Steroid 5ß-reductase (AKR1D1): Purification and characterization.

In mammals there are two 3-oxo-4-ene steroid reductases that generate either A/B-trans or A/B cis-ring junctions in the steroid nucleus known as steroid 5α- and 5ß- reductases, respectively. There is only one steroid 5ß- reductase in each species and these are members of the aldo-keto-reductase (AKR) protein superfamily. The corresponding human enzyme is AKR1D1, and it plays an essential role in bile-acid biosynthesis. Germline mutations in AKR1D1 give rise to bile-acid deficiency. Because of its central role in steroid metabolism and need for detailed structure-function studies there is a need to purify the enzyme to homogeneity and in high yield. We report the purification of milligram amounts of crystallographic quality homogeneous recombinant protein for structure-function studies and its characterization.

The androgen receptor.

The Androgen Receptor (AR) is a ligand (androgen) activated transcription factor and a member of the nuclear receptor (NR) superfamily. It is required for male sex hormone function. AR-FL (full-length) has the domain structure of NRs, an N-terminal domain (NTD) required for transactivation, a DNA-binding domain (DBD), a nuclear localization signal (NLS) and a ligand-binding domain (LBD). Paradoxes exist in that endogenous ligands testosterone (T) and 5α-dihydrotestosterone (DHT) have differential effects on male sexual development while binding to the same receptor and transcriptional specificity is achieved even though the androgen response elements (AREs) are identical to those seen for the progesterone, glucocorticoid and mineralocorticoid receptors. A high resolution 3-dimensional structure of AR-FL by either cryo-EM or X-ray crystallography has remained elusive largely due to the intrinsic disorder of the NTD. AR function is regulated by post-translational modification leading to a large number of proteoforms. The interaction of these proteoforms in multiprotein complexes with co-activators and co-repressors driven by interdomain coupling mediates the AR transcriptional output. The AR is a drug target for selective androgen receptor modulators (SARMS) that either have anabolic or androgenic effects. Protstate cancer is treated with androgen deprivation therapy or by the use of AR antagonists that bind to the LBD. Drug resistance occurs due to adaptive AR upregulation and the appearance of splice variants that lack the LBD and become constitutively active. Bipolar T treatment and NTD-antagonists could surmount these resistance mechanisms, respectively. These recent advances in AR signaling are described.

AKR1C3 Converts Castrate and Post-Abiraterone DHEA-S into Testosterone to Stimulate Growth of Prostate Cancer Cells via 5-Androstene-3ß,17ß-Diol.

Androgen receptor signaling inhibitors (ARSI) are used to treat castration-resistant prostate cancer (CRPC) to stop a resurgence of androgen receptor (AR) signaling. Despite early success, patients on ARSIs eventually relapse, develop drug resistance, and succumb to the disease. Resistance may occur through intratumoral steroidogenesis mediated by upregulation of aldo-keto reductase family 1C member 3 (AKR1C3). Patients treated with leuprolide (castrate) and those treated with leuprolide plus abiraterone (post-Abi) harbor a reservoir of DHEA-S which could fuel testosterone (T) biosynthesis via AKR1C3 to cause a resurgence of prostate cancer cell growth. We demonstrate that concentrations of DHEA-S found in castrate and post-Abi patients are (i) converted to T in an AKR1C3-dependent manner in prostate cancer cells, and (ii) in amounts sufficient to stimulate AKR1C3-dependent cell growth. We observed this in primary and metastatic prostate cancer cell lines, CWR22PC and DuCaP, respectively. Androgen measurements were made by stable isotope dilution LC-MS/MS. We demonstrate AKR1C3 dependence using stable short hairpin RNA knockdown and pharmacologic inhibitors. We also demonstrate that free DHEA is reduced to 5-androstene-3ß,17ß-diol (5-Adiol) by AKR1C3 and that this is a major metabolite, suggesting that in our cell lines 5-Adiol is a predominant precursor of T. We have identified a mechanism of ARSI resistance common to both primary and metastatic cell lines that is dependent on the conversion of DHEA to 5-Adiol on route to T catalyzed by AKR1C3. SIGNIFICANCE: We show that reservoirs of DHEA-S that remain after ARSI treatment are converted into T in primary and metastatic prostate cancer cells in amounts sufficient to stimulate cell growth. Pharmacologic and genetic approaches demonstrate that AKR1C3 is required for these effects. Furthermore, the route to T proceeds through 5-Adiol. We propose that this is a mechanism of ARSI drug resistance.

Association of multi-criteria derived air toxics hazard score with lung cancer incidence in a major metropolitan area.

Background: Lung cancer remains a major health problem world-wide. Environmental exposure to lung cancer carcinogens can affect lung cancer incidence. We investigated the association between lung cancer incidence and an air toxics hazard score of environmental carcinogen exposures derived previously under the exposome concept. Methods: Lung cancer cases diagnosed in Philadelphia and the surrounding counties between 2008 and 2017 were identified from the Pennsylvania Cancer Registry. Age-adjusted incidence rates at the ZIP code level were calculated based on the residential address at diagnosis. The air toxics hazard score, an aggregate measure for lung cancer carcinogen exposures, was derived using the criteria of toxicity, persistence, and occurrence. Areas with high incidence or hazard score were identified. Spatial autoregressive models were fitted to evaluate the association, with and without adjusting for confounders. Stratified analysis by smoking prevalence was performed to examine potential interactions. Results: We observed significantly higher age-adjusted incidence rates in ZIP codes that had higher air toxics hazard score values after controlling for demographic variables, smoking prevalence, and proximity to major highways. Analyzes stratified by smoking prevalence suggested that exposure to environmental lung carcinogens had a larger effect on cancer incidence in locations with higher smoking prevalence. Conclusion: The positive association between the multi-criteria derived air toxics hazard score and lung cancer incidence provides the initial evidence to validate the hazard score as an aggregate measure of carcinogenic exposures in the environment. The hazard score can be used to supplement the existing risk factors in identifying high risk individuals. Communities with higher incidence/hazard score may benefit from greater awareness of lung cancer risk factors and targeted screening programs.

Aldo-Keto Reductase 1C3 Inhibitor Prodrug Improves Pharmacokinetic Profile and Demonstrates In Vivo Efficacy in a Prostate Cancer Xenograft Model.

Aldo-keto reductase 1C3 (AKR1C3) is overexpressed in castration-resistant prostate cancer where it acts to drive proliferation and aggressiveness by producing androgens. The reductive action of the enzyme leads to chemoresistance development against various clinical antineoplastics across a range of cancers. Herein, we report the continued optimization of selective AKR1C3 inhibitors and the identification of 5r, a potent AKR1C3 inhibitor (IC50 = 51 nM) with >1216-fold selectivity for AKR1C3 over closely related isoforms. Due to the cognizance of the poor pharmacokinetics associated with free carboxylic acids, a methyl ester prodrug strategy was pursued. The prodrug 4r was converted to free acid 5r in vitro in mouse plasma and in vivo. The in vivo pharmacokinetic evaluation revealed an increase in systemic exposure and increased the maximum 5r concentration compared to direct administration of the free acid. The prodrug 4r demonstrated a dose-dependent effect to reduce the tumor volume of 22Rv1 prostate cancer xenografts without observed toxicity.

Insulin-Induced AKR1C3 Induces Fatty Acid Synthase in a Model of Human PCOS Adipocytes.

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women. In PCOS, insulin resistance and hyperandrogenism could drive the increased risk for cardiometabolic disease. Aldo-keto reductase family 1 member C3 (AKR1C3) is induced by insulin in PCOS adipocytes and is the predominant enzyme for potent androgen formation causing ligand-dependent androgen receptor (AR) activation. AR induces fatty acid synthase (FASN), a central enzyme for de novo lipogenesis. To investigate how insulin signaling induces AKR1C3 to promote lipid overload through induction of FASN, we used differentiated human Simpson-Golabi-Behmel syndrome adipocytes as a model for PCOS adipocytes. Induction of AKR1C3 and FASN was shown to be dependent on phosphoinositide 3-kinase/protein kinase B/ mammalian target of rapamycin/nuclear factor-erythroid 2-related factor 2 using pharmacological and genetic manipulation. FASN induction was shown to be AKR1C3 and AR dependent. Monofunctional AKR1C3 inhibitors, which competitively inhibit AKR1C3, did not block FASN induction, whereas bifunctional inhibitors, which competitively inhibit AKR1C3 and attenuate AR signaling by increasing AR degradation and ubiquitination, did suggesting a nonenzymatic role for AKR1C3 to stabilize AR. AKR1C3 and AR interacted as seen by co-immunoprecipitation, proximity ligation assay, and co-occupancy on FASN locus using chromatin immunoprecipitation-quantitative polymerase chain reaction assays in a ligand-dependent and ligand-independent manner. In the absence of androgens, bifunctional inhibitors prevented lipid droplet formation, whereas monofunctional inhibitors did not. We propose that AKR1C3 has 2 roles in PCOS: to catalyze potent androgen formation in adipocytes promoting hyperandrogenism and to induce FASN by stabilizing AR in the absence of androgens. AKR1C3 may be a therapeutic target for bifunctional inhibitors to reduce cardiometabolic disease in PCOS women.

Germline Mutations in Steroid Metabolizing Enzymes: A Focus on Steroid Transforming Aldo-Keto Reductases.

Steroid hormones synchronize a variety of functions throughout all stages of life. Importantly, steroid hormone-transforming enzymes are ultimately responsible for the regulation of these potent signaling molecules. Germline mutations that cause dysfunction in these enzymes cause a variety of endocrine disorders. Mutations in SRD5A2, HSD17B3, and HSD3B2 genes that lead to disordered sexual development, salt wasting, and other severe disorders provide a glimpse of the impacts of mutations in steroid hormone transforming enzymes. In a departure from these established examples, this review examines disease-associated germline coding mutations in steroid-transforming members of the human aldo-keto reductase (AKR) superfamily. We consider two main categories of missense mutations: those resulting from nonsynonymous single nucleotide polymorphisms (nsSNPs) and cases resulting from familial inherited base pair substitutions. We found mutations in human AKR1C genes that disrupt androgen metabolism, which can affect male sexual development and exacerbate prostate cancer and polycystic ovary syndrome (PCOS). Others may be disease causal in the AKR1D1 gene that is responsible for bile acid deficiency. However, given the extensive roles of AKRs in steroid metabolism, we predict that with expanding publicly available data and analysis tools, there is still much to be uncovered regarding germline AKR mutations in disease.

Role of Human Aldo-Keto Reductases and Nuclear Factor Erythroid 2-Related Factor 2 in the Metabolic Activation of 1-Nitropyrene via Nitroreduction in Human Lung Cells.

1-Nitropyrene (1-NP) is a constituent of diesel exhaust and classified as a group 2A probable human carcinogen. The metabolic activation of 1-NP by nitroreduction generates electrophiles that can covalently bind DNA to form mutations to contribute to cancer causation. NADPH-dependent P450 oxidoreductase (POR), xanthine oxidase (XO), aldehyde oxidase (AOX), and NAD(P)H/quinone oxidoreductase 1 (NQO1) may catalyze 1-NP nitroreduction. We recently found that human recombinant aldo-keto reductases (AKRs) 1C1-1C3 catalyze 1-NP nitroreduction. NQO1 and AKR1C1-1C3 are genes induced by nuclear factor erythroid 2-related factor 2 (NRF2). Despite this knowledge, the relative importance of these enzymes and NRF2 to 1-NP nitroreduction is unknown. We used a combination of pharmacological and genetic approaches to assess the relative importance of these enzymes and NRF2 in the aerobic nitroreduction of 1-NP in human bronchial epithelial cells, A549 and HBEC3-KT. 1-NP nitroreduction was assessed by the measurement of 1-aminopyrene (1-AP), the six-electron reduced metabolite of 1-NP, based on its intrinsic fluorescence properties (λex and λem). We found that co-treatment of 1-NP with salicylic acid, an AKR1C1 inhibitor, or ursodeoxycholate, an AKR1C2 inhibitor, for 48 h decreased 1-AP production relative to 1-NP treatment alone (control) in both cell lines. R-Sulforaphane or 1-(2-cyano-3,12,28-trioxooleana-1,9(11)-dien-28-yl)-1H-imidazole (CDDO-Im), two NRF2 activators, each increased 1-AP production relative to control only in HBEC3-KT cells, which have inducible NRF2. Inhibitors of POR, NQO1, and XO failed to modify 1-AP production relative to control in both cell lines. Importantly, A549 wild-type cells with constitutively active NRF2 produced more 1-AP than A549 cells with heterozygous expression of NFE2L2/NRF2, which were able to produce more 1-AP than A549 cells with homozygous knockout of NFE2L2/NRF2. Together, these data show dependence of 1-NP metabolic activation on AKR1Cs and NRF2 in human lung cells. This is the second example whereby NFE2L2/NRF2 is implicated in the carcinogenicity of diesel exhaust constituents.

An End Point-Specific Framework for Read-Across Analog Selection for Human Health Effects.

Integrating computational chemistry and toxicology can improve the read-across analog approach to fill data gaps in chemical safety assessment. In read-across, structure-related parameters are compared between a target chemical with insufficient test data and one or more materials with sufficient data. Recent advances have focused on enhancing the grouping or clustering of chemicals to facilitate toxicity prediction via read-across. Analog selection ascertains relevant features, such as physical-chemical properties, toxicokinetic-related properties (bioavailability, metabolism, and degradation pathways), and toxicodynamic properties of chemicals with an emphasis on mechanisms or modes of action. However, each human health end point (genotoxicity, skin sensitization, phototoxicity, repeated dose toxicity, reproductive toxicity, and local respiratory toxicity) provides a different critical context for analog selection. Here six end point-specific, rule-based schemes are described. Each scheme creates an end point-specific workflow for filling the target material data gap by read-across. These schemes are intended to create a transparent rationale that supports the selected read-across analog(s) for the specific end point under study. This framework can systematically drive the selection of read-across analogs for each end point, thereby accelerating the safety assessment process.

Reconstitution of human adrenocortical specification and steroidogenesis using induced pluripotent stem cells.

The mechanisms leading to adrenal cortex development and steroid synthesis in humans remain poorly understood due to the paucity of model systems. Herein, we recapitulate human fetal adrenal cortex specification processes through stepwise induction of human-induced pluripotent stem cells through posterior intermediate mesoderm-like and adrenocortical progenitor-like states to ultimately generate fetal zone adrenal-cortex-like cells (FZLCs), as evidenced by histomorphological, ultrastructural, and transcriptome features and adrenocorticotropic hormone (ACTH)-independent Δ5 steroid biosynthesis. Furthermore, FZLC generation is promoted by SHH and inhibited by NOTCH, ACTIVIN, and WNT signaling, and steroid synthesis is amplified by ACTH/PKA signaling and blocked by inhibitors of Δ5 steroid synthesis enzymes. Finally, NR5A1 promotes FZLC survival and steroidogenesis. Together, these findings provide a framework for understanding and reconstituting human adrenocortical development in vitro, paving the way for cell-based therapies of adrenal insufficiency.

Role of Human Aldo-Keto Reductases in the Nitroreduction of 1-Nitropyrene and 1,8-Dinitropyrene.

1-Nitropyrene (1-NP) and 1,8-dinitropyrene (1,8-DNP) are diesel exhaust constituents and are classified by the International Agency for Research on Cancer as probable (Group 2A) or possible (Group 2B) human carcinogens. These nitroarenes undergo metabolic activation by nitroreduction to result in the formation of DNA adducts. Human aldo-keto reductases (AKRs) 1C1-1C3 catalyze the nitroreduction of 3-nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one, 3-NBA), but the extent of AKR contribution toward the nitroreduction of additional nitroarenes, including 1-NP and 1,8-DNP, is currently unknown. In the present study, we investigated the ability of human recombinant AKRs to catalyze 1-NP and 1,8-DNP nitroreduction by measuring the formation of the respective six-electron reduced amine products in discontinuous ultraviolet-reverse phase high-performance liquid chromatography enzymatic assays. We found that AKR1C1-1C3 were able to catalyze the formation of 1-aminopyrene (1-AP) and 1-amino-8-nitropyrene (1,8-ANP) in our reactions with 1-NP and 1,8-DNP, respectively. We determined kinetic parameters (Km, kcat, and kcat/Km) and found that out of the three isoforms, AKR1C1 had the highest catalytic efficiency (kcat/Km) for 1-AP formation, whereas AKR1C3 had the highest catalytic efficiency for 1,8-ANP formation. Use of ultra-performance liquid chromatography high-resolution mass spectrometry verified amine product identity and provided evidence for the formation of nitroso- and hydroxylamino-intermediates in our reactions. Our study expands the role of AKR1C1-1C3, which are expressed in human lung cells, in the metabolic activation of nitroarenes that can lead to DNA adduct formation, mutation, and carcinogenesis.

Quantification of Plasma 8-Isoprostane by High-Performance Liquid Chromatography with Tandem Mass Spectrometry in a Case-Control Study of Lung Cancer.

AIM: 8-iso-prostaglandin F2α is a biomarker of lipid peroxidation, and one of the most commonly used measures of oxidative stress. It is an established biomarker of lung cancer risk. It is commonly measured by enzyme-linked immunosorbent assay. Given its importance, we developed a stable isotope dilution UPLC-tandem mass spectrometric method for the rapid determination of 8-isoprostane in blood. METHODS: We tested the discriminatory capability of the method in 49 lung cancer patients, 55 benign lung nodule patients detected by chest X-ray, and 41 patients with chronic obstructive pulmonary disease (COPD) or asthma. RESULTS: Significant differences were found in mean 8-isoprostane levels between the three groups (p = 0.027), and post-hoc tests found higher levels in the lung cancer patients than in patients with benign nodules (p = 0.032) and COPD/asthma (p = 0.014). The receiving operating characteristic area under the curve (AUC) was 0.69 for differentiating the lung cancer group from the benign nodule group, and 0.7 for differentiating from the COPD/asthma group. CONCLUSIONS: The UPLC-MS/MS-based method is an efficient analytical tool for measuring 8-isoprostane plasma concentrations. The results suggest exploring its utility as a marker for early lung cancer screening.

Peripheral blood leukocyte mitochondrial DNA content and risk of lung cancer.

Background: Previous studies of peripheral blood leukocyte mitochondrial DNA (mtDNA) content and risk of lung cancer have yielded inconsistent results, and no studies have evaluated the association between mtDNA content and post-resection lung cancer outcomes. Methods: Using a case-control study design, we evaluated the association between mtDNA content and risk of lung cancer in 465 cases and 378 controls. We also evaluated the association between mtDNA content and survival in 189 cases with surgically resected non-small cell lung cancer (NSCLC). Relative mtDNA content was measured using a quantitative real-time polymerase chain reaction (PCR) assay in peripheral blood genomic DNA. We calculated odds ratios (ORs) and 95% confidence intervals (CIs) using multivariable logistic regression, adjusting for age, gender, race, and smoking history. Results: mtDNA content was lower in cases compared to controls, with medians of 1.26 [interquartile range (IQR), 0.98-1.70)] and 1.79 (IQR, 1.34-2.10; P<0.001), respectively. Compared to the quartile of subjects with the highest mtDNA content, there was significantly higher likelihood of lung cancer in the second lowest quartile (OR 3.44; 95% CI: 2.06-5.75) and the lowest quartile (OR 6.36; 95% CI: 3.86-10.47). In patients with resected NSCLC, there was no association between lower mtDNA content and recurrence-free survival (RFS) [hazard ratio (HR) 0.89; 95% CI: 0.47-1.66] or overall survival (OS) (HR 0.71; 95% CI: 0.35-1.46). Conclusions: Thus, our results counter previous studies and find that lower mtDNA content is associated with lung cancer risk. Our results suggest that mtDNA content could potentially serve as a risk biomarker, but is not associated with survival outcomes in NSCLC.

Nitroreduction: A Critical Metabolic Pathway for Drugs, Environmental Pollutants, and Explosives.

Nitro group containing xenobiotics include drugs, cancer chemotherapeutic agents, carcinogens (e.g., nitroarenes and aristolochic acid) and explosives. The nitro group undergoes a six-electron reduction to form sequentially the nitroso-, N-hydroxylamino- and amino-functional groups. These reactions are catalyzed by nitroreductases which, rather than being enzymes with this sole function, are enzymes hijacked for their propensity to donate electrons to the nitro group either one at a time via a radical mechanism or two at time via the equivalent of a hydride transfer. These enzymes include: NADPH-dependent flavoenzymes (NADPH: P450 oxidoreductase, NAD(P)H-quinone oxidoreductase), P450 enzymes, oxidases (aldehyde oxidase, xanthine oxidase) and aldo-keto reductases. The hydroxylamino group once formed can undergo conjugation reactions with acetate or sulfate catalyzed by N-acetyltransferases or sulfotransferases, respectively, leading to the formation of intermediates containing a good leaving group which in turn can generate a nitrenium or carbenium ion for covalent DNA adduct formation. The intermediates in the reduction sequence are also prone to oxidation and produce reactive oxygen species. As a consequence, many nitro-containing xenobiotics can be genotoxic either by forming stable covalent adducts or by oxidatively damaging DNA. This review will focus on the general chemistry of nitroreduction, the enzymes responsible, the reduction of xenobiotic substrates, the regulation of nitroreductases, the ability of nitrocompounds to form DNA adducts and act as mutagens as well as some future directions.

Automating Predictive Toxicology Using ComptoxAI.

ComptoxAI is a new data infrastructure for computational and artificial intelligence research in predictive toxicology. Here, we describe and showcase ComptoxAI's graph-structured knowledge base in the context of three real-world use-cases, demonstrating that it can rapidly answer complex questions about toxicology that are infeasible using previous technologies and data resources. These use-cases each demonstrate a tool for information retrieval from the knowledge base being used to solve a specific task: The "shortest path" module is used to identify mechanistic links between perfluorooctanoic acid (PFOA) exposure and nonalcoholic fatty liver disease; the "expand network" module identifies communities that are linked to dioxin toxicity; and the quantitative structure-activity relationship (QSAR) dataset generator predicts pregnane X receptor agonism in a set of 4,021 pesticide ingredients. The contents of ComptoxAI's source data are rigorously aggregated from a diverse array of public third-party databases, and ComptoxAI is designed as a free, public, and open-source toolkit to enable diverse classes of users including biomedical researchers, public health and regulatory officials, and the general public to predict toxicology of unknowns and modes of action.

Air pollution and lung cancer survival in Pennsylvania.

OBJECTIVE: Lung cancer is a leading cause of cancer death in the United States. Exposure to outdoor air pollution (OAP) is associated with increased lung cancer incidence, however little is known about the association of OAP and survival after diagnosis. METHODS: We investigated the effects of OAP and lung cancer survival in Pennsylvania using data from Pennsylvania Cancer Registry. The study population consisted of 252,123 patients diagnosed between 1990 and 2017. The Environmental Protection Agency's ambient air monitoring network provided information on OAP exposure of NO2, O3, PM2.5, and PM10. Mean OAP exposures were calculated by interpolating exposure concentrations from the five nearest monitors within a 50-kilometer radius of each patient's residential address from date of diagnosis to date of death or last contact. Cox proportional-hazards models were used to estimate the hazard ratios (HR) for OAP exposures for overall and lung cancer-specific survival. Statistical analyses were stratified by SEER cancer stage groupings (localized, regional, and distant) and adjusted for individual-level and area-level covariates. RESULTS: Median survival time was 0.76 [CIs: 0.75, 0.77] years for the study population and for localized, regional, and distant site diagnosis were 2.2 [CIs: 2.17, 2.23], 1.13 [CIs: 1.12, 1.15], and 0.42 [CIs: 0.41, 0.43] years, respectively. NO2 indicated the greatest HR which increased with increasing magnitude of exposure across all cancer staging groups for deaths before 2-years post-diagnosis. HRs varied by stage and magnitude of OAP exposure with greatest overall effects shown in NO2 followed by PM2.5, O3, and PM10. A subgroup analysis of patients with treatment status information (2010-2017) showed similar associations of increasing HRs with increasing exposure. CONCLUSION: These findings supported the hypotheses that OAP can influence the carcinogenic process, impairing chemotherapy treatment, and provide important public health implications since environmental factors are not often considered in prognosis of survival after diagnosis.

Conversion of Classical and 11-Oxygenated Androgens by Insulin-Induced AKR1C3 in a Model of Human PCOS Adipocytes.

Polycystic ovary syndrome (PCOS) is the most prevalent endocrinopathy in women. A common symptom of PCOS is hyperandrogenism (AE); however, the source of these androgens is uncertain. Aldo-keto reductase family 1 member C3 (AKR1C3) catalyzes the formation of testosterone (T) and 5α-dihydrotestosterone (DHT) in peripheral tissues, which activate the androgen receptor (AR). AKR1C3 is induced by insulin in adipocytes and may be central in driving the AE in PCOS. We elucidated the conversion of both classical and 11-oxygenated androgens to potent androgens in a model of PCOS adipocytes. Using high-performance liquid chromatography (HPLC) discontinuous kinetic assays to measure product formation by recombinant AKR1C3, we found that the conversion of 11-keto-Δ4-androstene-3,17-dione (11K-4AD) to 11-ketotestosterone (11K-T) and 11-keto-5α-androstane-3,17-dione (11K-5AD) to 11-keto-5α-dihydrotestosterone (11K-DHT) were superior to the formation of T and DHT. We utilized a stable isotope dilution liquid chromatography high resolution mass spectrometric (SID-LC-HRMS) assay for the quantification of both classical and 11-oxygenated androgens in differentiated Simpson-Golabi-Behmel syndrome adipocytes in which AKR1C3 was induced by insulin. Adipocytes were treated with adrenal derived 11ß-hydroxy-Δ4-androstene-3,17-dione (11ß-OH-4AD), 11K-4AD, or Δ4-androstene-3,17-dione (4AD). The conversion of 11ß-OH-4AD and 11K-4AD to 11K-T required AKR1C3. We also found that once 11K-T is formed, it is inactivated to 11ß-hydroxy-testosterone (11ß-OH-T) by 11ß-hydroxysteroid dehydrogenase type 1 (HSD11B1). Our data reveal a unique role for HSD11B1 in protecting the AR from AE. We conclude that the 11-oxygenated androgens formed in adipocytes may contribute to the hyperandrogenic profile of PCOS women and that AKR1C3 is a potential therapeutic target to mitigate the AE of PCOS.

Identification of spatio-temporal clusters of lung cancer cases in Pennsylvania, USA: 2010-2017.

BACKGROUND: It is known that geographic location plays a role in developing lung cancer. The objectives of this study were to examine spatio-temporal patterns of lung cancer incidence in Pennsylvania, to identify geographic clusters of high incidence, and to compare demographic characteristics and general physical and mental health characteristics in those areas. METHOD: We geocoded the residential addresses at the time of diagnosis for lung cancer cases in the Pennsylvania Cancer Registry diagnosed between 2010 and 2017. Relative risks over the expected case counts at the census tract level were estimated using a log-linear Poisson model that allowed for spatial and temporal effects. Spatio-temporal clusters with high incidence were identified using scan statistics. Demographics obtained from the 2011-2015 American Community Survey and health variables obtained from 2020 CDC PLACES database were compared between census tracts that were part of clusters versus those that were not. RESULTS: Overall, the age-adjusted incidence rates and the relative risk of lung cancer decreased from 2010 to 2017 with no statistically significant space and time interaction. The analyses detected 5 statistically significant clusters over the 8-year study period. Cluster 1, the most likely cluster, was in southeastern PA including Delaware, Montgomery, and Philadelphia Counties from 2010 to 2013 (log likelihood ratio = 136.6); Cluster 2, the cluster with the largest area was in southwestern PA in the same period including Allegheny, Fayette, Greene, Washington, and Westmoreland Counties (log likelihood ratio = 78.6). Cluster 3 was in Mifflin County from 2014 to 2016 (log likelihood ratio = 25.3), Cluster 4 was in Luzerne County from 2013 to 2016 (log likelihood ratio = 18.1), and Cluster 5 was in Dauphin, Cumberland, and York Counties limited to 2010 to 2012 (log likelihood ratio = 17.9). Census tracts that were part of the high incidence clusters tended to be densely populated, had higher percentages of African American and residents that live below poverty line, and had poorer mental health and physical health when compared to the non-clusters (all p < 0.001). CONCLUSIONS: These high incidence areas for lung cancer warrant further monitoring for other individual and environmental risk factors and screening efforts so lung cancer cases can be identified early and more efficiently.

Characterization of the major single nucleotide polymorphic variants of aldo-keto reductase 1C3 (type 5 17ß-hydroxysteroid dehydrogenase).

Aldo-keto reductase (AKR) 1C3, also known as type 5 17ß-hydroxysteroid dehydrogenase and prostaglandin F synthase, is a member of the AKR superfamily that reduces aldehydes and ketones to primary and secondary alcohols. It plays an essential role in the peripheral formation of androgens and is implicated in several steroid hormone dependent diseases including prostate cancer, breast cancer, and polycystic ovary syndrome (PCOS). AKR1C3 has 14 nonsynonymous single nucleotide polymorphisms (nsSNPs) with different global frequencies and ethnic distributions. Association studies support their role in disease, but a detailed functional genomic analysis of these variants is lacking. One study examined five AKR1C3 nsSNPs for their ability to reduce exemestane, an aromatase inhibitor used to treat breast cancer, to 17ß-dihydroexemestane, and reported a 17-250-fold reduction in catalytic efficiency of H5Q, E77G, K104D, and R258C variants compared to wild type (WT). This observation provided the impetus to examine the impact of these variants on AKR1C3 function. Here, we purified AKR1C3 WT, and the top four most frequently occurring nsSNPs, H5Q, E77G, K104D, and R258C, from E. coli to expand upon their characterization and illuminate functional differences that could affect disease outcome and treatment. While we found negligible deviations in steady state kinetics, the K104D variant showed reduced thermal stability compared to WT. The presence of NAD(P)+ restored the stability of the variant. As it is unlikely that the apoenzyme will exist within the cell without cofactor bound the K104D is not expected to manifest a phenotype.