Obesity Represses CYP2R1, the Vitamin D 25-Hydroxylase, in the Liver and Extrahepatic Tissues.

Low plasma level of 25-hydroxyvitamin D (25-OH-D), namely vitamin D deficiency, is associated with obesity and weight loss improves 25-OH-D status. However, the mechanism behind obesity-induced vitamin D deficiency remains unclear. Here, we report that obesity suppresses the expression of cytochrome P450 (CYP) 2R1, the main vitamin D 25-hydroxylase, in both mice and humans. In humans, weight loss induced by gastric bypass surgery increased the expression of CYP2R1 in the s.c. adipose tissue suggesting recovery after the obesity-induced suppression. At the same time, CYP27B1, the vitamin D 1α-hydroxylase, was repressed by the weight loss. In a mouse (C57BL/6N) model of diet-induced obesity, the plasma 25-OH-D was decreased. In accordance, the CYP2R1 expression was strongly repressed in the liver. Moreover, obesity repressed the expression of CYP2R1 in several extrahepatic tissues, the kidney, brown adipose tissue, and testis, but not in the white adipose tissue. Obesity had a similar effect in both male and female mice. In mice, obesity repressed expression of the vitamin D receptor in brown adipose tissue. Obesity also upregulated the expression of the vitamin D receptor and CYP24A1 in the s.c. adipose tissue of a subset of mice; however, no effect was observed in the human s.c. adipose tissue. In summary, we show that obesity affects CYP2R1 expression both in the mouse and human tissues. We suggest that in mouse the CYP2R1 repression in the liver plays an important role in obesity-induced vitamin D deficiency. Currently, it is unclear whether the CYP2R1 downregulation in extrahepatic tissues could contribute to the obesity-induced low plasma 25-OH-D, however, this phenomenon may affect at least the local 25-OH-D concentrations. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

CYP-associated drug-drug interactions: A mission accomplished?

On the basis of official Finnish Medicines Authority (Fimea)-approved drug monographs, less than half of the approved small-molecule drugs between 2007 and 2016 were substrates, inhibitors or inducers of CYP enzymes, predominantly of CYP3A4. No significant unexpected, life-threatening, CYP-associated drug-drug interactions (CYP-DDIs) of newly approved drug entities have been observed in the last 10-15 years. The present analysis seems to suggest that tools to study and predict potentially significant CYP-DDIs are working and efficient.

Inhibition and induction of CYP enzymes in humans: an update.

The cytochrome P450 (CYP) enzyme family is the most important enzyme system catalyzing the phase 1 metabolism of pharmaceuticals and other xenobiotics such as herbal remedies and toxic compounds in the environment. The inhibition and induction of CYPs are major mechanisms causing pharmacokinetic drug-drug interactions. This review presents a comprehensive update on the inhibitors and inducers of the specific CYP enzymes in humans. The focus is on the more recent human in vitro and in vivo findings since the publication of our previous review on this topic in 2008. In addition to the general presentation of inhibitory drugs and inducers of human CYP enzymes by drugs, herbal remedies, and toxic compounds, an in-depth view on tyrosine-kinase inhibitors and antiretroviral HIV medications as victims and perpetrators of drug-drug interactions is provided as examples of the current trends in the field. Also, a concise overview of the mechanisms of CYP induction is presented to aid the understanding of the induction phenomena.

Pregnane X Receptor Activator Rifampin Increases Blood Pressure and Stimulates Plasma Renin Activity.

We conducted a clinical trial with 22 healthy volunteers to investigate the effects of pregnane X receptor (PXR) agonist rifampin on blood pressure (BP). The study was randomized, crossover, single-blind, and placebo-controlled. Rifampin 600 mg or placebo once daily was administered for a week and the 24-hour ambulatory BP was monitored at the end of each arm on the eighth day. Rifampin elevated the mean systolic and diastolic 24-hour BP (4.7 mmHg, P < 0.0001, and 3.0 mmHg, P < 0.001, respectively) as well as the mean heart rate (3.5 bpm, P = 0.038). The serum renin concentration and the plasma renin activity were increased. Although rifampin increased circulating 4ß-hydroxycholesterol (4ßHC) as expected, the plasma 4ßHC concentration strongly negatively correlated with 24-hour BP, especially systolic, in both rifampin and placebo arms (rifampin systolic BP, r = -0.69, P < 0.001; placebo systolic BP, r = -0.70, P < 0.001). The 4ßHC, an agonist for liver X receptor (LXR), induced renin expression modestly in LXR-α expressing Calu-6 cells but only at unphysiologically high 4ßHC concentrations. In conclusion, rifampin stimulates renin activity and has a hypertensive effect. This finding should be considered when designing interaction studies involving rifampin or other PXR agonists. Furthermore, PXR may represent a putative therapeutic target for the treatment of hypertension.

Fasting-Induced Transcription Factors Repress Vitamin D Bioactivation, a Mechanism for Vitamin D Deficiency in Diabetes.

Low 25-hydroxyvitamin D levels correlate with the prevalence of diabetes; however, the mechanisms remain uncertain. Here, we show that nutritional deprivation-responsive mechanisms regulate vitamin D metabolism. Both fasting and diabetes suppressed hepatic cytochrome P450 (CYP) 2R1, the main vitamin D 25-hydroxylase responsible for the first bioactivation step. Overexpression of coactivator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), induced physiologically by fasting and pathologically in diabetes, resulted in dramatic downregulation of CYP2R1 in mouse hepatocytes in an estrogen-related receptor α (ERRα)-dependent manner. However, PGC-1α knockout did not prevent fasting-induced suppression of CYP2R1 in the liver, indicating that additional factors contribute to the CYP2R1 repression. Furthermore, glucocorticoid receptor (GR) activation repressed the liver CYP2R1, suggesting GR involvement in the regulation of CYP2R1. GR antagonist mifepristone partially prevented CYP2R1 repression during fasting, suggesting that glucocorticoids and GR contribute to the CYP2R1 repression during fasting. Moreover, fasting upregulated the vitamin D catabolizing CYP24A1 in the kidney through the PGC-1α-ERRα pathway. Our study uncovers a molecular mechanism for vitamin D deficiency in diabetes and reveals a novel negative feedback mechanism that controls crosstalk between energy homeostasis and the vitamin D pathway.

Extranuclear Sirtuins and Metabolic Stress.

SIGNIFICANCE: Extranuclear sirtuins in cytosol (SIRT2) and mitochondria (SIRT3, SIRT4, and SIRT5) are key regulators of metabolic enzymes and the antioxidative defense mechanisms. They play an important role in the adjustment of metabolic pathways in alterations of the nutritional status. Recent Advances: Recent studies have shown that in addition to lysine deacetylation, sirtuins catalyze several different lysine deacylation reactions, removal of lipid modifications, and adenosine diphosphate-ribosylation. Large-scale studies have revealed hundreds of target proteins regulated by different sirtuin modifications. CRITICAL ISSUES: Sensing of the metabolic state and regulation of the sirtuin function and expression are critical components of the machinery, optimizing cellular functions in the switch from fed to fasting condition. Overfeeding, obesity, and metabolic diseases cause metabolic stress that dysregulates the sirtuins, which may play a role in the pathogenesis and complications of metabolic diseases such as type 2 diabetes, fatty liver disease, and cardiac diseases. In the current review, we will discuss the significance of the extranuclear sirtuins as metabolic regulators and in protection against the reactive oxygen species, and also how these sirtuins are regulated by metabolic status and their putative role in metabolic diseases. FUTURE DIRECTIONS: To efficiently utilize sirtuins as drug targets for treatment of the metabolic diseases, better understanding of the sirtuin functions, targets, regulation, and cross talk is needed. Furthermore, more studies in humans are needed to confirm the many observations mainly made in animal and cell models so far. Antioxid. Redox Signal. 28, 662-676.

Arsenic, cadmium, lead and mercury levels in blood of Finnish adults and their relation to diet, lifestyle habits and sociodemographic variables.

The Northern Finland Birth Cohort program (NFBC) is the epidemiological and longitudinal prospective general population research program, which was established to promote health and wellbeing of the population in northern Finland. The aim of present study, as a part of the NFBC program, was to analyze the blood levels of arsenic (B-As), cadmium (B-Cd), lead (B-Pb), total mercury (B-Hg) and selenium (B-Se); to compare these levels with threshold limits; to study sociodemographic factors; and to correlate these levels with calcium and haemoglobin. The study was comprised of 249 NFBC subjects, of which 123 were female and 126 were male (ages 31.1 ± 0.3 and 31.1 ± 0.4, respectively). All participants were asked to complete a questionnaire regarding diet and living habits. The geometric means (± SD) of B-As were 0.49 ± 2.80 µg/l and 0.44 ± 2.72 µg/l; B-Cd were 0.18 ± 4.02 µg/l and 0.12 ± 3.21 µg/l; B-Pb were 17.0 ± 1.8 µg/l and 9.06 ± 2.20 µg/l; B-Hg were 2.18 ± 2.02 µg/l and 1.85 ± 1.78 µg/l; and B-Se were 106.0 ± 1.3 and 94.3 ± 1.3 µg/l in males and females, respectively. Among the subjects in the present analysis, 23 % of males and 17.1 % of females had B-As levels above the ATSDR normal human levels of B-As in unexposed individuals (1.0 µg/l). The B-Pb geometric mean (12.44 µg/l) was approximately one eighth the CDC toxicological cut-off point of 100 µg/l. Twenty-one individuals (8.4 %) exceeded a B-Hg level of 5.8 µg/l. Fifty-eight females (47 %) had a B-Hg higher than 2.0 µg/l, the German Federal Environmental Agency cut-off point for women (18-69 years) who consume fish at least three times/month; therefore, their babies could be at risk of adverse effects during development.

Who watches the watchmen? Regulation of the expression and activity of sirtuins.

Sirtuins (SIRT1-7) are a family of nicotine adenine dinucleotide (NAD+)-dependent enzymes that catalyze post-translational modifications of proteins. Together, they regulate crucial cellular functions and are traditionally associated with aging and longevity. Dysregulation of sirtuins plays an important role in major diseases, including cancer and metabolic, cardiac, and neurodegerative diseases. They are extensively regulated in response to a wide range of stimuli, including nutritional and metabolic challenges, inflammatory signals or hypoxic and oxidative stress. Each sirtuin is regulated individually in a tissue- and cell-specific manner. The control of sirtuin expression involves all the major points of regulation, including transcriptional and post-translational mechanisms and microRNAs. Collectively, these mechanisms control the protein levels, localization, and enzymatic activity of sirtuins. In many cases, the regulators of sirtuin expression are also their substrates, which lead to formation of intricate regulatory networks and extensive feedback loops. In this review, we highlight the mechanisms mediating the physiologic and pathologic regulation of sirtuin expression and activity. We also discuss the consequences of this regulation on sirtuin function and cellular physiology.-Buler, M., Andersson, U., Hakkola, J. Who watches the watchmen? Regulation of the expression and activity of sirtuins.

Hypoxia-Inducible Factor Prolyl 4-Hydroxylase-2 Inhibition Protects Against Development of Atherosclerosis.

OBJECTIVE: Small-molecule hypoxia-inducible factor prolyl 4-hydroxylase (HIF-P4H) inhibitors are being explored in clinical studies for the treatment of anemia. HIF-P4H-2 (also known as PHD2 or EglN1) inhibition improves glucose and lipid metabolism and protects against obesity and metabolic dysfunction. We studied here whether HIF-P4H-2 inhibition could also protect against atherosclerosis. APPROACH AND RESULTS: Atherosclerosis development was studied in low-density lipoprotein (LDL) receptor-deficient mice treated with an oral HIF-P4H inhibitor, FG-4497, and in HIF-P4H-2-hypomorphic/C699Y-LDL receptor-mutant mice, all mice being fed a high-fat diet. FG-4497 administration to LDL receptor-deficient mice reduced the area of atherosclerotic plaques by ≈50% when compared with vehicle-treated controls and also reduced their weight gain, insulin resistance, liver and white adipose tissue (WAT) weights, adipocyte size, number of inflammation-associated WAT macrophage aggregates and the high-fat diet-induced increases in serum cholesterol levels. The levels of atherosclerosis-protecting circulating autoantibodies against copper-oxidized LDL were increased. The decrease in atherosclerotic plaque areas correlated with the reductions in weight, serum cholesterol levels, and WAT macrophage aggregates and the autoantibody increase. FG-4497 treatment stabilized HIF-1α and HIF-2α and altered the expression of glucose and lipid metabolism and inflammation-associated genes in liver and WAT. The HIF-P4H-2-hypomorphic/C699Y-LDL receptor-mutant mice likewise had a ≈50% reduction in atherosclerotic plaque areas, reduced WAT macrophage aggregate numbers, and increased autoantibodies against oxidized LDL, but did not have reduced serum cholesterol levels. CONCLUSIONS: HIF-P4H-2 inhibition may be a novel strategy for protecting against the development of atherosclerosis. The mechanisms involve beneficial modulation of the serum lipid profile and innate immune system and reduced inflammation.

Tumour suppressor protein p53 regulates the stress activated bilirubin oxidase cytochrome P450 2A6.

Human cytochrome P450 (CYP) 2A6 enzyme has been proposed to play a role in cellular defence against chemical-induced oxidative stress. The encoding gene is regulated by various stress activated transcription factors. This paper demonstrates that p53 is a novel transcriptional regulator of the gene. Sequence analysis of the CYP2A6 promoter revealed six putative p53 binding sites in a 3kb proximate promoter region. The site closest to transcription start site (TSS) is highly homologous with the p53 consensus sequence. Transfection with various stepwise deletions of CYP2A6-5'-Luc constructs--down to -160bp from the TSS--showed p53 responsiveness in p53 overexpressed C3A cells. However, a further deletion from -160 to -74bp, including the putative p53 binding site, totally abolished the p53 responsiveness. Electrophoretic mobility shift assay with a probe containing the putative binding site showed specific binding of p53. A point mutation at the binding site abolished both the binding and responsiveness of the recombinant gene to p53. Up-regulation of the endogenous p53 with benzo[α]pyrene--a well-known p53 activator--increased the expression of the p53 responsive positive control and the CYP2A6-5'-Luc construct containing the intact p53 binding site but not the mutated CYP2A6-5'-Luc construct. Finally, inducibility of the native CYP2A6 gene by benzo[α]pyrene was demonstrated by dose-dependent increases in CYP2A6 mRNA and protein levels along with increased p53 levels in the nucleus. Collectively, the results indicate that p53 protein is a regulator of the CYP2A6 gene in C3A cells and further support the putative cytoprotective role of CYP2A6.

Sodium valproate induces mitochondrial respiration dysfunction in HepG2 in vitro cell model.

Sodium valproate (VPA) is a potentially hepatotoxic antiepileptic drug. Risk of VPA-induced hepatotoxicity is increased in patients with mitochondrial diseases and especially in patients with POLG1 gene mutations. We used a HepG2 cell in vitro model to investigate the effect of VPA on mitochondrial activity. Cells were incubated in glucose medium and mitochondrial respiration-inducing medium supplemented with galactose and pyruvate. VPA treatments were carried out at concentrations of 0-2.0mM for 24-72 h. In both media, VPA caused decrease in oxygen consumption rates and mitochondrial membrane potential. VPA exposure led to depleted ATP levels in HepG2 cells incubated in galactose medium suggesting dysfunction in mitochondrial ATP production. In addition, VPA exposure for 72 h increased levels of mitochondrial reactive oxygen species (ROS), but adversely decreased protein levels of mitochondrial superoxide dismutase SOD2, suggesting oxidative stress caused by impaired elimination of mitochondrial ROS and a novel pathomechanism related to VPA toxicity. Increased cell death and decrease in cell number was detected under both metabolic conditions. However, immunoblotting did not show any changes in the protein levels of the catalytic subunit A of mitochondrial DNA polymerase γ, the mitochondrial respiratory chain complexes I, II and IV, ATP synthase, E3 subunit dihydrolipoyl dehydrogenase of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glutathione peroxidase. Our results show that VPA inhibits mitochondrial respiration and leads to mitochondrial dysfunction, oxidative stress and increased cell death, thus suggesting an essential role of mitochondria in VPA-induced hepatotoxicity.

SIRT5 is under the control of PGC-1α and AMPK and is involved in regulation of mitochondrial energy metabolism.

The sirtuins (SIRTs; SIRT1-7) are a family of NAD(+)-dependent enzymes that dynamically regulate cellular physiology. Apart from SIRT1, the functions and regulatory mechanisms of the SIRTs are poorly defined. We explored regulation of the SIRT family by 2 energy metabolism-controlling factors: peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and AMP-activated protein kinase (AMPK). Overexpression of PGC-1α in mouse primary hepatocytes increased SIRT5 mRNA expression 4-fold and also the protein in a peroxisome proliferator-activated receptor α (PPARα)- and estrogen-related receptor α (ERRα)-dependent manner. Furthermore, food withdrawal increased SIRT5 mRNA 1.3-fold in rat liver. Overexpression of AMPK in mouse hepatocytes increased expression of SIRT1, SIRT2, SIRT3, and SIRT6 <2-fold. In contrast, SIRT5 mRNA was down-regulated by 58%. The antidiabetes drug metformin (1 mM), an established AMPK activator, reduced the mouse SIRT5 protein level by 44% in cultured hepatocytes and by 31% in liver in vivo (300 mg/kg, 7 d). Metformin also induced hypersuccinylation of mitochondrial proteins. Moreover, SIRT5 overexpression increased ATP synthesis and oxygen consumption in HepG2 cells, but did not affect mitochondrial biogenesis. In summary, our results identified SIRT5 as a novel factor that controls mitochondrial function. Moreover, SIRT5 levels are regulated by PGC-1α and AMPK, which have opposite effects on its expression.-Buler, M., Aatsinki, S.-M., Izzi, V., Uusimaa, J., Hakkola, J. SIRT5 is under the control of PGC-1α and AMPK and is involved in regulation of mitochondrial energy metabolism.

Metformin induces PGC-1α expression and selectively affects hepatic PGC-1α functions.

BACKGROUND AND PURPOSE: The objective of this study was to determine how the AMPK activating antidiabetic drug metformin affects the major activator of hepatic gluconeogenesis, PPARγ coactivator 1α (PGC-1α) and liver functions regulated by PGC-1α. EXPERIMENTAL APPROACH: Mouse and human primary hepatocytes and mice in vivo were treated with metformin. Adenoviral overexpression, siRNA and reporter gene constructs were used for mechanistic studies. KEY RESULTS: Metformin increased PGC-1α mRNA and protein expression in mouse primary hepatocytes. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) (another AMPK activator) had the opposite effect. Metformin also increased PGC-1α in human primary hepatocytes; this effect of metformin was abolished by AMPK inhibitor compound C and sirtuin 1 siRNA. AMPK overexpression by AMPK-Ad also increased PGC-1α. Whereas metformin increased PGC-1α, it down-regulated gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Furthermore, metformin attenuated the increase in PEPCK and G6Pase mRNAs induced by PGC-1α overexpression, but did not affect PGC-1α-mediated induction of mitochondrial genes. Metformin down-regulated several key transcription factors that mediate the effect of PGC-1α on gluconeogenic genes including Krüppel-like factor 15, forkhead box protein O1 and hepatocyte NF 4α, whereas it increased nuclear respiratory factor 1, which is involved in PGC-1α-mediated regulation of mitochondrial proteins. CONCLUSIONS AND IMPLICATIONS: Down-regulation of PGC-1α is not necessary for suppression of gluconeogenic genes by metformin. Importantly, metformin selectively affects hepatic PGC-1α-mediated gene regulation and prevents activation of gluconeogenesis, but does not influence its regulation of mitochondrial genes. These results identify selective modulation of hepatic PGC-1α functions as a novel mechanism involved in the therapeutic action of metformin.

Function and regulation of the Cyp2a5/CYP2A6 genes in response to toxic insults in the liver.

The mouse hepatic cytochrome P450 (CYP) 2A5 and its human orthologue CYP2A6 catalyse the metabolism of a number of drugs and toxins, such as halothane and aflatoxin B1. The enzymes are named "Coumarin 7-hydroxylase" and "Nicotine Hydroxylase", respectively, by virtue of their high affinity and specific activity towards these compounds. Bilirubin, the breakdown product of haem, has been suggested to be the endogenous substrate for both enzymes. Uniquely, CYP2A5 and CYP2A6 are induced during pathological conditions associated with liver injury when the function of most other CYP enzymes is compromised, which suggests an exceptional mode of regulation of the corresponding genes. Regulation of these genes is indeed complex where the promoters interact with multiple stress-activated transcription factors. The Cyp2a5 promoter contains a "stress-responding" cluster of binding motifs, which interact with major mediators of toxic insults including nuclear factor-E2 p45-related factor 2 (Nrf2) and aryl hydrocarbon receptor (AhR). These interactions are crucial in the up-regulation of the genes under stress conditions. Additionally, elevated transcription is also achieved through mRNA stabilisation mediated by interaction of the stress activated heterogenous ribonucleoprotein A1 (hnRNP A1) with the 3'UTR of the CYP2A5/CYP2A6 mRNA. The up-regulation via enhanced transcription combined with mRNA stabilisation, as seen in some of the stress situations, leads to a particularly strong, fast and persistent response. This review brings together knowledge obtained from studies in our laboratories and others' on regulation of Cyp2a5/CYP2A6 genes in response to toxic insults and toxicological significance of their catalytic activities that may provide clues to a functional role of the enzymes in relation to liver toxicity.

Metformin reduces hepatic expression of SIRT3, the mitochondrial deacetylase controlling energy metabolism.

Metformin inhibits ATP production in mitochondria and this may be involved in the anti-hyperglycemic effects of the drug. Sirtuin 3 (SIRT3) is a mitochondrial protein deacetylase that regulates the function of the electron transport chain and maintains basal ATP yield. We hypothesized that metformin treatment could diminish mitochondrial ATP production through downregulation of SIRT3 expression. Glucagon and cAMP induced SIRT3 mRNA in mouse primary hepatocytes. Metformin prevented SIRT3 induction by glucagon. Moreover, metformin downregulated constitutive expression of SIRT3 in primary hepatocytes and in the liver in vivo. Estrogen related receptor alpha (ERRα) mediates regulation of Sirt3 gene by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). ERRα mRNA expression was regulated in a similar manner as SIRT3 mRNA by glucagon, cAMP and metformin. However, a higher metformin concentration was required for downregulation of ERRα than SIRT3. ERRα siRNA attenuated PGC-1α mediated induction of SIRT3, but did not affect constitutive expression. Overexpression of the constitutively active form of AMP-activated protein kinase (AMPK) induced SIRT3 mRNA, indicating that the SIRT3 downregulation by metformin is not mediated by AMPK. Metformin reduced the hepatocyte ATP level. This effect was partially counteracted by SIRT3 overexpression. Furthermore, metformin decreased mitochondrial SIRT3 protein levels and this was associated with enhanced acetylation of several mitochondrial proteins. However, metformin increased mitochondrial mass in hepatocytes. Altogether, our results indicate that metformin attenuates mitochondrial expression of SIRT3 and suggest that this mechanism is involved in regulation of energy metabolism by metformin in the liver and may contribute to the therapeutic action of metformin.

Heme and heme biosynthesis intermediates induce heme oxygenase-1 and cytochrome P450 2A5, enzymes with putative sequential roles in heme and bilirubin metabolism: different requirement for transcription factor nuclear factor erythroid- derived 2-like 2.

Cytochrome P450 2A5 (CYP2A5) oxidizes bilirubin to biliverdin and represents a putative candidate for maintaining bilirubin at safe but adequate antioxidant levels. Curiously, CYP2A5 is induced by both excessive heme and chemicals that inhibit heme synthesis. We hypothesized that heme homeostasis is a key modifier of Cyp2a5 expression via transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2) and characterized the coordination of CYP2A5 and heme oxygenase-1 (HMOX1) responses using wild-type and Nrf2(-/-) primary mouse hepatocytes. HMOX1 was rapidly elevated by exogenous hemin, thereby limiting the transactivation of Cyp2a5 until high heme (> 5µM) exposure. Nrf2 was mandatory for CYP2A5 but not for HMOX1 induction by heme. CYP2A5 was intensively and HMOX1 moderately elevated in heme synthesis blockades by succinylacetone and N-methyl protoporphyrin IX, and Nrf2 partially mediated the induction of CYP2A5. Immunoelectron microscopy revealed that CYP2A5 is targeted Nrf2 dependently both to the endoplasmic reticulum (ER) and mitochondria. However, excessive heme increased CYP2A5 predominantly in the ER. Phenobarbital, dibutyryl-cAMP, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) overexpression stimulate heme biosynthesis and induce CYP2A5. Acute but not chronic CYP2A5 induction by phenobarbital required Nrf2, whereas CYP2A5 induction by dibutyryl-cAMP and PGC-1α was potentiated by Nrf2 knockout. Collectively, heme homeostasis is established as a crucial regulator of hepatic Cyp2a5 expression mediated via Nrf2 activation, whereas Nrf2 is redundant for Hmox1 induction by heme. Similar subcellular targeting and coordination of CYP2A5 and HMOX1 responses suggest favorable conditions for enhanced CYP2A5-mediated bilirubin maintenance in altered heme homeostasis that predisposes to oxidative stress.

Characterization of human cytochrome P450 induction by pesticides.

Pesticides are a large group of structurally diverse toxic chemicals. The toxicity may be modified by cytochrome P450 (CYP) enzyme activity. In the current study, we have investigated effects and mechanisms of 24 structurally varying pesticides on human CYP expression. Many pesticides were found to efficiently activate human pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR). Out of the 24 compounds tested, 14 increased PXR- and 15 CAR-mediated luciferase activities at least 2-fold. While PXR was predominantly activated by pyrethroids, CAR was, in addition to pyrethroids, well activated by organophosphates and several carbamates. Induction of CYP mRNAs and catalytic activities was studied in the metabolically competent, human derived HepaRG cell line. CYP3A4 mRNA was induced most powerfully by pyrethroids; 50 µM cypermethrin increased CYP3A4 mRNA 35-fold. CYP2B6 was induced fairly equally by organophosphate, carbamate and pyrethroid compounds. Induction of CYP3A4 and CYP2B6 by these compound classes paralleled their effects on PXR and CAR. The urea herbicide diuron and the triazine herbicide atrazine induced CYP2B6 mRNA more than 10-fold, but did not activate CAR indicating that some pesticides may induce CYP2B6 via CAR-independent mechanisms. CYP catalyzed activities were induced much less than the corresponding mRNAs. At least in some cases, this is probably due to significant inhibition of CYP enzymes by the studied pesticides. Compared with human CAR activation and CYP2B6 expression, pesticides had much less effect on mouse CAR and CYP2B10 mRNA. Altogether, pesticides were found to be powerful human CYP inducers acting through both PXR and CAR.

Energy-sensing factors coactivator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and AMP-activated protein kinase control expression of inflammatory mediators in liver: induction of interleukin 1 receptor antagonist.

Obesity and insulin resistance are associated with chronic, low grade inflammation. Moreover, regulation of energy metabolism and immunity are highly integrated. We hypothesized that energy-sensitive coactivator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and AMP-activated protein kinase (AMPK) may modulate inflammatory gene expression in liver. Microarray analysis revealed that PGC-1α up-regulated expression of several cytokines and cytokine receptors, including interleukin 15 receptor α (IL15Rα) and, even more importantly, anti-inflammatory interleukin 1 receptor antagonist (IL1Rn). Overexpression of PGC-1α and induction of PGC-1α by fasting, physical exercise, glucagon, or cAMP was associated with increased IL1Rn mRNA and protein expression in hepatocytes. Knockdown of PGC-1α by siRNA down-regulated cAMP-induced expression of IL1Rn in mouse hepatocytes. Furthermore, knockdown of peroxisome proliferator-activated receptor α (PPARα) attenuated IL1Rn induction by PGC-1α. Overexpression of PGC-1α, at least partially through IL1Rn, suppressed interleukin 1ß-induced expression of acute phase proteins, C-reactive protein, and haptoglobin. Fasting and exercise also induced IL15Rα expression, whereas glucagon and cAMP resulted in reduction in IL15Rα mRNA levels. Finally, AMPK activator metformin and adenoviral overexpression of AMPK up-regulated IL1Rn and down-regulated IL15Rα in primary hepatocytes. We conclude that PGC-1α and AMPK alter inflammatory gene expression in liver and thus integrate energy homeostasis and inflammation. Induction of IL1Rn by PGC-1α and AMPK may be involved in the beneficial effects of exercise and caloric restriction and putative anti-inflammatory effects of metformin.

Cytochrome P450-mediated pulmonary metabolism of carcinogens: regulation and cross-talk in lung carcinogenesis.

Lung cancer is strongly associated with exogenous risk factors, in particular tobacco smoking and asbestos exposure. New research data are accumulating about the regulation of the metabolism of tobacco carcinogens and the metabolic response to oxidative stress. These data provide mechanistic details about why well known risk factors cause lung cancer. The purpose of this review is to evaluate the present knowledge of the role of cytochrome P450 (CYP) enzymes in the metabolism of tobacco carcinogens and associations with tobacco and asbestos carcinogenesis. Major emphasis is placed on human data and regulatory pathways involved in CYP regulation and lung carcinogenesis. The most exciting new research findings concern cross-talk of the CYP-regulating aryl hydrocarbon receptor with other transcription factors, such as nuclear factor-erythroid 2-related factor 2, involved in the regulation of xenobiotic metabolism and antioxidant enzymes. This cross-talk between transcription factors may provide mechanistic evidence for clinically relevant issues, such as differences in lung cancers between men and women and the synergism between tobacco and asbestos as lung carcinogens.

Cytochrome P450 2A5 constitutive expression and induction by heavy metals is dependent on redox-sensitive transcription factor Nrf2 in liver.

Mouse cytochrome P450 2A5 (CYP2A5) is upregulated in various pathophysiological liver diseases and induced by structurally variable hepatotoxic chemicals. A putative common feature for all of these conditions is altered cellular redox status. Nuclear factor erythroid 2-like 2 (Nrf2) is a transcription factor that is post-translationally regulated by oxidative stress and controls the transcription of numerous protective target genes. In the present study, we have extensively characterized the regulation of Cyp2a5 by Nrf2 and compared it to a well-characterized target gene Hmox1. The treatment of mouse primary hepatocytes with lead chloride, methylmercury chloride, or phenethyl isothiocyanate all leads to nuclear accumulation of Nrf2. Both CYP2A5 and HMOX1 were induced by all three compounds; however, HMOX1 responded more rapidly and transiently as compared to CYP2A5. Experiments in Nrf2(-/-) primary hepatocytes showed that Nrf2 is crucial for CYP2A5 induction but not for elevation of HMOX1. Both CYP2A5 and HMOX1 were upregulated by Nrf2 overexpression and downregulated by Keap1 or Bach1 overexpression. However, in all cases, CYP2A5 responded much more potently. Results in Nrf2-deficient animals showed that CYP2A5 expression is significantly attenuated in the absence of Nrf2, while expression of HMOX1 was unaffected. Therefore, Cyp2a5 joins the group of genes constitutively regulated by Nrf2. Our current results unequivocally show that expression of CYP2A5 is tightly controlled by Nrf2 in liver. Nrf2 is needed for constitutive expression of CYP2A5, and CYP2A5 is also sensitively upregulated by an increased level of Nrf2 protein. Therefore, CYP2A5 upregulation could be a useful indicator for hepatic activation of the Nrf2 pathway.