Search Results (218)

Metabolic resistance is commonly attributed to the overexpression of detoxification enzymes, whereas the metabolic systems that sustain detoxification remain less well resolved. Here, we integrated widely targeted metabolomics and transcriptomics to compare the clothianidin-resistant brown planthopper strain CLR with the susceptible strain CLS and validated candidate genes in an independent nitenpyram-resistant background. CLR and CLS exhibited distinct metabolomic profiles. Differential genes and metabolites converged on carbon metabolism, glycolysis/gluconeogenesis, the tricarboxylic acid cycle, glutathione metabolism, pentose and glucuronate interconversions, cytochrome P450-mediated xenobiotic metabolism, and ABC transporters. These changes were summarized into four interconnected modules involving glycolytic energy supply, protective sugar-derived metabolites, pyruvate–TCA–malate metabolism, and UGT-mediated glycosylation. Expression analysis identified a mitochondrial NADP-dependent isocitrate dehydrogenase gene and UDP-glucosyltransferase 2 (UGT2) as consistently upregulated in both resistant backgrounds. Silencing NADP reduced the LC₅₀ of nitenpyram and clothianidin by 1.84- and approximately 1.81-fold, respectively, whereas UGT2 silencing produced corresponding reductions of 1.89- and 1.83-fold. These findings indicate that neonicotinoid resistance in Nilaparvata lugens is supported by coordinated remodelling of central metabolism and detoxification and identify two candidate metabolic nodes for further resistance management research. Full article

Rhipicephalus microplus is a cattle ectoparasite responsible for economic losses in livestock systems. The present study aimed to evaluate the essential oil (EO) of Piper hemmendorffii and its major constituent, limonene, for their activity against R. microplus larvae. The EO of P. hemmendorffii and its major compound, limonene, were evaluated against pyrethroid-resistant R. microplus larvae using larval immersion bioassays, biochemical analyses of detoxification enzymes and acetylcholinesterase, and scanning electron microscopy (SEM). The EO showed higher larvicidal potency (LC₅₀ = 10.46 mg/mL; 95% CI: 9.86–11.09) compared to limonene (LC₅₀ = 31.62 mg/mL; 95% CI: 30.86–32.40). Piper hemmendorffii EO affected the following classes of enzymes investigated: α-Esterase (α-EST) activity increased at LC₂₅ and LC₅₀, reaching its highest level at LC₅₀, whereas β-esterase (β-EST) activity increased only at LC₂₅. Cytochrome P450 (CYP450)-associated heme content activity showed a significant increase exclusively at LC₅₀, suggesting a response of the surviving larvae to EO exposure, although this increase does not by itself demonstrate the activation of detoxification pathways. In contrast, limonene did not significantly affect the activity of the three evaluated enzymes. The inhibition of AChE by the EO suggests a possible effect on the cholinergic system, which may be associated with neurotoxic activity, whereas limonene had no significant effect. The absence of detectable ultrastructural changes by SEM suggests that treatment with either EO or limonene did not induce evident external morphological damage to the larval cuticle under the evaluated conditions. EO toxicity seems to be associated with changes in detoxification enzyme and AChE activities, suggesting the involvement of these biochemical pathways in larval response. Full article

Aflatoxin B1 (AFB1) is a highly toxic mycotoxin which can be carried over into animal products and cause deterioration of livestock productivity when fed to livestock and wildlife. This review proposes a biomarker-guided framework for improving the early assessment of AFB1 exposure and toxicological responses in animal models. Oral exposure leads to the absorption of AFB1, which is bioactivated in the liver to the reactive AFB1-exo-8,9-epoxide that causes DNA and protein adduct formation, inflammation, mitochondrial apoptosis, and other effects. Cytochrome P450 activation and glutathione-dependent detoxification are in balance in determining susceptibility species, and this balance is different for poultry, pigs, ruminants, and rodents. In addition to traditional liver enzymes and histopathology, we highlight mechanistically informative biomarkers such as metabolites of aflatoxin, DNA and albumin adduct, lipid peroxidation products, antioxidant indices, cytokines, apoptotic markers, as well as signals involved in the Nrf2/NFκB pathway. AFB1 also damages the integrity of the intestinal barrier, the maintenance of the intestinal gut microbiota, reproductive function, growth performance, and development, thus creating a gut–liver-systemic toxic cascade. Finally, an assessment of stage-targeted interventions such as aluminosilicate binders, adsorbents derived from yeast, probiotics and nano-enabled interventions is conducted as viable tools for the reduction in exposure and injury. This review offers targeted mitigation strategies for early diagnosis of aflatoxicosis in animal production systems based on a biomarker approach. Full article

Treatment-resistant mental health conditions are common in primary care and challenging for clinicians. Trial-and-error prescribing can prolong morbidity and increase adverse drug reactions (ADRs). Pharmacogenomic (PGx) testing enables individualised prescribing by identifying gene–drug interactions affecting psychotropic response. Thirty adults with treatment-resistant mental health conditions underwent PGx testing using a commercial panel (one lost to follow-up [n = 29]). Patients received PGx-guided treatment (n = 8) or standard care (n = 21). Phenotypes were assigned per CPIC and DPWG guidelines, with prescribing guided by clinical experience where guidelines were unavailable. Medication histories were reviewed for gene–drug concordance, ADRs, and treatment failures. Clinical improvement at eight weeks was defined as “marked” or “moderate” improvement and/or ADR resolution. Actionable genotypes were common, particularly CYP2D6 (27.5% poor/intermediate drug metabolising phenotype) and CYP2C19 (37.9%). Guideline-actionable gene–drug interactions occurred in 37% of patients, and eleven patients possessed actionable phenotype at multiple loci. Gene–drug interactions were identified in nine patients and guidance was fully implemented in six. Clinical benefit at 8 weeks was achieved in 6/8 patients with genotype-guided changes versus 8/21 receiving standard care. PGx-guided prescribing may support improved antidepressant response and tolerability while reducing trial-and-error prescribing for treatment-resistant patients in primary care. Full article

In drug-induced liver injury (DILI) prediction field, animal models and in vitro cell models are most commonly used. However, animal models require long experimental timelines and may exhibit species-specific differences compared with humans, whereas conventional two-dimensional (2D) cell culture models lack cell-to-cell and cell-to-extracellular matrix (ECM) interaction. Liver organoid models and liver organ-on-a-chip can better simulate the human liver microenvironment; however, the construction of liver organoids requires a long cycle and high costs, while liver organ-on-a-chip systems demand specialized equipment and professional technicians. Herein, we selected the human C3A cell line, characterized by its low cost and facile culture conditions to establish an in vitro three-dimensional (3D) liver model. Briefly, C3A cells were embedded in Matrigel and cultured for 7 days to allow model maturation. Compared with their 2D-cultured cell model, the established 3D model exhibited elevated mRNA expression levels of drug-metabolizing cytochrome P450 enzymes (CYPs). Moreover, the model displayed robust expression of key hepatic biomarkers, as well as bile duct biomarkers. To evaluate the model’s applicability for DILI prediction, we performed toxicity assessments using a panel of six well-characterized hepatotoxicants and three non-hepatotoxic compounds. Notably, the 3D C3A model achieved a sensitivity of 83.3%, specificity of 100%, and overall accuracy of 88.9%. Furthermore, treatment of this model with chlorpromazine, a well-characterized cholangiotoxic agent, resulted in suppressed expression of the bile duct biomarker cytokeratin 19 (CK19) and bile salt export pump (BSEP), accompanied by impaired bile acid transport capacity. Taken together, this study provided a simple, low-cost, easy to culture, and more readily scalable 3D hepatic model in comparison with conventional 2D primary human hepatocyte (PHHs) models and other advanced 3D liver models. Notably, the model displayed dual hepatic and biliary characteristics, supporting predictions of both DILI and drug-induced bile duct injury. It provided a promising in vitro platform for assessing drug-induced hepatobiliary toxicity, with potential to reduce reliance on animal experiments and accelerate early-stage screening of novel pharmaceutical candidates. Full article

Cytochrome P450 monooxygenases (CYPs/P450s) are versatile heme-containing enzymes involved in drug metabolism, steroidogenesis, secondary metabolite biosynthesis, and biotechnology. Although African researchers have contributed to P450 research for nearly four decades, no comprehensive assessment of African-led research has been conducted. This study presents a historical and bibliometric analysis of African-led P450 research published between 1987 and 2025 using PubMed and Web of Science. Of 1382 retrieved articles, 558 had African corresponding authors, and 149 included African co-authors. Research output increased markedly after 2011, with South Africa contributing 64% of African-led publications. Corresponding authors represented 31 African countries, while collaborations extended to 54 non-African countries, demonstrating strong international integration. Drug metabolism, pharmacology, and toxicology dominated the research landscape, followed by molecular biology, genomics, enzymology, and structural biology. African researchers have made important contributions to pharmacogenomics, steroidogenesis, insecticide resistance, microbial biotechnology, biosensors, and natural-product metabolism. Collectively, African-led publications have received more than 20,500 citations on Google Scholar, highlighting their global scientific impact. Despite this progress, important gaps remain in large-scale pharmacogenomics, structural biology, systems biology, industrial biotechnology, and omics-based studies. Overall, African researchers have made substantial contributions to global cytochrome P450 research and have created opportunities for future scientific growth and collaboration. Full article

Metabolic dysfunction-associated steatohepatitis (MASH; formerly non-alcoholic steatohepatitis, NASH) is characterized by oxidative stress, inflammatory activation, hepatocellular injury, and progressive liver dysfunction. However, the global transcriptomic landscape underlying stress-induced hepatic injury remains incompletely understood. In this study, we employed a methionine–choline-deficient (MCD) diet-induced murine model to characterize the phenotypic and transcriptomic alterations associated with liver injury. Male C57BL/6J mice were fed either a control or MCD diet, and hepatotoxicity was assessed by survival analysis, body and liver weight measurements, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, histopathological examination, RNA sequencing, quantitative real-time PCR (qRT-PCR), and tumor necrosis factor-alpha (TNF-α) enzyme-linked immunosorbent assay (ELISA). MCD feeding markedly reduced survival and body weight while inducing hepatomegaly and significant elevations in serum ALT and AST, indicating severe hepatocellular injury. Histopathological analysis demonstrated hepatic steatosis, hepatocellular ballooning, and lobular inflammation without histological evidence of fibrosis. Transcriptomic profiling revealed extensive gene expression remodeling, characterized by activation of inflammatory pathways, enrichment of MAPK-related signaling, dysregulation of lipid metabolism, suppression of antioxidant defense systems, impairment of cytochrome P450-mediated detoxification, and upregulation of apoptosis-associated genes. qRT-PCR further validated the differential expression of representative genes involved in inflammatory signaling (Tlr4, Nfkb1, Nlrp3, and Casp1), MAPK signaling (Fos), xenobiotic metabolism (Cyp4f18), lipid metabolism (Apoa4 and Lpl), extracellular matrix remodeling (Mmp12), and oxidative stress responses (Sod1 and Gstp1). In addition, elevated serum TNF-α levels provided protein-level evidence supporting activation of the TLR4/NF-κB/TNF-α/NLRP3 inflammatory axis. Although fibrosis-associated transcriptional responses were detected, the absence of histological fibrosis suggests transcriptional priming of fibrogenic pathways rather than established fibrogenesis. Collectively, these findings provide a transcriptomic framework linking oxidative stress, impaired detoxification, inflammatory activation, and stress-responsive signaling to MCD-induced hepatic injury. The MCD model provides a valuable experimental platform for characterizing hepatic stress-response transcriptomes and for generating hypotheses that can subsequently be evaluated in environmentally relevant toxicological models. Nevertheless, caution should be exercised when extrapolating these findings to obesity-associated human MASLD, as the MCD model lacks key metabolic features of the human disease, including obesity and insulin resistance. Therefore, the present findings should be interpreted primarily as transcriptomic signatures of stress-induced hepatic injury rather than as a direct representation of the pathophysiological processes underlying human obesity-associated MASLD. Full article

Despite increasing interest in fungal remediation systems for the treatment of persistent contaminants, the mechanisms governing fungal transformation of halogenated organic compounds remain poorly resolved. The aim of this study was to determine whether the transformation of halogenated nitrophenols is driven by isolated extracellular enzymes and cofactor-dependent oxidative activity or instead reflects coordinated system-level fungal metabolism. To address this question, we investigated the transformation of 2-chloro-4-nitrophenol (2C4NP) and 5-fluoro-2-nitrophenol (5F2NP) by ascomycete fungi Caldariomyces fumago (C. fumago) and Curvularia sp. under varying nutrient and cofactor conditions. Whole-culture transformation, crude supernatant activity, purified enzyme assays, intracellular detoxification responses, and genome-resolved functional annotation were integrated to evaluate the relative contributions of extracellular and intracellular processes. Transformation was strongly dependent on fungal species, substrate identity, nutrient availability, and cofactor composition. C. fumago achieved complete transformation of 2C4NP and up to 85.3% transformation of 5F2NP, whereas Curvularia sp. exhibited strict Na₃VO₄-dependent transformation of 5F2NP. Crude supernatants retained partial transformation capacity, achieving ~40–45% substrate depletion under conditions supporting whole-culture activity. Purified chloroperoxidase and laccase showed negligible independent activity and did not reproduce whole-culture transformation behavior. Lignin peroxidase activity was consistently induced during contaminant exposure and peaked during periods of maximum transformation. Cytochrome P450 inhibition did not prevent transformation. Baseline glutathione S-transferase activity was detected in both fungi, and comparative genome analysis identified conserved intracellular detoxification-associated enzyme alongside divergent extracellular oxidative enzyme repertoires. Together, these findings demonstrate that transformation of halogenated nitrophenols by fungi cannot be explained by isolated extracellular enzymes alone but is consistent with coordinated extracellular and intracellular system-level metabolism. These findings highlight an underexplored role for integrated fungal metabolic systems in bioremediation and provide a mechanistic basis for developing a scalable fungal platform for treatment of persistent halogenated contaminants. Full article

Asthma is a heterogeneous chronic airway disease in which oxidative stress (OS) plays a central mechanistic role beyond classical immune-mediated inflammation. Reactive oxygen and nitrogen species (ROS/RNS), generated by recruited inflammatory cells and activated airway structural cells, drive epithelial injury, mucus hypersecretion, airway remodeling, and modulate key transcription factors including nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. This review synthesizes current evidence on the multilevel redox-based determinants of asthma severity, spanning from genetic polymorphisms to circulating biomarkers. We examine serum antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), peroxiredoxins (PRDXs), and the thioredoxin (Trx) system as dynamic indicators of systemic redox status and disease severity, alongside oxidative enzymes including NADPH oxidases and dual oxidases (NOX/DUOX), xanthine oxidase (XO), and myeloperoxidase (MPO) that serve as upstream sources of airway oxidant burden. Functional genetic polymorphisms in antioxidant genes (SOD2, CAT, glutathione S-transferase mu 1/glutathione S-transferase theta 1 (GSTM1/GSTT1), heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1 (Nrf2/KEAP1)) and oxidative enzyme genes including nitric oxide synthase 1/2/3 (NOS1/2/3), MPO, cytochrome b-245 alpha chain (CYBA), and xanthine dehydrogenase (XDH) are reviewed as modulators of individual redox capacity and asthma susceptibility, with particular attention to gene–environment interactions. We further discuss oxidative damage biomarkers, including malondialdehyde (MDA), 8-isoprostanes, 4-hydroxynonenal, 8-oxo-7, 8-dihydro-2′-deoxyguanosine, protein carbonyls, 3-nitrotyrosine, and advanced oxidation protein products as indicators of lipid, DNA, and protein oxidation that correlate with disease activity and control. The roles of micronutrient cofactors in modulating antioxidant enzyme function and their potential as contextual biomarkers are also addressed. Additionally, emerging evidence on microRNAs (miRNAs) linked to OS biology in asthma is presented. Finally, we critically evaluate the challenges limiting clinical translation, including biomarker non-specificity, analytical variability, gene–environment complexity, and the absence of standardized reference ranges. This integrated framework supports the development of multilevel redox prognostic panels combining genetic, enzymatic, and oxidative damage readouts for improved asthma phenotyping, severity stratification, and personalized therapeutic approaches. Full article

Compounds from olive (Olea europaea L.) and pomegranate (Punica granatum L.) have many beneficial effects on human health. This review paper considers the inhibitory potential, under in vitro conditions, of bioactive components of olive and pomegranate on different enzyme systems. Research shows that olive polyphenols (oleuropein, hydroxytyrosol, luteolin, and oleocanthal), as well as pomegranate polyphenols (punicalagin, urolithin A, ellagic acid), inhibit cyclooxygenase and lipoxygenase enzymes, which are associated with inflammatory processes. They also show an inhibitory effect on acetylcholinesterase, butyrylcholinesterase, and β-secretase, which opens up the possibility of a strong neuroprotective effect. Olive and pomegranate polyphenols also have an inhibitory effect on enzymes involved in carbohydrate metabolism, such as amylase and glucosidase, and can help fight diabetes and regulate human metabolism. In addition, polyphenols and extracts of both plants showed an inhibitory effect on cytochrome P450 enzymes, which metabolize most drugs. These data open up the possibility of interactions with certain groups of drugs. The current evidence supports the view that olive and pomegranate polyphenols act as biologically versatile compounds with considerable pharmaceutical and nutraceutical potential. Future investigations integrating enzymology, metabolomics, molecular docking, and clinical validation will be essential for translating these promising in vitro findings into evidence-based therapeutic applications. Full article

Spermatogenesis is a highly coordinated biological process in which diploid spermatogonia undergo mitotic expansion, meiotic division, and terminal differentiation into haploid spermatozoa. This process is tightly regulated by intrinsic germ cell programs and extrinsic signals from Sertoli cells within the seminiferous epithelium. Among the signaling pathways governing male germ cell development, all-trans retinoic acid (RA), a bioactive metabolite of vitamin A, has emerged as a master regulator of meiotic initiation and spermatogonial differentiation in mammals. RA functions through nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which regulate transcriptional networks essential for germ cell progression, including the activation of Stimulated by Retinoic Acid 8 (STRA8), a key determinant of meiotic entry. Intratesticular RA homeostasis is maintained by a balance between synthesis via aldehyde dehydrogenase (ALDH) enzymes and degradation by cytochrome P450 family 26 (CYP26) enzymes, ensuring precise temporal and spatial control of germ cell development. While rodent models have defined core mechanisms of RA signaling, the canine testis provides a valuable comparative and translational system due to its physiological similarity to human spermatogenesis and relevance to reproductive management. Recent studies highlight conserved RA signaling pathways in dogs, including receptor-mediated transcriptional regulation, feedback control of RA metabolism, and post-transcriptional modulation via microRNAs. Importantly, pharmacological manipulation of RA signaling can reversibly disrupt spermatogenesis, supporting its potential applications in non-hormonal male contraception. This review integrates molecular, developmental, pharmacological, and comparative evidence and presents RA signaling as a central regulatory axis of spermatogenesis with important translational applications. Full article

Background: Cytochrome P450 3A4 (CYP3A4) is a key membrane-anchored drug-metabolizing enzyme. Its expression and purification in heterologous systems are severely hindered by low yield and detergent-induced structural inactivation. Although extracellular vesicles (EVs) provide an ideal natural lipid bilayer environment to stabilize membrane proteins, targeted loading remains challenging. The ESCRT and ALIX-binding region (EABR) of CEP55 can efficiently recruit core components of the endosomal sorting complex (ESCRT) to mediate membrane fission. Objectives: This study used the EABR motif to drive the targeted vesicular secretion of CYP3A4, thereby establishing a novel membrane protein engineering platform. Methods and Results: EABR was fused with fluorescent protein, confirming its specific mediation of vesicular secretion. Recombinant plasmids of EABR/CYP3A4 and its reverse mutant (R-EABR) were transfected into HEK293T cells. Western blot and midazolam-based metabolic assays showed that forward EABR significantly enhanced CYP3A4 expression and EV secretion, while R-EABR lost exocytosis function. EVs isolated by ultracentrifugation verified EABR’s role in recruiting ESCRT and improving CYP3A4 activity. Conclusions: Forward CEP55-EABR specifically and efficiently drives vesicular encapsulation of CYP3A4, enhancing its expression and secretion. This ESCRT-mediated strategy avoids destructive purification, provides a stable lipid-rich bioreactor for CYP3A4, and has great translational potential in high-throughput in vitro drug metabolism and screening platforms. Full article

Antiretroviral drugs (ARVDs) remain the cornerstone of HIV/AIDS management, but their therapeutic efficacy and safety are highly influenced by bioconversion processes such as hepatic metabolism and enzymatic transformation. Variability in metabolic pathways, mediated by cytochrome P450 enzymes and other liver-based systems, contributes to interindividual differences in drug response, toxicity, and resistance. Recent advances in artificial intelligence, particularly artificial neural networks (ANNs), offer promising tools for modeling and optimizing these complex bioconversion processes. ANNs are capable of learning nonlinear relationships from high-dimensional datasets, making them ideal for predicting the pharmacokinetic parameters, enzyme–substrate interactions, and metabolic stability of ARVDs. This review explores the emerging role of ANNs in understanding and optimizing the metabolic transformation of antiretroviral agents. Key applications are discussed, including prediction of drug–enzyme interactions, in silico modeling of hepatic clearance, and simulation of enzyme kinetics. The integration of molecular descriptors, omics data, and clinical parameters into ANN models allows for improved prediction accuracy and personalized therapy. Furthermore, ANN-based tools can aid in early-stage drug development by identifying metabolic liabilities and guiding structural modifications to enhance metabolic stability. Despite their potential, challenges such as data scarcity, model interpretability, and standardization remain. Future research should focus on hybrid models combining ANN with mechanistic pharmacokinetics, the incorporation of real-world patient data, and validation against experimental outcomes. Overall, ANNs represent a powerful approach to optimizing ARVDs bioconversion, with the potential to improve efficacy, reduce toxicity, and support the development of next-generation antiretroviral therapies Full article

The combination of thiopurine and methotrexate (MTX) is a standard co-therapy regimen for acute lymphoblastic leukemia (ALL). Despite its efficacy, this regimen is constrained by a narrow therapeutic window and considerable inter-individual variability, which heightens the risk of drug-induced liver injury (DILI). MTX-induced metabolic strain further destabilizes cytokine-sensitive thiopurine detoxification pathways during systemic inflammation. Conventional pharmacogenetic (PGx) testing for TPMT and NUDT15 variants is effective in predicting myelosuppression, but often fails to detect hepatotoxicity as an adverse effect, suggesting a clinically significant genotype-phenotype difference. This review examines the molecular determinants of DILI, emphasizing the role of secondary metabolic pathways and transporter dynamics as key modulators of risk. The study describes cytokine-mediated (IL-6, TNF-α) transcriptional suppression of cytochrome P450 enzymes and hepatic transporters (SLCO1B1, ABCC2/4) not merely as secondary modulators, but as the primary determinants of localized, tissue-specific drug exposure through disrupted nuclear receptor signaling (PXR, CAR, HNF4α). This mechanism promotes functional phenoconversion and toxic molecular shunting, leading to increased intrahepatic drug exposure. It synthesizes the current knowledge on the metabolism of thiopurine and MTX, focusing on the genetic and non-genetic factors influencing toxicity and their interactions. The review also critically evaluates the limitations of static PGx-guided dosing. It highlights the need for comprehensive, real-time risk assessment that integrates gene-environment interactions, multi-omics data, and clinical monitoring to improve precision therapy for ALL. This approach combines extended PGx profiling, transcriptomic monitoring, and clinical biomarker assessment to provide a transformative strategy for precision drug delivery. Full article

Background/Objectives: Pharmacogenomics (PGx) enables personalized therapy by identifying genetic variants that influence drug response. Despite the advantages of next-generation sequencing (NGS), few clinically validated, guideline-aligned panels comprehensively detect common, rare, and structurally complex pharmacogenetic variants. Methods: We developed and analytically validated Action PharmaKitDx, a targeted NGS panel covering 335 pharmacogenes, including all priority genes recommended by CPIC, DPWG, and CPNDS. Performance was assessed using Coriell HapMap and GeT-RM reference materials across multiple library preparation workflows and Illumina platforms. Clinical feasibility was evaluated in 41 patient samples from diverse specialties. Results were compared with established reference methods, including PCR-based assays, STR analysis, Sanger sequencing, and whole-exome sequencing. Results: Analytical validation: More than 99% of target bases achieved ≥30× coverage. Analytical accuracy, sensitivity, specificity, and positive predictive value exceeded 99.3%, with repeatability and reproducibility >99.7%. Concordance with GeT-RM haplotypes reached 98% after star-allele harmonization. The panel accurately detected complex variants, including CYP2D6 copy-number changes and hybrid alleles. Clinical validation: Full concordance with prior genotyping was observed in clinical samples. Beyond the initial testing indication, each sample harbored a mean of six actionable variants (range 2–10). Thirty-six rare (minor allele frequency <1%) potentially actionable variants were additionally identified. Conclusions: Action PharmaKitDx demonstrates high analytical performance and broad clinical applicability, supporting its implementation as a scalable solution for comprehensive pharmacogenetic testing and precision prescribing. Full article