WILEY InterScience® DISCOVER SOMETHING GREAT
Steroidogenic gene expression in H295R cells and the human adrenal gland: adrenotoxic effects of lindane in vitro
Agneta Oskarsson,1,2 Erik Ullerås,2* Kathryn E. Plant,1 Joy P. Hinson3 and Peter S. Goldfarb1
1 School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
2 Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden
3 Centre for Endocrinology, WHRI, Barts and The London, Queen Mary School of Medicine and Dentistry, University of London, Charterhouse Square, London EC1M 6BQ, UK
Received 21 April 2006; Revised 9 June 2006; Accepted 9 June 2006
ABSTRACT: The focus on the refinement, reduction and replacement of animal use in toxicity testing requires the de- velopment of cell-based systems that mimic the effects of xenobiotics in human tissues. The human adrenocortical carci- noma cell line, H295R, has been proposed as a model for studies on adrenal steroidogenesis and its disruption. In this study, expression profiles for nine adrenal steroidogenic genes were characterized in H295R cells using real-time RT-PCR. Treatment with forskolin increased cortisol secretion and stimulated transcription of all the steroidogenic genes except SULT2A1. The transcript profile from H295R cells in the presence and absence of forskolin was compared with the tran- script profile from human adrenal glands. The gene expression pattern observed in the forskolin-treated H295R cells was more similar to that in the human adrenal gland, than the expression pattern in untreated cells. To examine H295R cells as a possible in vitro system for the assessment of adrenal disruption using molecular endpoints, the insecticide lindane (y-hexachlorocyclohexane) was used. In vivo, lindane has been shown to inhibit testicular, ovarian and adrenal steroidogenesis. It was demonstrated that lindane reduced cortisol secretion, downregulated the expression of a subset of the genes encoding steroidogenic enzymes and repressed transcriptional activation of the steroidogenic acute regulatory protein (StAR) gene promoter. Thus the H295R cell line provides a good in vitro system for the analysis of the human adrenal steroidogenic pathway at the level of hormone production and gene expression. This in vitro test can be used for the rapid detection of adrenal endocrine disruption and as a tool for mechanistic studies. Copyright @ 2006 John Wiley & Sons, Ltd.
KEY WORDS: adrenal; steroid; CYP enzyme; mRNA expression; forskolin; lindane; hexachlorocyclohexane; alternative methods
Introduction
The adrenal cortex is a primary steroidogenic site where mineralocorticoids, glucocorticoids and adrenal androgens are synthesized in morphologically and biochemically distinct zones. The production of steroid hormones changes during normal development (Rehman et al., 2003), during pathological conditions and following exposure to adrenotoxicants (Harvey and Everett, 2003; Hilscherova et al., 2004; Zhang et al., 2005). The development of in vitro screening systems is required in
order to refine, reduce and replace animal use in toxicity testing. Well-characterized experimental models are needed to understand the molecular mechanisms behind these changes (Harvey and Everett, 2006; Hinson and Raven, 2006).
A useful model for studies on basal functions and regulation of the adrenal steroidogenesis is the H295R cell line (Gazdar et al., 1990; Rainey et al., 1994; Rehman et al., 2003). The H295R adrenocortical cell line is derived from a primary human adrenocortical carci- noma. The cells are pluripotent and have the ability to produce each of the zone-specific steroid groups. The cell line can be differentiated into more ‘glomerulosa-like’ cells by pre-treatment with angiotensin II or potassium, increasing the synthesis of aldosterone (Rainey et al., 2004). Pre-treatment with forskolin or dibutyryl cyclic AMP causes differentiation into ‘zona fasciculata-like’ cells, increasing the synthesis of cortisol and androgens. Forskolin is a specific adenylate cyclase activator and acts on steroidogenesis via cAMP and the protein kinase A pathway (Laurenza et al., 1989; Rainey et al., 1993).
* Correspondence to: Erik Ullerås, Swedish University of Agricultural Sciences, Department of Biomedical Sciences and Veterinary Public Health, Division of Pathology, Pharmacology and Toxicology, Box 7028, SE-750 07 Uppsala, Sweden.
E-mail: erik.ulleras@bvf.slu.se
Contract/grant sponsor: Swedish Fund for Research without Animal Experiments.
Contract/grant sponsor: Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning.
Contract/grant sponsor: Swedish Animal Welfare Agency.
Contract/grant sponsor: University of Surrey.
StAR,
CYP11A1
CYP17A1
CYP17A1
SULT2A1
Cholesterol
Pregenolone
17a-OH-Pregenolone
DHEA
DHEAS
3ßHSD
1
3ßHSD
1
3ßHSD
1
CYP17A1
CYP17A1
Progesterone
17a-OH-progesterone
Androstenedione
CYP21A2
1
!
1
CYP21A2
CYP19A1
11-Deoxycorticosterone
Į
11-Deoxycortisol
I
Testosterone Oestrone
CYP11B1
CYP11B1
CYP11B2
CYP17A1
Aldosterone
Corticosterone
Cortisol
Forskolin treatment in H295R cells greatly increases cortisol, DHEA, aldosterone and androstenedione pro- duction (Gazdar et al., 1990).
The steroidogenic pathways are shown in Fig. 1. The first reaction is catalysed by the cholesterol side-chain cleavage enzyme (CYP11A1) and is rate-limited by the transport of cholesterol from the outer to the inner mito- chondrial membranes. This transport is regulated by the steroidogenic acute regulatory protein (StAR). CYP17A1 converts pregnenolone to 17a-hydroxypregnenolone, from which cortisol is synthesized by the enzymes 3ß-hydroxysteroid dehydrogenase type II (HSD3B2), CYP21A2 and CYP11B1. Aldosterone is synthesized from pregnenolone by the same enzymes with the action of CYP11B2 as the last step. Dehydroepiandrosterone (DHEA) is converted to DHEAS by dehydroepian- drosterone sulfotransferase (SULT2A1). Aromatase (CYP19A1) is responsible for the conversion of andro- stenedione into estrone, or testosterone into 17ß-estradiol.
Real-time RT-PCR analysis is a sensitive and specific method that allows direct quantification of mRNA. The present study used quantitative real-time RT-PCR for expression profiling of genes encoding enzymes in the steroidogenic pathway in the human adrenocortical carcinoma cell line H295R. To validate the model, the transcript profile in H295R cells was compared with that from the adult human adrenal gland.
As a model substance to test the effect of xeno- biotics on steroidogenesis in H295R cells, the organochlorine compound lindane was used. Lindane (y- hexachlorocyclohexane, yHCH) is the most acutely toxic
of the hexachlorocyclohexane isomers. It has been used widely as an insecticide in agriculture, to limit malaria mosquitos and also in the treatment of lice (EFSA, 2005). The acute toxic effects of lindane in humans include neurological symptoms such as convulsions (Jaeger et al., 1984). A number of in vivo studies have indicated that lindane also has an effect on reproduction in both male and female laboratory animals (Sircar and Lahiri, 1989). In some cases these effects have been shown to be due to effects of lindane on steroid production in reproductive organs. One report (Walsh and Stocco, 2000) studying mouse Leydig cells, pinpoints this effect to the expres- sion of the StAR gene, which encodes the protein performing the rate limiting step of steroidogenesis. In the female reproductive system, lindane has been shown to inhibit the ovarian side-chain cleavage enzyme (CYP11A1) (Sircar and Lahiri, 1990) as well as placen- tal activity of the aromatase enzyme (CYP19) (Nativelle- Serpentini et al., 2003). In addition to the effects of lindane on reproduction, a few reports indicate that adrenal steroidogenesis may also be a target for toxicity. Studies in rats indicate that administration of lindane in- hibits the activity of steroidogenic enzymes and decreases the levels of plasma glucocorticoid (Shivanandappa et al., 1982; Lahiri and Sircar, 1991). In vitro studies using the mouse adrenal tumour cell line Y1 (Zisterer et al., 1996) demonstrate that lindane reduces the production of the steroid intermediate pregnenolone. This effect is not due to a reduced activity of the side chain cleavage enzyme (CYP11A1), suggesting an involvement of the up-stream regulator StAR. Given the demonstrated effects of
lindane on animal adrenal cell steroidogenesis, but mind- ful of the substantial differences between rodent and human adrenal function in vivo, these studies were extended to H295R cells as a potential model system for analysis of human adrenal endocrine disruption.
Materials and Methods
Cell Culture
Human adrenocortical NCI-H295R cells were obtained from ATCC (Manassas, VA, USA) and cultured in D-MEM:F-12 medium, containing glutamine and 15 mmol 1-1 HEPES, supplemented with 1% ITS+ Premix (VWR international, Poole, UK), 2% Ultroser SF (BioSepra SA, Cergy-Saint-Christophe, France) and 1% penicillin/streptomycin. The medium and antibiotics were purchased from Invitrogen Life Technologies (Paisley, UK). The cells were maintained as monolayer cultures at 37 °℃ in an atmosphere of 95% air and 5% CO2.
Cell Proliferation/Toxicity Assay
H295R cells were sub-cultured in 96-well plates at a density of 2 × 104 cells/well. After 72 h the cells were dosed daily with 10 u forskolin (Sigma) dissolved in dimethyl sulfoxide, DMSO (final concentration 0.1%) or vehicle alone, for a total of 72 h. Lindane, y- hexachlorocyclohexane (Sigma) was added at the indi- cated concentrations, during the last 24 h. To assess the effect on cell proliferation/toxicity, a colorimetric cell proliferation kit (Cell proliferation kit II(XTT), Roche) was used according to the instructions of the manufac- turer. Briefly, XTT labelling mixture was added to the cells and the absorbance at 492 nm was measured after 3 h. Staurosporin was used as a positive control for inhibition of cell proliferation.
Cell Exposure and Cortisol Measurement
H295R cells of passage 20-28 were sub-cultured on to 6-well plates at a density of 5 x 105 cells/well. The cells
were dosed ± forskolin and ± lindane as above. Cortisol levels in the media were measured by radioimmunoassay, using an antibody obtained from Bioclin (Cardiff, UK) and radiolabelled cortisol from Amersham International (Amersham, UK) following the protocol provided. The sensitivity of the assay was 5 pmol ml-1.
RNA Isolation and Quantitative RT-PCR Analysis
Cells treated as described above were harvested and total RNA was extracted using the RNeasy Mini Kit (Qiagen, Crawley, UK). RNA was quantitated using a RiboGreen RNA quantitation kit (Molecular Probes); typical yields were 8-15 µg per well. 5 µg of total RNA was treated with RNase free DNase I (Promega, South- ampton, UK) at 37 ℃ for 30 min to remove any contami- nating genomic DNA, after which the enzyme was denatured at 65 ℃ for 10 min. The purity and integrity of the RNA was checked by gel electrophoresis. 1 ug of DNase-treated RNA was subjected to first-strand cDNA synthesis in a 20 ul reaction volume in the presence of 0.22 µg of random hexamer primers and 50 U of Super- script II Reverse Transcriptase (Invitrogen, Paisley, UK), according to the manufacturer’s protocol.
Human adrenal total RNA was purchased from Ambion (one 55-year-old female donor) and from Clontech (pooled from 61 male/female donors, 15-61 years of age). RNA integrity was checked, DNase treat- ment was performed and cDNA was prepared as above. Three independent cDNA preparations were made from each RNA sample.
Quantitative real-time reverse transcriptase PCR (Q- PCR) reactions were set up using FAM/TAMRA dual labelled probes together with appropriate primer sets. Primers and probes (Table 1) were designed using the Primer Express Software (Applied Biosystems, Warrington, UK) and purchased from MWG-Biotech AG. The size and specificity of the PCR products was verified by agarose gel electrophoresis. TaqMan Universal PCR Mastermix was purchased from Applied Biosystems and reactions set up according to the manufacturer’s protocol, except that the final volume was 25 ul. In each Q-PCR reaction cDNA generated from 50 ng of total RNA was amplified using the following PCR conditions: 95 ℃
| Gene | Primer (sense) | Primer (antisense) | Probe |
|---|---|---|---|
| StAR (NM_000349) | TTGCTTTATGGGCTCAAGAATG | GGAGACCCTCTGAGATTCTGCTT | CATGCGCTGGCAGTACATGTGCAC |
| CYP11A1 (NM_000781) | CTTCTTCGACCCGGAAAATTT | CCGGAAGTAGGTGATGTTCTTGT | CCCAACCCGATGGCTGAGCAA |
| CYP17A1 (NM_000102) | GCTGACTCTGGCGCACACT | CCATCCTTGAACAGGGCAAA | TCGCCAGCCTTCGATGCAGCT |
| HSD3B2 (NM_000198.1) | GCGGCTAATGGGTGGAATCTA | CCTCATTTATACTGGCAGAAAGGAAT | TGATACCTTGTACACTTGTGCGTTAAGACCCA |
| CYP21A2 (NM_000500) | TCCCAGCACTCAACCAACCT | CAGCTCAGAATTAAGCCTCAATCC | CTCCCTTCCTGACCCTCCGCTGC |
| CYP11B1 (NM_000497) | TCCCGAGGGCCTCTAGGA | GGGACAAGGTCAGCAAGATCTT | TGCTGCTTAGCCTGGCAAACCCTG |
| CYP11B2 (NM_000498) | TTGTTCAAGCAGCGAGTGTTG | GCATCCTCGGGACCTTCTC | TCCTCTGCTTCCTGAGCTGTCCCCT |
| SULT2A1 (NM_003167) | CCTCCAGCGGTGGCTACA | AATCGTCCGACATGATGATGAC | TTGAAACCCTCACACCACGCAGGA |
| CYP19A1 (NM_000103) | ACCAGCATCGTGCCTGAAG | CCAAGAGAAAAAGGCCAGTGA | CCTGCTGCCACCATGCCAGTCC |
15 min followed by 40 cycles of 95 ℃ 15 s, 60 ℃ 1 min. Q-PCR analysis was performed in an ABI7000 SDS instrument and quantitation was carried out using the ABI proprietary software against a standard curve gener- ated from human genomic DNA, using triplicates for four concentrations. Standard curves (R2 ≥ 0.98) were included in each analysis. To ensure that the samples were free from genomic DNA contamination, a control reaction lacking RT enzyme was carried out for every RNA sample. Non-template controls were also run routinely.
StAR Reporter Vector, Transfection and Determination of Secretory Alkaline Phosphatase
A fragment of the StAR gene upstream regulatory region (-922 to +57) was amplified by PCR from human genomic DNA using Platinum PFx DNA polymerase (Invitrogen) and the sense primer: CTGCCACTGGCTAGCTGTTTGACC and antisense primer GGTGGTTCTTCGTCCTTCCTGAGC. A muta- tion was introduced by the sense primer, where C was changed to the underlined T. The PCR product was digested with NheI and HindIII and subcloned into NheI- HindIII-digested, phosphatized pSEAP2-Basic vector (Clontech). The identity and orientation of the StAR promoter insert in the plasmid was verified by restriction fragment analysis and DNA sequencing.
H295R cells were cultured in 24-well plates with 2.5 × 105 cells/well in 0.5 ml medium. After 48 h, the pSEAP-StAR reporter construct, prepared by Endo-Free Plasmid maxi Protocol (Qiagen, Crawley, UK), were transfected using Fugene 6 Transfection Reagent (Roche, Indianapolis, USA), according to the manufacturer’s instructions. The amount of DNA added was 0.2 ug/well and the Fugene:DNA ratio was 8:1. Then 24 h after transfection, the medium was replaced with medium con- taining either 0.1% DMSO as a vehicle-control or 10 µM forskolin. The cells were then incubated for 48 h, when the medium was replaced with medium containing DMSO, 10 µM forskolin or 10 UM forskolin plus the indi- cated concentrations of lindane. 100 ul of medium was collected 24 h after addition of lindane and stored at -20 ℃ for analysis of secretory alkaline phosphatase (SEAP) activity by the Aurora AP Chemiluminescent Reporter Gene Assay (MP Biomedicals).
Statistical Analysis
The results were analysed by Mann-Whitney U-test and P < 0.05 was taken as the level of significance. Prin- ciple component and hierarchical cluster analysis were carried out using the Multi-Variate Statistical Package (version 3.1).
| Lindane µM | DMSO | Forskolin | ||
|---|---|---|---|---|
| mean | ± SD | mean | ± SD | |
| 0 | 1.08 | 0.10 | 1.56ª | 0.07 |
| 1 | 1.05 | 0.08 | 1.49ª | 0.06 |
| 10 | 1.22 | 0.02 | 1.50ª | 0.06 |
| 100 | 1.16 | 0.07 | 1.27b | 0.05 |
a Significantly different from DMSO with corresponding lindane concentration.
b Significantly different from forskolin control.
Results
The Effect of Forskolin and Lindane on Cell Proliferation
In order to assess the effect of chemical treatments on the proliferation of the cells, a colorimetric assay, which measures mitochondrial function, was performed. The results, shown in Table 2, indicate that the addition of forskolin significantly increased the proliferation of the H295R cells. Addition of lindane did not show any significant effect on cell proliferation except for a slight decrease, to 82% of the control, at the highest concentra- tion (100 μM).
The Effect of Forskolin on Gene Expression in H295R Cells
Expression of the nine genes in the steroidogenic path- way with and without forskolin treatment is shown in Fig. 2A. The mRNA expression of all genes was up- regulated by forskolin, except SULT2A1, which was down-regulated. The effects of forskolin were most prominent for CYP11B1 and CYP17A1, which showed 5- to 6-fold higher expression than the controls. Gene expression of SULT2A1 after forskolin treatment was 60% of the control level.
Profile of Gene Expression in Human Adrenal Glands
In order to validate the cell model, mRNA levels of steroidogenic genes were compared in the cell line and normal human adrenal gland. Two commercially avail- able samples of human adrenal total RNA were analysed in a similar way as RNA from H295R cells. A similar pattern of expression was seen for the two samples (Fig. 2B). The only significant difference was for
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CYP19A1 (P = 0.03), for which the sample from Clontech, showed a significantly higher gene expression than the sample from Ambion.
To compare the overall expression profile of the steroidogenic genes in the human adrenal gland and the H295R cell line, principle component analysis (PCA) was carried out using data obtained for all nine genes. Two major axes were identified which account for 95.8% and
3.6% of the variance between the samples respectively (Fig. 2C). The two adrenal RNA samples plot close together on axis 1, indicating that there is little difference between them. RNA extracted from H295R cells treated with vehicle alone (DMSO) was clearly separated along the first axis from the adrenal samples; however, when the cells were dosed with forskolin, there was a shift along axis 1 indicating a reduction in the differences
between the cell line and adrenal gland mRNA expres- sion levels. Hierarchical cluster analysis confirmed this, with the Pearson coefficient between the cell line and the adrenal gland increasing from 0.24 to 0.6 upon treatment with forskolin. Taken together these results are consistent with the hypothesis that forskolin treatment in H295R cells induces an expression pattern for the steroidogenic genes closer to that of adult human adrenal tissue.
The Effect of Lindane on Cortisol Secretion and Gene Expression in H295R Cells
In order to analyse the overall effect of the putative human endocrine disruptor lindane on hormone secretion, the amount of cortisol in the cell culture medium was measured. Secretion of cortisol was increased approxi- mately four times by forskolin treatment (Fig. 3), corresponding well with the increased expression of steroidogenic genes. Exposure to lindane, however, significantly decreased the forskolin-induced secretion of cortisol. At the highest lindane concentration, cortisol secretion was reduced almost to baseline levels.
After having established that the H295 cell line is a suitable model for analysis of adult adrenal steroido- genesis, the effect of lindane treatment was tested on the expression of the genes encoding the enzymes involved in the steroidogenic pathway. The cells were exposed to increasing concentrations of lindane in the presence and absence of forskolin. Figure 4 shows that exposing the cells to lindane had a significant effect on the expression of all of the genes analysed. Forskolin-induced StAR gene expression was significantly inhibited by lindane at 1 and 10 um but there was no significant effect at the highest concentration. A concentration-dependent inhibi- tion by lindane of forskolin-induced gene expression was also observed for CYP17A1, SULT2A1, HSD3B2 and CYP19A1; in some cases the effect only being significant
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The Effect of Lindane on Activation of the StAR Promoter
Since StAR-mediated cholesterol transport is the rate limiting step in steroidogenesis, the mechanism of action of lindane on StAR gene expression was examined using a reporter construct. A DNA fragment encompassing the StAR promoter region was cloned and inserted in a reporter vector encoding secreted alkaline phosphatase (SEAP). The resulting reporter plasmid was transiently transfected into H295R cells and the effect of forskolin ± lindane was studied based on the measurement of the secreted reporter protein (SEAP). In Fig. 5 it is shown that addition of forskolin activates the StAR promoter, resulting in a 6-fold induction. At higher concentrations of lindane, the forskolin-induced activation of the StAR gene promoter was significantly repressed.
Discussion
This study evaluated the H295R cell line as an in vitro model for the screening and mechanistic analysis of adrenotoxicants. The secretion of cortisol was verified as a relevant endpoint, being modulated by both forsko- lin and lindane. Forskolin treatment of the H295R cell line resulted in a 4-fold increase of cortisol secretion. There was a concomitant induction by forskolin of all but one of the genes with a key role in the adrenal steroidogenesis.
The analysis of the steroidogenic gene expression in H295R cells and human adult adrenal displayed a gener- ally good correlation. It was shown that the addition of forskolin makes the steroidogenic transcript profile in H295R cells more similar to the profile in the adult adrenal gland.
The CYP11 enzymes, A1, B1 and B2, are located in the mitochondria. It has previously been demonstrated
that the H295R cell line has a low expression of CYP11B1 (Muller-Vieira et al., 2005). Forskolin treat- ment, however, caused a 6-fold increase in the CYP11B1 mRNA level. This indicates that the cell line can be used as a model to study the effects of modulators on CYP11B1. Forskolin, which is generally considered as an inducer of glucocorticoid synthesis, also up-regulated gene expression of CYP11B2, which catalyses the last step in the aldosterone synthesis (Fig. 1). An induction of aldosterone synthesis, in addition to glucocorticoid and androgen synthesis, after forskolin treatment of H295R cells, has previously been reported (Rainey et al., 1994).
CYP17A1 catalyses the synthesis of both cortisol and androgens. It was expressed in the DMSO-treated cells and induced to a high degree by forskolin. This enzyme is not present in rodents, which is a drawback when using rodents as experimental animals in studies of ad- renal steroidogenesis. In H295R cells, the mRNA level of CYP17A1 was induced 5-fold following forskolin exposure. The expression of CYP17A1 was thus highest relative to the other genes, which was also the case in the human adrenal total RNA samples.
The midgestational human fetal adrenal secretes large amounts of DHEAS, whose synthesis is catalysed by SULT2A1. Rehman et al. (2003) reported 13-fold higher levels of SULT2A1 transcript in human fetal compared with adult adrenals. A down-regulation of SULT2A1 after forskolin treatment was found, in contrast to the other genes which were all up-regulated. The reason for this has not yet been elucidated, but it is possible that in the absence of forskolin, H295R cells display a more fetal- like phenotype as characterized by a high expression of SULT2A1. On the other hand, forskolin has been shown to increase both DHEA and DHEAS levels (Rainey et al., 1994), a result which has yet to be reconciled with our finding of SULT2A1 gene down-regulation.
CYP19 (aromatase) activity is not usually observed in the human adrenal gland or is perhaps expressed at too low a level to be detected enzymatically. However, adrenocortical adenomas are known to produce oestrogen and CYP19 mRNA has occasionally been detected in the normal adrenal (Bouraima et al., 2003). Interestingly, the Ambion RNA sample from a 55-year-old female showed a significantly lower CYP19A1 transcript level than the pooled sample from Clontech. In the H295R cells an approximate 4-fold increase was found in CYP19A1 gene expression in the presence of forskolin. CYP19 enzyme activity and gene expression has been shown to be up- regulated by cAMP (Watanabe and Nakajin, 2004). Our data further support the previous suggestion (Heneweer et al., 2004) that H295R cells should also be a good model for studies on the specific effects of xenobiotics on CYP19 activity/expression.
In order to further evaluate H295R cells as a model system the effects of the insecticide lindane were ana- lysed. Lindane has previously been shown to display
toxic effects on a variety of organs and physiological processes in experimental animals (see introduction). This study showed that lindane exposure significantly down- regulates the secretion of cortisol from H295R cells. This effect was most evident in the forskolin-induced cells.
To further examine the mechanism behind the reduc- tion in the synthesis of this essential hormone, the study next analysed whether lindane would have any effect on the level of steroidogenic gene expression in the H295R cells. For a majority of the genes analysed, the effect of lindane was restricted to the highest concentration (100 µM). At this concentration there was also a small but significant effect on the proliferation of the H295R cells. Although this effect is small, it could be taken to indicate that part of the reduction in gene expression in lindane- treated H295R cells is due to non-specific cytotoxic effects. However, the reduction in gene expression observed for CYP17A1 and HSD3B2 (which are both required for the production of cortisol) demonstrated a linear dependence across the whole range of lindane concentrations used, suggesting that the effect is not due simply to general cytotoxicity.
The rate limiting step in steroidogenesis is the StAR- mediated transport of cholesterol across the mitochondrial membrane and it has previously been shown that lindane treatment reduces the levels of StAR mRNA and protein in Leydig cells (Walsh and Stocco, 2000). In this study using human adrenal cells, it was demonstrated that lindane significantly reduced the forskolin induced mRNA level of StAR. This was observed at 1 and 10 UM concentrations, while there is no significant effect at 100 µM. It is conceivable that this may be due to the fact that the xenobiotic substance lindane affects multiple cellular targets at the highest concentration and causes counteracting effects, such as a reduced rate of transcrip- tion as well as a stabilization of mRNA. Indeed it has been shown that while PCB126 has no effect on the tran- scriptional activity of the CYP11B1 and CYP11B2 gene promoters it can cause stabilization of their mRNAs at a post-transcriptional level (Lin et al., 2006). In this model system, it was demonstrated that lindane significantly attenuates the forskolin induced activation of the StAR promoter. This underscores the importance of correlating the diverse mechanistic effects of xenobiotics with the net effect on a measured toxicological end-point of bio- logical relevance.
It should also be noted that, while there is a good cor- relation in this study between expression of the relevant steroidogenic genes and cortisol secretion, lindane may also have effects on other cellular pathways. Our prelimi- nary data (not shown) from a microarray-based analysis of the H295R transcriptome following lindane exposure has revealed further potential targets. One example is the expression of the gene for glucose 6-phosphate de- hydrogenase (G6PDH) which also appears to be down- regulated by lindane. G6PDH is the major regulator of
the pentose phosphate pathway which supplies NADPH to all the enzymatic steps of adrenal steroid biosynthesis. Further studies will address the significance of this and other potential mechanisms for the adrenotoxic effects of lindane.
A well-characterized in vitro system with a pattern of steroidogenic gene expression similar to the human ad- renal gland will be a useful screening tool for studies on environmental adrenotoxicants and on therapeutic drugs with adrenal side-effects. In particular, the need for test- ing strategies to assess chemicals with adrenal endocrine- disrupting properties has been highlighted (Harvey and Everett, 2003) and indeed the H295R cell line has previ- ously been proposed as a relevant and useful experimen- tal model for studying the effects of such xenobiotics (Heneweer et al., 2004; Zhang et al., 2005). This study has now quantified the mRNA expression levels of nine key genes in the adrenal steroidogenesis pathways and demonstrated that for these genes, exposure of the cells to forskolin produces an expression pattern more closely resembling that in the adult adrenal gland. It also analysed the effect of the pesticide lindane on adrenal cortisol secretion in vitro and partially revealed the molecular basis of this effect. The findings suggest that the H295R cell line can be used successfully to analyse the whole human adrenal steroidogenic pathway at the level of hormone production and gene expression and the modulation by adrenotoxic endocrine disruptors.
Acknowledgements-Funding for this project was provided by the Swedish Fund for Research without Animal Experiments, the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, the Swedish Animal Welfare Agency and the University of Surrey. We acknowledge helpful advice from Dr Nick Plant on the PCA and cluster analysis.
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