Metabolic effects of nuclear receptor activation in vivo after 28-day oral exposure to three endocrine-disrupting chemicals.

Environmental exposure to endocrine-disrupting chemicals (EDCs) can lead to metabolic disruption, resulting in metabolic complications including adiposity, dyslipidemia, hepatic lipid accumulation, and glucose intolerance. Hepatic nuclear receptor activation is one of the mechanisms mediating metabolic effects of EDCs. Here, we investigated the potential to use a repeated dose 28-day oral toxicity test for identification of EDCs with metabolic endpoints. Bisphenol A (BPA), pregnenolone-16α-carbonitrile (PCN), and perfluorooctanoic acid (PFOA) were used as reference compounds. Male and female wild-type C57BL/6 mice were orally exposed to 5, 50, and 500 µg/kg of BPA, 1000, 10 000, and 100 000 µg/kg of PCN and 50 and 300 µg/kg of PFOA for 28 days next to normal chow diet. Primary endpoints were glucose tolerance, hepatic lipid accumulation, and plasma lipids. After 28-day exposure, no changes in body weight and glucose tolerance were observed in BPA-, PCN-, or PFOA-treated males or females. PCN and PFOA at the highest dose in both sexes and BPA at the middle and high dose in males increased relative liver weight. PFOA reduced plasma triglycerides in males and females, and increased hepatic triglyceride content in males. PCN and PFOA induced hepatic expression of typical pregnane X receptor (PXR) and peroxisome proliferator-activated receptor (PPAR)α target genes, respectively. Exposure to BPA resulted in limited gene expression changes. In conclusion, the observed changes on metabolic health parameters were modest, suggesting that a standard repeated dose 28-day oral toxicity test is not a sensitive method for the detection of the metabolic effect of EDCs.

Adverse outcome pathway for pregnane X receptor-induced hypercholesterolemia.

Pharmaceuticals and environmental contaminants contribute to hypercholesterolemia. Several chemicals known to cause hypercholesterolemia, activate pregnane X receptor (PXR). PXR is a nuclear receptor, classically identified as a sensor of chemical environment and regulator of detoxification processes. Later, PXR activation has been shown to disrupt metabolic functions such as lipid metabolism and recent findings have shown PXR activation to promote hypercholesterolemia through multiple mechanisms. Hypercholesterolemia is a major causative risk factor for atherosclerosis and greatly promotes global health burden. Metabolic disruption by PXR activating chemicals leading to hypercholesterolemia represents a novel toxicity pathway of concern and requires further attention. Therefore, we constructed an adverse outcome pathway (AOP) by collecting the available knowledge considering the molecular mechanisms for PXR-mediated hypercholesterolemia. AOPs are tools of modern toxicology for systematizing mechanistic knowledge to assist health risk assessment of chemicals. AOPs are formalized and structured linear concepts describing a link between molecular initiating event (MIE) and adverse outcome (AO). MIE and AO are connected via key events (KE) through key event relationships (KER). We present a plausible route of how PXR activation (MIE) leads to hypercholesterolemia (AO) through direct regulation of cholesterol synthesis and via activation of sterol regulatory element binding protein 2-pathway.

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.

The EDCMET Project: Metabolic Effects of Endocrine Disruptors.

Endocrine disruptors (EDs) are defined as chemicals that mimic, block, or interfere with hormones in the body's endocrine systems and have been associated with a diverse array of health issues. The concept of endocrine disruption has recently been extended to metabolic alterations that may result in diseases, such as obesity, diabetes, and fatty liver disease, and constitute an increasing health concern worldwide. However, while epidemiological and experimental data on the close association of EDs and adverse metabolic effects are mounting, predictive methods and models to evaluate the detailed mechanisms and pathways behind these observed effects are lacking, thus restricting the regulatory risk assessment of EDs. The EDCMET (Metabolic effects of Endocrine Disrupting Chemicals: novel testing METhods and adverse outcome pathways) project brings together systems toxicologists; experimental biologists with a thorough understanding of the molecular mechanisms of metabolic disease and comprehensive in vitro and in vivo methodological skills; and, ultimately, epidemiologists linking environmental exposure to adverse metabolic outcomes. During its 5-year journey, EDCMET aims to identify novel ED mechanisms of action, to generate (pre)validated test methods to assess the metabolic effects of Eds, and to predict emergent adverse biological phenotypes by following the adverse outcome pathway (AOP) paradigm.

Regulation of hepatic energy metabolism by the nuclear receptor PXR.

The pregnane X receptor (PXR) is a nuclear receptor that is traditionally thought to be specialized for sensing xenobiotic exposure. In concurrence with this feature PXR was originally identified to regulate drug-metabolizing enzymes and transporters. During the last ten years it has become clear that PXR harbors broader functions. Evidence obtained both in experimental animals and humans indicate that ligand-activated PXR regulates hepatic glucose and lipid metabolism and affects whole body metabolic homeostasis. Currently, the consequences of PXR activation on overall metabolic health are not yet fully understood and varying results on the effect of PXR activation or knockout on metabolic disorders and weight gain have been published in mouse models. Rifampicin and St. John's wort, the prototypical human PXR agonists, impair glucose tolerance in healthy volunteers. Chronic exposure to PXR agonists could potentially represent a risk factor for diabetes and metabolic syndrome. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

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.

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.

Inhibition of activin receptor 2 signalling ameliorates metabolic dysfunction-associated steatotic liver disease in western diet/L-NAME induced cardiometabolic disease.

OBJECTIVE: Blockade of activin 2 receptor (ACVR2) signaling has been shown to improve insulin sensitivity and aid in weight loss. Inhibition of ACVR2 signaling restores cardiac function in multiple heart failure models. However, its potential in the treatment of obesity-related cardiometabolic disease remains unknown. Here, we investigated targeting ACVR2 signaling in cardiometabolic disease manifested with metabolic dysfunction-associated steatotic liver disease (MASLD). METHODS: Mice were fed a high-fat, high-sugar diet combined with the administration of nitric oxide synthase inhibitor L-NAME in drinking water, which causes hypertensive stress. For the last eight weeks, the mice were treated with the soluble ACVR2B decoy receptor (sACVR2B-Fc). RESULTS: sACVR2B-Fc protected against the development of comorbidities associated with cardiometabolic disease. This was most pronounced in the liver where ACVR2 blockade attenuated the development of MASLD including cessation of pro-fibrotic activation. It also significantly reduced total plasma cholesterol levels, impeded brown adipose tissue whitening, and improved cardiac diastolic function. In vitro, ACVR2 ligands activin A, activin B and GDF11 induced profibrotic signaling and the proliferation of human cardiac fibroblasts. CONCLUSIONS: Blockade of ACVR2B exerts broad beneficial effects for therapy of cardiometabolic disease. By reducing obesity, ameliorating cardiovascular deterioration and restraining MASLD, blockade of ACVR2B signaling proves a potential target in MASLD and its comorbidities.

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.

Pregnane X receptor activation remodels glucose metabolism to promote NAFLD development in obese mice.

OBJECTIVE: Both obesity and exposure to chemicals may induce non-alcoholic fatty liver disease (NAFLD). Pregnane X Receptor (PXR) is a central target of metabolism disrupting chemicals and disturbs hepatic glucose and lipid metabolism. We hypothesized that the metabolic consequences of PXR activation may be modified by existing obesity and associated metabolic dysfunction. METHODS: Wildtype and PXR knockout male mice were fed high-fat diet to induce obesity and metabolic dysfunction. PXR was activated with pregnenolone-16α-carbonitrile. Glucose metabolism, hepatosteatosis, insulin signaling, glucose uptake, liver glycogen, plasma and liver metabolomics, and liver, white adipose tissue, and muscle transcriptomics were investigated. RESULTS: PXR activation aggravated obesity-induced liver steatosis by promoting lipogenesis and inhibiting fatty acid disposal. Accordingly, hepatic insulin sensitivity was impaired and circulating alanine aminotransferase level increased. Lipid synthesis was facilitated by increased liver glucose uptake and utilization of glycogen reserves resulting in dissociation of hepatosteatosis and hepatic insulin resistance from the systemic glucose tolerance and insulin sensitivity. Furthermore, glucagon-induced hepatic glucose production was impaired. PXR deficiency did not protect from the metabolic manifestations of obesity, but the liver transcriptomics and metabolomics profiling suggest diminished activation of inflammation and less prominent changes in the overall metabolite profile. CONCLUSIONS: Obesity and PXR activation by chemical exposure have a synergistic effect on NAFLD development. To support liver fat accumulation the PXR activation reorganizes glucose metabolism that seemingly improves systemic glucose metabolism. This implies that obese individuals, already predisposed to metabolic diseases, may be more susceptible to harmful metabolic effects of PXR-activating drugs and environmental chemicals.

Streptozotocin-induced Diabetes Represses Hepatic CYP2R1 Expression but Induces Vitamin D 25-Hydroxylation in Male Mice.

Vitamin D deficiency [ie, low plasma 25-hydroxyvitamin D (25-OH-D)] associates with the prevalence of metabolic diseases including type 1 diabetes; however, the molecular mechanisms are incompletely understood. Recent studies have indicated that both fasting and metabolic diseases suppress the cytochrome P450 (CYP) 2R1, the major hepatic vitamin D 25-hydroxylase. We specifically studied the effect of a mouse model of type 1 diabetes on the regulation of Cyp2r1 and vitamin D status. We show that streptozotocin-induced diabetes in mice suppresses the expression of the Cyp2r1 in the liver. While insulin therapy normalized the blood glucose levels in the diabetic mice, it did not rescue the diabetes-induced suppression of Cyp2r1. Similar regulation of Cyp2r1 was observed also in the kidney. Plasma 25-OH-D level was not decreased and was, in contrast, higher after 4 and 8 weeks of diabetes. Furthermore, the vitamin D 25-hydroxylase activity was increased in the livers of the diabetic mice, suggesting compensation of the Cyp2r1 repression by other vitamin D 25-hydroxylase enzymes. Cyp27b1, the vitamin D 1α-hydroxylase, expression in the kidney and the plasma 1α,25-dihydroxyvitamin D level were higher after 4 weeks of diabetes, while both were normalized after 13 weeks. In summary, these results indicate that in the mouse model of type 1 diabetes suppression of hepatic Cyp2r1 expression does not result in reduced hepatic vitamin D 25-hydroxylase activity and vitamin D deficiency. This may be due to induction of other vitamin D 25-hydroxylase enzymes in response to diabetes.

Nuclear Receptor PXR in Drug-Induced Hypercholesterolemia.

Atherosclerosis is a major global health concern. The central modifiable risk factors and causative agents of the disease are high total and low-density lipoprotein (LDL) cholesterol. To reduce morbidity and mortality, a thorough understanding of the factors that influence an individual's cholesterol status during the decades when the arteria-narrowing arteriosclerotic plaques are forming is critical. Several drugs are known to increase cholesterol levels; however, the mechanisms are poorly understood. Activation of pregnane X receptor (PXR), the major regulator of drug metabolism and molecular mediator of clinically significant drug-drug interactions, has been shown to induce hypercholesterolemia. As a major sensor of the chemical environment, PXR may in part mediate hypercholesterolemic effects of drug treatment. This review compiles the current knowledge of PXR in cholesterol homeostasis and discusses the role of PXR in drug-induced hypercholesterolemia.

Pregnane X Receptor‒4ß-Hydroxycholesterol Axis in the Regulation of Overweight- and Obesity-Induced Hypertension.

Background Mechanisms mediating hypertensive effects of overweight and obesity have not been fully elucidated. We showed previously that activation of pregnane X receptor (PXR) by rifampicin elevates 24-hour blood pressure (BP) and plasma 4ß-hydroxycholesterol (4ßHC), agonist for liver X receptor (LXR). Methods and Results In combined "PXR activation data set" (n=62) of 4 clinical trials, 1 week rifampicin dosing increased office systolic BP (SBP) by 3.1 mm Hg, DBP 1.8 mm Hg, and mean arterial pressure 2.2 mm Hg in comparison with placebo (P<0.01). Plasma 4ßHC had negative correlation with SBP both in rifampicin (r=-0.46, P=0.0002) and placebo (r=-0.45, P=0.0003) arms, although 4ßHC was elevated >3-fold by rifampicin. In "non-intervention data set" (n=102) of patients with obesity and healthy volunteers (body mass index, 19.2-55.2 kg/m2), 4ßHC had negative correlations (P<0.00001) with office SBP (r=-0.51), diastolic BP (r=-0.50), and mean arterial pressure (r=-0.54). Lean women had higher 4ßHC than men, with increasing weight repressing 4ßHC (r=-0.62, P<0.00001) in both sexes. In multiple linear regression analysis, the only statistically significant predictor for SBP was 4ßHC. Six-day PXR agonist dosing elevated SBP in rats (n=7-8/group). PXR activation elevated 4ßHC and after PXR agonist was withdrawn and elevated 4ßHC was left to act alone, SBP was reduced on days 7 to 14 in comparison with control rats. Conclusions PXR activation elevates SBP. Elevated circulating 4ßHC lowers SBP in rats, and higher 4ßHC is an independent predictor of lower SBP in humans. PXR-4ßHC-LXR is novel BP-regulating pathway deregulated in overweight and obesity by repressed 4ßHC, with implications for sex-specific BP regulation. Registration URL: https://www.clinicaltrials.gov; Unique identifiers: NCT00985270, NCT01293422, NCT01690104, NCT02329405, and NCT01330251.

Rifampicin induces the bone form of alkaline phosphatase in humans.

Pregnane X receptor (PXR) is a xenobiotic-sensing nuclear receptor that regulates drug metabolism in the liver and intestine. In our clinical trials on healthy volunteers to discover novel metabolic functions of PXR activation, we observed that rifampicin, a well-established ligand for human PXR, 600 mg daily for a week, increased the plasma alkaline phosphatase (ALP) significantly compared with the placebo. Further analysis with lectin affinity electrophoresis revealed that especially the bone form of ALP was elevated. To investigate the mechanism(s) of bone ALP induction, we employed osteoblast lineage differentiated from human primary bone marrow-derived mesenchymal stromal cells. Rifampicin treatment increased ALP activity and mRNA level of bone biomarker genes (ALP, MGP, OPN and OPG). PXR expression was detected in the cells, but the expression was very low compared with the human liver. To further investigate the potential role of PXR in the ALP induction, we treated mice and rats with a rodent PXR ligand pregnenolone 16α-carbonitrile (PCN). However, PCN treatment did not increase plasma ALP activity or bone ALP mRNA expression. In conclusion, rifampicin treatment induces the bone form of ALP in the serum of healthy human volunteers. Further studies are required to establish the mechanism of this novel finding.

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.

Activation of pregnane X receptor induces atherogenic lipids and PCSK9 by a SREBP2-mediated mechanism.

BACKGROUND AND PURPOSE: Many drugs and environmental contaminants induce hypercholesterolemia and promote the risk of atherosclerotic cardiovascular disease. We tested the hypothesis that pregnane X receptor (PXR), a xenobiotic-sensing nuclear receptor, regulates the level of circulating atherogenic lipids in humans and utilized mouse experiments to identify the mechanisms involved. EXPERIMENTAL APPROACH: We performed serum NMR metabolomics in healthy volunteers administered rifampicin, a prototypical human PXR ligand or placebo in a crossover setting. We used high-fat diet fed wild-type and PXR knockout mice to investigate the mechanisms mediating the PXR-induced alterations in cholesterol homeostasis. KEY RESULTS: Activation of PXR induced cholesterogenesis both in pre-clinical and clinical settings. In human volunteers, rifampicin increased intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL) and total cholesterol and lathosterol-cholesterol ratio, a marker of cholesterol synthesis, suggesting increased cholesterol synthesis. Experiments in mice indicated that PXR activation causes widespread induction of the cholesterol synthesis genes including the rate-limiting Hmgcr and upregulates the intermediates in the Kandutsch-Russell cholesterol synthesis pathway in the liver. Additionally, PXR activation induced plasma proprotein convertase subtilisin/kexin type 9 (PCSK9), a negative regulator of hepatic LDL uptake, in both mice and humans. We propose that these effects were mediated through increased proteolytic activation of sterol regulatory element-binding protein 2 (SREBP2) in response to PXR activation. CONCLUSION AND IMPLICATIONS: PXR activation induces cholesterol synthesis, elevating LDL and total cholesterol in humans. The PXR-SREBP2 pathway is a novel regulator of the cholesterol and PCSK9 synthesis and a molecular mechanism for drug- and chemical-induced hypercholesterolemia.

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.