CYP1B1 mRNA Research Articles

Epigenetic modifications in the GH-dependent Prlr, Hnf6, Cyp7b1, Adh1 and Cyp2a4 genes.

Many sex differences in liver gene expression originate in the brain, depend on GH secretion and may underlie sex disparities in hepatic disease. Because epigenetic mechanisms may contribute, we studied promoter methylation and microRNA abundance in the liver, associated with expression of sexual dimorphic genes in mice with selective disruption of the dopamine D2 receptor in neurons (neuroDrd2KO), which decreases hypothalamic Ghrh, pituitary GH, and serum IGFI and in neonatally androgenized female mice which have increased pituitary GH content and serum IGFI. We evaluated mRNA levels of the female predominant genes prolactin receptor (Prlr), alcohol dehydrogenase 1 (Adh1), Cyp2a4, and hepatocyte nuclear transcription factor 6 (Hnf6) and the male predominant gene, Cyp7b1. Female predominant genes had higher mRNA levels compared to males, but lower methylation was only detected in the Prlr and Cyp2a4 female promoters. In neuroDrd2KO mice, sexual dimorphism was lost for all genes; the upregulation (feminization) of Prlr and Cyp2a4 in males correlated with decreased methylation of their promoters, and the downregulation (masculinization) of Hnf-6 mRNA in females correlated inversely with its promoter methylation. Neonatal androgenization of females evoked a loss of sexual dimorphism only for the female predominant Hnf6 and Adh1 genes, but no differences in promoter methylation were found. Finally, mmu-miR-155-5p, predicted to target Cyp7b1 expression, was lower in males in association with higher Cyp7b1 mRNA levels compared to females and was not modified in neuroDrd2KO or TP mice. Our results suggest specific regulation of gene sexually dimorphic expression in the liver by methylation or miRNAs.

Evaluation of in vitro absorption, distribution, metabolism, and excretion and assessment of drug-drug interaction of rucaparib, an orally potent poly(ADP-ribose) polymerase inhibitor

1. The absorption, distribution, metabolism, elimination, and drug-drug interaction (DDI) potential of the poly(ADP-ribose) polymerase (PARP) inhibitor rucaparib was characterised in vitro.2. Rucaparib showed moderate cellular permeability, moderate human plasma protein binding (70.2%), and slow metabolism in human liver microsomes (HLMs). In HLMs, cytochrome P450 (CYP) 1A2 and CYP3A contributed to the metabolism of rucaparib to its major metabolite M324 with estimated fractions of metabolism catalysed by CYP (fm,CYP) of 0.27 and 0.64, respectively. Rucaparib reversibly inhibited CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3As (IC50, 3.55, 12.9, 5.42, 41.6, and 17.2–22.9 µM [2 substrates], respectively), but not CYP2B6 or CYP2C8 (>190 µM). No time-dependent inhibition of any CYP was observed. In cultured human hepatocytes, rucaparib showed concentration-dependent induction of CYP1A2 mRNA and downregulation of CYP3A4 and CYP2B6 mRNA. In transfected cells expressing drug transporters, rucaparib was a substrate for P-gp and BCRP, but not for OATP1B1, OATP1B3, OAT1, OAT3, or OCT2. Rucaparib inhibited P-gp and BCRP (IC50, 169 and 55 µM, respectively) and slightly inhibited OATP1B1, OATP1B3, OAT1, and OAT3 (66%, 58%, 58%, and 42% inhibition, respectively) at 300 µM. Rucaparib inhibited OCT1, OCT2, MATE1, and MATE2-K (IC50, 4.3, 31, 0.63, and 0.19 μM, respectively).3. DDI risk assessment using static models suggested potential CYP-related DDIs, with rucaparib as a perpetrator. Caution is advised when co-administering rucaparib with sensitive substrates of MATEs, OCT1, and OCT2.

Disruption of cytochrome P450 enzymes in the liver and small intestine in chicken embryos in ovo exposed to glyphosate.

Glyphosate is the active component of several commercial formulations as in Roundup®. The present study was investigated the toxic effects of pure glyphosate or Roundup® on the liver and small intestine of chick embryos. On day 6, a total of 180 fertile eggs injected with deionized water (control group), 10mg pure glyphosate, or 10mg of the active ingredient glyphosate in Roundup®/kg egg mass. The results showed an increase in relative weights of the liver in embryos that treated with Roundup®. Furthermore, oxidative stress was observed in the embryos treated with glyphosate or Roundup®, increased total superoxide dismutase, and content of malondialdehyde in the liver and intestine; moreover, decrease of glutathione peroxidase in the liver with increased in the intestine compared with the control. Besides, glutamic-pyruvic transaminase was increased in Roundup® group compared with other groups. Moreover, histopathological alterations in the liver and intestine tissues were observed in treated groups. Suppression of hepatic CYP1A2, CYP1A4, CYP1B1, and MDR1 mRNA expression after exposed to Roundup®. Furthermore, inhibition of CYP1A4 in the duodenum, CYP1A4, and MRP2 in the jejunum in embryos exposed to glyphosate or Roundup®. In addition, glyphosate treatment caused an increase of CYP3A5, CYP1C1, and IFNY mRNA expression in the jejunum and CYP1A2 expression in the ileum, while IFN-Y gene increase in embryos treated with Roundup®. In conclusion, in ovo exposure to glyphosate caused histopathological alterations and induced oxidative stress in the liver and small intestines. Moreover, the expression of cytochrome P450, MDR1, and MRP2 transporters was also modulated in the liver and small intestines for chick embryos.

MRNA as a Novel Treatment Strategy for Hereditary Spastic Paraplegia Type 5

Hereditary spastic paraplegia type 5 is a neurodegenerative disease caused by loss-of-function mutations in the CYP7B1 gene encoding the oxysterol 7-α-hydroxylase involved in bile acid synthesis in the liver. Lack of CYP7B1 leads to an accumulation of its oxysterol substrates, in particular 25-hydroxycholesterol and 27-hydroxycholesterol that are able to cross the blood-brain barrier and have neurotoxic properties. A potential therapeutic strategy for SPG5 is the replacement of CYP7B1 by administration of mRNA. Here, we studied the intravenous application of formulated mouse and human CYP7B1 mRNA in mice lacking the endogenous Cyp7b1 gene. A single-dose injection of either mouse or human CYP7B1 mRNA led to a pronounced degradation of oxysterols in liver and serum within 2 days of treatment. Pharmacokinetics indicate a single injection of human CYP7B1 mRNA to be effective in reducing oxysterols for at least 5 days. Repetitive applications of mRNA were safe for at least 17 days and resulted in a significant reduction of neurotoxic oxysterols not only in liver and serum but also to some extent in the brain. Our study highlights the potential to use mRNA as a novel therapy to treat patients with SPG5 disease.

Tissue distribution and characterization of feline cytochrome P450 genes related to polychlorinated biphenyl exposure

Cats have been known to be extremely sensitive to chemical exposures. To understand these model species' sensitivity to chemicals and their toxicities, the expression profiles of xenobiotic-metabolizing enzymes should be studied. Unfortunately, the characterization of cytochrome P450 (CYP), the dominant enzyme in phase I metabolism, in cats has not extensively been studied. Polychlorinated biphenyls (PCBs) are known as CYP inducers in animals, but the information regarding the PCB-induced CYP expression in cats is limited. Therefore, in the present study, we aimed to elucidate the mRNA expression of the CYP1–CYP3 families in the cat tissues and to investigate the CYP mRNA expression related to PCB exposure. In cats, the greatest abundance of CYP1–CYP3 (CYP1A2, CYP2A13, CYP2C41, CYP2D6, CYP2E1, CYP2E2, CYP2F2, CYP2F5, CYP2J2, CYP2U1, and CYP3A132) was expressed in the liver, but some extrahepatic isozymes were found in the kidney (CYP1A1), heart (CYP1B1), lung (CYP2B11 and CYP2S1) and small intestine (CYP3A131). In cats, CYP1A1, CYP1A2 and CYP1B1 were significantly upregulated in the liver as well as in several tissues exposed to PCBs, indicating that these CYPs were distinctly induced by PCBs. The strong correlations between 3,3′,4,4′-tetrachlorobiphenyl (CB77) and CYP1A1 and CYP1B1 mRNA expressions were noted, demonstrating that CB77 could be a potent CYP1 inducer. In addition, these CYP isoforms could play an essential role in the PCBs biotransformation, particularly 3–4 Cl-PCBs, because a high hydroxylated metabolite level of 3–4 Cl-OH-PCBs was observed in the liver.

Metazachlor: Mode of action analysis for rat liver tumour formation and human relevance

In a 2-year study the herbicide metazachlor (BAS 479H) was shown to significantly increase the incidence of liver tumours in female Wistar rats at a dietary level of 8000 ppm. As metazachlor is not a genotoxic agent, a series of in vivo and in vitro investigative studies were undertaken to elucidate the mode of action (MOA) for metazachlor-induced female rat liver tumour formation. Male and female Wistar rats were given diets containing 0 (control), 200 and 8000 ppm metazachlor for 3, 7, 14 and 28 days. The treatment of male rats with 200 and 8000 ppm metazachlor and female rats with 8000 ppm metazachlor resulted in significant increases in relative liver weight, which was associated with a centrilobular hepatocyte hypertrophy. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 8000 ppm metazachlor for 3 and 7 days and in female rats given 200 ppm metazachlor for 7–28 days and 8000 ppm metazachlor for 3–28 days. Significant increases in relative liver weight, centrilobular hepatocyte hypertrophy and hepatocyte RDS were also observed in male and female Wistar rats given and 500 ppm sodium phenobarbital (NaPB) for 3–28 days. The treatment of female Wistar rats with either 8000 ppm metazachlor for 7 days or with 500 ppm NaPB for 3 and 7 days resulted in the nuclear translocation of the hepatic constitutive androstane receptor (CAR). Treatment of male and female Wistar rats with 8000 ppm metazachlor for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content, CYP2B subfamily-dependent enzyme activities and mRNA levels, together with some increases in CYP3A enzyme activity and mRNA levels. The treatment of male Wistar rat hepatocytes with metazachlor (concentration range 0.5–50 μM) and NaPB (500 μM) for 4 days resulted in increased CYP2B enzyme activities and mRNA levels; with metazachlor and NaPB also producing significant increases in hepatocyte RDS levels. Studies were also performed with hepatocytes from male Sprague-Dawley wild type (WT) rats and CAR knockout (CAR KO) rats. While both treatment with metazachlor and NaPB for 4 days increased CYP2B enzyme activities and mRNA levels in WT rat hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both metazachlor and NaPB only increased RDS in WT but not in CAR KO rat hepatocytes. The treatment of hepatocytes from two male human donors with 0.5–25 μM metazachlor or 500 μM NaPB for 4 days resulted in increases in CYP2B6 and CYP3A4 mRNA levels but had no effect on hepatocyte RDS. EGF as concurrently used positive control demonstrated the expected RDS response in all rat and human hepatocyte cultures. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that metazachlor is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for metazachlor-induced female rat liver tumour formation has been established. Based on the lack of effect of metazachlor on RDS in human hepatocytes, it is considered that the MOA for metazachlor-induced rat liver tumour formation is qualitatively not plausible for humans.

AHR and GPER mediate the stimulatory effects induced by 3-methylcholanthrene in breast cancer cells and cancer-associated fibroblasts (CAFs)

BackgroundThe chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1, which is involved in the oncogenic activation of environmental pollutants as well as in the estrogen biosynthesis and metabolism. 3MC was shown to induce estrogenic responses binding to the estrogen receptor (ER) α and stimulating a functional interaction between AHR and ERα. Recently, the G protein estrogen receptor (GPER) has been reported to mediate certain biological responses induced by endogenous estrogens and environmental compounds eliciting an estrogen-like activity.MethodsMolecular dynamics and docking simulations were performed to evaluate the potential of 3MC to interact with GPER. SkBr3 breast cancer cells and cancer-associated fibroblasts (CAFs) derived from breast tumor patients were used as model system. Real-time PCR and western blotting analysis were performed in order to evaluate the activation of transduction mediators as well as the mRNA and protein levels of CYP1B1 and cyclin D1. Co-immunoprecipitation studies were performed in order to explore the potential of 3MC to trigger the association of GPER with AHR and EGFR. Luciferase assays were carried out to determine the activity of CYP1B1 promoter deletion constructs upon 3MC exposure, while the nuclear shuttle of AHR induced by 3MC was assessed through confocal microscopy. Cell proliferation stimulated by 3MC was determined as biological counterpart of the aforementioned experimental assays. The statistical analysis was performed by ANOVA.ResultsWe first ascertained by docking simulations the ability of 3MC to interact with GPER. Thereafter, we established that 3MC activates the EGFR/ERK/c-Fos transduction signaling through both AHR and GPER in SkBr3 cells and CAFs. Then, we found that these receptors are involved in the up-regulation of CYP1B1 and cyclin D1 as well as in the stimulation of growth responses induced by 3MC.ConclusionsIn the present study we have provided novel insights regarding the molecular mechanisms by which 3MC may trigger a physical and functional interaction between AHR and GPER, leading to the stimulation of both SkBr3 breast cancer cells and CAFs. Altogether, our results indicate that 3MC may engage both GPER and AHR transduction pathways toward breast cancer progression.

High-content analysis of constitutive androstane receptor (CAR) translocation identifies mosapride citrate as a CAR agonist that represses gluconeogenesis

The constitutive androstane receptor (CAR) plays an important role in hepatic drug metabolism and detoxification but has recently been projected as a potential drug target for metabolic disorders due to its repression of lipogenesis and gluconeogenesis. Thus, identification of physiologically-relevant CAR modulators has garnered significant interest. Here, we adapted the previously characterized human CAR (hCAR) nuclear translocation assay in human primary hepatocytes (HPH) to a high-content format and screened an FDA-approved drug library containing 978 compounds. Comparison of hCAR nuclear translocation results with the Tox21 hCAR luciferase reporter assay database in 643 shared compounds revealed significant overlap between these two assays, with approximately half of hCAR agonists also mediating nuclear translocation. Further validation of these compounds in HPH and/or using published data from literature demonstrated that hCAR translocation exhibits a higher correlation with the induction of hCAR target genes, such as CYP2B6, than the luciferase assay. In addition, some CAR antagonists which repress CYP2B6 mRNA expression in HPH, such as sorafenib, rimonabant, and CINPA1, were found to translocate hCAR to the nucleus of HPH. Notably, both the translocation assay and the luciferase assay identified mosapride citrate (MOS), a gastroprokinetic agent that is known to reduce fasting blood glucose levels in humans, as a novel hCAR activator. Further studies with MOS in HPH uncovered that MOS can repress the expression of gluconeogenic genes and decrease glucose output from hepatocytes, providing a previously unidentified liver-specific mechanism by which MOS modulates blood glucose levels.

Adenosine Deaminases Acting on RNA Downregulate the Expression of Constitutive Androstane Receptor in the Human Liver-Derived Cells by Attenuating Splicing.

Adenosine deaminases acting on RNA (ADARs) enzymes-catalyzing adenosine-to-inosine RNA editing possibly modulates gene expression and function. In this study, we investigated whether ADARs regulate the expression of human constitutive androstane receptor (CAR), which controls the expression of various drug-metabolizing enzymes. CAR mRNA and protein levels in human hepatocellular carcinoma-derived HepG2 cells were increased by knockdown of ADAR1 and slightly increased by ADAR2, indicating that ADARs negatively regulate CAR expression. Increased luciferase activity of a reporter plasmid containing the CYP3A4 promoter region by phenobarbital was augmented by transfection of siRNA for ADAR1 (siADAR1) but not by siADAR2. In addition, the knockdown of ADAR1 resulted in the enhanced induction of CYP2B6 and CYP3A4 mRNA by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime and phenobarbital, respectively. These results suggest that ADAR1-mediated downregulation of CAR affects its downstream cytochrome P450 expression. When the transcription was inhibited by α-amanitin, the degradation of CAR mRNA was attenuated by knockdown of ADAR1, suggesting that the increase in CAR mRNA level by ADAR1 knockdown is a post-transcriptional event. Finally, we found that ADAR1 knockdown promotes the splicing of CAR as a mechanism of the increased expression of CAR by ADAR1 knockdown. In conclusion, this study revealed that ADAR1 plays a role in modulating xenobiotic metabolism potency via regulation of CAR. SIGNIFICANCE STATEMENT: This study revealed that adenosine deaminase acting on RNA 1 (ADAR1) and ADAR2, which catalyze adenosine-to-inosine RNA editing, downregulate the expression of constitutive androstane receptor (CAR) in human liver-derived cells by attenuating splicing. The downregulation of CAR by ADARs affected its downstream cytochrome P450 expression. ADARs would play a role in modulating xenobiotic metabolism potency via regulation of CAR.

Abstract 13: Structure elucidation, metabolism, and drug interaction potential of ACP-5862, an active, major, circulating metabolite of acalabrutinib

Abstract Acalabrutinib (Calquence®) is a potent, selective, orally administered, covalent inhibitor of Bruton tyrosine kinase (BTK) that received accelerated approval for relapsed/refractory mantle cell lymphoma from the US FDA in October 2017. Profiling of acalabrutinib metabolites in human plasma revealed a late-eluting, +16 Da metabolite circulating at concentrations higher than parent drug. Metabolite regiochemistry could not be determined by mass spectrometry. In vitro metabolism and preparative HPLC was used to generate a pure sample of the metabolite for structural characterization by NMR. Confirmatory chemical synthesis revealed a pyrrolidine ring-opened ketone. The structure of the metabolite, designated ACP-5862, and a smaller -2 Da peak, identified as dehydropyrrolidine, M25, inferred a common carbinolamide intermediate in their genesis. Both metabolites retained the butynamide electrophile responsible for the inactivation of BTK. In vitro studies on the inhibition of BTK and related Tec and Src kinases revealed that ACP-5862 was active against BTK with similar selectivity and potency to acalabrutinib (Kaptein et al, 2019) This work then investigated the in vitro metabolism and drug transport features of acalabrutinib, and the metabolite ACP-5862, to establish the potential for clinical drug-drug interactions (DDI) via CYPs, UGTs and drug transporters. CYP reaction phenotyping indicated CYP3A4 was responsible for both the formation and further metabolism of ACP-5862. Km and Vmax values for the formation of ACP-5862 using rCYP3A4 were 2.78 μM and 4.13 pmol/pmol CYP/min, respectively. The in vitro intrinsic clearance of ACP-5862 was 23.6 μL/min/mg. Acalabrutinib weakly inhibited CYP2C8, CYP2C9 and CYP3A4 in vitro, and ACP-5862 weakly inhibited CYP2C9 and CYP2C19, with no inhibition of CYP1A2, CYP2B6, or CYP2D6. Similarly, UGT1A1, UGT2B7, and aldehyde oxidase were not inhibited. Neither parent or ACP-5862 strongly induced CYP1A2, CYP2B6, or CYP3A4 mRNA. Acalabrutinib and ACP-5862 were substrates of MDR1 and BCRP in vitro, but were not substrates of OATP1B1 or OATP1B3. Acalabrutinib was not a substrate of OAT1, OAT3, and OCT2. Based on static PK model calculations, acalabrutinib may cause a modest increase in exposure to coadministered BCRP substrates by inhibition of intestinal BCRP, but with no inhibition of BCRP at the systemic level. The PK of substrates of MDR1, MATE1, MATE2-K, OATP1B1, OATP1B3, OAT1, OAT3, and OCT2 are not likely to be altered by acalabrutinib or ACP-5862. These data were combined with clinical DDI data (Izumi et al, 2017) to simulate DDI in the presence of CYP3A inhibitors and inducers. PBPK models confirmed that acalabrutinib and ACP-5862 were not likely to perpetrate CYP2C8 or CYP3A4 mediated drug interactions (Zhou et al., 2019). Overall, acalabrutinib and major metabolite, ACP-5862 have a favorable drug interaction profile. Citation Format: Terry Podoll, Paul G. Pearson, Jerry Evarts, Tim Ingallinera, Hao Sun, Stephen Byard, Adrian J. Fretland, J. Greg Slatter. Structure elucidation, metabolism, and drug interaction potential of ACP-5862, an active, major, circulating metabolite of acalabrutinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 13.

The alterations of bile acids in rats with high-fat diet/streptozotocin-induced type 2 diabetes and their negative effects on glucose metabolism

PurposeBile acids (BAs) as a kind of endogenous and signaling molecules altered under the circumstance of T2DM, which could impact on the relevant pathways to further affect the glucose metabolism and insulin secretion and might be associated with the T2DM development and restoration. However, the potential mechanisms still need more various and multifaceted studies. Here, we explored the alterations of BAs features and their mechanisms, and discussed the potential effects of the altered BAs on the glucose metabolic disorder via the relevant signaling pathways. Main methodsThe high-fat diet (HFD) feeding combining with injection of low-dose streptozotocin (STZ) was employed for inducing the T2DM rat model. Based on that, we investigated the alterations of the concentrations and compositions of BAs and their mechanisms, and explored the effects of the altered BAs on the glucose metabolic disorder via farnesoid X receptor (Fxr) and G protein-coupled bile acid receptor (Tgr5)-mediated pathways. Key findingsIn rats with T2DM, the BAs in rats with T2DM exhibited characteristic alterations, especially the increased ratio of 12α-OH to non-12α-OH BAs in serum, which could be ascribed to the up-regulated Cyp8b1 mRNA expression ratio in the liver. Moreover, Additionally, the altered BAs had negative effects on glucose metabolic disorder via inhibiting the Trg5/Fxr-mediated pathways in colon, liver and pancreas in rats with T2DM. SignificanceBAs in rats with T2DM exhibited the characteristic alterations, which could provide a cue for searching biomarkers of the T2DM diagnosis, and the altered BAs might aggravate the glucose metabolic disorder.

Potential of Teucrium chamaedrys L. to modulate apoptosis and biotransformation in colorectal carcinoma cells

Ethnopharmacological relevanceTeucrum chamaedrys L. is one of the known medicinal plants, useful for treatment of various health problems, especially digestive. In this study, we investigated methanol, ethyl-acetate and acetone extracts of T. chamaedrys in respect to their anticancer properties in SW480 colorectal cancer cells. Materials and methodsCytotoxicity and proapoptotic potential were assessed by MTT cell viability assay and AO/EB double staining. Molecular mechanisms of induced apoptosis were determined by monitoring Fas receptor protein expression through immunofluorescence, Caspase 8 and 9 activity, as well as concentrations of O2.- spectrophotometrically. Additionally, mRNA expression of biotransformation enzymes (CYP1A1, CYP1B1, GSTP1) and membrane transporters (MRP1 and MRP2) involved in drug resistance were investigated by qPCR method. Qualitative analysis of individual phenolic compounds was performed by reversed phase HPLC-MS analysis. ResultsMethanol extract shows the best cytotoxicity and selectivity compared to ethyl-acetate and acetone extracts, mainly causing apoptosis of SW480 cells, without affecting normal HaCaT keratinocytes. The increased expression of Fas receptor protein and caspase 8 activity indicate that the death receptor-mediated pathway plays a crucial role in the observed apoptosis. The increased caspase 9 activity and O2.- concentration suggest that mitochondria are also involved in the apoptosis. T. chamaedrys methanol extract inhibits mRNA expression of CYP1A1, CYP1B1, GSTP1, MRP1 and MRP2 in SW480 cells. ConclusionsInduction of apoptosis and inhibition of CYP1A1, CYP1B1, GSTP1, MRP1 and MRP2 mRNA expression implies that T. chamaedrys can serve as a valuable source of bioactive compounds as dietary supplements or selective anticancer agents, with the ability to induce apoptosis and modulate drug resistance in colorectal cancer cells.

Cholesterol enrichment in liver mitochondria impairs oxidative phosphorylation and disrupts the assembly of respiratory supercomplexes

Mitochondrial cholesterol accumulation is a hallmark of alcoholic and non-alcoholic fatty liver diseases and impairs the function of specific solute carriers through changes in membrane physical properties. However, its impact on mitochondrial respiration and organization of respiratory supercomplexes has not been determined so far. Here we fed mice a cholesterol-enriched diet (HC) supplemented with sodium cholate to examine the effect of cholesterol in mitochondrial function. HC feeding increased liver cholesterol content, which downregulated Srebp2 and Hmgcr expression, while sodium cholate administration decreased Cyp7a1 and Cyp8b1 mRNA levels, suggesting the downregulation of bile acid synthesis through the classical pathway. HC-fed mice exhibited increased expression of Stard1 and Mln64 and enhanced mitochondrial free cholesterol levels (2–3 fold), leading to decreased membrane fluidity. Mitochondria from HC-fed mice displayed increased cholesterol loading in both outer and inner mitochondrial membranes. Cholesterol loading decreased complex I and complex II-driven state 3 respiration and mitochondrial membrane potential. Decreased respiratory and uncoupling control ratio from complex I was also observed after in situ enrichment of mouse liver mitochondria with cholesterol or enantiomer cholesterol, the mirror image of natural cholesterol. Moreover, in vivo cholesterol loading decreased the level of complex III2 and the assembly of respiratory supercomplexes I1+III2+IV and I1+III2. Moreover, HC feeding caused oxidative stress and mitochondrial GSH (mGSH) depletion, which translated in hepatic steatosis and liver injury, effects that were rescued by replenishing mGSH with GSH ethyl ester. Overall, mitochondrial cholesterol accumulation disrupts mitochondrial functional performance and the organization of respiratory supercomplexes assembly, which can contribute to oxidative stress and liver injury.

European Commission releases strategy addressing pharmaceuticals in the environment

Models of inflammation and infection, such as bacterial lipopolysaccharide (LPS), cause suppression of cytochrome P450 expression in various species, although the mechanisms involved are poorly understood. The effects of LPS...

Diallyl Trisulfide Enhances Benzo[a]pyrene-induced CYP1A1 Expression and Metabolic Activation in Hepatic HepG2 Cells.

Benzo[a]pyrene (BaP), an environmental pollutant produced by combustion processes, induces expression of cytochrome P450 (CYP) 1A1 via the activation of aryl hydrocarbon receptor (AHR). Induced CYP1A1 is involved in BaP metabolism, resulting in either detoxification or metabolic activation in a context-dependent manner. The effect of diallyl trisulfide (DATS), a garlic-derived organosulfur compound, on BaP metabolism has not been investigated. The combined effect of DATS and BaP on BaP metabolism in hepatocyte-derived HepG2 cells was examined. DATS enhanced BaP-induced CYP1A1 and CYP1B1 mRNA expression, BaP hydroxylation and BaP-DNA adduct formation. Combined treatment of BaP and DATS also increased reactive oxygen species levels. DATS enhanced BaP-induced AHR recruitment and histone H3 acetylation on the CYP1A1 promoter. DATS combined treatment enhances BaP metabolic activation through an AHR-modulating mechanism.

The effect of chronic kidney disease on CYP2B expression and activity in male Wistar rats.

Chronic kidney disease (CKD) is characterized by progressive reduction in kidney function over time. CKD affects greater than 10% of the population and its incidence is on the rise due to the growing prevalence of its risk factors. Previous studies demonstrated CKD alters nonrenal clearance of drugs in addition to reducing renal clearance. We assessed the function and expression of hepatic CYP2B enzymes using a rat model of CKD. CKD was induced in Wistar rats by supplementing their chow with adenine and confirmed through the detection of elevated uremic toxins in plasma. Liver enzymes AST and ALT were unchanged by the adenine diet. Bupropion was used as a probe substrate for hepatic CYP2B function using rat liver microsomes. The resulting metabolite, hydroxy‐bupropion, and bupropion were quantified by ultra‐performance liquid chromatography coupled to time‐of‐flight mass spectrometry. Level of mRNA and protein were determined by RT‐PCR and Western blot, respectively. The results of our study demonstrate that CYP2B1 is downregulated in a rat model of CKD. CYP2B1 mRNA level was significantly decreased (88%, P < 0.001) in CKD relative to control. Similarly, maximal enzymatic velocity (V max) for CYP2B was decreased by 46% in CKD relative to control (P < 0.0001). Previous studies involving patients with CKD demonstrated altered bupropion pharmacokinetics compared to control. Hence, our results suggest that these alterations may be mediated by attenuated CYP2B hepatic metabolism. This finding may partially explain the alterations in pharmacokinetics and nonrenal drug clearance frequently observed in patients with CKD.

RNA Editing Enzymes Modulate the Expression of Hepatic CYP2B6, CYP2C8, and Other Cytochrome P450 Isoforms.

A-to-I RNA editing, the most frequent type of RNA editing in mammals, is catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes. Recently, we found that there is a large interindividual variation in the expression of ADAR1 protein in the human livers. In this study, we investigated the possibility that A-to-I RNA editing may modulate the expression of cytochrome P450 (P450), causing interindividual variations in drug metabolism potencies. We found that knockdown of ADAR1 or ADAR2 in HepaRG cells resulted in the decreased expression of CYP2B6 and CYP2C8 mRNA and protein. Knockdown of ADARs significantly decreased the stability of CYP2B6 mRNA but not CYP2C8 mRNA. Luciferase assays revealed that the 3'-untranslated region of CYP2B6 and the promoter region of CYP2C8 would be involved in the decrease in their expression by the knockdown of ADARs. We found that the decreased expression of the hepatocyte nuclear factor 4α (HNF4α) protein by the knockdown of ADARs was one of the reasons for the decreased transactivity of CYP2C8. The mRNA levels of other P450 isoforms, such as CYP2A6, 2C9, 2C19, 2D6, and 2E1, which are known to be regulated by HNF4α, were also decreased by ADAR1 or ADAR2 knockdown. Exceptionally, the CYP3A4 mRNA level was significantly increased by ADAR1 knockdown, suggesting the possibility that the change could be due to the change in the expression or function of other regulatory factors. In conclusion, this study revealed that the RNA editing enzymes ADAR1 and ADAR2 are novel regulatory factors of P450-mediated drug metabolism in the human liver.

Determinants of Cytochrome P450 2D6 mRNA Levels in Healthy Human Liver Tissue.

Cytochrome P450 2D6 (CYP2D6) is a major drug‐metabolizing enzyme that exhibits large interindividual variability. Recent studies suggest that differential transcriptional regulation of CYP2D6 in part may be responsible for the variability. In this study, we characterized potential determinants of CYP 2D6 transcript levels in healthy human liver tissue samples (n = 115), including genetic polymorphisms in CYP2D6 and the genes encoding transcription regulators for CYP2D6 expression; mRNA expression of the transcription factors and their known target genes; and hepatic levels of bile acids and retinoids, agents that modulate the expression/activity of the transcription factors. Their associations with CYP2D6 mRNA levels in the tissues were examined. Results from multivariable linear regression analysis revealed CYP8B1 mRNA level and rs3892097, the single‐ nucleotide polymorphism defining the nonfunctional CYP2D6*4 allele, as the two most significant predictors of CYP2D6 mRNA levels in the liver tissue samples, explaining 30% of the variability.

Marinobufagenin, a mammalian endogenous cardiotonic steroid, is synthesized from the intermediates in the classical bile acid pathway in Cyp27A1 knockout mice

ObjectivesThe bioactive steroidal Na/K‐ATPase ligand marinobufagenin (MBG) participates in renal Na+ transport, cell homeostasis, regulates blood pressure and arterial wall collagen production. MBG biosynthesis in mammals is controlled by the Cyp27a1 enzyme (cytochrome P450, family 27, subfamily a, polypeptide 1), which initiates the acidic bile acid (BA) pathway in rodent adrenal cortex. Nevertheless, the knockout of Cyp27a1 in mice did not reduce MBG production. In addition, the homozygous Cyp27a1(−/−) mice (HO) exhibited an upregulation of adrenal Akr1d1 enzyme (aldo‐keto reductase family 1, member D1) vs. wild type mice (WT). Akr1d1 participates in the final steps of both acidic and classical BA pathways. We hypothesized (i) that MBG may be synthesized from the intermediates in the classical BA pathway under Akr1d1 control, and (ii) that inhibition of Akr1d1 in HO will decrease MBG production.MethodsAll‐trans retinoic acid (ATRA) suppresses Akr1d1 and the enzymes controlled classical BA pathway. Four‐to‐six months old male HO and wild type mice (WT) were fed ATRA diet (150 mg/kg of diet) or control diet (CTRL) for 10 days (n=8/group). Adrenal Cyp27a1 and Akr1d1, and hepatic Cyp27a1, Akr1d1, Cyp7a1, and Cyp8b1 mRNA (by qPCR), and MBG excretion were assessed before (baseline, BL) and after ATRA diet. Data were analyzed by t‐test or 2‐way ANOVA and are presented as mean±SE.ResultsAdrenal and hepatic Cyp27a1 mRNAs were down‐regulated by 80% in HO vs. WT (p&lt;0.01). Adrenal Akr1d1 mRNA was 3.5‐fold overexpressed in HO vs. WT (p&lt;0.01) (Figure 1A). Hepatic mRNAs were overexpressed in HO vs. WT (Figure 1B; Table). ATRA diet decreased expression of hepatic Akr1d1 mRNA in both strains vs. CTRL (HO: by 180%, p&lt;0.01; WT: by 170%, p&lt;0.01). Hepatic Cyp7a1 and Cyp8b1 mRNAs were also significantly down‐regulated by ATRA in both WT and HO but remained significantly higher in HO than in WT (Table). Baseline urine MBG level was 2‐fold higher in HO vs. WT (p&lt;0.05); inhibition of Akr1d1 by ATRA was associated with a decrease in urine MBG in HO (5.3±0.5 vs. 2.9±0.2 pmol/24hr; BL vs. ATRA; p&lt;0.05), but not in WT (2.1±0.4 vs. 2.6±0.9 pmol/24hr; BL vs. ATRA, n/s).ConclusionsThe up‐regulation of hepatic Akr1d1, Cyp7a1 and Cyp8b1 enzymes in Cyp27a1(−/−) mice may compensate for a lack of Cyp27a1 and promote production of a bioactive steroid MBG from the intermediates in the classical BA pathway. Inhibition of Akr1d1 resulted in a decrease of MBG production in Cyp27a1(−/−) mice only. MBG production was not changed by ATRA in WT with normal expression of Cyp27a1, which indicates that Cyp27a1 is a predominant enzyme in MBG biosynthesis.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rooibos (Aspalathus linearis) and honeybush (Cyclopia species) modulate the oxidative stress associated injury of diesel exhaust particles in human umbilical vein endothelial cells

BackgroundPrevious evidence show foods and beverages rich in polyphenolic compounds to have favourable effects on the cardiovascular system. HypothesisThe current study assessed the modulation of oxidative stress and associated inflammation induced by diesel exhaust particles (DEP - SRM 2975) by pre-treatment of human umbilical vein endothelial cells (HUVECs) with aqueous extracts of rooibos [fermented (FR) as well as green form (GR)] and honeybush [fermented form (FH)]. Study designHUVEC are either exposed to DEP (10 µg/ml) for 4 h or pre-treated with 40 and 60 µg/ml of FR or GH or FR, or 50 µg/ml orientin (OR) for 6 h prior to DEP exposure. MethodsIn vitro antioxidant capacity of the extracts was assessed and the polyphenol contents were also assessed by HPLC. ROS, cell viability, lactate dehydrogenase leakage, lipid peroxidation, GSH:GSSG ratios, conjugated diene and protein carbonyl levels were determined as indices of oxidative stress and cytotoxicity. RT-qPCR and western blot were used to assess inflammatory cytokines and antioxidant genes expression. ResultsDEP caused a dose and time-dependent increase in ROS production, significant (p < 0.001) increase in protein carbonyl (PC) formation, thiobarbituric acid reactive substances and conjugated dienes levels (p < 0.01) and a significant reduction in glutathione (GSH) redox status. Pre-incubation with either the herbal extracts or orientin attenuated these effects. The significant increase in IL-1α, IL-6, IL-8, VCAM-1 and ATF4 gene expression caused by DEP (10 µg/ml) were also attenuated by the presence of the FR, GR and FH extracts, and OR . Pre-treatment with the rooibos extracts or flavone orientin enhanced cell viability, reduced LDH leakage, enhanced mRNA expression of NQO1 and Nrf2, but repressed CYP1B1 mRNA induced by DEP. Western blot showed both the herbal tea extracts and orientin to enhance NQO1 and γGSC protein induction by DEP. ConclusionTaken together, the herbal extracts offer protection against DEP-induced oxidative stress and inflammatory response.