Search Results (1,096)

Background/Objectives: Metabolic enzymes are crucial for the detoxification of exogenously administered drugs, especially enzymes expressed in the intestine and the liver. Recent advancements in analytical methodologies enable sensitive and specific quantitative measurements of proteins, facilitating a more accurate evaluation of their expression and relative contribution to drug metabolism. Methods: The aim of the study was to characterize the protein expression levels of 16 Cytochrome P450s (CYP450s) and 2 carboxylesterases (CESs) in human liver and intestinal tissues using absolute quantification by HPLC-MS/MS. Human hepatocytes (HHEP) and human liver microsomes (HLM) were utilized, along with a novel intestinal preparation from cryopreserved human intestinal mucosa (CHIM), to perform proteomic analyses. Results: A comprehensive evaluation of 16 CYP450s and 2 CES enzyme expression in human liver and intestinal tissues is provided to reflect their relative abundance. Among the various in vitro systems evaluated, 14 of 16, 15/16, and 7/16 CYP450 of the isoforms analyzed were detected in HHEP, HLM, and CHIM, respectively. In hepatic systems, CYP2C9 exhibited the highest expression among CYP450 isoforms, a trend consistently observed in both HHEP and HLM. CYP3A4 was the most abundantly expressed isoform in CHIM preparations. Across all systems tested, CES1 and CES2 showed the highest overall protein expression levels, surpassing those of the CYP450s. Conclusions: Our findings demonstrate that the absolute quantification method employed is reliable, producing consistent results across two different in vitro hepatic systems (HHEP and HLM). This study supports the utility of absolute quantification approaches for accurately profiling drug-metabolizing enzymes and provides new, valuable insights to improve in vitro/in vivo extrapolation and more informed predictive pharmacokinetic modeling strategies. Full article

Background: The cytochrome P450 family 21 subfamily A member 2 gene (CYP21A2) encodes 21-hydroxylase, a key enzyme in adrenal steroid biosynthesis. Despite its physiological importance, the diversity of CYP21A2 transcript variants and their tissue-specific expression in domestic animals, including cattle, remains largely unexplored. This study aimed to characterize CYP21A2 transcription in adrenal glands and ovaries and assess the potential transcriptional activity of its pseudogene, CYP21A1P. Methods: CYP21A2 transcription was investigated in adrenal and ovarian tissues of 12 healthy cows using semi-quantitative PCR and Sanger sequencing. Real-time PCR was performed to confirm expression levels. Melting curve analysis and electrophoresis were used to validate distinct amplicons corresponding to different transcript variants. Extended amplicons were sequenced to identify transcripts corresponding to reference sequences and potential pseudogene products. Results: A single transcript variant (NM_001013596.1) was consistently detected in adrenal glands, whereas ovaries expressed two variants: NM_001013596.1 and XM_024983378.2. Semi-quantitative analysis showed significantly higher CYP21A2 expression in adrenal glands compared to ovaries (p < 0.01). In ovarian samples, the NM_001013596.1 variant was more abundant than the XM_024983378.2 (p < 0.01). Sanger sequencing revealed two products matching CYP21A2 reference transcripts and an additional, longer product containing sequence motifs specific to the pseudogene CYP21A1P, indicating its transcriptional activity. Conclusions: These results provide the first evidence of tissue-specific expression and differential abundance of CYP21A2 transcript variants in cattle and suggest the transcription of the CYP21A1P pseudogene. The findings reveal the complexity of CYP21A2 expression in steroidogenic tissues and suggest potential regulatory roles for transcript and pseudogene variants in bovine physiology. Full article

Researchers strive to exploit kinetic potentials of multistep reactions by positioning enzymes in a regulated fashion. Therein, the proliferating cell nuclear antigen (PCNA) from Sulfolobus solfataricus is a promising biomolecular tool due to its extraordinary architecture. PCNA is a circular DNA sliding clamp, which can bind and move along DNA and thus, be applied for the immobilization and transport of biomolecules on versatile DNA scaffolds. Additionally, its heterotrimeric character facilitates the colocalization of enzyme cascades with defined stoichiometry. This study provides insights into the in vitro binding behavior of PCNA and its potential as protein scaffold for DNA functionalization and controlled biocatalysis: (1) PCNA was capable of binding circular DNA and wireframe DNA nanostructures. (2) DNA binding was predominantly mediated by the PCNA1 subunit. (3) PCNA assembly around DNA was compromised when cysteines were introduced at the PCNA–PCNA interfaces to stabilize the ring via disulfide bonds. (4) A two-enzyme cascade, comprising a pseudo-monomeric cytochrome P450 BM3 monooxygenase and a monomeric alcohol dehydrogenase (ADH), was successfully fused to PCNA, retaining catalytic activity. (5) When immobilized on DNA, the cascade performance was not assessable, due to nearly complete loss of ADH activity in proximity to DNA. Full article

The unique cytochrome P450 BM3 from Priestia megaterium (syn. Bacillus megaterium) is renowned for its versatile high catalytic activity. The cyp102A1-LG23 gene encoding its CYP102A1-LG23 mutant variant was expressed in Escherichia coli and Mycolicibacterium smegmatis. The in vivo activity of the heterologous enzyme was assessed with respect to androstenedione (AD), androstadienedione (ADD), testosterone (TS) and dehydroepiandrosterone (DHEA). Alongside 7β-hydroxylation, the heterologous enzyme catalyzed the mono- and dihydroxylation of C19 steroids. For the first time, the formation of 7β-, 6β- and 11α-hydroxylated derivatives of ADD using a bacterial enzyme, as well as the hydroxylation of DHEA at the C7α and C7β positions, and its dihydroxylation with the formation of the 7α,15α-dihydroxylated derivative using the mutant cytochrome P450 BM3 were demonstrated. The steroid structures were confirmed using mass spectrometry and ¹H NMR spectroscopy. The advantages of using mycolicibacteria as a bacterial chassis for gene expression were also shown. The results demonstrate the unusual properties of the mutant cytochrome P450 BM3-LG23 and open up prospects for its application in the biotechnological production of valuable hydroxysteroids. Full article

Environmental pollution, driven by industrialization, urbanization, and agricultural practices, has intensified global ecological degradation. Among the most concerning pollutants is PFOS, a synthetic compound known for its chemical stability, environmental persistence, and bioaccumulative potential. Widely utilised in industrial and consumer products, PFOS infiltrates ecosystems and food chains, posing substantial risks to human and animal health. Upon exposure, PFOS disrupts lipid metabolism, damages cellular membranes, and alters signaling pathways through partial metabolism by cytochrome P450 enzymes. Accumulating evidence links PFOS to oxidative stress, mitochondrial dysfunction, endocrine disruption, neurotoxicity, and immunotoxicity. Critically, PFOS contributes to the development and progression of prostate, breast, and ovarian cancers via mechanisms such as hormonal interference, chronic inflammation, and epigenetic modifications. Epidemiological studies further associate elevated PFOS serum levels with increased cancer risk, particularly in occupationally and environmentally exposed populations. This review brings together the latest knowledge on PFOS emissions, mechanistic toxicity, and cancer-causing potential, highlighting the urgent need for focused research and improved regulatory measures to safeguard public health. Full article

This study investigates the mechanisms underlying osteocyte injury in a high glucose (HG) environment and explores potential therapeutic targets and diagnostic markers for diabetic osteoporosis, a common complication of type 2 diabetes mellitus (T2DM). Hyperglycemia induces oxidative stress through the reactive oxygen species (ROS) production, which impair osteocytes and accelerate bone loss. To examine these effects, MLO-Y4 cells and primary mouse osteocytes were cultured under normal glucose and HG conditions, with additional treatments using N-acetylcysteine (NAC, ROS scavenger) and rapamycin (autophagy promoter and mTOR inhibitor). Cell viability, ROS levels, and the autophagy and apoptosis markers expression (Beclin1, LC3, p62, Bax, Bcl2, cytochrome C, and caspase3) were assessed using CCK8/ATP level assay, flow cytometry, Western blot, qRT-PCR, immunofluorescence, and TUNEL staining. The results showed that HG inhibits cell proliferation, induces insulin resistance, generates ROS, alters antioxidant enzymes, and promotes oxidative stress, leading to mTOR activation, subsequent autophagy inhibition, and osteocyte apoptosis. NAC mitigated these effects, while rapamycin prevented HG-induced apoptosis by inhibiting mTOR activation and promoting autophagy. This suggests that ROS-induced mTOR activation impairs autophagy and hinders the clearance of damaged osteocytes, triggering apoptosis. This research provides foundational evidence and novel insights into diabetic osteoporosis pathogenesis and potential therapies. Full article

Background/Objectives: Although several studies have been reported on the modulation of Cytochrome P450 by resveratrol, inconsistencies in the results obtained require further investigation. Here, we report the results of in vivo and in vitro experiments investigating the effect of resveratrol on CYP1A2, which participates in the biotransformation of several drugs used for the treatment of human malignancies. Methods: Male Wistar rats were exposed to resveratrol through diet (1%) for 30 days, and the hepatic CYP1A2 activity and protein concentration were assayed at the end of the treatment. Additionally, the capacity of the phytochemical to interfere with the induction of CYP1A2 by benzo[a]pyrene (50 mg/kg body weight) was also studied. The inhibition of CYP1A2 activity in rat liver microsomal and recombinant human enzymes by resveratrol, as well as its inhibitory kinetics and type of inhibition, were compared. Results: No significant increase in the protein concentration of hepatic CYP1A2 was found in resveratrol-treated rats, but it induces CYP1A2 activity and enhances the induction effect of benzo[a]pyrene. In silico and in vitro experiments demonstrated that resveratrol binds to the active site of human CYP1A2 through hydrophobic interactions with PHE125, PHE226, PHE260, and ALA317, and hydrogen bonds with SER122 and ASP313. It inhibits human recombinant CYP1A2 activity as well as that in rat liver microsomes, with IC₅₀ values of 46 µM and 485 µM, respectively. Resveratrol showed a mixed type of inhibition of recombinant human protein and a competitive inhibition of rat liver microsomal CYP1A. Conclusions: We can conclude that resveratrol is an in vitro inhibitor of CYP1A2, but it increases the benzo[a]pyrene CYP induction effect in vivo. Full article

Natural product (NP) use by patients alongside their conventional cancer therapies is ubiquitous. This common, yet often hidden, practice can potentially contribute to significant patient harm, given the narrow therapeutic window of most anticancer drugs. This review takes on this challenge directly, moving past theoretical concerns to summarize current clinical evidence on interactions between widely used NPs and modern cancer treatments, including chemotherapy, targeted therapy, and immunotherapy. We break down the key pharmacokinetic (PK) mechanisms, such as the disruption of cytochrome P450 enzymes, and the pharmacodynamic (PD) effects that can either help or hinder treatment. By examining both well-established clinical interactions and those supported by preliminary or preclinical findings, we highlight how NPs may alter the effectiveness of anticancer medications and where evidence remains uncertain. Lack of reliable safety information for NPs along with widespread use of these products by patient populations has the potential to impact clinical care and patient outcomes significantly, frequently causing harm. We advocate for improved patient-provider communication and additional evidence-based research to address this gap in literature. The majority of reported interactions are based on preclinical or limited clinical evidence. A more rigorous evidence base including real-world data and clinical trials is urgently needed to guide practice. Full article

CYP24A1, a mitochondrial cytochrome P450 enzyme, plays a critical role in the catabolism of active vitamin D metabolites and is a key regulator of local vitamin D signaling in the small intestine. While traditionally studied in the context of renal physiology, increasing evidence highlights its distinct regulatory mechanisms and functional significance within the intestinal epithelium. This review explores the molecular architecture, tissue-specific expression patterns, and multifactorial regulation of CYP24A1 in enterocytes, encompassing nuclear receptor signaling, epigenetic and post-transcriptional control, and environmental influences such as inflammation, diet, and the gut microbiota. We discuss how intestinal CYP24A1 modulates the expression of vitamin D target genes involved in transcellular calcium absorption and epithelial barrier function, and how its dysregulation contributes to gastrointestinal disorders including inflammatory bowel diseases, celiac disease, microbiota dysbiosis, and colorectal cancer. In addition, we examine preclinical and translational evidence supporting CYP24A1 as a potential therapeutic target. Emerging strategies such as selective enzyme inhibitors, microbiota modulation, RNA-based technologies, and personalized supplementation approaches are considered in the context of restoring local vitamin D bioactivity and mineral homeostasis. Together, this review underscores the clinical importance of intestinal CYP24A1 and highlights novel opportunities for targeted interventions in vitamin D-responsive gastrointestinal pathologies. Full article

Candida spp. are common components of normal microbiota in the oral cavity. However, Candida albicans can be a primary cause of superficial infections in the oral cavity and esophagus, especially in immunocompromised individuals. While these infections are rarely life-threatening, they can significantly impair quality of life and, in severe cases, progress to hematogenous dissemination. Oral candidiasis often exhibits as pseudomembranous, erythematous (atrophic), chronic hyperplastic, denture stomatitis, or angular cheilitis. Esophageal candidiasis is typically diagnosed by upper endoscopy, which involves histological examination and brushing. Clinical guidelines recommend topical antifungal agents for mild oral candidiasis, and systemic agents for moderate-to-severe disease or when topical therapy fails. However, azole antifungals pose a substantial risk of drug–drug interactions, primarily due to the inhibition of cytochrome P450 enzymes and drug transporters, which dramatically alters the pharmacokinetics of co-administered drugs. Additionally, amphotericin B, a polyene macrolide antibiotic, may cause nephrotoxicity and electrolyte disturbances (e.g., hypokalemia and hypomagnesemia). Moreover, the co-administration of nephrotoxic drugs may augment the toxicity associated with amphotericin B. Therefore, this review aimed to provide a comprehensive overview of the management of oral and esophageal candidiasis from the viewpoint of clinical pharmacology, with a particular focus on drug–drug interactions and adverse effect profiles. Full article

Salmon farming has been affected by various bacterial diseases, and the use of antibiotics (such as oxytetracycline “OTC”) to control these diseases has become necessary and thus routine. This study aimed to determine how the gill cells are affected by OTC in Salmo salar. Gill tissue culture was performed in periods of 0.5, 1, 3, 6, 12, and 24 h, assessing the enzymatic activity and mRNA expression of catalase (CAT), cytochrome p450, glutathione peroxidase (GPx), glutathione reductase (Gr), and superoxide dismutase (SOD), HSP70 and HSP90, in response to two doses of OTC: 0.25 (low), and 3 µL/mL (high). The results indicated that the enzymatic activity of SOD and CAT showed low enzyme activity at both doses. At the same time, GR presented varied response patterns depending on the time and dose of OTC used, contrary to GPx, which just increased the enzyme activity at early times. Although the mRNA expression presented the most precise pattern of expression, they were not in line with the enzymatic activities. The HSP70 and HSP90 mRNA expression response (as a cellular damage marker) increased mRNA levels at low and high doses, respectively, but at different times, alluding to a differentiated response given by the size of the chaperone. These results suggest an oxidative response of the gills to OTC exposure and constitute significant information on the amount of OTC used in aquaculture and on methods for improving the optimal dose of drugs, fish health, and, consequently, environmental health. Full article

Mythimna separata (Walker) (Lepidoptera: Noctuidae) is a major migratory pest causing severe damage to cereal crops such as maize, wheat, and rice across Asia, and is also found in many parts of Oceania. With increasing insecticide resistance, botanical alternatives are urgently needed. This study evaluated the insecticidal potential of hypaconitine, a C19-diterpenoid alkaloid from Aconitum coreanum, against M. separata larvae. Hypaconitine exhibited significant stomach toxicity and strong antifeedant activity. It also caused pronounced growth inhibition, prolonged larval and pupal development, reduced pupation and adult emergence, induced morphological deformities, and significantly shortened adult longevity. Crucially, biochemical assays revealed sustained, time- and concentration-dependent upregulation of key detoxification enzymes—carboxylesterase (CarE), glutathione S-transferase (GST), and cytochrome P450 (CYP450)—over 72 h, indicating that hypaconitine imposes severe metabolic fitness costs rather than being readily detoxified. These effects collectively demonstrate that hypaconitine’s insecticidal efficacy arises not only from direct toxicity but also from exploiting the physiological trade-offs inherent in xenobiotic defense. Its multi-modal action—combining larvicidal, antifeedant, growth-regulatory, and metabolism-disrupting effects—presents a novel strategy for bioinsecticide development with a lower risk of resistance evolution. These findings highlight hypaconitine as a promising candidate for sustainable, integrated management of M. separata and other resistant lepidopteran pests. Full article

Objectives: Research on the antimicrobial effect of Hypericum plant constituents is very rarely accompanied by studies of the cytotoxic effect on cell lines. In the current study, besides microbiological tests, an investigation of the cytotoxicity of Hypericum active ingredients on five non-tumorigenic cell lines, as well as research into the effect on other factors of host homeostasis, was performed. Methods: The main methods applied included an MTT assay, the broth microdilution method (BMD), real-time PCR, live cell imaging with Hoechst dye, Western blot, an enzyme-linked immunosorbent assay (ELISA), and skin irritation test on rabbits. Results: The mean inhibitory concentrations (IC₅₀) of six selected agents—previously phytochemically characterized extracts and compounds—ranged from 0.63 to 48 µg/mL. Due to their strong antimicrobial effect and favorable cytotoxic profile, the extract RochC from Hypericum rochelii and the compound olympiforin B from Hypericum olympicum were selected for subsequent studies at their previously determined minimum inhibitory concentrations (MICs) against Staphylococcus aureus—0.625 and 1 µg/mL, respectively. These doses were lower than their IC₅₀ values and the maximum tolerated concentrations (MTCs), according to ISO 10993-5, Annex C, for fibroblast cells, including a human gingival line. The MIC values of RochC and Olympiforin B against the cariogenic Streptococcus mutans were 6 and 3 µg/mL, respectively, values lower than the IC₅₀ values of the gingival cells. Olympiforin B inhibited the gene expression of the staphylococcal biofilm-related genes icaA and icaD, while RochC induced icaA and had a versatile effect on icaD. The MIC values for lactobacilli strains were higher than for S. aureus. The phytoconstituents did not cause cytopathic effects or apoptosis in CCL-1 fibroblasts at 2 × MIC. However, the agents at 1 × MIC significantly induced Atg5 and Atg7, proteins related to autophagy. Cytochrome P450 was not induced in liver cells, with the exception of a dose of 2 × MIC of RochC. The agents did not irritate rabbit skin in vivo at a dose of even 10 × MIC. Conclusions: The extract and compound have potential for further pharmacological development. Full article

Cytochrome P450 enzymes (CYPs) are versatile biocatalysts capable of performing selective oxidation reactions valuable for industrial and pharmaceutical applications. However, their catalytic efficiency is often constrained by dependence on costly electron donors, the requirement for redox partners, and uncoupling reactions that divert reducing power toward reactive oxygen species. Improving electron transfer efficiency through optimized redox partner interactions is therefore critical for developing effective CYP-based biocatalysts. In this study, we investigated the interaction between CYP119, a thermophilic CYP from Sulfolobus acidocaldarius, and putidaredoxin (Pdx), the redox partner of P450cam. Using rational design and computational modeling with PyRosetta 3, 14 CYP119 variants were modeled and analyzed by docking simulations on the Rosie Docking Server. Structural analysis identified three key mutations (N34E, D77R, and N34E/D77R) for site-directed mutagenesis. These mutations (N34E, D77R, and N34E/D77R) enhanced Pdx binding affinity by 20-, 3-, and 12-fold, respectively, without affecting substrate binding. Catalytic assays using lauric acid and indirect assays to monitor electron transfer revealed that, despite improved complex formation, the N34E variant showed reduced electron transfer efficiency compared to D77R. These findings highlight the delicate balance between redox partner binding affinity and catalytic turnover, emphasizing that fine-tuning electron transfer interfaces are essential for engineering efficient CYP biocatalysts. Full article

Adamantane is a unique tricyclic hydrocarbon with a rigid, diamond-like structure. It is widely distributed in natural hydrocarbons and has significant potential applications in medicine, materials science, and pharmaceuticals. Despite its high chemical stability, biocatalytic methods using cytochrome P450 enzymes and microorganisms, such as Pseudomonas strains and actinobacteria, demonstrate high regional specificity in the hydroxylation and modification of adamantane, primarily at tertiary C–H bonds. This review summarises the latest data on the mechanisms and pathways of the microbial transformation of adamantane and its derivatives, including the key metabolites and enzymatic systems involved. The advantages of biocatalysis—namely, high selectivity, environmental friendliness and mild reaction conditions—are highlighted as a promising approach to the synthesis of functional adamantane derivatives for use in the development of innovative drugs and materials. The limitations of current methods and prospects for development are also discussed, including searching for new microorganisms and regulating enzymatic activity to increase the efficiency of biotransformation. Full article