Stephan Steckelbroeck

Inhibition of human cytochrome P450 aromatase activity by butyltins

Organotin compounds are widely used as antifouling agents and bioaccumulate in the food chain. Tributyltin chloride (TBT) has been shown to induce imposex in female gastropods. On the basis of this observation it has been suggested that TBT acts as an endocrine disrupter inhibiting the conversion of androgens to estrogens mediated by the aromatase cytochrome P450 enzyme. However, to date, the molecular basis of TBT-induced imposex and in particular its putative inhibitory effects on human aromatase cytochrome P450 activity have not been investigated. Therefore, we examined the effects of the organotin compounds tetrabutyltin (TTBT), TBT, dibutyltin dichloride (DBT) and monobutyltin trichloride (MBT) on human placental aromatase activity. TBT was found to be a partial competitive inhibitor of aromatase activity with an IC(50) value of 6.2 microM with 0.1 microM androstenedione as substrate. TBT impaired the affinity of the aromatase to androstenedione but did not affect electron transfer from NADPH to aromatase via inhibiting the NADPH reductase. DBT acted as a partial but less potent inhibitor of human aromatase activity (65% residual activity), whereas TTBT and MBT had no effect. The residual activity of TBT-saturated aromatase was 37%. In contrast, human 3beta-HSD type I activity was only moderately inhibited by TBT (80% residual activity). Moreover, neither TTBT or DBT nor MBT inhibited the 3beta-HSD type I activity. Together, these results suggest that the environmental pollutants TBT and DBT, both present in marine organisms, textile and plastic products, may have specific impacts on the metabolism of sex hormones in humans.

Steroid sulfatase (STS) expression in the human temporal lobe: enzyme activity, mRNA expression and immunohistochemistry study.

Dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) are suggested to be important neurosteroids. We investigated steroid sulfatase (STS) in human temporal lobe biopsies in the context of possible cerebral DHEA(S) de novo biosynthesis. Formation of DHEA(S) in mature human brain tissue has not yet been studied. 17α‐Hydroxylase/C17‐20‐lyase and hydroxysteroid sulfotransferase catalyze the formation of DHEA from pregnenolone and the subsequent sulfoconjugation, respectively. Neither their mRNA nor activity were detected, indicating that DHEA(S) are not produced within the human temporal lobe. Conversely, strong activity and mRNA expression of DHEAS desulfating STS was found, twice as high in cerebral neocortex than in subcortical white matter. Cerebral STS resembled the characteristics of the known placental enzyme. Immunohistochemistry revealed STS in adult cortical neurons as well as in fetal and adult Cajal‐Retzius cells. Organic anion transporting proteins OATP‐A, ‐B, ‐D, and ‐E showed high mRNA expression levels with distinct patterns in cerebral neocortex and subcortical white matter. Although it is not clear whether they are expressed at the blood–brain barrier and facilitate an influx rather than an efflux, they might well be involved in the transport of steroid sulfates from the blood. Therefore, we hypothesize that DHEAS and/or other sulfated 3β‐hydroxysteroids might enter the human temporal lobe from the circulation where they would be readily converted via neuronal STS activity.

Tibolone Metabolism in Human Liver Is Catalyzed by 3α/3β-Hydroxysteroid Dehydrogenase Activities of the Four Isoforms of the Aldo-Keto Reductase (AKR)1C Subfamily

Tibolone [[7α,17α]-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one] is used to treat climacteric symptoms and prevent osteoporosis. It exerts tissue-selective effects via site-specific metabolism into 3α- and 3β-hydroxymetabolites and a Δ4-isomer. Recombinant human cytosolic aldo-keto reductases 1C1 and 1C2 (AKR1C1 and AKR1C2) produce 3β-hydroxytibolone, and the liver-specific AKR1C4 produces predominantly 3α-hydroxytibolone. These observations may account for the appearance of 3β-hydroxytibolone in target tissues and 3α-hydroxytibolone in the circulation. Using liver autopsy samples (which express AKR1C1-AKR1C4), tibolone was reduced via 3α- and 3β-hydroxysteroid dehydrogenase (HSD) activity. 3β-Hydroxytibolone was exclusively formed in the cytosol and was inhibited by the AKR1C2-specific inhibitor 5β-cholanic acid-3α, 7α-diol. The cytosolic formation of 3α-hydroxytibolone was inhibited by an AKR1C4-selective inhibitor, phenolphthalein. The ratio of these stereoisomers was 4:1 in favor of 3β-hydroxytibolone. In HepG2 cell cytosol and intact cells (which do not express AKR1C4), tibolone was exclusively reduced to 3β-hydroxytibolone and was blocked by the AKR1C1-AKR1C3 inhibitor flufenamic acid. In primary hepatocytes (which express AKR1C1-AKR1C4), time-dependent reduction of tibolone into 3β- and 3α-hydroxytibolone was observed again in a 4:1 ratio. 3β-HSD activity was inhibited by both 5β-cholanic acid-3α,7α-diol and flufenamic acid, implicating a role for AKR1C2 and AKR1C1. By contrast, the formation of 3α-hydroxytibolone was exclusively inhibited by phenolphthalein implicating AKR1C4 in this reaction. 3β- and 3α-Hydroxytibolone were rapidly metabolized into polar metabolites (>85%). The formation of minor amounts of tibolone was also observed followed by AKR1C-catalyzed epimerization. The low hepatic formation of 3α-hydroxytibolone suggests that AKR1C4 is not the primary source of this metabolite and instead it maybe formed by an intestinal or enterobacterial 3α-HSD.

Effects of butyltins on human 5α-reductase type 1 and type 2 activity

Butyltins are widely used biocides and accumulate in the food chain. Tributyltin is an imposex-inducing endocrine disrupter in animals. Imposex is characterized by the development of additional male sex organs on females. In a previous study, we identified tributyltin as an inhibitor of human cytochrom P450 aromatase activity. The present work focuses on the impact of butyltins on human androgen metabolism. Activation of androgens is mediated by two human 5alpha-reductase isoenzymes. 5alpha-Reductase type 1 was completely inhibited by tributyltin chloride (IC50=19.9 microM) and dibutyltin dichloride (IC50=32.9 microM), whereas 5alpha-reductase type 2 was only inhibited by tributyltin chloride (IC50=10.8 microM). Both isoenzymes were not affected by tetrabutyltin or monobutyltin indicating that at least two butyl groups bound to the positively charged Sn are required for the interaction of butyltins with the enzymes. Tributyltin inhibited 5alpha-reductase type 1 competitively whereas an irreversible inhibition was evident for the type 2 isoenzyme. In contrast to the distinct effects on 5alpha-reductases, reductive brain 17beta-hydroxysteroid dehydrogenase activity was not inhibited by any butyltin. Insufficient activation of androgens is responsible for developmental disorders of the male reproductive system such as hypospadias. At pharmacologic levels butyltins might contribute to the onset of developmental disorders of the male reproductive system. At present, however, it is unknown whether these levels are reached after acute or chronic exposure to butyltins.

Characterization of the dehydroepiandrosterone (DHEA) metabolism via oxysterol 7α-hydroxylase and 17-ketosteroid reductase activity in the human brain

Dehydroepiandrosterone and its sulphate are important factors for vitality, development and functions of the CNS. They were found to be subjects to a series of enzyme‐mediated conversions within the rodent CNS. In the present study, we were able to demonstrate for the first time that membrane‐associated dehydroepiandrosterone 7α‐hydroxylase activity occurs within the human brain. The cytochrome P450 enzyme demonstrated a sharp pH optimum between 7.5 and 8.0 and a mean KM value of 5.4 µm, corresponding with the presence of the oxysterol 7α‐hydroxylase CYP7B1. Real‐time RT–PCR analysis verified high levels of CYP7B1 mRNA expression in the human CNS. The additionally observed conversion of dehydroepiandrosterone via cytosolic 17β‐hydroxysteroid dehydrogenase activity could be ascribed to the activity of an enzyme with a broad pH optimum and an undetectably high KM value. Subsequent experiments with cerebral neocortex and subcortical white matter specimens revealed that 7α‐hydroxylase activity is significantly higher in the cerebral neocortex than in the subcortical white matter (p < 0.0005), whereas in the subcortical white matter, 17β‐hydroxysteroid dehydrogenase activity is significantly higher than in the cerebral neocortex (p < 0.0005). No sex differences were observed. In conclusion, the high levels of CYP7B1 mRNA in brain tissue as well as in a variety of other tissues in combination with the ubiquitous presence of 7α‐hydroxylase activity in the human temporal lobe led us to assume a neuroprotective function of the enzyme such as regulation of the immune response or counteracting the deleterious effects of neurotoxic glucocorticoids, rather than a distinct brain specific function such as neurostimulation or neuromodulation.

Allopregnanolone serum levels and expression of 5α-reductase and 3α-hydroxysteroid dehydrogenase isoforms in hippocampal and temporal cortex of patients with epilepsy

In the human central nervous system, progesterone is rapidly metabolised to 5α-dihydroprogesterone which subsequently is further reduced to allopregnanolone (AP). These conversions are catalysed by 5α-reductase and 3α-hydroxysteroid dehydrogenase (3α-HSD). Although different isoforms of both enzymes have been identified in the brain, our knowledge of their expression in the human brain remains limited. The aim of the present study was to investigate the mRNA expression of 5α-reductase 1 as well as 3α-HSD 1, 2, 3 and 20α-HSD in brain tissue from patients with pharmacoresistant temporal lobe epilepsy (TLE). Specimens were derived from either the hippocampus or the temporal lobe cortex and from the tumor-free approach corridor tissue of patients with brain tumors. Quantification of different mRNAs was achieved by real time PCR. In addition, we provide data on simultaneous evaluation of serum AP concentrations. We could demonstrate that 3α-HSD 1 was not expressed in the hippocampus and temporal lobe of patients with TLE. In the hippocampus and temporal lobe, the expression levels of 3α-HSD 2 were about 20% of that in liver tissue, those of 3α-HSD 3 about 7% and those of 20α-HSD about 2%, respectively. In patients with TLE, expression of 3α-HSD 2 was significantly higher in the hippocampus than in temporal lobe cortex tissue (P<0.006). AP concentrations did not correlate significantly with the mRNA expression levels of 5α-reductase 1, 3α-HSD 2 and 3 and 20α-HSD in any of the patient groups under investigation. In conclusion, the present study demonstrates mRNA expression of 5α-reductase 1 and 3α-HSD 2 and 3 and 20α-HSD in the hippocampus and temporal lobe of epileptic patients. These findings provide further molecular biological evidence for the formation and metabolism of neuroactive steroids in the human brain.

Expression of the 17β-hydroxysteroid dehydrogenase type 5 mRNA in the human brain

An enzyme-mediated metabolism of androgens and estrogens including 17beta-HSD activity in the brain of vertebrates was discovered approximately 30 years ago. Mainly 5alpha-reductase and aromatase have been studied in detail. Recently we could demonstrate reductive and oxidative 17beta-HSD activity as well as considerable mRNA expression of the 17beta-HSD types 3 and 4 in the human brain. In the present study, we report on 17beta-HSD type 5 mRNA expression in brain tissue of women and men. Data analysis did not reveal sex specific differences, but we determined a significantly higher mRNA concentration in the subcortical white matter (SC) than in the cerebral cortex (CX). Investigation of reductive 17beta-HSD in vitro activity with 2 microM androstenedione as the substrate revealed no sex specific differences. Testosterone formation was significantly higher in SC than in CX. Moreover, enzyme activity was significantly higher in brain tissue of adults compared to that of children.

Four Additional CLCN5 Exons Encode a Widely Expressed Novel Long CLC-5 Isoform but Fail to Explain Dent’s Phenotype in Patients without Mutations in the Short Variant

Background: Dent’s disease is caused by mutations in the CLCN5 gene coding for the chloride channel CLC-5. However, sequencing of CLCN5 exonic regions in some patients presenting with low-molecular-weight proteinuria and hypercalciuria – the hallmarks of Dent’s disease – failed to identify causative mutations. Aim: Given the observation that some species harbour a CLCN5 mRNA encoding an extended CLC-5 aminoterminus compared with the so far known human form, we worked on the presumption that an orthologous (longer) CLCN5 transcript is also present in humans and that our patients may have mutations herein. Methods: Extensive databank mining, reverse transcription polymerase chain reaction (RT-PCR) and automated sequencing were used in the search for novel CLCN5 transcripts. The human CLCN5 gene was investigated in 7 patients out of five families by direct automated sequencing of PCR-amplified DNA products. Results: Two new human CLCN5 transcripts expressed in kidney and various other tissues could be identified. These arise from a novel site of transcription initiation, alternative splicing and the use of four additional CLCN5 exons. If being translated, both these mRNAs would lead to an enlarged CLC-5 protein consisting of 816 amino acids by adding 70 aminoterminal residues to the so far known 746-amino-acid-long isoform. Sequence analysis of the henceforward 17 CLCN5 exons revealed no mutation in the patients with a phenotype resembling Dent’s disease. Conclusions: Despite the identification of further targets to explain Dent’s disease, the molecular defect in our patients remains to be elucidated. Hence, their phenotype may be explained by mutations that affect so far unknown regulating elements of the CLCN5 gene or another gene(s), probably encoding CLC-5 accessory protein(s).

Non-stereo-selective cytosolic human brain tissue 3-ketosteroid reductase is refractory to inhibition by AKR1C inhibitors

Cerebral 3α-hydroxysteroid dehydrogenase (3α-HSD) activity was suggested to be responsible for the local directed formation of neuroactive 5α,3α-tetrahydrosteroids (5α,3α-THSs) from 5α-dihydrosteroids. We show for the first time that within human brain tissue 5α-dihydroprogesterone and 5α-dihydrotestosterone are converted via non-stereo-selective 3-ketosteroid reductase activity to produce the respective 5α,3α-THSs and 5α,3β-THSs. Apart from this, we prove that within the human temporal lobe and limbic system cytochrome P450c17 and 3β-HSD/Δ(5-4) ketosteroid isomerase are not expressed. Thus, it appears that these brain regions are unable to conduct de novo biosynthesis of Δ(4)-3-ketosteroids from Δ(5)-3β-hydroxysteroids. Consequently, the local formation of THSs will depend on the uptake of circulating Δ(4)-3-ketosteroids such as progesterone and testosterone. 3α- and 3β-HSD activity were (i) equally enriched in the cytosol, (ii) showed equal distribution between cerebral neocortex and subcortical white matter without sex- or age-dependency, (iii) demonstrated a strong and significant positive correlation when comparing 46 different specimens and (iv) exhibited similar sensitivities to different inhibitors of enzyme activity. These findings led to the assumption that cerebral 3-ketosteroid reductase activity might be catalyzed by a single enzyme and is possibly attributed to the expression of a soluble AKR1C aldo-keto reductase. AKR1Cs are known to act as non-stereo-selective 3-ketosteroid reductases; low AKR1C mRNA expression was detected. However, the cerebral 3-ketosteroid reductase was clearly refractory to inhibition by AKR1C inhibitors indicating the expression of a currently unidentified enzyme. Its lack of stereo-selectivity is of physiological significance, since only 5α,3α-THSs enhance the effect of GABA on the GABA(A) receptor, whereas 5α,3β-THSs are antagonists.