Toward the identification of a function of the "orphan" enzyme DHRS7

The short chain dehydrogenase DHRS7 has been previously described as a possible tumor suppressor, regulated during prostate cancer progression, with the potential of being a marker of prostate cancer. However, the function of DHRS7 and substrates with good affinity to be of potential physiological meaning remains unknown leaving it still classified as an “orphan” enzyme. These observations furthered the need to identify physiologically relevant substrates and understand the mechanisms affected by DHRS7 in endogenously expressing cell lines. In this thesis, in vitro assays were performed to help to characterize the activity of DHRS7. They showed DHRS7 has 3α and 20β reductase activities on the carbonyl of steroidal substrates, and interestingly revealed conversion of the main ligand of the androgen receptor dihydrotestosterone (DHT) toward the inactive 5α-androstane3α,17β-diol (3α-Adiol). This activity was further characterized through androgen receptor (AR) transactivation activity in an overexpressing system and biochemically through kinetic enzyme turnover assays. Moreover, this activity allowed to develop a novel screening lysate assay for substrates and inhibitors identification. However, no other promising physiologically relevant substrates were revealed. In the second part, the phenotypic changes upon DHRS7 silencing were investigated in endogenous cell models by functional cancer assays, mass spectrometry and untargeted proteomics supported by cell cycle analysis, immunofluorescence, real time qPCR and western blot. These results disproved the modulation of the endogenous AR in the prostate cancer cell line LNCaP under DHRS7 depletion but supported the hypothesis of DHRS7 having a tumor suppressor role with protein changes observed for cell cycle, adhesion and migration relevant to the phenotype. Interestingly, protein changes involved in mechanisms relevant for tumor biogenesis were observed. In conclusion, the results presented in this thesis extend the knowledge about DHRS7 in vitro activity, provide the characterization of an in vitro tool to test hypothesized substrates and inhibitors and suggest further investigation toward androgen receptor independent mechanisms

Testing strategy for mutagenicity assessment for occupational exposure limit setting of pharmaceutical intermediates

Pharmaceutical intermediates (IM) are substances which are used in the synthesis of the active pharmaceutical ingredient. They are not intended for human administration, yet employees may get in contact with them during the manufacturing process. In the context of occupational health, IM are usually considered as substances with no pharmacological activity, however, hazard assessment is necessary to identify potential intrinsic hazards which would cause unwanted adverse effects. In particular, a potential carcinogenic effect influences the protection strategy at the workplace. DNA reactive substances as detected in the Ames test may, even if present at very low levels, lead to mutations and therefore, potentially cause cancer. The use of in silico methods to predict mutagenicity is increasingly acknowledged and implemented in the recently released ICH M7 guideline for limitation of DNA reactive impurities. In this paper we investigate the possibility to apply (quantitative) structure-activity-relationships ((Q)SARs) during hazard identification to reduce the number of Ames tests needed for hazard assessment of IM while maintaining high standard of protection of the employees. Ames test outcomes for 188 substances used in the pharmaceutical production were compared with their in silico predictions using two different (Q)SAR methodologies (knowledge based and statistical) complemented by expert knowledge. The results of the analysis showed that a negative prediction provides a high confidence that the IM is not mutagenic in the Ames test with the negative predictive value of 97 %. On the other hand the positive predictive value was only 57% and therefore considered too low to reliably consider positive predicted IM to be mutagenic. In order to avoid any unnecessary burden for occupational health purposes caused by falsely positive predicted IM, all positive predicted IM and those with insufficient coverage by the in silico systems are submitted to an Ames test to verify or reject the prediction. It is shown that the described in silico prediction approach ensures appropriate protection strategy of the employees. Resources for performing Ames tests which do not add value to hazard assessment could be reduced

DHRS7 (SDR34C1) - A new player in the regulation of androgen receptor function by inactivation of 5α-dihydrotestosterone?

DHRS7 (SDR34C1) has been associated with potential tumor suppressor effects in prostate cancer; however, its function remains largely unknown. Recent experiments using purified recombinant human DHRS7 suggested several potential substrates, including the steroids cortisone and Δ4-androstene-3,17-dione (androstenedione). However, the substrate and cofactor concentrations used in these experiments were very high and the physiological relevance of these observations needed to be further investigated. In the present study, recombinant human DHRS7 was expressed in intact HEK-293 cells in order to investigate whether glucocorticoids and androgens serve as substrates at sub-micromolar concentrations and at physiological cofactor concentrations. Furthermore, the membrane topology of DHRS7 was revisited using redox-sensitive green-fluorescent protein fusions in living cells. The results revealed that (1) cortisone is a substrate of DHRS7; however, it is not reduced to cortisol but to 20β-dihydrocortisone, (2) androstenedione is not a relevant substrate of DHRS7, (3) DHRS7 catalyzes the oxoreduction of 5α-dihydrotestosterone (5αDHT) to 3α-androstanediol (3αAdiol), with a suppressive effect on androgen receptor (AR) transcriptional activity, and (4) DHRS7 is anchored in the endoplasmic reticulum membrane with a cytoplasmic orientation. Together, the results show that DHRS7 is a cytoplasmic oriented enzyme exhibiting 3α/20β-hydroxysteroid dehydrogenase activity, with a possible role in the modulation of AR function. Further research needs to address the physiological relevance of DHRS7 in the inactivation of 5αDHT and AR regulation

Workshop Report Classification of Dermal Sensitizers in Pharmaceutical Manufacturing

Workers in pharmaceutical development are particularly at risk for occupational skin irritation and contact dermatitis, due to contacts with highly reactive intermediates (IM) and drug substances (DS). We examined, if alternative methods could replace presently used animal tests for hazard identification in pharmaceutical development and manufacturing, without apparent loss of worker safety and in line with regulations. The status of alternative methods for regulatory toxicology for consumer products such as cosmetics has recently been reviewed by the Organisation for Economic Co-operation and Development and the European Commission’s Joint Research Center for the European Chemicals Agency. They concluded that prediction of skin sensitization potential, extent and quality by in vitro methods, for regulatory assessments, will depend on the regulatory purpose and the level of confidence required. Some alternative methods are currently in validation. Current Globally Harmonized System regulations on classification, labelling and packaging of substances and mixtures depend on human data and animal tests, whereas alternative methods may provide supportive evidence. Since the levels of workplace skin exposure to DS and IM in pharmaceutical manufacturing and research labs are usually not known, it is not possible to conduct a quantitative risk assessments based on threshold calculations for contact sensitizers

The Potential Tumor-Suppressor DHRS7 Inversely Correlates with EGFR Expression in Prostate Cancer Cells and Tumor Samples

Prostate cancer (PCa), one of the most common malignancies in men, typically responds to initial treatment, but resistance to therapy often leads to metastases and death. The dehydrogenase/reductase 7 (DHRS7, SDR34C1) is an “orphan” enzyme without known physiological function. DHRS7 was previously found to be decreased in higher-stage PCa, and siRNA-mediated knockdown increased the aggressiveness of LNCaP cells. To further explore the role of DHRS7 in PCa, we analyzed the proteome of LNCaP cells following DHRS7 knockdown to assess potentially altered pathways. Although DHRS7 is able to inactivate 5α-dihydrotestosterone, DHRS7 knockdown did not affect androgen receptor (AR) target gene expression, and its effect on PCa cells seems to be androgen-independent. Importantly, proteome analyses revealed increased expression of epidermal growth factor receptor (EGFR), which was confirmed by RT-qPCR and Western blotting. Comparison of AR-positive LNCaP with AR-negative PC-3 and DU145 PCa cell lines revealed a negative correlation between DHRS7 and EGFR expression. Conversely, EGFR knockdown enhanced DHRS7 expression in these cells. Importantly, analysis of patient samples revealed a negative correlation between DHRS7 and EGFR expression, both at the mRNA and protein levels, and DHRS7 expression correlated positively with patient survival rates. These results suggest a protective role for DHRS7 in PCa

Exocrine pancreas glutamate secretion help to sustain enterocyte nutritional needs under protein restriction

Glutamine (Gln) is the most concentrated amino acid in blood and considered conditionally essential. Its requirement is increased during physiological stress, such as malnutrition or illness, despite its production by muscle and other organs. In the malnourished state Gln has been suggested to have a trophic effect on the exocrine pancreas and small intestine. However, the Gln transport capacity, the functional relationship of these two organs and the potential role of the Gln-glutamate (Glu) cycle are unknown. We observed that pancreatic acinar cells express lower levels of Glu than Gln transporters. Consistent with this expression pattern the rate of Glu influx into acinar cells was approximately 6-fold lower than that of Gln. During protein restriction, acinar cell glutaminase expression was increased and Gln accumulation maintained. Moreover, Glu secretion by acinar cells into pancreatic juice and thus into the lumen of the small intestine was maintained. In the intestinal lumen, Glu absorption was preserved and glutamate dehydrogenase expression was augmented, potentially providing the substrates for increasing energy production via the TCA cycle. Our findings suggest that one mechanism by which Gln exerts a positive effect on exocrine pancreas and small intestine involves the Gln metabolism in acinar cells and the secretion of Glu into the small intestine lumen. The exocrine pancreas acinar cells not only avidly accumulate Gln; but metabolize Gln to generate energy and to synthesize Glu for secretion in the pancreatic juice. Secreted Glu is suggested to play an important role during malnourishment in sustaining small intestinal homeostasis