wango EO, Makawiti DW, Einer-Jensen N, Oduor-Okelo D. Effects of graded doses of the pesticide Heptachlor on body weight, mating success, estrous cycle, gestation length and litter size in laboratory rats. Comp Biochem Physicol Pharmacol Toxicol Endocrinol 1995;83:79–89. Polettini A, Crippa O, Ravagli A, Saragoni A. A fatal case of poisoning with cantharidin. Forensic Sci Int 1992;56:37–43. Ray AC, Post LO, Hurst JM, Edwards WC, Reagor JC. Evaluation of an analytical method for the diagnosis of cantharidin toxicosis due to ingestion of blister beetles (Epicauta lemniscata) by horses and sheep. Am J Vet Res 1980;41: 932–3. Ray AC, Tamulinas SH, Reagor JC. High pressure liquid chromatographic determination of cantharidin, using a derivatization method in specimens from animals acutely poisoned by ingestion of blister beetles, Epicauta lemniscata. Am J Vet Res 1979;40:498–504. Schmitz DG. Cantharidin toxicosis in horses. J Vet Intern Med 1989;3:208–15.
44
N.-F. Twu et al. / Experimental and Toxicologic Pathology 64 (2012) 37–44
Stocco DM. StAR protein and the regulation of steroid hormone biosynthesis. Annu Rev Physiol 2001;63:193–213. Stocco C, Telleria C, Gibori G. The molecular control of corpus luteum formation, function, and regression. Endocr Rev 2007;28:117–49. Tagwireyi D, Ball DE, Loga PJ, Moyo S. Cantharidin poisoning due to “Blister beetle” ingestion. Toxicon 2000;38:1865–9. Tosac L, Crochet S, Ferre P, Foufelle F, Tesseraud S, Dupont J. AMP-activated protein kinase activation modulates progesterone secretion in granulosa cells from hen preovulatory follicles. J Endocrinol 2006;190:85–97. Walsh LP, McCormick C, Martin C, Stocco DM. Roundup inhibits steroidogenesis by disrupting steroidogenic acute regulatory (StAR) protein expression. Environ Health Perspect 2000a;108:769–76.
Walsh LP, Webster DR, Stocco DM. Dimethoate inhibits steroidogenesis by disrupting transcription of the steroidogenic acute regulatory (StAR) gene. J Endocrinol 2000b;167:253–63. Wang GS. Medical uses of Mylabris in ancient China and recent studies. J Ethnopharmacol 1989;26:147–62. Wu LS, Chen JC, Sheu SY, Huang CC, Kuo YH, Chiu CH, et al. Isocupressic acid blocks progesterone production from bovine luteal cells. Am J Chin Med 2002;30:533–41. Yu CC, Chen WY, Li PS. Protein phosphatase inhibitor cantharidin inhibits steroidogenesis and steroidogenic acute regulatory protein expression in cultured rat preovulatory follicles. Life Sci 2001;70:57–72.
Contents lists available at ScienceDirect
Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Cantharidin and norcantharidin inhibit caprine luteal cell steroidogenesis in vitro Nae-Fang Twu a , Ramanujam Srinivasan c , Chung-Hsi Chou b , Leang-Shin Wu c , Chih-Hsien Chiu c,∗ a
Department of Obstetrics and Gynecology, Taipei Veterans General Hospital, National Yang-Ming University, Taiwan School of Veterinary Medicine, National Taiwan University, Taipei 10617, Taiwan c Department of Animal Science and Technology, College of Bio-Resources and Agriculture, National Taiwan University, Taipei 10617, Taiwan b
a r t i c l e
i n f o
Article history: Received 3 March 2010 Accepted 2 June 2010 Keywords: Cantharidin Norcantharidin Caprine Luteal cells Steroidogenesis
a b s t r a c t Cantharidin and its analog norcantharidin are active constituents of Mylabris, have been demonstrated to ailments for a variety of cancers. But several reports of cantharidin’s natural or accidental toxicoses in field animals and humans showed a strong connection between cantharidin and its abortifacient and aphrodisiac properties. However, their exact cellular mechanisms in steroidogenesis remains poorly understood. Thus this study was aimed to explore the effects of cantharidin on luteal cell steroidogensis and to compare its effect with that of norcantharidin. For this purpose, luteal cells isolated from corpora lutea of native Taiwan goats were maintained in vitro and treated for 4 and 24 h with cantharidin and norcantharidin (0.1, 1.0, and 10 g ml−1 ) to assess their steroidogenic effects. Progesterone (P4 ) levels and steroidogenic enzyme expression were assessed by enzyme immunoassay and Western blot methods, respectively. In caprine luteal cells, cantharidin and norcantharidin repressed basal P4 production, as well as that mediated by ovine luteinizing hormone (oLH), 8-bromo-cyclic AMP (8-Br-cAMP), 22Rhydroxycholesterol (22R-OHC) and pregnenolone (P5 ). They also inhibited the expression of steroidogenic acute regulatory (StAR) protein, cytochrome P450 cholesterol side-chain cleavage (P450scc) enzyme, and 3-hydroxysteroid dehydrogenase (3-HSD) enzyme. Additionally, the greater inhibitory effect was detected using cantharidin, when it is compared with that of norcantharidin. Our results suggest that ingestion of cantharidin may decrease luteal steroidogenesis, and the decline in luteal P4 levels may disrupt reproductive functions in humans as well as animals. © 2010 Elsevier GmbH. All rights reserved.
1. Introduction Cantharidin (Fig. 1A) and its demethylated analog, norcantharidin (Fig. 1B), are natural toxins produced by Chinese blister beetles (Mylabris phalerata or Mylabris cichorii) and Spanish flies (Cantharis vesicatoria). Blister beetles are most commonly found in southern Europe, Africa and Asia (Oaks et al., 1960; Wang, 1989; Tagwireyi et al., 2000; Moed et al., 2001). It has been reported that the use of dried bodies of the Chinese blister beetle belonging to Mylabris species, as a traditional Chinese medicine since antiquity, and also used as a folk medicine still today (Wang, 1989; Tagwireyi et al., 2000). The preparations from these beetles have been used by herbalists in some parts of South Africa as abortifacients and for their claimed aphrodisiac effects in males (Harrisberg et al., 1984; Tagwireyi et al., 2000).
∗ Corresponding author at: Laboratory of Animal Physiology, Department of Animal Science and Technology, College of Bio-Resources and Agriculture, National Taiwan University, Taipei 10617, Taiwan. Tel.: +886 2 3366 4158; fax: +886 02 2733 7095. E-mail address: [email protected] (C.-H. Chiu). 0940-2993/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2010.06.003
Cantharidin toxicoses, often due to the ingestion of feed containing blister beetles, have been documented in a variety of animals, including mammals, birds, and frogs, and continue to present a problem for ranchers (Schmitz, 1989). The presence of cantharidin has been confirmed in field ruminants, especially in horses and sheep diagnosed by chromatographic identification of their stomach contents (Ray et al., 1980). Experimentally-induced cantharidin toxicosis has been studied in experimental animals (rabbit, rat, goat, sheep, and horse) given them cantharidin or dried preparations of blister beetles, and renal dysfunction in sheep and horse has been reported frequently (Ray et al., 1979). In addition, there are several reports of human poisonings resulting from the ingestion of these toxins (Tagwireyi et al., 2000). Both cantharidin and norcantharidin have potential clinical utility as inhibitors of protein phosphatase 1 and 2A (PP1, PP2A) (McCluskey et al., 2003). The possibility that phosphatases might influence secretory processes has also attracted attention recently, and phosphatase-linked mechanisms have been implicated extensively in several endocrine tissues, including islets of langerhans and chromaffin and pituitary cells (Gagliardino et al., 1991; Green and Orme-Johnson, 1991; Iriuchijima et al., 1992; Polettini et al., 1992; Abayasekara et al., 1996; Murphy and Jones, 1996; Ciereszko et al., 2001). Both cantharidin and norcantharidin have
38
N.-F. Twu et al. / Experimental and Toxicologic Pathology 64 (2012) 37–44
Fig. 1. The chemical structures of cantharidin (A) and norcantharidin (B).
been used to assess the role of phosphatases in steroidogenesis (Abayasekara et al., 1996; Ciereszko et al., 2001). However, the molecular mechanisms through by which cantharidin and its analogue norcantharidin exert their effects on steroidogenesis are not fully understood. We have reported previously that the establishment of a luteal cell line (tsCLC-D) by transformation of a temperature-sensitive A209 (tsA209) mutant of simian virus 40 (SV40), a stable cell line derived from corpus luteum of Taiwan goat. We proposed that these cells retaining steroidogenic capacity of tsA209 could make them useful experimental tool to study the regulation of steroidogenesis (Chiu et al., 2008a). Moreover, these cell lines are thought to be very useful tool for the evaluation of luteal function in the field of reproductive toxicology and asses the pathological conditions in animals, especially under the effects of steroidogenically active drugs. The importance of steroidogenic acute regulatory (StAR) protein, cytochrome P450 cholesterol side-chain cleavage (P450scc) enzyme, and 3-hydroxysteroid dehydrogenase (3-HSD) enzyme in basal and hormone-regulated steroidogenesis are well-known (Stocco, 2001; Stocco et al., 2007). However, the effect of cantharidin and norcantharidin on the expression of these enzymes has yet to be determined. In the present study, primary caprine luteal cells and stable caprine luteal cells (tsCLC-D) (Chiu et al., 2008a) were used to investigate the effects of cantharidin and norcantharidin on basal and hormonally-induced progesterone (P4 ) production. In addition, we determined the effects of these toxins on the expression of StAR protein, P450scc enzyme, and 3-HSD enzyme. 2. Materials and methods 2.1. Reagents and chemicals Cell culture medium 199 (M199) with Earle’s salts and lglutamine (without sodium bicarbonate), trypsin–EDTA (0.25%), penicillin-G, streptomycin sulfate, fetal bovine serum (FBS) and trypan blue stain were purchased from Invitrogen Corporation (Grand Island, NY, USA). Ovine luteinizing hormone (NIDDK-oLH-1-3) was a kind gift from the NIDDK National Hormone Pituitary Program (Bethesda, MD, USA). Collagenase 1A, 8-bromo-cyclic AMP (8-BrcAMP), DNase 1, and other general chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Pregnenolone and 22hydroxycholesterol (22R-OHC) were purchased from Steraloids, Inc. (Newport, RI, USA). Cantharidin was purchased from Sigma Chemical Co., and norcantharidin was a generous gift from Dr. Guang-Sheng Wang (Department of Physiology, National YangMing University, Taiwan).
and Use Committee, College of Medicine, National Taiwan University, and all experimental procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory animals. Estrous cycles were assessed by monitoring serum P4 levels daily as described previously (Chiu et al., 2008a). In brief, blood samples were collected from each animal daily and the serum P4 concentration was measured by enzyme immunoassay. Adult females with regular estrous cycles were checked daily to define the phases of the cycle as detailed subsequently. Day 1 of the estrous cycle was defined as the first day of low serum P4 (<2 ng ml−1 ) following by the midcycle peak P4 . Ovaries (n = 3) were collected surgically from goats in spontaneous estrus on day 9–12 and transported to the laboratory in ice-cold PBS immediately after collection. Corpora lutea were dissected and washed thoroughly with sterile saline. The corpus luteum was digested in 5 ml of Hank’s balanced salt solution containing collagenase 1 (400 U ml−1 ) and DNase I (200 U ml−1 ). Cell viability was determined by the trypan blue dye exclusion method (approximately 85–90%). 2.3. Primary luteal cell and tsCLC-D cell line maintenance As we demonstrated in our earlier study, the tsCLC-D cell line responds to 8-Br-cyclic AMP, 22 R-OHC and pregnenolone (P5 ) treatment with an increase in progesterone (P4 ) biosynthesis. And also can express StAR protein, P450scc enzyme and 3-HSD enzyme as similar to normal luteal cells. Their lost responses to gonadotropin, enforced us to use of primary luteal cells which were derived from mid luteal phase in this study. First, the isolated primary luteal cells were resuspended in M199 medium (105 cells ml−1 ), then seeded in 48-well plates (Coster, Cambridge, MA) and grown for 24 h at 37 ◦ C with 5% CO2 . The temperaturesensitive tsCLC-D (Chiu et al., 2008a) cells were grown in T-25 cell culture flasks (Coster) with M199 medium, supplemented with 5% FBS for 24 h at 34 ◦ C with 5% CO2 . At the time of seeding, the nonadherent cells were removed by aspiration and the healthy cells were collected by trypsinization. The trypsinized cell pellets gently resuspended in M199 medium (105 cells ml−1 ) were seeded in 48-well plates and grown for 24 h at 39 ◦ C with 5% CO2 . 2.4. Treatment of luteal cells The time-course effects of cantharidin and norcantharidin on basal P4 secretion and the influence of cantharidin and norcantharidin on oLH- and cAMP-stimulated P4 production were performed by using primary caprine luteal cells. The effects of cantharidin and norcantharidin on 22R-OHC- and pregnenolonesupported P4 production, Western blot study of StAR protein, P450scc enzyme, and 3-HSD enzyme expression, cellular proliferation and viability and cellular imaging studies were performed by using tsCLC-D cells. All experiments in this study were performed in serum-free M199 media. At the time of experiments, experimental cultures were replaced with serum-free M199 media. Stimulation of cells were performed using a maximal stimulatory doses of oLH (100 ng ml−1 ), the cyclic AMP analog 8-Br-cAMP (100 M). In some experiments the optimal doses of 22R-OHC (1 g ml−1 ) a steroidogenic substrate for P450scc enzyme or pregnenolone (1 g ml−1 ) a steroidogenic substrate for 3-HSD enzyme. After 4 or 24 h, culture media were collected and stored at −20 ◦ C until the enzyme immunoassay (EIA) was performed.
2.2. Animals and cell collection 2.5. P4 assay Native crossbred adult female Taiwanese goats (1–3 years of age) were maintained in good health with normal estrous cycles. Animals were given alfalfa hay and mineral block and water ad libitum. All experimental protocols were approved by the Animal Care
The progesterone assay was modified from a direct EIA using G7, an IgM monoclonal antibody with specific affinity of 1.1 × 1010 M−1 as described previously (Guo et al., 2001; Wu et al., 2002).
N.-F. Twu et al. / Experimental and Toxicologic Pathology 64 (2012) 37–44
It exhibits cross-reactivity of <0.01% with bovine serum albumin and other steroids including pregnenolone, testosterone, estradiol, and estrone. Aliquots (50 l) of diluted medium and horseradish peroxide-linked progesterone conjugate (150 l) were added to microtiter plates coated with 200 l of G7 (representing a 1:40,000 dilution). After incubation at room temperature with gentle shaking for 15 min and three washes with Tween-20 in 0.01 M phosphate buffer at pH 7.0, the color was developed with 200 l of 3.7 mM O-phenylenediamine in 0.03% H2 O2 for 15 min. The reaction was stopped by the addition of 50 l of 8N H2 SO4 . The optical density (OD) of each sample was assessed using a dual wavelength reader (Dynatech, Denkendort, Germany) at a wavelength set between 490 and 630 nm. The concentration of P4 was determined by comparing the sample values with a P4 standard curve. The coefficients of variation within and between assays were 7 and 12%, respectively. The sensitivity of the assay was 0.3 pg ml−1 (Guo et al., 2001; Chun et al., 2002). All the standards and samples were assayed in duplicate. 2.6. Western blot analysis After treatments, the cells were rinsed twice with cold PBS and harvested. Then the cells were resuspended in cold lysis buffer (2% SDS, 50 mM Tris, pH 6.8, 5 mM EDTA, 1% 2-mercaptoethanol, 5% glycerol) and whole cell extracts were prepared as described previously (Tosac et al., 2006). Samples containing 50 g of protein were loaded individually on 12% SDS-polyacrylamide gels (SDS-PAGE) as described previously (Tosac et al., 2006). Following electrophoresis, the separated proteins were electro-transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated overnight at 4 ◦ C in PBS containing 0.05% Tween-20 (PBST) and 5% fat-free milk, followed by incubation with appropriate amounts of StAR polyclonal (the epitope-specific polyclonal antibody was produced in a rabbit immunized against a synthetic 26 amino acid peptide (82AMQRALGILKDQEGWKKESRANGDE107) derived from the coding sequences reported for the bovine StAR gene (Gene Bank Accession No. Q28918)) (Chiu et al., 2008b), P450scc and 3-HSD polyclonal antibodies (polyclonal antibodies against P450scc and 3b-HSD conserved recombinant protein was produced by transformation of E. coli BL21 (DE3) with the recombinant plasmid containing the clone of full-length cDNA for P450scc and 3b-HSD in the prokaryotic expression vector pET29a) (Chiu et al., 2008c) in PBST-fat-free milk for 1 h at room temperature. After four washes with PBST, the membrane was incubated for 1 h with a 1:5000 dilution of corresponding anti-mouse peroxidase-conjugated IgG. The membrane was washed again with PBST, and bound antibodies were visualized by the ECL system (Amersham Pharmacia Inc., NJ Biotech, Piscataway, NJ) using Kodak X-OMAT film (Eastman Kodak Co., Rochester, NY). Monoclonal mouse anti-goat -actin and secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). 2.7. 3 H-thymidine incorporation and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays Cellular proliferation was assessed by using the 3 H-thymidine incorporation assay. For proliferation assays, tsCLC-D cells were seeded (1 × 104 cells well−1 ) onto 96-well plates (Nunc, Wiesbaden, Germany) in 200 l M 199 medium supplemented with 5% FBS at 37 ◦ C with 5% CO2 . After 24 h, the culture media was replaced with 200 l of M 199 medium with or without cantharidin or norcantharidin (0.1, 1.0, or 10 l ml−1 ), after which the cells were cultured for 8 h in the presence of 0.5 Ci well−1 [3H]TdR (Amersham-Buchler, Braunschweig, Germany). The cells were collected on fiberglass filter paper with a 96-well harvester (Phar-
39
macia, Freiburg, Germany), and radioactivity was quantified with a 96-well -plate liquid scintillation counter (Pharmacia). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay were used to determine the number of living cells in the sample. In brief, the cells were seeded on 96-well plates with M199 medium supplemented with 5% FBS for 24 h at 37 ◦ C with 5% CO2 , at a density of 1 × 104 cells ml−1 . That would ensure exponential growth for the duration of the assay. After 24 h, the growth medium was replaced with serum-free M199 medium. Six duplicate wells were set up for each sample, and untreated cells served as controls. Cantharidin or norcantharidin were initially dissolved in dimethylsulfoxide (DMSO), which was present at <1% final concentration during assays. The cells were then treated for 24 h with cantharidin or norcantharidin at the concentrations of 0.1, 1, and 10 g ml−1 , after which 10 l MTT (Sigma) was added to each well and the cells were incubated for 4 h at 37 ◦ C. The medium was removed, and MTT stabilization solution (dimethylsulfoxide:ethanol = 1:1) was added to each well and shaken for 10 min until all crystals were dissolved. The OD was measured at 595 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Wells without cells were used to define zero absorbance. Each assay was performed in triplicate. 2.8. Data analysis All experimental values in the figures were expressed as mean ± SEM of triplicates of three individual experiments, except Western blot studies. Statistical analysis was performed using ANOVA (two-factor with replications) followed by Duncan’s multiple comparison (Duncan, 1995). A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Effect of cantharidin and norcantharidin on basal P4 secretion by primary caprine luteal cells To determine the short-term (4 h) and long-term (24 h) effects of cantharidin and norcantharidin on basal P4 secretion, primary caprine luteal cells were cultured with various doses (0.1, 1.0, and 10 g ml−1 ) of the toxins. In this study, the basal P4 secretion was dose-dependently reduced by cantharidin in both 4 and 24 h (Fig. 2A). Norcantharidin reduced basal P4 secretion after 24 h, but the effect was not dose-dependent (Fig. 2B). 3.2. Effect of cantharidin and norcantharidin on oLH- and 8-Br-cAMP-regulated P4 secretion by primary caprine luteal cells To determine the influence of cantharidin and norcantharidin on oLH- and cAMP-stimulated P4 production by primary caprine luteal cells, the cells were incubated with or without cantharidin or norcantharidin (0.1, 1.0, or 10 g ml−1 ), along with or without oLH (100 ng ml−1 ) or 8-Br-cAMP (100 M). The cAMP analog activates PKA directly and induces maximal P4 production. Under oLH and 8-Br-cAMP-stimulated conditions, both cantharidin (Fig. 2C) and norcantharidin (Fig. 2D) produced dose-dependent inhibition of P4 production after 24 h of incubation. 3.3. Effect of cantharidin and norcantharidin on basal; and 22R-OHC- and pregnenolone-supported P4 secretion by tsCLC-D cells We next tested the effects of cantharidin and norcantharidin on basal level; and 22R-OHC- and pregnenolone-supported P4 production in tsCLC-D cells. Cells were treated for 24 h with cantharidin and norcantharidin (0.01, 0.1, 1.0, or 10 g ml−1 ) alone or in the
40
N.-F. Twu et al. / Experimental and Toxicologic Pathology 64 (2012) 37–44
Fig. 2. Effects of cantharidin and norcantharidin on basal, oLH-, and cAMP-stimulated P4 synthesis. Primary caprine luteal cells were cultured in serum-free medium for 24 h with increasing concentrations (0.1, 1, and 10 g ml−1 ) of cantharidin or norcantharidin in the absence (A and B) or presence (C and D) of oLH (100 ng ml−1 ) or (cAMP 100 M). The media was collected after 4 and 24 h, and P4 concentrations were determined by EIA as described in Section 2. All data are the mean ± SEM of triplicates of three individual experiments; each experiment was performed triplicate. Bars with different letters are significantly different at p < 0.05.
presence or absence of 1 g ml−1 22R-OHC or pregnenolone. 22ROHC bypasses the action of StAR protein and supports the P4 synthesis. A dose-dependent inhibited basal level P4 production in tsCLC-D cells by cantharidin and norcantharidin are shown in Fig. 3. As shown in Fig. 4B, norcantharidin barely observed inhibition of 22R-OHC-supported P4 production, but not cantharidin (Fig. 4A) and both were shown their maximal inhibition evident at 10 g ml−1 doses. Both these inhibitors did not interfere with steroidogenic enzymes directly since they did not prevent P4 production by lueal cells, which were supplied with 22R-OHC as a substrate. Cantharidin and norcantharidin also dose-dependently inhibited pregnenolone-supported P4 production in tsCLC-D cells (Fig. 4C and D). The maximal inhibitory effects of both compounds were found at 10 g ml−1 dose. 3.4. Effects of cantharidin and norcantharidin on StAR, P450scc, and 3ˇ-HSD expression As determined by Western blot analysis (Fig. 5), cantharidin produced dose-dependent (1 and 10 g ml−1 ) inhibition of StAR protein, P450scc enzyme, and 3-HSD enzyme expression. While dose-dependent inhibition of StAR protein and P450scc enzyme expression was seen with norcantharidin (1 and 10 g ml−1 ), but
Fig. 3. Effects of cantharidin and norcantharidin on basal P4 secretion in tsCLC-D cells. The tsCLC-D cells were cultured in serum-free medium with increasing concentrations (0.01, 0.1, 1, and 10 g ml−1 ) of cantharidin or norcantharidin. The media was collected after 24 h, and P4 concentrations were determined by EIA. All data are the mean ± SEM of triplicates of three individual experiments; each experiment was performed triplicate. Bars with different letters are significantly different at p < 0.05.
N.-F. Twu et al. / Experimental and Toxicologic Pathology 64 (2012) 37–44
41
Fig. 4. Effects of cantharidin and norcantharidin on 22R-OHC- and pregnenolone-supported P4 synthesis in tsCLC-D cells. Cells were cultured in serum-free medium for 24 h with increasing concentrations (0.01, 0.1, 1, and 10 g ml−1 ) of cantharidin or norcantharidin and co-incubated with 22R-OHC (1 g ml−1 ; A and B) or pregnenolone (1 g ml−1 ; C and D). The media was collected after 24 h, and P4 concentrations were determined. All data are the mean ± SEM of triplicates of three individual experiments; each experiment was performed in triplicate. Bars with different letters are significantly different at p < 0.05.
the inhibition of 3-HSD enzyme expression was only observed in the lower norcantharidin dose. After coincubation with 22R-OHC (1 g ml−1 ), P450scc enzyme expression was inhibited by both the 1 and 10 g ml−1 concentrations of cantharidin, but only by the higher concentration of norcantharidin (Fig. 6). After coincubation with pregnenolone (1 g ml−1 ), inhibition of 3-HSD enzyme expression was observed in response to 10 g ml−1 concentration of cantharidin, whereas norcantharidin was ineffective at both doses. 3.5. Effect of cantharidin and norcantharidin on tsCLC-D cellular proliferation and viability In order to explore whether cantharidin and norcantharidin effects tsCLC-D cell population with two fundamentally different parameters such as proliferation and viability, we performed 3 Hthymidine assay and MTT assay. The 3 H-thymidine assay detects an increase in the proportion of cells in S phase within the target population, whereas the MTT assay detects an increase in total cell number measured by detection of cellular metabolic activity. First, the 3 H-thymidine uptake assay was subjected to determine whether the inhibition of tsCLC-D cell population reflects a decrease in the rate of DNA synthesis. As depicted in Fig. 7A, both cantharidin and norcantharidin produced significant (p < 0.05), rapid (24 h) dose-dependent inhibition of mitogen-induced cell proliferation. However, the effect of cantharidin was more pronounced than that of norcantharidin. Next we assessed the cell viability by using a metabolic assay, MTT assay. As seen in Fig. 7B, the cytotoxic effects were evident
at the highest (10 g ml−1 ) concentration of cantharidin, but not detected for norcantharidin at any concentration. In contrast, treatment with norcantharidin at any concentrations (0.1–10 g ml−1 ) had no apparent effects on cell viability. Cytotoxicity also was confirmed with morphological and ultra-structural analysis, after incubation of tsCLC-D cells for 24 h with 10 g ml−1 cantharidin and norcantharidin. Typical extracellular membrane-bound apoptotic bodies surrounded by ruptured cells, decreased intracellular volume, ruptured intercellular organelles, and condensed chromatin were detected in the cantharidin-treated cells, but not in the norcantharidin-treated or control cells (data not shown).
4. Discussion The deleterious effects of naturally occurring toxins from variety of sources on female reproductive functions are major serious growing problem for human and wild life. The presence of functionally normal corpus luteum (CL) is essential for the maintenance of gestation in mammals. The major secretory product of the CL is the C21 steroid, progesterone (P4 ). This suggests the normal levels of P4 secretion are essential for the implantation and subsequent development of feto-placental unit. Thus, the perturbation in the secretion and/or action of P4 by exogenous compounds can profoundly affect the steroidogenic capacity of the corpus luteum. In this regard, the abnormal luteal function cause failure of implantation and embryonic wastage by effecting the estrus cycle, pregnancy as well as the developing young ones (Oduma et al., 1995) or early embryonic deaths.
42
N.-F. Twu et al. / Experimental and Toxicologic Pathology 64 (2012) 37–44
Fig. 5. Effects of cantharidin and norcantharidin on expression of StAR protein, P450scc, and 3-HSD enzymes in tsCLC-D cells. The tsCLC-D cells were cultured in serum-free medium for 24 h with increasing concentrations (0, 1, and 10 g ml−1 ) of cantharidin or norcantharidin. After treatment, total cellular protein was extracted and loaded (50 g) in SDS-PAGE and immunobloted with appropriate amounts of StAR, P450scc and 3B-HSD antibodies, as described in Section 2. The blot is representative of three separate experiments with similar experimental patterns.
In the present study, we investigated the effects of cantharidin and norcantharidin on progesterone (P4 ) production and the effects of those toxins on the expression of StAR protein, P450scc enzyme, and 3-HSD enzyme by using primary caprine luteal cells and caprine luteal cell line (tsCLC-D) (Chiu et al., 2008a). We assessed the effects of cantharidin and norcantharidin on oLH- and 8-Br-cAMP-stimulated P4 production. In our results the suppressed oLH-stimulated P4 productions by cantharidin demonstrate that the inhibition may be due to partial inhibition of adenylate cyclase, resulting in the reduction of cAMP formation. Furthermore, these finding corroborates with the inhibition of StAR
Fig. 6. Effects of cantharidin and norcantharidin on 22R-OHC-supported P450scc enzyme (upper panel) and pregnenolone-induced 3-HSD enzyme (lower panel) expression in tsCLC-D cells. The tsCLC-D cells were cultured in serum-free medium for 24 h with increasing concentrations (0.1, 1, and 10 g ml−1 ) of cantharidin or norcantharidin and co-incubated with 22R-OHC or pregnenolone (1 g ml−1 ). After treatment, total cellular protein was extracted and loaded (50 g) in SDS-PAGE and immunobloted with appropriate amounts of P450scc and 3B-HSD antibodies as described in Section 2. The blot is representative of three separate experiments with similar experimental patterns.
protein expression by cantharidin and also supports the previous studies (Jones et al., 2000), thereby showed that both steroidogenesis and StAR protein activities were blocked by the inhibition of protein phosphatase 1 and 2A (PP1, PP2A). Our results of the inhibited 8-Br-cAMP-stimulated P4 production by cantharidin suggest that the blocked cAMP-mediated steroid production by preventing the expression of StAR protein and this also supports the earlier studies (Burns et al., 2000). It has been demonstrated that both cantharidin and norcantharidin are strong inhibitors of PP1 and PP2A (Liu et al., 2008). These studies implicate that the important roles of PP1/2A in stimulus-dependent StAR protein expression, and thus of steroidogenesis. However, the
Fig. 7. Effects of cantharidin and norcantharidin on tsCLC-D cell proliferation and viability. The tsCLC-D cells were exposed to cantharidin or norcantharidin (0.1, 1.0, and 10 g ml−1 ) in a regular medium for 24 h following addition of 3 H-thymidine for 24 h (A). Cell viability was measured by MTT assay (B). Each bar represents the mean ± SEM of triplicates of three individual experiments; each experiment was performed in triplicate. Bars with different letters are significantly different at p < 0.05.
N.-F. Twu et al. / Experimental and Toxicologic Pathology 64 (2012) 37–44
hormone-regulated steroidogenesis is dependent upon serine or threonine phosphorylation (Green and Orme-Johnson, 1991), but the role of phosphatases in the regulation of P4 secretion by luteal cells were unknown. In the present study, the inhibition of P4 secretion by cantharidin and norcantharidin, inhibitors of PP1/PP2A can perhaps best be explained in terms of preventing the dephosphorylation of a protein which is essential for steroidogenesis and which is thought to be involved in a phosphorylation/dephosphorylation cycle. This was also been supported by the presence and activity of PP1 and PP2A in rat luteal cells and the consequences of inhibiting PP activity in terms of P4 secretion (Abayasekara et al., 1996). More over the inhibition of PP1 and PP2A by okadaic acid enhanced FSH-mediated P4 secretion in primary granulosa cells (Gonzalez Reyes et al., 1997) and rat preovulatory follicles (Yu et al., 2001). Our results are consistent with previous studies, which suggest that the regulation of the serine/threonine phosphorylation balance is involved in luteal cell steroidogenesis. These results could also imply that cantharidin had no direct influence on the activities of steroidogenic enzymes (P450scc or 3-HSD) since cantharidin did not prevent P4 synthesis by lueal cells supplied with 22R-OHC, as a substrate. This is because 22R-OHC freely crosses the aqueous space between outer and inner mitochondrial membranes (Abayasekara et al., 1996). StAR protein promotes the transport of cholesterol from the outer mitochondrial membrane to the inner membrane, where the P450scc complex is located. Therefore, the suppression of StAR protein expression resulted in reduced steroid production. Our study was strongly supported by other previous studies have reported that organophosphate compounds (Dimethoate, Roundup, diethylumbelliferyl phosphate) inhibit steroidogenesis by blocking StAR protein expression in MA-10 cells (Walsh et al., 2000a,b; Manna et al., 2003). Comparing the results of 3 H-thymidine assay and MTT assay, the data clearly demonstrate that cantharidin and norcantharidin have inhibitory effect on DNA synthesis rather than apoptotic effects and also these results high correspondence concerning the decrease of luteal cell population through mitotic arrest. In caprine luteal cells, the inhibition of P4 production by cantharidin and norcantharidin was primarily due to the result of inhibition of steroidogenesis, providing evidence for the ability of these toxins to modulate gonadotropin-stimulated regulation of luteal function. Our results also suggest that the ingestion of cantharidin may decrease the ovarian steroidogenesis, and the decline in serum P4 levels may lead to a disruption of reproductive functions in humans, as well as other animals. 5. Conclusions We concluded that, cantharidin and norcantharidin repressed basal as well as oLH, 8-Br-cAMP, and 22R-OHC-induced P4 production in caprine luteal cells. Our evidence indicates the reduction in P4 production is mediated through the inhibition of StAR protein, P450scc enzyme, and 3-HSD enzyme expression. Interestingly, cantharidin more effectively inhibited caprine luteal cell steroidogenesis, as compared to norcantharidin. This study broadens our understanding of the possible intermediate targets influenced by cantharidin and its analog norcantharidin. In the present study, we established an in vitro cell system to investigate the effects of the closely related, naturally occurring toxins cantharidin and norcantharidin on luteal cell steroidogenesis. Both cantharidin and norcantharidin were found to inhibit the production of P4 and steroidogenic protein expression in luteal cells. In contrast to cantharidin, the inhibition of luteal cell steroidogenesis by norcantharidin is low. Steroidogenesis is regulated, at least in part, by the involvement of protein phosphorylation.
43
Acknowledgements The authors thank Dr. Guang-Sheng Wang for providing the norcantharidin. This study was supported by the research grants from the National Science Council, Republic of China (NSC93-2313-B002-113 and NSC95-2313-B-002-060-MY3). References Abayasekara DR, Ford SL, Persaud SJ, Jones PM. Role of phosphoprotein phosphatases in the corpus luteum. II. Control of progesterone secretion by isolated rat luteal cells. J Endocrinol 1996;150:213–21. Burns CJ, Gyles SL, Persaud SJ, Sugden D, Whitehouse BJ, Jones PM. Phosphoprotein phosphatases regulate steroidogenesis by influencing StAR gene transcription. Biochem Biophys Res Commun 2000;273:35–9. Chiu CH, Guo IC, Lin JH, Wu LS. Characterization of a stable steroidogenic caprine luteal cell line transformed by a temperature-sensitive simian virus 40. Chin J Physiol 2008a;51:369–75. Chiu CH, Wu LS, Jong DS. Production and application of a polyclonal peptide antiserum for universal detection of StAR protein. Chin J Physiol 2008b;51: 54–61. Chiu CH, Wei HW, Wu LS. Generation and utilization of P450 cholesterol side-chain cleavage enzyme and 3beta-hydroxysteroid dehydrogenase antibodies for universal detection. J Immunoassay Immunochem 2008c;29:152–60. Chun WB, Cheng WF, Wu LS, Yang PC. The use of plasma progesterone profiles to predict the reproductive status of anestrous gilts and sows. Theriogenology 2002;58:1165–74. ´ Ciereszko R, Opałka M, Kaminska B, Wojtczak M, Okrasa S, Dusza L. Luteotrophic action of prolactin during the early luteal phase in pigs: the involvement of protein kinases and phosphatases. Reprod Biol 2001;1:62–83. Duncan DB. Multiple range and multiple F test. Biometrics 1995;11:1–42. Gagliardino JJ, Krinks MH, Gagliardino EE. Identification of the calmodulin-regulated protein phosphatase, calcineurin, in rat pancreatic islets. Biochim Biophys Acta 1991;1091:370–3. Gonzalez Reyes J, Santana P, Gonzalez Robaina I, Cabrera Oliva J, Estevez F, Hernandez I, et al. Effect of the protein phosphatase inhibitor okadaic acid on FSH-induced granulosa cell steroidogenesis. J Endocrinol 1997;152: 131–9. Green EG, Orme-Johnson NR. Inhibition of steroidogenesis in rat adrenal cortex cells by a threonine analogue. J Steroid Biochem Mol Biol 1991;40:421–9. Guo IC, Wu LS, Lin JH, Chung BC. Differential inhibition of progesterone synthesis in bovine luteal cells by estrogens and androgens. Life Sci 2001;68:1851– 65. Harrisberg J, Deseta JC, Cohen L, Temlett J, Milne FJ. Cantharidine poisoning with neurological complications. S Afr Med J 1984;65:614–5. Iriuchijima T, Michimata T, Ogiwara T, Mizuma H, Yamada M, Murakami M, et al. Inhibitory effects of okadaic acid on thyrotropin and prolactin secretion from rat anterior pituitaries. Neuropeptides 1992;21:207–10. Jones PM, Sayed SB, Persaud SJ, Burns CJ, Gyles S, Whitehouse BJ. Cyclic AMP-induced expression of steroidogenic acute regulatory protein is dependent upon phosphoprotein phosphatase activities. J Mol Endocrinol 2000;24:233–9. Liu FY, Li Y, Peng YM, Ye K, Li J, Liu YH, et al. Norcantharidin ameliorates proteinuria, associated tubulointerstitial inflammation and fibrosis in protein overload nephropathy. Am J Nephrol 2008;28:465–77. Manna PR, Eubank DW, Lalli E, Sassone-Corsi P, Stocco DM. Transcriptional regulation of the mouse steroidogenic acute regulatory protein gene by the cAMP response-element binding protein and steroidogenic factor 1. J Mol Endocrinol 2003;30:381–97. McCluskey A, Ackland SP, Bowyer MC, Baldwin ML, Garner J, Walkom CC, et al. Cantharidin analogues: synthesis and evaluation of growth inhibition in a panel of selected tumour cell lines. Bioorg Chem 2003;31:68–79. Moed L, Shwayder TA, Chang MW. Cantharidin revisited: a blistering defense of an ancient medicine. Arch Dermatol 2001;137:1357–60. Murphy LI, Jones PM. Phospho-serine/threonine phosphatases in rat islets of Langerhans: identification and effect on insulin secretion. Mol Cell Endocrinol 1996;117:195–202. Oaks WW, Ditunno JF, Magnani T, Levy HA, Mills LC. Cantharidin poisoning. Arch Intern Med 1960;105:574–82. Oduma JA, Wango EO, Makawiti DW, Einer-Jensen N, Oduor-Okelo D. Effects of graded doses of the pesticide Heptachlor on body weight, mating success, estrous cycle, gestation length and litter size in laboratory rats. Comp Biochem Physicol Pharmacol Toxicol Endocrinol 1995;83:79–89. Polettini A, Crippa O, Ravagli A, Saragoni A. A fatal case of poisoning with cantharidin. Forensic Sci Int 1992;56:37–43. Ray AC, Post LO, Hurst JM, Edwards WC, Reagor JC. Evaluation of an analytical method for the diagnosis of cantharidin toxicosis due to ingestion of blister beetles (Epicauta lemniscata) by horses and sheep. Am J Vet Res 1980;41: 932–3. Ray AC, Tamulinas SH, Reagor JC. High pressure liquid chromatographic determination of cantharidin, using a derivatization method in specimens from animals acutely poisoned by ingestion of blister beetles, Epicauta lemniscata. Am J Vet Res 1979;40:498–504. Schmitz DG. Cantharidin toxicosis in horses. J Vet Intern Med 1989;3:208–15.
44
N.-F. Twu et al. / Experimental and Toxicologic Pathology 64 (2012) 37–44
Stocco DM. StAR protein and the regulation of steroid hormone biosynthesis. Annu Rev Physiol 2001;63:193–213. Stocco C, Telleria C, Gibori G. The molecular control of corpus luteum formation, function, and regression. Endocr Rev 2007;28:117–49. Tagwireyi D, Ball DE, Loga PJ, Moyo S. Cantharidin poisoning due to “Blister beetle” ingestion. Toxicon 2000;38:1865–9. Tosac L, Crochet S, Ferre P, Foufelle F, Tesseraud S, Dupont J. AMP-activated protein kinase activation modulates progesterone secretion in granulosa cells from hen preovulatory follicles. J Endocrinol 2006;190:85–97. Walsh LP, McCormick C, Martin C, Stocco DM. Roundup inhibits steroidogenesis by disrupting steroidogenic acute regulatory (StAR) protein expression. Environ Health Perspect 2000a;108:769–76.
Walsh LP, Webster DR, Stocco DM. Dimethoate inhibits steroidogenesis by disrupting transcription of the steroidogenic acute regulatory (StAR) gene. J Endocrinol 2000b;167:253–63. Wang GS. Medical uses of Mylabris in ancient China and recent studies. J Ethnopharmacol 1989;26:147–62. Wu LS, Chen JC, Sheu SY, Huang CC, Kuo YH, Chiu CH, et al. Isocupressic acid blocks progesterone production from bovine luteal cells. Am J Chin Med 2002;30:533–41. Yu CC, Chen WY, Li PS. Protein phosphatase inhibitor cantharidin inhibits steroidogenesis and steroidogenic acute regulatory protein expression in cultured rat preovulatory follicles. Life Sci 2001;70:57–72.