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Ecotoxicology and Environmental Safety
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ECOTOXICOLOGY ENVIRONMENTAL SAFETY
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Organotin exposure stimulates steroidogenesis in H295R Cell via cAMP pathway
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Xueting Yana,b, Bin Hea,b,*, Lihong Liua,b, Guangbo Qua,b, Jianbo Shia,b, Chunyang Liaoª,b, Ligang Hua,c,*, Guibin Jianga,b
a State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
b University of Chinese Academy of Sciences, Beijing 100049, China
” Institute of Environment and Health, Jianghan University, Wuhan, Hubei 430056, China
ARTICLE INFO
Keywords:
Organotin compounds Steroid hormone Steroidogenesis H295R Cell Cyclic AMP
ABSTRACT
Organotin compounds (OTs) are used in a range of industrial products, such as antifouling paints, agricultural pesticides and stabilizers. Owing to potential endocrine-disrupting effects, human exposure to such compounds is a concern. Nevertheless, little is known about the adverse effect of OTs on adrenocortical function in or- ganisms. In this study, the human adrenocortical carcinoma cell (H295R) model was used to investigate effects of OTs on steroidogenesis and potential causes for such endocrine disruption was examined. H295R cells were exposed to several commonly used OTs, including triphenyltin (TPT), tributyltin (TBT), dibutyltin (DBT), and monobutyltin (MBT), and the production level of steroid hormones were quantified. TPT and TBT decreased the production levels of 17ß-estradiol, aldosterone, and cortisol, but increased that of testosterone. Furthermore, the expression levels of ten major steroidogenic genes (HMGR, StAR, CYP11A1, 3ßHSD2, CYP17, CYP19A1, CYP21, CYP11B1, CYP11B2, and 17ßHSD) were examined and both up-regulation of CYP11B2 and down-regulation of StAR, 3ßHSD2, CYP19A1, CYP21 and CYP11B1 by TPT and TBT were observed. Intracellular levels of ATP and cyclic adenosine monophosphate (cAMP) and the activity of adenylate cyclase (AC) decreased in the H295R cells treated with TPT and TBT. No obvious changes in H295R were found with the treatment of DBT and MBT. These results suggest that OTs may stimulate steroidogenesis in vitro via inhibition of cAMP signaling pathway.
1. Introduction
Organotin compounds (OTs), in particular triphenyltin (TPT) and tributyltin (TBT), have extensively been used as stabilizers and anti- fouling agents (Hoch, 2001). Marine pollution resulting from the use of OTs is of great concern due to their adverse biological effects on non- target marine lifes, such as imposex in gastropod even by low con- centration of TBT (1 ng L-1, as Sn) (Alzieu, 2000). Dibutyltin (DBT) and monobutyltin (MBT) are commonly used as stabilizers for PVC plastics and their toxicity is less than that of TBT. Although being banned from paints in 2008, OTs were found in the coastal environment and biota at concentrations ranging from undetected to hundreds of ng L-1 as Sn for water and from 100 to 1500 ng Sn g-1 wet weight for biota (Antizar- Ladislao, 2008; Jiang et al., 2001). OTs can be accumulated in marine organisms and transferred to humans through food chain (Howell and Behrends, 2010). It was reported that TBT presented in human blood at concentrations in a range of 50-400 nM (Whalen et al., 1999).
Toxicological effects of OTs both in vitro and in vivo have been documented and the imposex and masculinization of female gastropod appeared to be the main biological impact to marine organisms (Cima and Ballarin, 2012; Graceli et al., 2013; Pagliarani et al., 2013). As endocrine-disrupting chemicals (EDC), OTs induce alteration in ex- pression of hormones and activity of steroidogenic enzymes and steroid receptors span from the aquatic species to terrestrial organisms (Kopp et al., 2017; McGinnis and Crivello, 2011; Sanderson and van den Berg, 2003; Tabb and Blumberg, 2006). It has been demonstrated that TBT altered the levels of testosterone and oestradiol so that induced a po- tential masculinization in clams (Morcillo and Porte, 2000). The en- hancive production of testosterone in both female Nucella lapillus and Hinia reticulata was found for exposure to TBT (Bettin et al., 1996). The reproductive function, including sexual development, spermatogenesis, fertilization, embryonic development and larval metamorphosis, were seriously influenced by TBT in freshwater snail and ascidians (Giusti et al., 2013; Mansueto et al., 2003). TBT inhibiting intracellular
* Corresponding authors at: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
E-mail addresses: bhe@rcees.ac.cn (B. He), lghu@rcees.ac.cn (L. Hu).
cholesterol biosynthesis that resulting in abnormal sex steroid produc- tion and in turn testicular dysfunction has been reported in male Syrian hamsters (Kanimozhi et al., 2014). TBT was also found disturbing the reproductive endocrine system in cow granulosa cells through in- creasing progesterone (Schoenfelder et al., 2003). Treatment of bovine adrenal fasciculata-reticularis cells with 100 nM TBT inhibited the production of cortisol and androstenedione, but induced the accumu- lation of intermediate steroids, indicating the inhibition of the P450s activities by TBT (Yamazaki et al., 2005). OTs can act as a specific in- hibitor of aromatase enzyme that converts androgen to estrogen (Matthiessen and Gibbs, 1998). For instance, TBT suppressed 3ß-hy- droxysteroid dehydrogenase (3ßHSD) and aromatase (CYP19A1) ac- tivity at a half maximal inhibitory concentration (IC50) of micromolar level in human placental cell line JEG-3 (Cao et al., 2017). Evidences also showed that OTs elicited their endocrine disrupting effects through interactions with nuclear and steroid receptors, such as estrogen re- ceptor (ER), retinoid X receptors (RXR), and peroxisome proliferator- activated receptor (PPAR) (Brtko and Dvorak, 2015; Milton et al., 2017).
Although there seem to be studies on endocrine-disrupting effects of OTs, the underlying mechanism remains largely unknown. In vitro ex- periments were conducted in this study to examine the target biological macromolecule of OTs and related action mechanism. In practice, in vitro model is more often used for preliminary evaluation of adverse effects of toxicants and understanding the mechanism than in vivo one. H295R cell, a human adrenocortical carcinoma cell line, is suggested as a feasible model for screening the effects of chemicals on steroidogen- esis because of its ability to express the gene, enzymes and hormones involved in adrenal and gonadal steroidogenesis (Fig. S1) (Sanderson, 2006). To date, the H295R steroidogenesis assay has been used to test the toxicity of persistent organic pollutants, including brominated flame retardants, organophosphate flame retardants, polychlorinated biphenyls, bisphenols, and tris(2,3-dibromopropyl) isocyanurate (Ding et al., 2007; Feng et al., 2016; Li et al., 2016; Liu et al., 2012; Tremoen et al., 2014).
The objective of this study was to evaluate the effect of several OTs (TPT, TBT, DBT, and MBT) on steroidogenesis pathway in H295R cells. Production of four steroid hormones (testosterone, 17ß-estradiol, al- dosterone, and cortisol) and expression of ten steroidogenic genes (HMGR, StAR, CYP11A1, 3ßHSD2, CYP17, CYP19A1, CYP21, CYP11B1, CYP11B2, and 17ßHSD) were examined. Intracellular levels of cyclic adenosine monophosphate (cAMP) and ATP and activity of adenylate cyclase (AC) were measured to further elucidate the steroidogenic pathway of such chemicals.
2. Materials and methods
2.1. Chemicals
The standards of TPT, TBT, DBT, and MBT were purchased from Dr Ehrenstorfer GmbH (Augsburg, Germany). Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (DMEM/F12), phenol red-free DMEM/ F12, trypsin, and fetal bovine serum (FBS, charcoal stripped) were purchased from HyClone (Logan, USA). Insulin-transferrin-selenium-G (ITES-G) and penicillin-streptomycin were bought from Gibco (Grand Island, USA), and Nu-Serum was bought from BD Bioscience (Bedford, USA). All chemicals used for complementary DNA (cDNA) preparation were from Takara (Shiga, Japan). Sucrose was from Amresco (Shanghai, China); 2-[4-(2-Hydroxyethyl) - 1-piperazinyl]ethane- sulfonic acid (HEPES) was obtained from J & K Technology Co., Ltd. (Beijing, China); ethylenediaminetetraacetic acid (EDTA) was supplied by Sinopharm Chemical Reagent (Shanghai, China); ethanol, trizol, chloroform, benzamidine, protease inhibitor, ATP, GTP, pyruvate ki- nase, myokinase, phosphoenolpyruvate were from Sigma-Aldrich (USA). All other chemicals were analytical grade or better. Ethanol was used as solvent for making OT standard solutions and the final
concentration of solvent in the exposure media was less than 0.1% (v/ v). Ultrapure water (18.2 MQ2 cm) obtained from a Milli-Q Advanced A10 system (Millipore, USA) was used throughout the experiment.
2.2. H295R cell culture
The H295R cells were purchased from Cell Resource Center (Beijing, China). The culture medium was DMEM/F12 supplemented with 2.5% Nu-Serum, 1% penicillin-streptomycin, and 1% ITES-G. The H295R cells were cultured at 37 ℃ in 5% CO2. The culture medium was re- placed every 3 days and cells were digested with trypsin once a week for subculturing.
2.3. Cell viability assay
The H295R cells were seeded at a density of 3 x 105 cells mL-1 in a 96-well plate. The exposure medium consisted of phenol red-free DMEM/F12 medium supplemented with 1% ITES-G, 1% penicillin — streptomycin, and 2.5% FBS (charcoal stripped). Cells were allowed to attach plate for 24h before the test chemicals were added to the medium. Cytotoxicities of all chemicals were tested at a series of con- centrations of OTs (10, 50, and 100 nM) using Alamar Blue assay after 48 h of exposure.
2.4. Quantification of steroid hormones
For the measurement of hormone, cells were seeded in 12-well plates at a concentration of 1 x 106 cells mL-1 in 1 mL of cell medium per well. After 48 h of exposure to various OTs (10, 50, and 100 nM), the medium was collected to measure the hormone levels. Testosterone, 17ß-estradiol, cortisol, and aldosterone were measured using Radioimmunoassay (RIA) kits obtained from Beijing North Institute of Biological Technology (Beijing, China) according to manufacturers’ specifications.
2.5. RNA isolation and cDNA preparation
H295R cells were dispersed in 6-well plates at a density of 1 x 106 cells mL-1 in 2 mL of medium per well. The culture medium was re- placed after cell attachment, and then cells were exposed to 10, 50, and 100 nM of OTs for 48 h. After removal of the medium, cells were wa- shed thrice with cold phosphate buffered solution (PBS) and lysed by addition of 1 mL per well of Trizol reagent for total RNA isolation. Cells were dissociated by repeated pipetting for five minutes, and collected to a microcentrifuge tube. Phase separation was applied for RNA extrac- tion by adding 0.2 mL chloroform to each tube. The mixture was agi- tated with vortex and centrifuged at 12,000 g for 15 min at 4 ℃ after incubate for 5 min. For RNA precipitation, the upper aqueous phase was carefully pipetted into a 1.5 ml tube, and incubated with 0.5 mL of isopropyl alcohol for 5 min at 4 ℃. RNA pellet was obtained by cen- trifugation at 12,000 g for 10 min at 4 ℃, and then washed once with cold 75% ethanol. The supernatant was discarded after centrifugation at 7500 g for 5 min at 4 ℃, and the pellet was air dried for 10 min. Finally, the isolated RNA was redissolved in RNase-free water and stored at -80℃ until further analysis. The RNA concentration and purity was determined by measuring the ratio of absorbance at 260 nm and 280 nm with a Nanodrop-2000 spectrophotometer (Thermo Scientific, USA).
cDNA was synthesized from 2 µg of total RNA using SuperScript system (Invitrogen, USA) with 0.5 µg oligo-(dT)20 and RNase-free water to a final volume of 15 uL. RNA and primers were denatured at 70 ℃ for 5 min, and immediately cooled on ice for a few minutes. The reverse transcription was conducted by incubating a 10 uL mixture in- cluding 5 uL of 5 x RT buffer, 1 µL of M-MLV Reverse Transcriptase, 1.5 uL of dNTP, .5 uL of RNase, and 2 uL of RNase-free water at 42 ℃ for 1 h, and then terminated at 87 ℃ for 5 min. The cDNA was stored at
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- 20 ℃ until used.
2.6. Real-time polymerase chain reaction (PCR) analysis
Quantitative PCR was performed with the SYBR Green qPCR mix (Promega, USA) on a real-time PCR system (Bio-Rad, USA). Primer sequences for gene in the experiment were retrieved from literature (Table S1, ESI+). The thermal cycling was 95 ℃ for 3 min, 45 cycles at 95 ℃ for 15 s and 60 ℃ for 1 min. The 2-44Ct method was used to calculate fold changes in gene transcript expression (Livak and Schmittgen, 2001). Gene transcription levels were measured in tripli- cate for each group and each exposure was repeated for three times. The housekeeping gene (ß-actin) expression was used for normalization of the gene expression data.
2.7. Quantification of levels of ATP and cAMP and activity of AC
The assay of cellular ATP in H295R was performed using a com- mercial assay kit (ATP Determination Kit, Life Technologies, USA), based on the luciferin-luciferase reaction by following the manufac- turer’s recommendation (Yamada et al., 2014). Briefly, the cells were treated with 0, 10, 50, and 100 nM of OTs for 24 h, and then washed and lysed with 0.1% Triton X-100/PBS. Cell lysates (100 uL) were added to 100 µL of a luciferin/luciferase mixture and incubated for 30 min at room temperature. The ATP content was quantified by measuring the luminescence on a luminometer (Synergy H1 Microplate Reader, Biotek, USA).
The content of intracellular cAMP in H295R was analyzed by en- zyme-linked immunosorbent assay (ELISA) using a cAMP ELISA kit (Cayman Chemical Company, USA) (Liu et al., 2005). Briefly, H295R cells were exposed to OTs (10, 50, and 100 nM) for 24h and then collected by removal of the supernatant. The cAMP content was mea- sured in accordance with the manufacturer’s instruction.
The adenylate cyclase (AC) activity was determined by measuring the amount of cAMP produced in the reaction medium as previously
described (Wang et al., 2015). Briefly, cells were exposed to OTs as mentioned above and collected for future homogenization in buffer (20 mM HEPES pH 7.4, 5.0 mM benzamidine, 1.0 mM EDTA, 0.25 mM sucrose, and 10 µg mL-1 protease inhibitor). Cell membranes were collected by two steps of differential centrifugation (1000 g for 5 min at 4 ℃ and 40,000 g for 20 min at 4 ℃). The amount of membrane proteins isolated from H295R cells was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA). For the AC activity assay, 30 µg of membrane protein was incubated in the reaction buffer (50 mM Tris-HCl pH 7.4, 5.0 mM MgCl2, 2.5 mM phosphoenolpyruvate, 1.0 mM ATP, .5 mM EDTA, 0.1 µM GTP, .2 IU pyruvate kinase, and 0.1 unit of myokinase) at 37 ℃ for 15 min. The converted cAMP amount in membrane was determined by an ELISA kit. All data were shown as mean ± standard deviation (SD) of three independent experiments. Statistical analysis was performed with the Student’s t-test. P < 0.05 was considered as statistically significant.
3. Results and discussion
Because of deleterious effects on biota, such as hepatotoxicity, neurotoxicity, teratogenicity, immunotoxicity, and imposex, environ- mental contamination by TPT and TBT and their degradation products (DBT and MBT) is of public health concern (Graceli et al., 2013). Malformation, masculinization and hormonal imbalance in marine mammals were recognized biomarkers of the OT contamination. In this study, the expression levels of four steroid hormones and ten major genes in the H295R cell treated with OTs (TPT, TBT, DBT and MBT) were investigated. In order to examine the disrupting effect on human cells, the exposure doses of OTs used in the present study were adopted from the average total butyltin concentrations (65 nM) reported in human blood samples from Michigan, USA (Kannan et al., 1999). The viability of H295R cells was investigated after being treated with 10, 50, and 100 nM of OTs. All treated groups showed no significant de- crease in cell viability after 48 h of OT treatments (data not shown).
3.1. Effect of OTs on steroid hormones
High exposure doses of TPT and TBT decreased the expression of 17ß-estradiol, cortisol, and aldosterone in a dose-dependent manner in H295R (Fig. 1). In the 100 nM TPT treatment group, levels of produc- tion of 17ß-estradiol, cortisol, and aldosterone in the H295R cells were significantly decreased to 40%, 66% and 61%, respectively. Levels of production of testosterone in the H295R cells were elevated with ex- posure to high concentrations of TPT (50 and 100 nM). Similar pattern in the variation of hormone levels by TBT was observed (Fig. 1). Ex- posure to 100 nM TBT reduced the production levels of 17ß-estradiol, cortisol, and aldosterone in H295R to 24%, 57% and 55%, respectively, but increased the production level of testosterone to 140%. The in- crease pattern of testosterone in H295R was consistent with those ob- served in the previous studies, which showed that TBT exposure in- creased the testosterone levels in Nucella lapillus, Hinia reticulate, Hexaplex trunculus, and Bolinus brandaris (Abidli et al., 2012; Santos et al., 2005). The increase of androgen along with the decrease of es- trogen by OTs disrupted the androgen-estrogen balance in H295R, which may be corresponding with the masculinization observed in gastropod with exposure to OTs. To the contrary, DBT and MBT at the exposed concentrations (10, 50, and 100 nM) did not change the pro- duction of steroid hormones considered in the present study (Fig. 1).
3.2. Effect of OTs on steroidogenic genes
The abovementioned results indicate the significant alteration in steroidogenesis in H295R exposed to OTs, as showed by the up or down regulation of production levels of steroid hormones. Given hormone productions are directly determined by steroidogenic genes, we further investigated the expression of several major steroidogenic genes, in- cluding HMGR, StAR, CYP11A1, 3ßHSD2, CYP17, CYP19A1, CYP21, CYP11B1, CYP11B2, and 17ßHSD. Fig. 2 gives an overview of changes in the gene expression levels in the H295R cells treated with OTs. High exposure doses of TPT and TBT increased the expression of CYP11B2, but reduced the expressions of StAR, 3ßHSD2, CYP19A1, CYP21 and CYP11B1. The enzymes transcribed by these genes play key roles in steroid hormone biosynthesis in H295R.
The CYP11B2 enzyme is important in the aldosterone biosynthesis, and its modulation could influence various physiological processes regulated by cortisol and aldosterone (Gomez-Sanchez et al., 2014). Our observations partially corresponded to those found for PCB 126 and PCB 110 where a significant increase in the basal expression of CYP11B2 gene in H295R cells was observed (Li et al., 2004; Xu et al., 2006). StAR is a key gene in the steroid hormone synthesis, which enhances the metabolism of cholesterol to pregnenolone. Several pre- vious studies have shown that environmental toxicants such as lindane, o,p’-DDD, and manganese block steroidogenesis via suppression of the StAR gene expression, and our result that StAR expression was reduced by OTs is consistent with that of these studies (Lin et al., 2012; Cheng et al., 2005; Walsh and Stocco, 2000). The enzyme 3ßHSD2 is a ne- cessary enzyme in the conversion of pregnenolone to progesterone and biosynthesis of sex hormones. Among the steroidogenic genes examined in the present study, 3ßHSD2 was the most down-regulated by 100 nM TBT. Our results correlated well with a former study that the expression of 3ßHSD2 in H295R was suppressed by the sediment extracts mainly containing polyaromatic hydrocarbons and persistent chlorinated che- micals (Payne and Hales, 2004). CYP19A1, namely aromatase, is a member of the cytochrome P450 superfamily, and acts as estrogen synthetase to catalyze many reactions involved in steroidogenesis. In general, aromatase participates in the aromatization of androgens to estrogens so that affects sexual development. In our study, the result of RT-PCR indicated that CYP19A1 gene was down-regulated by exposure to high concentrations of TPT and TBT. It can be inferred that OTs inhibit the activity of aromatase, resulting increase of androgens along with decrease of estrogens. CYP21 is required for the synthesis of both aldosterone and cortisol. Our result showed that 100 nM TBT reduced the synthesis of cortisol in H295R, which might be related with the suppression in expression of CYP21. An in vitro study conducted with bovine adrenal fasciculata-reticularis cells also showed lower cortisol secretion following treatment with 1-1000 nM of TBT (Yamazaki et al., 2005). The CYP11B1 gene expression levels were decreased by OTs investigated in this study, which may directly lead to the cortisol re- duction. In addition, exposure to 50 nM of TPT decreased the expres- sion level of CYP17 in H295R (Fig. 2). The activity of CYP17 enzyme in human luteinizing granulosa cell was inhibited by 2,3,7,8-
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tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), which in turn led to a decrease in estradiol production (Moran et al., 2003).
All of the changes in the gene expression levels observed in the present study suggested that TPT and TBT perturbed the hormone synthesis and steroidogenesis in H295R. The regulation of hormone- related gene expression was more notable in the H295R cell treated with TPT and TBT than with DBT and MBT. As shown in Fig. 2, DBT and MBT did not alter the expressions of the most genes involved in ster- oidogenesis, which is in accordance with the finding that both chemi- cals barely changed the expression of hormone levels as stated above (Fig. 1).
3.3. Effect of OTs on cellular levels of ATP and cAMP and activity of AC
To further explore the mechanism of disturbance on steroidogenesis caused by OTs, we determined the levels of ATP and cAMP and activity of AC in H295R upon treatment with OTs at a series of concentrations of 0, 10, 50, and 100 nM for 24 h. ATP serves as a source of energy for cell growth and biological processes. Fig. 3A shows that the levels of ATP were significantly decreased by approximately 45% and 40% after 24 h of exposure to 100 nM TBT and TPT, respectively. Earlier study in the human induced pluripotent stem cell found that treatment with 50 nM of TBT reduced the ATP content, which is consistent with our results (Yamada et al., 2016). However, the measurement of ATP levels in H295R exposed to DBT and MBT showed no decline over the control.
The second-messenger cAMP is an important modulator in diverse cellular functions and it is synthesized from ATP by AC located on inner side of the plasma membrane. Protein kinase A (PKA), known as a cAMP-dependent protein kinase, has several functions in cell, including regulation for metabolism of glycogen, steroid, and lipid. It is
| Treatment | Concentration/nM | |||
|---|---|---|---|---|
| 0 | 10 | 50 | 100 | |
| MBT | 1.01 ± 0.20 | 1.14 ± 0.31 | 0.98 ± 0.05 | 1.09 ± 0.21 |
| DBT | 1.00 ± 0.08 | 0.93 ± 0.15 | 1.17 ± 0.26 | 0.87 ± 0.18 |
| TBT | 1.00 ± 0.11 | 0.76 ± 0.15* | 0.66 ± 0.32* | 0.47 ± 0.19* |
| TPT | 1.01 ± 0.18 | 1.16 ± 0.23 | 0.72 ± 0.12* | 0.50 ± 0.10* |
P < 0.05 (* ) indicates significant difference between the treatment and control groups.
unequivocal that the cAMP/PKA pathway involves in the steroid hor- mone biosynthesis in the human adrenal cell (Sewer and Watermen, 2001). Pesticides, including atrazine, vinclozolin, forskolin, and iso- butyl methylxanthine, have been found to disrupt the aromatase ac- tivity in steroidogenesis through increasing the intracellular cAMP level (Sanderson et al., 2002). In this study, exposure to 100 nM TBT and TPT significantly depressed cAMP level by 55% and 60%, respectively (Fig. 3B). The decrease in intracellular levels of ATP and cAMP fol- lowing exposure to TBT and DBT at 200 nM and 10 uM, respectively, has been shown in natural killer cell (Whalen and Loganathan, 2001). As shown in Table 1, the AC activity was reduced by 24% with 10 nM TBT, 34% with 50 nM TBT, and 53% with 100 nM TBT, respectively. Similarly, TPT caused as much as a 50% decrease in the AC activity after 24 h of exposure (Table 1). The intracellular levels of ATP and cAMP and activity of AC were inhibited by TBT and TPT in a dose- dependent manner. Nevertheless, no significant effect on these end- points was observed with exposure to either DBT or MBT. This may be explained by the fact that TPT and TBT are more toxic than DBT and MBT (both are metabolites of TBT). Overall, the inhibition in the levels of ATP and cAMP and the activity of AC by OTs was observed in H295R that resulted in the repression of cAMP/PKA pathway, which finally disrupted the steroidogenesis. Thus, we speculate that the up and/or down regulation of steroid hormones and the related genes induced by OTs was mediated by disrupting the cAMP signaling cascade.
4. Conclusion
In summary, this study indicates that OTs, especially TPT and TBT, perturbed steroid hormones and expression of steroidogenic genes in H295R cell. The elevation of testosterone and reduction of 17ß-estra- diol in H295R is consistent with the masculinization of gastropod re- ported in previous study. Our results suggest the mechanism of OT- induced disruption via modulation of cAMP/PKA pathway, which could interfere with the steroid hormone level and enzyme activity. These findings may contribute to elucidate the molecular mechanism involved in the toxic effects of OTs and provide the basis for assess the potential health risks of such chemicals in vivo. Further studies are needed to investigate the toxicological effects of OTs on steroidogenesis in vivo.
Acknowledgements
This study was supported by the National Nature Science Foundation of China (21277151, 21577153, 21537004, 21522706 and 21777179) and The Thousand Talents Plan for Young Professionals, China.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2018.03.028.
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