Multisite Evaluation and Validation of Optical Genome Mapping for Prenatal Genetic Testing.

Prenatal diagnostic testing of amniotic fluid, chorionic villi, or more rarely, fetal cord blood is recommended following a positive or unreportable noninvasive cell-free fetal DNA test, abnormal maternal biochemical serum screen, abnormal ultrasound, or increased genetic risk for a cytogenomic abnormality based on family history. Although chromosomal microarray is recommended as the first-tier prenatal diagnostic test, in practice, multiple assays are often assessed in concert to achieve a final diagnostic result. The use of multiple methodologies is costly, time consuming, and labor intensive. Optical genome mapping (OGM) is an emerging technique with application for prenatal diagnosis because of its ability to detect and resolve, in a single assay, all classes of pathogenic cytogenomic aberrations. In an effort to characterize the potential of OGM as a novel alternative to traditional standard of care (SOC) testing of prenatal samples, OGM was performed on a total of 200 samples representing 123 unique cases, which were previously tested with SOC methods (92/123 = 74.7% cases tested with at least two SOCs). OGM demonstrated an overall accuracy of 99.6% when compared with SOC methods, a positive predictive value of 100%, and 100% reproducibility between sites, operators, and instruments. The standardized workflow, cost-effectiveness, and high-resolution cytogenomic analysis demonstrate the potential of OGM to serve as a first-tier test for prenatal diagnosis.

Safety, Efficacy, and Cost of 0.4-mg Versus 2-mg Intranasal Naloxone for Treatment of Prehospital Opioid Overdose.

BACKGROUND: Intranasal naloxone is commonly used to treat prehospital opioid overdose. However, the optimal dose is unclear, and currently, no study exists comparing the clinical effect of intranasal naloxone at different doses. OBJECTIVE: The goal of this investigation was to compare the safety, efficacy, and cost of 0.4- versus 2-mg intranasal naloxone for treatment of prehospital opioid overdose. METHODS: A retrospective, cross-sectional study was performed of 218 consecutive adult patients receiving intranasal naloxone in 2 neighboring counties in Southeast Michigan: one that used a 0.4-mg protocol and one that used a 2-mg protocol. Primary outcomes were response to initial dose, requirement of additional dosing, and incidence of adverse effects. Unpooled, 2-tailed, 2-sample t-tests and χ2 tests for homogeneity were performed with statistical significance defined as P <0.05. RESULTS: There was no statistically significant difference between the 2 populations in age, mass, gender, proportion of exposures suspected as heroin, response to initial dose, required redosing, or total number of doses by any route. The overall rate of adverse effects was 2.1% under the lower-dose protocol and 29% under the higher-dose protocol (P < 0.001). The lower-dose protocol was 79% less costly. CONCLUSION AND RELEVANCE: Treatment of prehospital opioid overdose using intranasal naloxone at an initial dose of 0.4 mg was equally effective during the prehospital period as treatment at an initial dose of 2 mg, was associated with a lower rate of adverse effects, and represented a 79% reduction in cost.

Appropriate Air Medical Services Utilization and Recommendations for Integration of Air Medical Services Resources into the EMS System of Care: A Joint Position Statement and Resource Document of NAEMSP, ACEP, and AMPA.

This update to the 2013 joint position statement, Appropriate and Safe Utilization of Helicopter Emergency Medical Services, provides guidance for air medical services utilization based on currently available evidence. Air medical services utilization considerations fall into three major categories: clinical considerations, safety considerations, and system integration and quality assurance.Clinically, air medical services should accomplish one or more of three primary patient-centered goals: initiation or continuation of locally unavailable advanced or specialty care; expedited delivery to definitive care for time-sensitive interventions; and/or extraction from physically remote or otherwise inaccessible locations that limit timely access to necessary care. Ground-EMS (GEMS) transport is preferred when it is able to provide the necessary level of care and timely transport to definitive care.Risk identification and safety of both the patient and crew must be uniformly balanced against the anticipated degree of patient medical benefit. While auto-ready and auto-launch practices may increase access to air medical services, they also risk over-use, and so must be rigorously reviewed. Safety is enhanced during multi-agency emergency responses by coordinated interagency communication, ideally through centralized communication centers. Helicopter shopping and reverse helicopter shopping both create significant safety risks and their use is discouraged.Regional EMS systems must integrate air medical services to facilitate appropriate utilization in alignment with the primary patient goals while being cognizant of local indications, resources, and needs. To maximize consistent, informed air medical services utilization decisions, specific indications for and limitations to air medical services utilization that align with local and regional system and patient needs should be identified, and requests routed through centralized coordinating centers supported by EMS physicians.To limit risk and promote appropriate utilization of air medical services, GEMS clinicians should be encouraged to cancel an air medical services response if it is not aligned with at least one of the three primary patient-centered goals. Similarly, air medical services clinicians should be empowered to redirect patient transport to GEMS. Air medical services should not routinely be used solely to allow GEMS to remain in their primary service area.

Possible exercised-induced rhabdomyolysis associated with terbinafine.

Introduction. Terbinafine is reported to be associated with rhabdomyolysis. We present a patient taking terbinafine who may have developed exercise-induced rhabdomyolysis. Case Report. A healthy 40-year-old female developed onychomycosis of the right first toe for which she was taking terbinafine. After an increase in her exercise regimen, she began experiencing notable myalgias of the triceps. During outpatient evaluation, the patient was found to have elevated and worsening creatine kinase (CK) and aspartate transaminase. At evaluation in the emergency department, CK was <5000 IU/L and had decreased. She did not have electrolyte abnormalities, kidney injury or kidney failure. Discussion. Patients may be at risk for exercise-induced rhabdomyolysis while on terbinafine and may need to be cautioned regarding the intensity of exercise.

Epinephrine Administered for Anaphylaxis Unmasking a Type 1 Brugada Pattern on Electrocardiogram.

BACKGROUND: Brugada pattern on electrocardiography (ECG) can manifest as type 1 (coved pattern) and type 2 (saddleback pattern). Brugada syndrome represents an ECG with Brugada pattern in a patient with symptoms or clinical factors, including syncope, cardiac arrest, ventricular dysrhythmias, and family history. Brugada syndrome is caused by a genetic channelopathy, but the Brugada pattern may be drug-induced. Epinephrine-induced Brugada pattern has not been reported previously. CASE REPORT: A 63-year-old man developed anaphylaxis secondary to a bee sting, had a transient loss of consciousness, and self-administered intramuscular epinephrine. He subsequently presented to the emergency department and was found to have a type 1 Brugada pattern on ECG that resolved during observation. A historic ECG was reviewed that demonstrated a baseline type 2 Brugada pattern. His anaphylaxis was managed with steroids and antihistamines. He was observed without subsequent dysrhythmic events on telemetry or any further symptoms. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: The differential diagnosis for syncope includes dysrhythmia, such as Brugada syndrome. Among other possible drugs, epinephrine may induce a type 1 Brugada pattern. Patients with Brugada pattern on ECG should be referred immediately to electrophysiology for consideration of implantation of a cardioverter-defibrillator device, given the association of Brugada pattern with sudden cardiac arrest and ventricular dysrhythmias.

Disseminated Histoplasmosis with Miliary Histoplasmosis, Neurohistoplasmosis, and Histoplasma capsulatum Bacteremia in Probable Neurosarcoidosis.

INTRODUCTION: Neurosarcoidosis, either isolated or as part of systemic sarcoidosis, is an uncommon entity and has diagnostic uncertainty. Treatment for neurosarcoidosis can increase the risk of infections, including fungal infections such as disseminated histoplasmosis. Neurosarcoidosis may further predispose patients to infections of the central nervous system. CASE PRESENTATION: A 54-year-old male with a history of probable neurosarcoidosis on methotrexate and infliximab presented with encephalopathy, hypoxia, and reported fevers. The patient was found to have disseminated histoplasmosis involving the lungs (miliary histoplasmosis), central nervous system (neurohistoplasmosis), and bloodstream. The Histoplasma capsulatum infection was treated with amphotericin and then voriconazole. DISCUSSION: Patients with neurosarcoidosis are suspected to have blood-brain barrier dysfunction. Lumbar puncture should be considered as part of initial investigative studies for infection. Empiric antimicrobial therapy for a patient with neurosarcoidosis on immunosuppressive agents may need to include antifungal agents.

Sanguineous Pericardial Effusion and Cardiac Tamponade in the Setting of Graves' Disease: Report of a Case and Review of Previously Reported Cases.

Introduction. Pericardial effusion in the setting of hyperthyroidism is rare. We present a patient with Graves' disease who developed a sanguineous pericardial effusion and cardiac tamponade. Case Description. A 76-year-old man presenting with fatigue was diagnosed with Graves' disease and treated with methimazole. Two months later, he was hospitalized for uncontrolled atrial fibrillation. Electrocardiography showed diffuse low voltage and atrial fibrillation with rapid ventricular rate. Chest radiograph revealed an enlarged cardiac silhouette and left-sided pleural effusion. Thyroid stimulating hormone was undetectable, and free thyroxine was elevated. Diltiazem and heparin were started, and methimazole was increased. Transthoracic echocardiography revealed a large pericardial effusion with cardiac tamponade physiology. Pericardiocentesis obtained 1,050 mL of sanguineous fluid. The patient progressed to thyroid storm, treated with propylthiouracil, potassium iodine, hydrocortisone, and cholestyramine. Cultures and cytology of the pericardial fluid were negative. Thyroid hormone markers progressively normalized, and he improved clinically and was discharged. Discussion. We found 10 previously reported cases of pericardial effusions in the setting of hyperthyroidism. Heparin use may have contributed to the sanguineous nature of our patient's pericardial effusion, but other reported cases occurred without anticoagulation. Sanguineous and nonsanguineous pericardial effusions and cardiac tamponade may be due to hyperthyroidism.

A Possible Association of Diindolylmethane with Pulmonary Embolism and Deep Venous Thrombosis.

Introduction. 3,3'-Diindolylmethane is available as a supplement in the United States for "cancer prevention" and "augmentation of physical fitness." A derivative of indole-3-carbinol found in plants, diindolylmethane, binds to receptors associated with the sex steroid pathways and has unclear effects on estrogen and androgen physiology. We present a patient who had been taking diindolylmethane and developed right lower extremity deep venous thrombosis and bilateral pulmonary embolism. Case Presentation. A 65-year-old man presented with swelling, erythema, and warmth of his right lower extremity for three to four weeks. He had been taking diindolylmethane one tablet daily for three to four months. Risk factors for venous thromboembolism included tobacco use, personal history of possible pulmonary embolism, body mass index, and age. Imaging studies found extensive deep venous thrombosis in his right lower extremity and bilateral pulmonary embolism with probable right middle lobe infarction. Follow-up imaging showed chronic deep venous thrombosis in his right lower extremity. Discussion. As suggested in this single case, patients who take diindolylmethane may be at greater risk for venous thromboembolism. Further reports and studies are necessary in order to elucidate this possible association. Clinicians should question patients about supplements in the setting of venous thromboembolism.

Arthritis, Infectious Tenosynovitis, and Tendon Rupture in a Patient With Rheumatoid Arthritis and Psoriasis.

Multiple comorbidities, advanced age, tobacco use, and alcohol abuse made a proper diagnosis difficult in a patient with polyarticular septic arthritis, infectious tenosynovitis, and ruptures in the tendons of both thumbs.

Neuropathic pain following sagittal split ramus osteotomy of the mandible: prevalence, risk factors, and clinical course.

PURPOSE: To estimate the prevalence of, risk factors for, and clinical course of neuropathic pain (NPP) after sagittal split ramus osteotomy (SSRO) of the mandible in a large cohort of patients. MATERIALS AND METHODS: A retrospective cohort of all patients who underwent SSRO at 2 medical centers within Kaiser Permanente Northern California from January 2007 through September 2012 was assembled. Demographic, clinical, and surgical factors were abstracted from medical records and relevant comorbidities were identified. The prevalence of NPP in the cohort was calculated and the clinical signs, symptoms, temporal characteristics, and treatment response in affected patients were noted. RESULTS: The authors identified 1,778 patients who underwent SSRO and excluded 107 patients according to predefined criteria. The remaining 1,671 patients had a median age of 24 years (interquartile range, 19 to 35 yr) and 62.4% were women. Seven patients developed NPP after SSRO, which was an overall prevalence of 0.42%. All patients with NPP in this cohort were women and had a median age of 49 years. The risk factors for developing NPP after this surgery were older age (P = .0098), depression (P = .0100), and female gender. NPP developed an average of 30 days postoperatively (range, 18 to 56 days) and persisted for a median duration of 52 days (range, 30 to 69.5 days). All patients responded favorably to anticonvulsant (n = 6) or tricyclic (n = 1) medications, and no patients developed chronic postsurgical pain. CONCLUSIONS: NPP was an infrequent complication after SSRO, occurring in 1 of 238 patients in this cohort. The short duration and positive response to medication are reassuring findings. The results of this investigation highlight the need for prospective studies to further understand the spectrum of postoperative NPP.

PD-0332991, a potent and selective inhibitor of cyclin-dependent kinase 4/6, demonstrates inhibition of proliferation in renal cell carcinoma at nanomolar concentrations and molecular markers predict for sensitivity.

BACKGROUND: PD-0332991 is an inhibitor of cyclin-dependent kinases (CDK) 4 and 6, and was evaluated to determine its anti-proliferative effects in 25 renal cell carcinoma (RCC) cell lines. MATERIALS AND METHODS: Half-maximal inhibitory concentrations (IC50) of PD-0332991 were determined with cell line proliferation assays, as were its effects on the cell cycle, apoptosis, and retinoblastoma (RB) phosphorylation. Molecular markers for response prediction, including p16, p15, cyclin D1 (CCND1), cyclin E1 (CCNE1), E2F transcription factor 1 (E2F1), RB, CDK4 and CDK6, were studied using array comparative genomic hybridization (CGH) and gene expression. RESULTS: IC50 values for PD-0332991 ranged from 25.0 nM to 700 nM, and the agent demonstrated G0/G1 cell-cycle arrest, induction of late apoptosis, and blockade of RB phosphorylation. Through genotype and expression data p16, p15 and E2F1 were identified as having significant association between loss and sensitivity to PD-0332991: p16 (p=0.021), p15 (p=0.047), and E2F1 (p=0.041). CONCLUSION: PD-0332991 has antiproliferative activity in RCC cell lines, and molecular markers predict for sensitivity to this agent.

First report of a de novo 18q11.2 microdeletion including GATA6 associated with complex congenital heart disease and renal abnormalities.

Deletions of the long arm of chromosome 18 have been previously reported in many patients. Most cases involve the more distal regions of the long arm (18q21.1->qter). However, proximal interstitial deletions involving 18q11.2 are extremely rare. Here we report on a 14-month-old female with a 4.7 Mb (19,667,062-24,401,876 hg19) de novo interstitial deletion within chromosomal band 18q11.2, which includes GATA6 and 24 other RefSeq genes. The clinical features of our patient include complex congenital heart defects, a double outlet right ventricle, a subaortic ventricular septal defect, D-malposed great arteries, an atrial septal defect, a dysplastic aortic valve and patent ductus arteriosus. In addition, she had renal anomalies-a duplicated collecting system on the left and mild right hydronephrosis. These heart and renal defects are not reported in other patients with 18q proximal interstitial deletions. Heterozygous point mutations in GATA6, encoding for a zinc finger transcription factor, have been shown to cause congenital heart defects. Given the well-established biological role of GATA6 in cardiac development, a deletion of GATA6 is very likely responsible for our patient's complex congenital heart defects. This is the smallest and most proximal 18q11.2 deletion involving GATA6 that is associated with complex congenital heart disease and renal anomalies.

Atypical rearrangement involving 3'-IGH@ and a breakpoint at least 400 Kb upstream of an intact MYC in a CLL patient with an apparently balanced t(8;14)(q24.1;q32) and negative MYC expression.

The t(8;14)(q24.1;q32), the cytogenetic hallmark of Burkitt's lymphoma, is also found, but rarely, in cases of chronic lymphocytic leukemia (CLL). Such translocation typically results in a MYC-IGH@ fusion subsequently deregulating and overexpressing MYC on der 14q32. In CLL, atypical rearrangements resulting in its gain or loss, within or outside of IGH@ or MYC locus, have been reported, but their clinical significance remains uncertain. Herein, we report a 67 year-old male with complex cytogenetic findings of apparently balanced t(8;14) and unreported complex rearrangements of IGH@ and MYC loci. His clinical, morphological and immunophenotypic features were consistent with the diagnosis of CLL.Interphase FISH studies revealed deletions of 11q22.3 and 13q14.3, and an extra copy of IGH@, indicative of rearrangement. Karyotype analysis showed an apparently balanced t(8;14)(q24.1;q32). Sequential GPG-metaphase FISH studies revealed abnormal signal patterns: rearrangement of IGH break apart probe with the 5'-IGH@ on derivative 8q24.1 and the 3'-IGH@ retained on der 14q; absence of MYC break apart-specific signal on der 8q; and, the presence of unsplit 5'-MYC-3' break apart probe signals on der 14q. The breakpoint on 8q24.1 was found to be at least 400 Kb upstream of 5' of MYC. In addition, FISH studies revealed two abnormal clones; one with 13q14.3 deletion, and the other, with concurrent 11q deletion and atypical rearrangements. Chromosome microarray analysis (CMA) detected a 7.1 Mb deletion on 11q22.3-q23.3 including ATM, a finding consistent with FISH results. While no significant copy number gain or loss observed on chromosomes 8, 12 and 13, a 455 Kb microdeletion of uncertain clinical significance was detected on 14q32.33. Immunohistochemistry showed co-expression of CD19, CD5, and CD23, positive ZAP-70 expression and absence of MYC expression. Overall findings reveal an apparently balanced t(8;14) and atypical complex rearrangements involving 3'-IGH@ and a breakpoint at least 400 Kb upstream of MYC, resulting in the relocation of the intact 5'-MYC-3' from der 8q, and apposition to 3'-IGH@ at der 14q. This case report provides unique and additional cytogenetic data that may be of clinical significance in such a rare finding in CLL. It also highlights the utility of conventional and sequential metaphase FISH in understanding complex chromosome anomalies and their association with other clinical findings in patients with CLL. To the best of our knowledge, this is the first CLL reported case with such an atypical rearrangement in a patient with a negative MYC expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin treatment alters eicosanoid levels in several organs of the mouse in an aryl hydrocarbon receptor-dependent fashion.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) adversely affects many mammalian organs and tissues. These effects are mediated by the aryl hydrocarbon receptor (AHR). CYP1A1, CYP1A2 and CYP1B1 are upregulated by the liganded AHR. These (and other) cytochromes P450 can metabolize arachidonic acid into a variety of bioactive eicosanoids. Towards investigating a potential role of eicosanoids in TCDD toxicity, arachidonic acid, two other unsaturated long-chain fatty acids, and up to twenty-five eicosanoids were measured in five organs/tissues of male and female wild-type and Ahr null mice treated or untreated with TCDD. TCDD generally increased the levels of the four dihydroxyeicosatrienoic acids (DHETs) and (where measured) 5,6-epoxyeicosatrienoic acid and 18-, 19- and 20-hydroxyeicosatrienoic acids (HETEs) in the serum, liver, spleen and lungs, but not the heart, of both sexes, and increased the levels in the serum, liver and spleen of several metabolites that are usually considered products of lipoxygenase activity, but which may also be generated by cytochromes P450. TCDD also increased the levels of the esterified forms of these eicosanoids in the liver in parallel with the corresponding free forms. The levels of prostanoids were generally not affected by TCDD. The above changes did not occur in Ahr null mice, and are therefore mediated by the AHR. TCDD increased the mRNA levels of Cyp1a1, Cyp1a2, Cyp1b1 and the Pla2g12a form of phospholipase A(2) to varying degrees in the different organs, and these increases correlated with some but not all the changes in eicosanoids levels in the organs, suggesting that other enzymes may also be involved.

CYP2S1 is negatively regulated by corticosteroids in human cell lines.

Cytochrome P450s are monooxygenase proteins involved in the metabolism of both exogenous and endogenous compounds. CYP2S1 can metabolize eicosanoids in the absence of both NADPH and NADPH cytochrome P450 reductase, and can also activate the anticancer agent 1 AQ4N [1,4-bis{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxy anthracene-9,10-dione]. CYP2S1 is mainly expressed in extrahepatic tissues such as the trachea, lung, stomach, small intestine, spleen, skin, breast, kidney and placenta. Furthermore, increased expression of CYP2S1 occurs in several tumors of epithelial origin, making the characterization of CYP2S1 regulation relevant to the treatment of disease. We report that the synthetic glucocorticoid receptor ligand dexamethasone (DEX) represses CYP2S1 expression. The ED(50) is between 1 nM and 3 nM and maximal repression is reached by 48 h. Other corticosteroids are also effective at repressing CYP2S1. We show that repression by DEX is mediated by the glucocorticoid receptor and requires histone deacetylase activity.

Comparison of mibefradil and derivative NNC 55-0396 effects on behavior, cytochrome P450 activity, and tremor in mouse models of essential tremor.

NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2, 3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride], is a mibefradil derivative that retains potent in vitro T-type calcium channel antagonist efficacy. We compared the two compounds for behavioral toxicity, effects on cytochrome P450 activity, and efficacy against tremor in the γ-aminobutyric acid type A (GABAA) receptor subunit α1-null mouse, and the harmaline tremor model of essential tremor in wild-type mice. NNC 55-0396 was better tolerated than mibefradil in the horizontal wire test of sedation/motor function, with 3/6 failing at 300 and 30mg/kg respectively. To assess for a potential interaction with harmaline, mice were given the drugs, followed by harmaline or vehicle, and tested 30min later in the inverted wire grid test. Mibefradil exacerbated, whereas NNC 55-0396 ameliorated harmaline-induced test deficits. In mouse liver microsomes, NNC 55-0396 was a less potent inhibitor of harmaline O-demethylation than mibefradil (Ki: 0.95 and 0.29µM respectively), and also less potent at inhibiting testosterone 6-ß-hydroxylation (Ki: 0.71 and 0.12µM respectively). In the GABAA α1-null model, NNC 55-0396 but not mibefradil, (each at 20mg/kg), suppressed tremor while NNC 55-0396 at 12.5mg/kg suppressed harmaline-induced tremor by half by 20-100min, whereas mibefradil at the same dose did not significantly affect tremor. In contrast to mibefradil, NNC 55-0396 is well tolerated and suppresses tremor, and exerts less cytochrome P450 inhibition. These results suggest potential clinical utility for NNC 55-0396 or similar derivatives as a T-type calcium antagonist.

Human CYP2S1 metabolizes cyclooxygenase- and lipoxygenase-derived eicosanoids.

CYP2S1 is a recently described dioxin-inducible cytochrome P450. We previously demonstrated that human CYP2S1 oxidizes a number of carcinogens but only via the peroxide shunt. In this article, we investigated whether human CYP2S1 can metabolize cyclooxygenase- and lipoxygenase-derived lipid peroxides in a NADPH-independent fashion. Human CYP2S1 metabolizes prostaglandin G(2) (PGG(2)) (K(m) = 0.267 ± 0.072 µM) into several products including 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid (12-HHT). It also metabolizes prostaglandin H(2) (PGH(2)) (K(m) = 11.7 ± 2.8 µM) into malondialdehyde, 12-HHT, and thromboxane A(2) (TXA(2)). The turnover to 12-HHT by human CYP2S1 (1.59 ± 0.04 min(-1)) is 40-fold higher than that of TXA(2) (0.04 min(-1)). In addition to PGG(2) and PGH(2) metabolism, human CYP2S1 efficiently metabolizes the hydroperoxyeicosatetraenoic acids (5S-, 12S-, and 15S-) and 13S-hydroperoxyoctadecadienoic acid into 5-oxo-eicosatetraenoic acid (turnover = 16.7 ± 0.3 min(-1)), 12-oxo-eicosatetraenoic acid 1 (11.5 ± 0.9 min(-1)), 15-oxo-eicosatetraenoic acid (16.9 ± 0.8 min(-1)), and 13-octadecadienoic acid (20.2 ± 0.9 min(-1)), respectively. Other cytochromes P450 such as CYP1A1, 1A2, 1B1, and 3A4 underwent similar conversions but at slower rates. The fatty acid hydroperoxides were also converted by human CYP2S1 to several epoxyalcohols. Our data indicate that fatty acid endoperoxides and hydroperoxides represent endogenous substrates of CYP2S1 and suggest that the enzyme CYP2S1 may play an important role in the inflammatory process because some of the products that CYP2S1 produces play important roles in inflammation.

Role of epigenetic mechanisms in differential regulation of the dioxin-inducible human CYP1A1 and CYP1B1 genes.

The aryl hydrocarbon receptor (AhR) mediates induction of CYP1A1 and CYP1B1 by 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (dioxin) via binding to xenobiotic-responsive elements (XREs) in their enhancer regions. CYP1A1 and CYPIB1 were both inducible by dioxin in human MCF-7 cells. However, only CYP1A1 was inducible in human HepG2 cells. Further experiments focused on providing an explanation for this last observation. Dioxin induced the recruitment of AHR and the transcriptional coactivators p300 and p300/cAMP response element-binding protein binding protein-associated factor (PCAF) to the CYP1B1 enhancer in HepG2 cells but failed to induce recruitment of RNA polymerase II (polII) or the TATA binding protein (TBP) and acetylations of histones 3 and 4 or methylation of histone 3 at the promoter. Because p300 was required for dioxin induction of the aforementioned histone modifications at the CYP1B1 promoter and for induction of CYP1B1 transcription (in MCF-7 cells), the recruitments of p300 and AhR, although necessary, are not sufficient for eliciting the above responses to dioxin. Cytosine residues within CpG dinucleotides at the enhancer, including those within the XREs, were partially methylated, whereas those at the promoter were fully methylated. Treatment of HepG2 cells with 5-aza-2'-deoxycytidine led to partial demethylation of the promoter, restored polII and TBP binding, and CYP1B1 inducibility. Thus, the deficiency of CYP1B1 induction in HepG2 cells is ascribable to cytosine methylation at the promoter, which prevents recruitment of TBP and polII. It is noteworthy that our data indicate that stable recruitment of p300 and PCAF to the CYP1B1 gene does not require their tethering to the promoter and to the enhancer.

Fatty acid hydroperoxides support cytochrome P450 2S1-mediated bioactivation of benzo[a]pyrene-7,8-dihydrodiol.

In the accompanying report (p. 1031), we showed that a novel dioxin-inducible cytochrome P450, CYP2S1, efficiently metabolizes benzo[a]pyrene-trans-7,8-dihydrodiol (BaP-7,8-diol) into the highly mutagenic and carcinogenic benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (BaP-diol-t-epoxide), using cumene hydroperoxide in lieu of NADPH/O(2). Lipid hydroperoxide-supported P450 oxidation has been reported in several cases. However, it has not yet been described for the bioactivation of BaP-7,8-diol. In this report, we demonstrate that CYP2S1 can use various fatty acid hydroperoxides to support epoxidation of BaP-7,8-diol at a much higher rate than with cumene hydroperoxide. Kinetic analyses with several fatty acid hydroperoxides revealed that 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE) was the most potent oxidant tested (K(m), 3.4 +/- 0.8 microM; turnover, 4.51 +/- 0.13 min(-1)), followed by 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (K(m), 2.8 +/- 0.7 microM; turnover, 3.7 +/- 0.1 min(-1)), 5S-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (K(m), 2.7 +/- 0.8 microM; turnover, 3.69 +/- 0.09 min(-1)), and 15S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (K(m), 11.6 +/- 0.3 microM; turnover, 0.578 +/- 0.030 min(-1)). The antioxidant butylated hydroxyanisole inhibited CYP2S1-catalyzed epoxidation by 100%, suggesting that epoxidation proceeds by a free radical mechanism. Other cytochromes P450, including CYP1A1, CYP1B1, CYP1A2, and CYP3A4, were also able to epoxidize BaP-7,8-diol using various fatty acid hydroperoxides, although at slower rates than CYP2S1. The cytotoxicity of BaP-7,8-diol significantly increased in mammalian cells overexpressing CYP2S1, and BaP-diol-t-epoxide formation in these cells also increased in the presence of 13-HpODE. Together, these results suggest that fatty acid hydroperoxides can serve as physiological cofactors in supporting in vivo CYP2S1-catalyzed oxidation of BaP-7,8-diol, and that fatty acid hydroperoxides and CYP2S1 may play important roles in benzo[a]pyrene-induced carcinogenesis.

Functional characterization of human cytochrome P450 2S1 using a synthetic gene-expressed protein in Escherichia coli.

Human cytochrome P450 2S1 was recently identified and shown to be inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin and hypoxia. It is highly expressed in epithelial cells of tissues that are exposed to the environment and in many tumors of epithelial origin. The biological function of CYP2S1 has not yet been determined, although its possible role in carcinogen metabolism has been suggested. In this report, we investigated its ability to metabolize carcinogens. To obtain a large quantity of active enzyme for substrate screening, we overexpressed CYP2S1 in Escherichia coli (200 nM culture), using a synthetic gene approach. High-level expression allowed us to achieve purification of CYP2S1 with high specific content and purity (16 nmol/mg). Despite high-level expression, we found that CYP2S1 was not readily reduced by cytochrome P450 reductase, and thus no activity was found using NADPH. However, the oxidative activity of CYP2S1 was supported by cumene hydroperoxide or H(2)O(2), such that CYP2S1 oxidized many important environmental carcinogens, including benzo[a]pyrene, 9,10-dihydro-benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene-7,8-dihydrodiol, aflatoxin B1, naphthalene, and styrene, with high turnover. Most substrates tested were converted to detoxification products, except in the case of benzo[a]pyrene-7,8-dihydrodiol, which was converted into the very potent carcinogenic metabolite 7,8-dihydrodiol-trans-9,10-epoxide at a relatively efficient rate (K(m) = 12.4 +/- 2 microM, turnover = 2.3 min(-1)). This metabolite formation was also supported both in vitro and in vivo by fatty acid hydroperoxides described in the accompanying report (p. 1044). Together, these data indicate that CYP2S1 contributes to the metabolism of environmental carcinogens via an NADPH independent activity.