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.
•We reviewed the status of alternative methods for skin sensitization hazard.•For some of the alternative in vitro tests OECD test protocols are available.•Replace without apparent loss of worker ...health, in line with current regulation?•Worker protection in pharmaceutical manufacturing depends on human and animal data.
Workers in development and manufacturing of pharmaceuticals are at risk for occupational contact dermatitis (OCD) of irritative (ICD) or allergic (ACD) origin, due to contacts with reactive intermediates (IM) and drug substances (DS). We examined, if alternative methods could replace presently used animal tests for identification of ACD in pharmaceutical development and manufacturing, without apparent loss of worker health, in line with regulations. The status of alternative methods for regulatory toxicology for consumer products has recently been reviewed by the Organisation for Economic Co-operation and Development (OECD) and the European Commission’s Joint Research Center (JRC) for the European Chemicals Agency (ECHA). 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 level of confidence required. Some alternative methods are currently in validation. Current Globally Harmonized System (GHS) regulations on classification, labeling and packaging of substances and mixtures depend on human and animal data, whereas alternative methods may provide supportive evidence. Since the levels of workplace skin exposure to DS and IM in manufacturing of pharmaceuticals are usually not known, it is not possible to conduct quantitative risk assessments based on threshold calculations for contact sensitizers.
Contract Development and Manufacturing Organizations (CDMOs) that manufacture large, diverse portfolio of chemical and pharmaceutical substances require pragmatic risk‐based decisions with respect to ...the safe carry‐over between different chemical entities, as well as for worker protection. Additionally, CDMOs may not have access to primary study data, or data are generally lacking for a specific substance. While pharmaceuticals require the establishment of health‐based exposure limits (HBELs) (e.g., occupational exposure limits OELs and permitted daily exposure PDE limits), the limits for nonhazardous substances could be set in a protective and pragmatic way by using default values, when internally required. Because there is no aligned definition provided by authorities, nor agreed default values for nonhazardous substances, we provide a decision tree in order to help qualified experts (such as qualified toxicologists) to identify the group of nonhazardous substances and to assign default HBEL values for specific routes of exposure. The nonhazardous substances discussed within this publication are part of the following subgroups: (I) inactive pharmaceutical ingredients, (II) pharmaceutical excipients or cosmetic ingredients, (III) substances Generally Recognized as Safe (GRAS), and (IV) food ingredients, additives, and contact materials. The proposed default limit values are 1 mg/m3 for the OEL and 50 mg/day for the PDE oral and intravenous (IV) route.
Contract Development and Manufacturing Organizations (CDMOs) work with a large, diverse portfolio of pharmaceutical substances, thus, often need a pragmatic approach to apply risk‐based decisions for patient and worker safety during manufacturing in multipurpose facilities. This work provides guidance on identifying and setting default health‐based exposure limits (HBELs) for defined nonhazardous substances in pharmaceutical manufacturing in line with regulatory guidelines.
Pharmaceutical intermediates (IM) are used in the synthesis of active pharmaceutical ingredients. They are not intended for human administration, yet employees may be exposed to IM during the ...manufacturing process. In the context of occupational health, hazard assessment of IM is needed to identify potential intrinsic hazards which could cause unwanted adverse effects. In particular, a carcinogenic potential influences the protection strategy in the workplace. DNA reactive substances 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 the limitation of DNA reactive impurities. In this study we investigate the possibility to apply (quantitative) structure–activity-relationships ((Q)SARs) during hazard identification to reduce the number of Ames tests needed for a hazard assessment of IM while maintaining high standards of protection of 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 for mutagenicity 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 additional or new information for the purpose of hazard assessment could be reduced.
•Combined in silico approach for mutagenicity prediction to ensure worker safety.•High negative predictive value of 97% achieved.•Number of Ames tests for intermediates in production reduced.•Decision tree to derive an exposure limit for intermediates presented.
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.
•DHRS7 Exhibits 3α/20β-HSD activity.•DHRS7 has low activity to convert cortisone to 20β-dihydrocortisone.•DHRS7 converts 5α-dihydrotestosterone to 3α-androstanediol.•DHRS7 and AKR1C2 similarly ...suppress androgen receptor transcriptional activity.•DHRS7 is an endoplasmic reticulum membrane protein facing the cytoplasmic compartment.
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.
In order to assess the occupational health hazard of intermediates (IM), hazard data were traditionally generated through experimental animal testing, which are more and more replaced by alternative ...methods considering animal welfare and costs. Indeed, there are a number of other ways to assess the health hazards: comparing substances with similar structures, grouping them together into logical categories, doing specialized computer modelling, using weight of evidence approach (WoE) and integrated testing strategy (ITS). We have evaluated the current state of scientific knowledge from the literature and from in silico evaluation tools. We have proposed new ITS for Novartis Pharma own pharmaceutical IM for occupational health purposes which defines the tiered approach in performing in silico, in vitro and in vivo studies which support risk management to prevent workplace diseases that could be triggered by IM and reduces the number of in vivo tests required.
Immunofluorescence of pancreatic tissue suggested that the Naâº-dependent Glu transporter EAAT1 (Slc1a3) localizes proximal to the apical membrane of acinar cells and may be involved in the secretion ...of Glu.
