Metabolic activation of WHO-congeners PCB28, 52, and 101 by human CYP2A6: evidence from in vitro and in vivo experiments.

Despite extensive research on the metabolism of polychlorinated biphenyls (PCBs), knowledge gaps persist regarding their isoform-specific biotransformation pathways. This study aimed to elucidate the role of different cytochrome P450 enzymes in PCB metabolism, focusing on WHO-congeners 2,4,4'-trichlorobiphenyl (PCB28), 2,2',5,5'-tetrachlorobiphenyl (PCB52), and 2,2',4,5,5'-pentachlorobiphenyl (PCB101). Utilizing engineered HEK293 cell lines, we investigated the in vitro metabolism of these PCBs by CYP1A2, CYP2C8, CYP2C9, CYP3A4, CYP2A6, and CYP2E1, revealing robust production of hydroxylated metabolites. Our results show that CYP2A6 plays a major role in the metabolism of these congeners responsible for predominant formation of para-position hydroxylated metabolites, with concentrations reaching up to 1.61 µg/L (5,89 nM) for PCB28, 316.98 µg/L (1,03 µM) for PCB52, and 151.1 µg/L (441 nM) for PCB101 from a 20 µM parent PCB concentration. Moreover, concentration-dependent cytotoxic and cytostatic effects induced by reactive intermediates of the PCB hydroxylation pathway were observed in HEK293CYP2A6 cells, for all three congeners tested. CYP2A6 was specifically capable of activating PCBs 28 and 101 to genotoxic metabolites which produced genetic defects which were propagated to subsequent generations, potentially contributing to carcinogenesis. In a clinical study examining CYP2A6 enzyme activity in formerly exposed individuals with elevated internal PCB levels, a participant with increased enzyme activity showed a direct association between the phenotypic activity of CYP2A6 and the metabolism of PCB28, confirming the role of CYP2A6 in the in vivo metabolism of PCB28 also in humans. These results altogether reinforce the concept that CYP2A6 plays a pivotal role in PCB congener metabolism and suggest its significance in human health, particularly in the metabolism of lower chlorinated, volatile PCB congeners.

SLC22A11 Inserts the Uremic Toxins Indoxyl Sulfate and P-Cresol Sulfate into the Plasma Membrane.

Chronic kidney disease (CKD) is a global health concern affecting millions worldwide. One of the critical challenges in CKD is the accumulation of uremic toxins such as p-cresol sulfate (pCS) and indoxyl sulfate (IS), which contribute to systemic damage and CKD progression. Understanding the transport mechanisms of these prominent toxins is essential for developing effective treatments. Here, we investigated whether pCS and IS are routed to the plasma membrane or to the cytosol by two key transporters, SLC22A11 and OAT1. To distinguish between cytosolic transport and plasma membrane insertion, we used a hyperosmolarity assay in which the accumulation of substrates into HEK-293 cells in isotonic and hypertonic buffers was measured in parallel using LC-MS/MS. Judging from the efficiency of transport (TE), pCS is a relevant substrate of SLC22A11 at 7.8 ± 1.4 µL min-1 mg protein-1 but not as good as estrone-3-sulfate; OAT1 translocates pCS less efficiently. The TE of SLC22A11 for IS was similar to pCS. For OAT1, however, IS is an excellent substrate. With OAT1 and p-aminohippuric acid, our study revealed an influence of transporter abundance on the outcomes of the hyperosmolarity assay; very high transport activity confounded results. SLC22A11 was found to insert both pCS and IS into the plasma membrane, whereas OAT1 conveys these toxins to the cytosol. These disparate transport mechanisms bear profound ramifications for toxicity. Membrane insertion might promote membrane damage and microvesicle release. Our results underscore the imperative for detailed structural inquiries into the translocation of small molecules.

Pharmacogenetic and drug interaction aspects on ketamine safety in its use as antidepressant - implications for precision dosing in a global perspective.

Ketamine and its enantiomer S-ketamine (esketamine) are known to produce rapid-onset antidepressant effects in major depression. Intranasal esketamine has recently come onto the market as an antidepressant. Besides experience from short-term use in anaesthesia and analgesia, the experience with ketamine as long-term medication is rather low. The use of ketamine and esketamine is limited due to potential neurotoxicity, psychotomimetic side effects, potential abuse and interindividual variability in treatment response including cessation of therapy. Therefore, taking a look at individual patient risks and potential underlying variability in pharmacokinetics may improve safety and dosing of these new antidepressant drugs in clinical practice. Differential drug metabolism due to polymorphic cytochrome P450 (CYP) enzymes and gene-drug interactions are known to influence the efficacy and safety of many drugs. Ketamine and esketamine are metabolized by polymorphic CYP enzymes including CYP2B6, CYP3A4, CYP2C9 and CYP2A6. In antidepressant drug therapy, usually multiple drugs are administered which are substrates of CYP enzymes, increasing the risk for drug-drug interactions. We reviewed the potential impact of polymorphic CYP variants and common drug-drug interactions in antidepressant drug therapy affecting ketamine pharmacokinetics, and the role for dose optimization. The use of ketamine or intranasal esketamine as antidepressants demands a better understanding of the factors that may impact its metabolism and efficacy in long-term use. In addition to other clinical and environmental confounders, prior information on the pharmacodynamic and pharmacokinetic determinants of response variability to ketamine and esketamine may inform on dose optimization and identification of individuals at risk of adverse drug reactions.

Does Intracellular Metabolism Render Gemcitabine Uptake Undetectable in Mass Spectrometry?

The ergothioneine transporter ETT (formerly OCTN1; human gene symbol SLC22A4) is a powerful and highly specific transporter for the uptake of ergothioneine (ET). Recently, Sparreboom et al. reported that the ETT would transport nucleosides and nucleoside analogues such as cytarabine and gemcitabine with the highest efficiency. In our assay system, we could not detect any such transport. Subsequently, Sparreboom suggested that the intracellular metabolization of the nucleosides occurs so fast that the original compounds cannot be detected by LC-MS/MS after inward transport. Our current experiments with 293 cells disprove this hypothesis. Uptake of gemcitabine was easily detected by LC-MS/MS measurements when we expressed the Na+/nucleoside cotransporter CNT3 (SLC28A3). Inward transport was 1280 times faster than the intracellular production of gemcitabine triphosphate. The deoxycytidine kinase inhibitor 2-thio-2'-deoxycytidine markedly blocked the production of gemcitabine triphosphate. There was no concomitant surge in intracellular gemcitabine, however. This does not fit the rapid phosphorylation of gemcitabine. Uptake of cytarabine was very slow, but detection by MS was still possible. When the ETT was expressed and incubated with gemcitabine, there was no increase in intracellular gemcitabine triphosphate. We conclude that the ETT does not transport nucleosides.

Impact of steroid hormone levels on estradiol-mediated regulation of cytochrome P450 2B6 compared to 1B1 in breast cancer cells.

Pharmacogenetic variants of the steroid hormone-metabolizing enzyme cytochrome P450 2B6 (CYP2B6) were reported to be associated with breast cancer (BC) risk and prognosis. CYP2B6 expression is inducible by estradiol (E2) but induction was demonstrated only under steroid hormone-deprived medium conditions. Physiological conditions, however, even under endocrinological BC treatment, do not correspond to complete steroid hormone depletion. The aim of this study was to investigate the E2-mediated CYP2B6 and CYP1B1 regulation under various steroid hormone conditions, including physiological concentrations, in human oestrogen receptor positive (T47D, MCF-7) and negative (MDA-MB-231) BC cell lines. We confirm that steroid-deprived pre-cultivation led to CYP2B6 upregulation in T47D, but not in MCF-7. However, when pre-cultivated with steroid-containing medium CYP2B6 was downregulated in T47D and MCF-7, while the addition of physiological E2 concentrations to steroid-deprived medium resulted in a downregulation in T47D. In contrast, CYP1B1 was never downregulated in any culture condition. Thus, we show that E2-mediated CYP2B6 regulation in BC cells depends on steroid hormone exposure in a cell line-specific manner. Our data indicates the importance of being careful with conclusions drawn from CYP2B6 induction findings in vitro, as we demonstrate potential influences of hormonal changes on CYP2B6 expression, which could impact steroid hormone homeostasis and, consequently, BC risk.

Uncoupling of Cytochrome P450 2B6 and stimulation of reactive oxygen species production in pharmacogenomic alleles affected by interethnic variability.

Cytochrome P450 mediated substrate metabolism is generally characterized by the formation of reactive intermediates. In vitro and in vivo reaction uncoupling, results in the accumulation and dissociation of reactive intermediates, leading to increased ROS formation. The susceptibility towards uncoupling and altered metabolic activity is partly modulated by pharmacogenomic alleles resulting in amino acid substitutions. A large variability in the prevalence of these alleles has been demonstrated in CYP2B6, with some being predominantly unique to African populations. The aim of this study is to characterize the uncoupling potential of recombinant CYP2B6*1, CYP2B6*6 and CYP2B6*34 metabolism of specific substrates. Therefore, functional effects of these alterations on enzyme activity were determined by quantification of bupropion, efavirenz and ketamine biotransformation using HPLC-MS/MS. Determination of H2O2 levels was performed by the AmplexRed/horseradish peroxidase assay. Our studies of the amino acid substitutions Q172H, K262R and R487S revealed an exclusive use of the peroxide shunt for the metabolism of bupropion and ketamine by CYP2B6*K262R. Ketamine was also identified as a trigger for the peroxide shunt in CYP2B6*1 and all variants. Concurrently, ketamine acted as an uncoupler for all enzymes. We further showed that the expressed CYP2B6*34 allele results in the highest H2O2 formation. We therefore conclude that the reaction uncoupling and peroxide shunt are directly linked and can be substrate specifically induced with K262R carriers being most likely to use the peroxide shunt and R487S carrier being most prone to reaction uncoupling. This elucidates the functional diversity of pharmacogenomics in drug metabolism and safety.

Nutrimetric Validation of Solanidine as Dietary-Derived CYP2D6 Activity Marker In Vivo.

CYP2D6 is involved in the metabolism of many drugs. Its activity is affected by pharmacogenetic variability leading to highly polymorphic phenotypes between individuals, affecting safety and efficacy of drugs. Recently, solanidine, a steroidal alkaloid from potatoes, and its metabolites, has been identified as a dietary-derived activity marker for CYP2D6. The intraday variability in plasma within individuals has not been studied yet in healthy subjects. As part of a CYP phenotyping cocktail study with 20 healthy participants, plasma concentrations of solanidine, 4-OH-solanidine and 3,4-secosolanidine-3,4-dioic acid (SSDA) were determined using a sensitive liquid chromatography-mass spectrometry method in urine and in plasma at timepoints 0, 2.5, 5, 8, and 24 hours after intake of test substances. The participants were phenotyped for CYP2D6 with oral metoprolol (12.5 mg) with 15 plasma sampling points over 24 hours (DRKS00028922). Metabolic ratios (MRs) of metabolite to parent plasma concentrations were formed from single timepoints and the area under the curve (AUC). All participants were genotyped for CYP2D6. The intra-individual variability of the CYP2D6 metabolite SSDA was highly stable with a median SD of 11.62% over 24 hours. MR SSDA/solanidine was more variable (median SD 31.90%) but correlated significantly at all measured timepoints with AUC MR α-OH-metoprolol/metoprolol. The AUC MR SSDA/solanidine showed a significant linear relationship with the genetically predicted CYP2D6 activity score. This study substantiates the MR SSDA/solanidine as CYP2D6 activity marker. The high correlation with metoprolol MR indicates a valid prediction of the CYP2D6 phenotype at any timepoint during the study day.

Hyperosmolarity stimulates transporter-mediated insertion of estrone sulfate into the plasma membrane, but inhibits the uptake by SLC10A1 (NTCP).

Many drugs are largely hydrophobic molecules; a transporter might conceivably insert these into the plasma membrane. At least 18 transporters from diverse families have been reported to transport the model compound estrone sulfate alias estrone-3-sulfate (E3S). Out of these, we recently examined SLC22A11 (OAT4). We concluded from a comparison of E3S and uric acid transport that SLC22A11 does not translocate E3S into the cytosol, but into the plasma membrane. Here we present a hyperosmolarity alias hypertonicity assay to differentiate transport mechanisms. Human transporters were expressed heterologously in 293 cells. Solute uptake into intact cells was measured by LC-MS. Addition of mannitol or sucrose led to rapid cell shrinkage, but cell viability after 60 min in hyperosmolar buffer was not impaired. A decrease in substrate accumulation with increasing osmolarity as observed here for several substrates and the transporters SLC22A11, ETT (SLC22A4), OCT2 (SLC22A2), OAT3 (SLC22A8), and MATE1 (SLC47A1) suggests regular substrate translocation into the cytosol. An increase as observed for E3S transport by SLC22A11, OAT3, MATE1, SLC22A9, and SLC10A6 implies insertion into the membrane. In marked contrast to the other E3S transporters, the bile acid transporter SLC10A1 (NTCP, Na+ taurocholate co-transporting polypeptide) showed a decrease in the accumulation of E3S in hyperosmolar buffer; the same was observed with taurocholic acid. Indeed, our data from several functional assays strongly suggest that the transport mechanism is identical for both substrates. Apparently, a unique transport mechanism has been established for SLC10A1 by evolution that ensures the transport of amphipathic, detergent-like molecules into the cytosol.