CYP2E1 overexpression protects COS-7 cancer cells against ferroptosis.

Ferroptosis is a recently described form of regulated cell death initiated by the iron-mediated one-electron reduction of lipid hydroperoxides (LOOH). Cytochrome P450 2E1 (CYP2E1) induction, a consequence of genetic polymorphisms or/and gene induction by xenobiotics, may promote ferroptosis by contributing to the cellular pool of LOOH. However, CYP2E1 induction also increases the transcription of anti-ferroptotic genes that regulate the activity of glutathione peroxidase 4 (GPX4), the main ferroptosis inhibitor. Based on the above, we hypothesize that the impact of CYP2E1 induction on ferroptosis depends on the balance between pro- and anti-ferroptotic pathways triggered by CYP2E1. To test our hypothesis, ferroptosis was induced with class 2 inducers (RSL-3 or ML-162) in mammalian COS-7 cancer cells that don't express CYP2E1 (Mock cells), and in cells engineered to express human CYP2E1 (WT cells), and the impact on viability, lipid peroxidation and GPX4 was assessed. CYP2E1 overexpression protected COS-7 cancer cells against ferroptosis, evidenced by an increase in the IC50 and a decrease in lipid ROS in WT versus Mock cells after exposure to class 2 inducers. CYP2E1 overexpression produced an 80% increase in the levels of the GPX4 substrate glutathione (GSH). Increasing GSH in Mock cells protected cells against ferroptosis by ML-162. Depleting GSH, or inhibiting Nrf2 in WT cells reverted the protective effect mediated by CYP2E1, causing a decrease in the IC50 and an increase in lipid ROS after exposure to ML-162. These results show that CYP2E1 overexpression protects COS-7 cancer cells against ferroptosis, an effect probably mediated by Nrf2-dependent GSH induction.

Antioxidant and pro-oxidant mechanisms of (+) catechin in microsomal CYP2E1-dependent oxidative stress.

The objectives of this work were to evaluate the effects of catechin on cytochrome P450 2E1 (CYP2E1)-dependent oxidative stress. Microsomes co-expressing human CYP2E1 with NADPH cytochrome P450 reductase and cytochrome b5 were incubated with NADPH and DTPA at pH 7.0. Superoxide anion generation was specifically detected by spin-trapping with DEPMPO. Generation of the DEPMPO-OOH adduct was not observed in the absence of CYP2E1 and in the presence of superoxide dismutase (SOD) or catechin, while catalase was ineffective. Reactive oxygen species generation was detected with 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) by the EPR-detection of its oxidation product, 3-carboxy-proxyl radical (CP●). CP● generation was not observed in the absence of CYP2E1 and in the presence of SOD, while catalase was ineffective. In contrast, catechin increased CPH oxidation, an effect that was not observed in the absence of CYP2E1 or in the presence of SOD (but not catalase), and was not associated with an increase in oxygen consumption. Catechin also increased the non-specific oxidation of the probes CPH and hydroethidine by the superoxide anion-generating system xanthine plus xanthine oxidase. Catechin oxidized CPH in the presence of horseradish peroxidase plus hydrogen peroxide, a catechin radical-generating system. In conclusion, catechin exhibits both antioxidant (superoxide-scavenging) and pro-oxidant effects under CYP2E1-dependent oxidative stress.

Iron overload prevents oxidative damage to rat brain after chlorpromazine administration.

The hypothesis tested is that Fe administration leads to a response in rat brain modulating the effects of later oxidative challenges such as chlorpromazine (CPZ) administration. Either a single dose (acute Fe overload) or 6 doses every second day (sub-chronic Fe overload) of 500 or 50 mg Fe-dextran/kg, respectively, were injected intraperitoneally (ip) to rats. A single dose of 10 mg CPZ/kg was injected ip 8 h after Fe treatment. DNA integrity was evaluated by quantitative PCR, lipid radical (LR·) generation rate by electron paramagnetic resonance (EPR), and catalase (CAT) activity by UV spectrophotometry in isolated brains. The maximum increase in total Fe brain was detected after 6 or 2 h in the acute and sub-chronic Fe overload model, respectively. Mitochondrial and nuclear DNA integrity decreased after acute Fe overload at the time of maximal Fe content; the decrease in DNA integrity was lower after sub-chronic than after acute Fe overload. CPZ administration increased LR· generation rate in control rat brain after 1 and 2 h; however, CPZ administration after acute or sub-chronic Fe overload did not affect LR· generation rate. CPZ treatment did not affect CAT activity after 1-4 h neither in control rats nor in acute Fe-overloaded rats. However, CPZ administration to rats treated sub-chronically with Fe showed increased brain CAT activity after 2 or 4 h, as compared to control values. Fe supplementation prevented brain damage in both acute and sub-chronic models of Fe overload by selectively activating antioxidant pathways.

1,3-Butadiene-induced mitochondrial dysfunction is correlated with mitochondrial CYP2E1 activity in Collaborative Cross mice.

Cytochrome P450 2E1 (CYP2E1) metabolizes low molecular weight hydrophobic compounds, including 1,3-butadiene, which is converted by CYP2E1 to electrophilic epoxide metabolites that covalently modify cellular proteins and DNA. Previous CYP2E1 studies have mainly focused on the enzyme localized in the endoplasmic reticulum (erCYP2E1); however, active CYP2E1 has also been found in mitochondria (mtCYP2E1) and the distribution of CYP2E1 between organelles can influence an individual's response to exposure. Relatively few studies have focused on the contribution of mtCYP2E1 to activation of chemical toxicants. We hypothesized that CYP2E1 bioactivation of 1,3-butadiene within mitochondria adversely affects mitochondrial respiratory complexes I-IV. A population of Collaborative Cross mice was exposed to air (control) or 200ppm 1,3-butadiene. Subcellular fractions (mitochondria, DNA, and microsomes) were collected from frozen livers and CYP2E1 activity was measured in microsomes and mitochondria. Individual activities of mitochondrial respiratory complexes I-IV were measured using in vitro assays and purified mitochondrial fractions. In air- and 1,3-butadiene-exposed mouse samples, mtDNA copy numbers were assessed by RT-PCR, and mtDNA integrity was assessed through a PCR-based assay. No significant changes in mtDNA copy number or integrity were observed; however, there was a decrease in overall activity of mitochondrial respiratory complexes I, II, and IV after 1,3-butadiene exposure. Additionally, higher mtCYP2E1 (but not erCYP2E1) activity was correlated with decreased mitochondrial respiratory complex activity (in complexes I-IV) in the 1,3-butadiene-exposed (not control) animals. Together, these results represent the first in vivo link between mitochondrial CYP2E1 activity and mitochondrial toxicity.

Subcellular localization of rat CYP2E1 impacts metabolic efficiency toward common substrates.

Cytochrome P450 2E1 (CYP2E1) detoxifies or bioactivates many low molecular-weight compounds. Most knowledge about CYP2E1 activity relies on studies of the enzyme localized to endoplasmic reticulum (erCYP2E1); however, CYP2E1 undergoes transport to mitochondria (mtCYP2E1) and becomes metabolically active. We report the first comparison of in vitro steady-state kinetic profiles for erCYP2E1 and mtCYP2E1 oxidation of probe substrate 4-nitrophenol and pollutants styrene and aniline using subcellular fractions from rat liver. For all substrates, metabolic efficiency changed with substrate concentration for erCYP2E1 reflected in non-hyperbolic kinetic profiles but not for mtCYP2E1. Hyperbolic kinetic profiles for the mitochondrial enzyme were consistent with Michaelis-Menten mechanism in which metabolic efficiency was constant. By contrast, erCYP2E1 metabolism of 4-nitrophenol led to a loss of enzyme efficiency at high substrate concentrations when substrate inhibited the reaction. Similarly, aniline metabolism by erCYP2E1 demonstrated negative cooperativity as metabolic efficiency decreased with increasing substrate concentration. The opposite was observed for erCYP2E1 oxidation of styrene; the sigmoidal kinetic profile indicated increased efficiency at higher substrate concentrations. These mechanisms and CYP2E1 levels in mitochondria and endoplasmic reticulum were used to estimate the impact of CYP2E1 subcellular localization on metabolic flux of pollutants. Those models showed that erCYP2E1 mainly carries out aniline metabolism at all aniline concentrations. Conversely, mtCYP2E1 dominates styrene oxidation at low styrene concentrations and erCYP2E1 at higher concentrations. Taken together, subcellular localization of CYP2E1 results in distinctly different enzyme activities that could impact overall metabolic clearance and/or activation of substrates and thus impact the interpretation and prediction of toxicological outcomes.

Prooxidant and antioxidant properties of salicylaldehyde isonicotinoyl hydrazone iron chelators in HepG2 cells.

BACKGROUND: Salicylaldehyde isonicotinoyl hydrazone (SIH) is an iron chelator of the aroylhydrazone class that displays antioxidant or prooxidant effects in different mammalian cell lines. Because the liver is the major site of iron storage, elucidating the effect of SIH on hepatic oxidative metabolism is critical for designing effective hepatic antioxidant therapies. METHODS: Hepatocyte-like HepG2 cells were exposed to SIH or to analogs showing greater stability, such as N'-[1-(2-Hydroxyphenyl)ethyliden]isonicotinoyl hydrazide (HAPI), or devoid of iron chelating properties, such as benzaldehyde isonicotinoyl hydrazone (BIH), and toxicity, oxidative stress and antioxidant (glutathione) metabolism were evaluated. RESULTS: Autoxidation of Fe(2+)in vitro increased in the presence of SIH or HAPI (but not BIH), an effect partially blocked by Fe(2+) chelation. Incubation of HepG2 cells with SIH or HAPI (but not BIH) was non-toxic and increased reactive oxygen species (ROS) levels, activated the transcription factor Nrf2, induced the catalytic subunit of γ-glutamate cysteine ligase (Gclc), and increased glutathione concentration. Fe(2+) chelation decreased ROS and inhibited Nrf2 activation, and Nrf2 knock-down inhibited the induction of Gclc in the presence of HAPI. Inhibition of γ-glutamate cysteine ligase enzymatic activity inhibited the increase in glutathione caused by HAPI, and increased oxidative stress. CONCLUSIONS: SIH iron chelators display both prooxidant (increasing the autoxidation rate of Fe(2+)) and antioxidant (activating Nrf2 signaling) effects. GENERAL SIGNIFICANCE: Activation by SIH iron chelators of a hormetic antioxidant response contributes to their antioxidant properties and modulates the anti- and pro-oxidant balance.

N-acetylcysteine inhibits the up-regulation of mitochondrial biogenesis genes in livers from rats fed ethanol chronically.

BACKGROUND: Chronic ethanol (EtOH) administration to experimental animals induces hepatic oxidative stress and up-regulates mitochondrial biogenesis. The mechanisms by which chronic EtOH up-regulates mitochondrial biogenesis have not been fully explored. In this work, we hypothesized that oxidative stress is a factor that triggers mitochondrial biogenesis after chronic EtOH feeding. If our hypothesis is correct, co-administration of antioxidants should prevent up-regulation of mitochondrial biogenesis genes. METHODS: Rats were fed an EtOH-containing diet intragastrically by total enteral nutrition for 150 days, in the absence or presence of the antioxidant N-acetylcysteine (NAC) at 1.7 g/kg/d; control rats were administered isocaloric diets where carbohydrates substituted for EtOH calories. RESULTS: EtOH administration significantly increased hepatic oxidative stress, evidenced as decreased liver total glutathione and reduced glutathione/glutathione disulfide ratio. These effects were inhibited by co-administration of EtOH and NAC. Chronic EtOH increased the expression of mitochondrial biogenesis genes including peroxisome proliferator-activated receptor gamma-coactivator-1 alpha and mitochondrial transcription factor A, and mitochondrial DNA; co-administration of EtOH and NAC prevented these effects. Chronic EtOH administration was associated with decreased mitochondrial mass, inactivation and depletion of mitochondrial complex I and complex IV, and increased hepatic mitochondrial oxidative damage, effects that were not prevented by NAC. CONCLUSIONS: These results suggest that oxidative stress caused by chronic EtOH triggered the up-regulation of mitochondrial biogenesis genes in rat liver, because an antioxidant such as NAC prevented both effects. Because NAC did not prevent liver mitochondrial oxidative damage, extra-mitochondrial effects of reactive oxygen species may regulate mitochondrial biogenesis. In spite of the induction of hepatic mitochondrial biogenesis genes by chronic EtOH, mitochondrial mass and function decreased probably in association with mitochondrial oxidative damage. These results also predict that the effectiveness of NAC as an antioxidant therapy for chronic alcoholism will be limited by its limited antioxidant effects in mitochondria, and its inhibitory effect on mitochondrial biogenesis.

Effect of garlic-derived organosulfur compounds on mitochondrial function and integrity in isolated mouse liver mitochondria.

The objectives of this work were to evaluate the direct effects of diallysulfide (DAS) and diallyldisulfide (DADS), two major organosulfur compounds of garlic oil, on mitochondrial function and integrity, by using isolated mouse liver mitochondria in a cell-free system. DADS produced concentration-dependent mitochondrial swelling over the range 125-1000µM, while DAS was ineffective. Swelling experiments performed with de-energized or energized mitochondria showed similar maximal swelling amplitudes. Cyclosporin A (1µM), or ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA, 1mM) were ineffective in inhibiting DADS-induced mitochondrial swelling. DADS produced a minor (12%) decrease in mitochondrial membrane protein thiols, but did not induce clustering of mitochondrial membrane proteins. Incubation of mitochondria with DADS (but not DAS) produced an increase in the oxidation rate of 2',7' dichlorofluorescein diacetate (DCFH-DA), together with depletion of reduced glutathione (GSH) and increased lipid peroxidation. DADS (but not DAS) produced a concentration-dependent dissipation of the mitochondrial membrane potential, but did not induce cytochrome c release. DADS-dependent effects, including mitochondrial swelling, DCFH-DA oxidation, lipid peroxidation and loss of mitochondrial membrane potential, were inhibited by antioxidants and iron chelators. These results suggest that DADS causes direct impairment of mitochondrial function as the result of oxidation of the membrane lipid phase initiated by the GSH- and iron-dependent generation of oxidants.

Increased oxidative stress and cytotoxicity by hydrogen sulfide in HepG2 cells overexpressing cytochrome P450 2E1.

The main objectives of this work were to evaluate the effects of hydrogen sulfide on oxidative stress and cytotoxicity parameters in HepG2 cells and to assess the extent to which cytochrome P450 2E1 (CYP2E1) activity modulates the effects of hydrogen sulfide on oxidative stress and cytotoxicity. Sodium hydrosulfide (NaHS) caused time- and concentration-dependent cytotoxicity in both non-P450-expressing HepG2 cells (C34 cells) and CYP2E1-overexpressing HepG2 cells (E47 cells); however, NaHS-dependent cytotoxicity was higher in E47 than C34 cells. Cytotoxicity by NaHS in C34 and E47 cells was mainly necrotic in nature and associated with an early decrease in mitochondrial membrane potential. NaHS caused increased oxidation of lipophilic (C11-BODIPY(581/591)) and hydrophilic (DCFH-DA) probes only in E47 cells, at a time point prior to overt cytotoxicity. Trolox, an amphipathic antioxidant, partially inhibited both the cytotoxicity and the increased oxidative stress detected in E47 cells exposed to NaHS. Cell-permeable iron chelators and CYP2E1 inhibitors significantly inhibited the oxidation of C11-BODIPY(581/591) in E47 cells in the presence of NaHS. NaHS produced lipid peroxidation and cytotoxicity in E47 cells supplemented with a representative polyunsaturated fatty acid (docosahexaenoic acid) but not in C34 cells; these effects were inhibited by α-tocopherol, a lipophilic antioxidant. These data suggest that CYP2E1 enhances H(2)S-dependent cytotoxicity in HepG2 cells through the generation of iron-dependent oxidative stress and lipid peroxidation.

CYP2E1 overexpression inhibits microsomal Ca2+-ATPase activity in HepG2 cells.

Cytochrome P450 2E1 (CYP2E1) is a microsomal enzyme that generates reactive oxygen species during its catalytic cycle. We previously found an important role for calcium in CYP2E1-potentiated injury in HepG2 cells. The possibility that CYP2E1 may oxidatively damage and inactivate the microsomal Ca2+-ATPase in intact liver cells was evaluated, in order to explain why calcium is elevated during CYP2E1 toxicity. Microsomes were isolated by differential centrifugation from two liver cell line: E47 cells (HepG2 cells transfected with the pCI neo expression vector containing the human CYP2E1 cDNA, which overexpress active microsomal CYP2E1), and control C34 cells (HepG2 cells transfected with the pCI neo expression vector alone, which do not express significantly any cytochrome P450). The Ca2+-dependent ATPase activity was determined by measuring the accumulation of inorganic phosphate from ATP hydrolysis. CYP2E1 overexpression produced a 45% decrease in Ca2+-dependent ATPase activity (8.6 nmol Pi/min/mg protein in C34 microsomes versus 4.7 nmol Pi/min/mg protein in microsomes). Saturation curves with Ca2+ or ATP showed that CYP2E1 overexpression produced a decrease in Vmax but did not affect the Km for either Ca2+ or ATP. The decrease in activity was not associated with a decrease in SERCA protein levels. The ATP-dependent microsomal calcium uptake was evaluated by fluorimetry using fluo-3 as the fluorogenic probe. Calcium uptake rate in E47 microsomes was 28% lower than in C34 microsomes. Treatment of E47 cells with 2mM N-acetylcysteine prevented the decrease in microsomal Ca2+-ATPase found in E47 cells. These results suggest that CYP2E1 overexpression produces a decrease in microsomal Ca2+-ATPase activity in HepG2 cells mediated by reactive oxygen species. This may contribute to elevated cytosolic calcium and to CYP2E1-potentiated injury.

S-adenosyl methionine protects ob/ob mice from CYP2E1-mediated liver injury.

Pyrazole treatment to induce cytochrome P-450 2E1 (CYP2E1) was recently shown to cause liver injury in ob/ob mice but not in lean mice. The present study investigated the effects of S-adenosyl-l-methionine (SAM) on the CYP2E1-dependent liver injury in ob/ob mice. Pyrazole treatment of ob/ob mice for 2 days caused necrosis, steatosis, and elevated serum transaminase and triglyceride levels compared with saline ob/ob mice. Administration of SAM (50 mg/kg body wt ip every 12 h for 3 days) prevented the observed pathological changes as well as the increase of apoptotic hepatocytes, caspase 3 activity, and serum TNF-alpha levels. SAM administration inhibited CYP2E1 activity but not CYP2E1 content. The pyrazole treatment increased lipid peroxidation, 4-hydroxynonenal and 3-nitrotyrosine protein adducts, and protein carbonyls. These increases in oxidative and nitrosative stress were prevented by SAM. Treatment of ob/ob mice with pyrazole lowered the endogenous SAM levels, and these were elevated after SAM administration. Mitochondrial GSH levels were very low after pyrazole treatment of the ob/ob mice; this was associated with elevated levels of malondialdehyde and 4-hydroxynonenal and 3-nitrotyrosine protein adducts in the mitochondria. All these changes were prevented with SAM administration. SAM protected against pyrazole-induced increase in serum transaminases, necrosis, triglyceride levels, caspase-3 activity, and lipid peroxidation even when administered 1 day after pyrazole treatment. In the absence of pyrazole, SAM lowered the slightly elevated serum transaminases, triglyceride levels, caspase-3 activity, and lipid peroxidation in obese mice. In conclusion, SAM protects against and can also reverse or correct CYP2E1-induced liver damage in ob/ob mice.

Role of intracellular calcium and phospholipase A2 in arachidonic acid-induced toxicity in liver cells overexpressing CYP2E1.

Liver cells (HepG2 and primary hepatocytes) overexpressing CYP2E1 and exposed to arachidonic acid (AA) were previously shown to lose viability together with enhanced lipid peroxidation. These events were blocked in cells pre-incubated with antioxidants (alpha-tocopherol, glutathione ethyl ester), or in HepG2 cells not expressing CYP2E1. The goal of the current study was to evaluate the role of calcium and calcium-activated hydrolases in these CYP2E1-AA interactions. CYP2E1-expressing HepG2 cells treated with AA showed an early increase in cytosolic calcium and partial depletion of ionomycin-sensitive calcium stores. These changes in calcium were blocked by alpha-tocopherol. AA activated phospholipase A2 (PLA2) in CYP2E1-expressing liver cells, and this was inhibited by PLA2 inhibitors or alpha-tocopherol. PLA2 inhibitors prevented the cell death caused by AA, without affecting CYP2E1 activity or lipid peroxidation. AA toxicity and PLA2 activation were inhibited in calcium-depleted cells, but not by removal of extracellular calcium alone. Removal of extracellular calcium inhibited the early increase in cytosolic calcium caused by AA. CYP2E1 overexpressing HepG2 cells exposed to AA showed a decrease in mitochondrial membrane potential, which was prevented by the PLA2 inhibitors. These results suggest that AA-induced toxicity to CYPE1-expressing cells: (i) is associated with release of Ca2+ from intracellular stores that depends mainly on oxidative membrane damage; (ii) is associated with activation of PLA2 that depends on intracellular calcium and lipid peroxidation; (iii) does not depend on increased influx of extracellular calcium, and (iv) depends on the effect of converging events (lipid peroxidation, intracellular calcium, activation of PLA2) on mitochondria to induce bioenergetic failure and necrosis. These interactions may play a role in alcohol liver toxicity, which requires polyunsaturated fatty acids, and involves induction of CYP2E1.

Role of phosphatidylinositol 3-kinase/AKT as a survival pathway against CYP2E1-dependent toxicity.

The objective of this work was to evaluate the possible role of PI3-kinase/AKT as a survival pathway against CYP2E1-dependent toxicity. E47 cells (HepG2 cells transfected with human CYP2E1 cDNA) exposed to 25 microM iron-nitrilotriacetate+5 microM arachidonic acid (AA+Fe) developed higher toxicity than C34 cells (HepG2 cells transfected with empty plasmid). Toxicity was associated with increased oxidative stress and activation of calcium-dependent hydrolases calpain and phospholipase A2. Treatment of E47, but not C34 cells, with arachidonic acid and iron (AA+Fe) led to a decrease in the phosphorylation state of AKT. 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), a specific inhibitor of PI3-kinase, produced a further decrease of phosphorylated AKT in AA+Fe-treated E47 cells. LY294002 and down-regulation of endogenous AKT with small interference RNAs increased the toxicity of AA+Fe in E47 cells. Toxicity of AA+Fe in rat hepatocytes was also increased by LY294002. LY294002 did not affect phospholipase A2 or calpain activation, CYP2E1 activity, or lipid peroxidation elicited by AA+Fe. alpha-Tocopherol prevented both AA+Fe and AA+Fe+LY294002-induced toxicity and decrease of phosphorylated AKT. LY294002 potentiated AA+Fe-induced loss of mitochondrial membrane potential and ATP, whereas overexpression of constitutively active AKT partially prevented mitochondrial impairment and toxicity. Mitochondrial permeability transition inhibitors prevented both AA+Fe and AA+Fe+LY294002-induced toxicity and decrease of mitochondrial membrane potential. These results suggest that: i) AA+Fe+CYP2E1-induced oxidative stress decreases AKT activation; ii) AKT inactivation induces mitochondrial impairment associated with opening of the permeability transition pore but is not dependent on the activation state of bad, glycogen synthase kinase-3beta, mammalian target of rapamycin, or bcl-xL; and iii) PI3-kinase/AKT may serve as a survival pathway against CYP2E1-dependent toxicity.

Role of cytochrome P450 in phospholipase A2- and arachidonic acid-mediated cytotoxicity.

Phospholipases A2 (PLA2) comprise a set of extracellular and intracellular enzymes that catalyze the hydrolysis of the sn-2 fatty acyl bond of phospholipids to yield fatty acids and lysophospholipids. The PLA2 reaction is the primary pathway through which arachidonic acid (AA) is released from phospholipids. PLA2s have an important role in cellular death that occurs via necrosis or apoptosis. Several reports support the hypothesis that unesterified arachidonic acid in cells is a signal for the induction of apoptosis. However, most of the biological effects of arachidonic acid are attributable to its metabolism by mainly three different groups of enzymes: cytochromes P450, cyclooxygenases, and lipoxygenases. In this review we will focus on the role of cytochrome P450 in AA metabolism and toxicity. The major pathways of arachidonic acid metabolism catalyzed by cytochrome P450 generate metabolites that are subdivided into two groups: the epoxyeicosatrienoic acids, formed by CYP epoxygenases, and the arachidonic acid derivatives that are hydroxylated at or near the omega-terminus by CYP omega-oxidases. In addition, autoxidation of AA by cytochrome P450-derived reactive oxygen species produces lipid hydroperoxides as primary oxidation products. In some cellular models of toxicity, cytochrome P450 activity exacerbates PLA2- and AA-dependent injury, mainly through the production of oxygen radicals that promote lipid peroxidation or production of metabolites that alter Ca2+ homeostasis. In contrast, in other situations, cytochrome P450 metabolism of AA is protective, mainly by lowering levels of unesterified AA and by production of metabolites that activate antiapoptotic pathways. Several lines of evidence point to the combined action of phospholipase A2 and cytochrome P450 as central in the mechanism of cellular injury in several human diseases, such as alcoholic liver disease and myocardial reperfusion injury. Inhibition of specific PLA2 and cytochrome P450 isoforms may represent novel therapeutic strategies against these diseases.

[Medical and surgical management of noniatrogenic traumatic tracheobronchial injuries]. / Manejo médico-quirúrgico de las lesiones traqueobronquiales traumáticas no iatrogénicas.

OBJECTIVE: To describe the medical and surgical management of noniatrogenic traumatic tracheobronchial injuries. PATIENTS AND METHOD: From January 1993 to July 2004, 15 cases of traumatic tracheobronchial injury were treated in our department. The diagnosis was established by bronchoscopy and a computed tomography chest scan was performed on all patients. Surgical treatment was selected for patients with unstable vital signs, an open tracheal wound, associated esophageal lesions, progression of subcutaneous or mediastinal emphysema, mediastinitis or suspicious mediastinal secretions on imaging tests, or difficulties with mechanical ventilation due to the traumatic tracheobronchial injury. RESULTS: The mean (SD) age of the patients was 35.5 (18.9) years and 12 (80%) were male. Of the 15 cases, 13 (86.7%) had penetrating trauma and 2 (13.3%) blunt trauma. The most common location of the injury was in the bronchi (9 cases; 60%), followed by the cervical trachea (4 cases; 26.6%), followed by both the thoracic trachea and bronchi (2 cases; 13.4%). The most common initial symptom was subcutaneous emphysema, which presented in 11 (73.3%) patients. Chest (12 cases; 86.7%) and orthopedic injuries (9 cases; 60%) were the most common associated injuries. Surgery was the treatment of choice in 11 (73.3%) cases and conservative medical treatment in 4 (26.7%). An irreversible brain injury caused the death of 1 patient receiving conservative treatment. CONCLUSIONS: Tracheobronchial injuries may be treated conservatively if they meet strict selection criteria. Size and location should not be used as selection criteria for surgical treatment.

Manejo médico-quirúrgico de las lesiones traqueobronquiales traumáticas no iatrogénicas / Medical and surgical management of noniatrogenic traumatic tracheobronchial injuries

Objetivo: Describir el manejo médico-quirúrgico de las lesiones traqueobronquiales traumáticas no iatrogénicas. Pacientes y método: De enero de 1993 a julio de 2004 se registraron en nuestro servicio 15 casos de lesiones traqueobronquiales traumáticas. En todos los pacientes el diagnóstico se estableció por broncoscopia y a todos se le realizó una tomografía computarizada de tórax. Se eligió tratamiento quirúrgico cuando había inestabilidad vital del paciente, herida traqueal abierta, lesiones esofágicas asociadas, progresión de enfisema subcutáneo o mediastínico, mediastinitis o colecciones mediastínicas sospechosas en pruebas de imagen o dificultades en la ventilación mecánica por la lesión traqueobronquial traumática. Resultados: La edad media (± desviación estándar) de los pacientes fue de 35,5 ± 18,9 años y 12 (80%) eran varones. Se registraron 13 traumatismos cerrados (86,7%) y 2 abiertos (13,3%). La localización más frecuente de la lesión fue bronquial (9 casos; 60%), seguida de tráquea cervical (4 casos; 26,6%) y tráquea toracicobronquial (2 casos; 13,4%). El síntoma inicial más frecuente fue el enfisema subcutáneo, que presentaron 11 pacientes (73,3%). Las lesiones asociadas más frecuentes fueron torácicas, con 12 casos (86,7%), seguidas de ortopédicas, con 9 (60%). El tratamiento de elección fue quirúrgico en 11 casos (73,3%) y médico conservador en 4 (26,7%). Falleció una paciente tratada de forma conservadora por lesión cerebral irreversible. Conclusiones: Las lesiones traqueobronquiales pueden tratarse de forma conservadora si cumplen criterios estrictos de selección. El tamaño o la localización no debe ser un criterio para la elección del tratamiento quirúrgico

Objective: To describe the medical and surgical management of noniatrogenic traumatic tracheobronchial injuries. Patients and method: From January 1993 to July 2004, 15 cases of traumatic tracheobronchial injury were treated in our department. The diagnosis was established by bronchoscopy and a computed tomography chest scan was performed on all patients. Surgical treatment was selected for patients with unstable vital signs, an open tracheal wound, associated esophageal lesions, progression of subcutaneous or mediastinal emphysema, mediastinitis or suspicious mediastinal secretions on imaging tests, or difficulties with mechanical ventilation due to the traumatic tracheobronchial injury. Results: The mean (SD) age of the patients was 35.5 (18.9) years and 12 (80%) were male. Of the 15 cases, 13 (86.7%) had penetrating trauma and 2 (13.3%) blunt trauma. The most common location of the injury was in the bronchi (9 cases; 60%), followed by the cervical trachea (4 cases; 26.6%), followed by both the thoracic trachea and bronchi (2 cases; 13.4%). The most common initial symptom was subcutaneous emphysema, which presented in 11 (73.3%) patients. Chest (12 cases; 86.7%) and orthopedic injuries (9 cases; 60%) were the most common associated injuries. Surgery was the treatment of choice in 11 (73.3%) cases and conservative medical treatment in 4 (26.7%). An irreversible brain injury caused the death of 1 patient receiving conservative treatment. Conclusions: Tracheobronchial injuries may be treated conservatively if they meet strict selection criteria. Size and location should not be used as selection criteria for surgical treatment

[Importance of routine mediastinal staging in women with non-small cell lung cancer]. / Importancia de la estadificación mediastínica, sistemática en mujeres con carcinoma broncogénico, no microcítico.

OBJECTIVE: To study the specific importance of mediastinal staging in women with nonsmall cell lung cancer. PATIENTS AND METHODS: Between July 1981 and September 2003 we surgically staged 2172 patients with nonsmall cell lung cancer who met the inclusion criteria for resectability and operability. A subgroup of 108 women was studied. Cervical mediastinoscopy was performed in all cases, with the addition of anterior mediastinotomy in cases with left upper lobe involvement. All patients underwent a preoperative computed tomography chest scan. RESULTS: Cervical mediastinoscopy was performed on all 108 patients, 26 of whom also underwent anterior mediastinotomy. Positive findings were recorded in 44 (40.7%) of the 108 cases: 39 of the 108 mediastinoscopies (36.1%), 9 of the 26 mediastinotomies (34.6%), and in 5 cases (19.2%) both mediastinoscopy and mediastinotomy. Nodal involvement was found in 13% of cases in clinical stage IA and 30.8% of cases in clinical stage IB. The percentage of positive findings was significantly higher for cases with adenocarcinoma or large cell carcinoma (P<.05). We performed 67 thoracotomies: 46 patients underwent lobectomy (42.6% of the 108), 7 bilobectomy (6.5%), 9 pneumonectomy (8.3%), and 5 exploratory thoracotomy (4.6%). The agreement between clinical staging after mediastinoscopy and pathological staging after thoracotomy was 47% (stage IA) and 57% (stage IB). CONCLUSIONS: Routine mediastinoscopy is indicated for all women with nonsmall cell lung cancer, regardless of clinical stage.

Inhibition of CYP2E1 catalytic activity in vitro by S-adenosyl-L-methionine.

The objective of this work was to evaluate the possible in vitro interactions of S-adenosyl-l-methionine (SAM) and its metabolites S-(5'-Adenosyl)-l-homocysteine (SAH), 5'-Deoxy-5'-(methylthio)adenosine (MTA) and methionine with cytochrome P450 enzymes, in particular CYP2E1. SAM (but not SAH, MTA or methionine) produced a type II binding spectrum with liver microsomal cytochrome P450 from rats treated with acetone or isoniazid to induce CYP2E1. Binding was less effective for control microsomes. SAM did not alter the carbon monoxide binding spectrum of P450, nor denature P450 to P420, nor inhibit the activity of NADPH-P450 reductase. However, SAM inhibited the catalytic activity of CYP2E1 with typical substrates such as p-nitrophenol, ethanol, and dimethylnitrosamine, with an IC(50) around 1.5-5mM. SAM was a non-competitive inhibitor of CYP2E1 catalytic activity and its inhibitory actions could not be mimicked by methionine, SAH or MTA. However, SAM did not inhibit the oxidation of ethanol to alpha-hydroxyethyl radical, an assay for hydroxyl radical generation. In microsomes engineered to express individual human P450s, SAM produced a type II binding spectrum with CYP2E1-, but not with CYP3A4-expressing microsomes, and SAM was a weaker inhibitor against the metabolism of a specific CYP3A4 substrate than a specific CYP2E1 substrate. SAM also inhibited CYP2E1 catalytic activity in intact HepG2 cells engineered to express CYP2E1. These results suggest that SAM interacts with cytochrome P450s, especially CYP2E1, and inhibits the catalytic activity of CYP2E1 in a reversible and non competitive manner. However, SAM is a weak inhibitor of CYP2E1. Since the K(i) for SAM inhibition of CYP2E1 activity is relatively high, inhibition of CYP2E1 activity is not likely to play a major role in the ability of SAM to protect against the hepatotoxicity produced by toxins requiring metabolic activation by CYP2E1 such as acetaminophen, ethanol, carbon tetrachloride, thioacetamide and carcinogens.

Importancia de la estadificación mediastínica, sistemática en mujeres con carcinoma broncogénico, no microcítico / Importance of routine mediastinal staging in women with nonsmall cell lung cancer

OBJETIVO: Investigar la importancia específica de la estadificación mediastínica de mujeres con carcinoma broncogénico, no microcítico. PACIENTES Y MÉTODOS: Entre julio de 1981 y septiembre de 2003 estadificamos quirúrgicamente a 2.172 pacientes con carcinoma broncogénico no microcítico que cumplían criterios de resecabilidad y operabilidad. Se realizó mediastinoscopia cervical de forma habitual, y mediastinotomía anterior en caso de afectación del lóbulo superior izquierdo. Todos los pacientes tenían una tomografía axial computarizada de tórax preoperatoria. Se estudió al subgrupo de 108 mujeres. RESULTADOS: Realizamos mediastinoscopia cervical en 108 casos y mediastinotomía anterior en 26. Fueron positivas 44 (40,7%) —39 (36,1%) mediastinoscopias, 9 (34,6%) mediastinotomías y en 5 (19,2%) casos ambas—. Se objetivó afectación ganglionar en un 13% de los casos en estadio IA y en un 30,8% en estadio IB. El porcentaje de positividad fue significativamente mayor en caso de adenocarcinoma o carcinoma de células grandes (p < 0,05). Realizamos 67 toracotomías: 46 (42,6%) lobectomías, 7 (6,5%) bilobectomías, 9 (8,3%) neumonectomías y 5 (4,6%) toracotomías exploradoras. La concordancia entre estadificación clínica posmediastinoscopia y patológica postoracotomía fue del 47% (IA) y del 57% (IB). CONCLUSIONES: La mediastinoscopia sistemática está indicada en todas las mujeres con carcinoma broncogénico no microcítico independientemente del estadio clínico

OBJECTIVE: To study the specific importance of mediastinal staging in women with nonsmall cell lung cancer. PATIENTS AND METHODS: Between July 1981 and September 2003 we surgically staged 2172 patients with nonsmall cell lung cancer who met the inclusion criteria for resectability and operability. A subgroup of 108 women was studied. Cervical mediastinoscopy was performed in all cases, with the addition of anterior mediastinotomy in cases with left upper lobe involvement. All patients underwent a preoperative computed tomography chest scan. RESULTS: Cervical mediastinoscopy was performed on all 108 patients, 26 of whom also underwent anterior mediastinotomy. Positive findings were recorded in 44 (40.7%) of the 108 cases: 39 of the 108 mediastinoscopies (36.1%), 9 of the 26 mediastinotomies (34.6%), and in 5 cases (19.2%) both mediastinoscopy and mediastinotomy. Nodal involvement was found in 13% of cases in clinical stage IA and 30.8% of cases in clinical stage IB. The percentage of positive findings was significantly higher for cases with adenocarcinoma or large cell carcinoma (P<.05). We performed 67 thoracotomies: 46 patients underwent lobectomy (42.6% of the 108), 7 bilobectomy (6.5%), 9 pneumonectomy (8.3%), and 5 exploratory thoracotomy (4.6%). The agreement between clinical staging after mediastinoscopy and pathological staging after thoracotomy was 47% (stage IA) and 57% (stage IB). CONCLUSIONS: Routine mediastinoscopy is indicated for all women with nonsmall cell lung cancer, regardless of clinical stage