Impact of DCM-Causing Genetic Background on Long-Term Response to Cardiac Resynchronization Therapy.

BACKGROUND: Patients with nonischemic dilated cardiomyopathy (DCM), severe left ventricular (LV) dysfunction, and complete left bundle branch block benefit from cardiac resynchronization therapy (CRT). However, a large heterogeneity of response to CRT is described. Several predictors of response to CRT have been identified, but the role of the underlying genetic background is still poorly explored. OBJECTIVES: In the present study, the authors sought to define differences in LV remodeling and outcome prediction after CRT when stratifying patients according to the presence or absence of DCM-causing genetic background. METHODS: From our center, 74 patients with DCM subjected to CRT and available genetic testing were retrospectively enrolled. Carriers of causative monogenic variants in validated DCM-causing genes, and/or with documented family history of DCM, were classified as affected by genetically determined disease (GEN+DCM) (n = 25). Alternatively, by idiopathic dilated cardiomyopathy (idDCM) (n = 49). The primary outcome was long-term LV remodeling and prevalence of super response to CRT (evaluated at 24-48 months after CRT); the secondary outcome was heart failure-related death/heart transplant/LV assist device. RESULTS: GEN+DCM and idDCM patients were homogeneous at baseline with the exception of QRS duration, longer in idDCM. The median follow-up was 55 months. Long-term LV reverse remodeling and the prevalence of super response were significantly higher in the idDCM group (27% in idDCM vs 5% in GEN+DCM; P = 0.025). The heart failure-related death/heart transplant/LV assist device outcome occurred more frequently in patients with GEN+DCM (53% vs 24% in idDCM; P = 0.028). CONCLUSIONS: Genotyping contributes to the risk stratification of patients with DCM undergoing CRT implantation in terms of LV remodeling and outcomes.

New perspectives in diagnosis and risk stratification of non-ischaemic dilated cardiomyopathy.

Dilated cardiomyopathy is a primitive heart muscle condition, characterized by structural and functional abnormalities, in the absence of a specific cause sufficient to determine the disease. It is, though, an 'umbrella' term that describes the final common pathway of different pathogenic processes and gene-environment interactions. Performing an accurate diagnostic workup and appropriate characterization of the patient has a direct impact on the patient's outcome. The physician should adapt a multiparametric approach, including a careful anamnesis and physical examination and integrating imaging data and genetic testing. Aetiological characterization should be pursued, and appropriate arrhythmic risk stratification should be performed. Evaluations should be repeated thoroughly at follow-up, as the disease is dynamical over time and individual risk might evolve. The goal is an all-around characterization of the patient, a personalized medicine approach, in order to establish a diagnosis and therapy tailored for the individual patient.

Lipid-Lowering Drug Therapy: Critical Approach for Implementation in Clinical Practice.

Increased levels of low-density lipoprotein cholesterol (LDL-C) are recognized as a primary risk factor for atherosclerotic cardiovascular disease, which remains the leading cause of death worldwide. Lowering LDL-C levels clearly reduces the risk of cardiovascular events, with benefits related to both absolute reduction and duration of treatment; however, a threshold below which low LDL-C levels can be dangerous has never been established. Since the discovery of statins, cardiovascular research has focused on developing new lipid-lowering agents. Ezetimibe and proprotein convertase subtilisin-kexin type 9 inhibitors have been found to further reduce LDL-C values and subsequent cardiovascular risk. Novel recently approved inclisiran and bempedoic acid, currently being tested in cardiovascular outcomes studies, are further expanding our pharmacological armamentarium, enabling the clinician to diminish residual risk related to LDL-C. Moreover, new agents are paving the way to successful treatment of homozygous familial hypercholesterolemia. This review summarizes the main characteristics of current and emerging lipid-lowering therapies to assist with comprehensive evidence-based decision making.

Early prognostic stratification and identification of irreversibly shocked patients despite primary percutaneous coronary intervention.

BACKGROUND: Despite prognostic improvements in ST-elevation myocardial infarction (STEMI), patients presenting with cardiogenic shock (CS) have still high mortality. Which are the relevant early prognostic factors despite revascularization in this high-risk population is poorly investigated. METHODS: We analyzed STEMI patients treated with primary percutaneous coronary intervention (PCI) and enrolled at the University Hospital of Trieste between 2012 and 2018. A decision tree based on data available at first medical contact (FMC) was built to stratify patients for 30-day mortality. Multivariate analysis was used to explore independent factors associated with 30-day mortality. RESULTS: Among 1222 STEMI patients consecutively enrolled, 7.5% presented with CS. CS compared with no-CS patients had worse 30-day mortality (33% vs 3%, P < 0.01). Considering data available at FMC, CS patients with a combination of age ≥76 years, anterior STEMI and an expected ischemia time > 3 h and 21 min were at the highest mortality risk, with a 30-day mortality of 85.7%. In CS, age (OR 1.246; 95% CI 1.045-1,141; P = 0.003), final TIMI flow 2-3 (OR 0.058; 95% CI 0.004-0.785; P = 0.032) and Ischemia Time (OR = 1.269; 95% CI 1.001-1.609; P = 0.049) were independently associated with 30-day mortality. CONCLUSIONS: In a contemporary real-world population presenting with CS due to STEMI, age is a relevant negative factor whereas an early and successful PCI is positively correlated with survival. However, a subgroup of elderly patients had severe prognosis despite revascularization. Whether pPCI may have an impact on survival in a very limited number of irreversibly critically ill patients remains uncertain and the identification of irreversibly shocked patients remains nowadays challenging.

Prevalence and predictors of persistent sinus rhythm after elective electrical cardioversion for atrial fibrillation.

AIMS: To evaluate the prevalence and predictors of persistent sinus rhythm in a recent cohort of unselected patients undergoing electrical cardioversion for atrial fibrillation. METHODS: We enrolled all consecutive patients undergoing elective electrical cardioversion for atrial fibrillation between January 2017 and December 2018. We analysed baseline clinical and echocardiographic data as well as pharmacological antiarrhythmic therapy. Primary endpoint was the maintenance of sinus rhythm at 12 months after electrical cardioversion. RESULTS: Of the 300 patients enrolled, 270 (90%) had successful electrical cardioversion and among them, 201 patients have 12-month follow-up data (mean age 70 ±â€Š10 years; 74% men). At 12 months, only 45.7% were in sinus rhythm. Patients without sinus rhythm compared with persistent sinus rhythm at 12 months had a lower baseline left ventricle ejection fraction (LVEF) (49.1 ±â€Š16 vs. 59.7 ±â€Š9%, P = 0.02) and had more frequently a history of atrial fibrillation more than 12 months (55 vs. 34% P = 0.003). At the multivariate analysis, only the duration of the disease beyond 12 months (OR 0.26, 95% CI: 0.08-0.88, P = 0.032), LVEF (OR 1.06, 95% CI: 1.01-1.12, P = 0.012) and the presence of sinus rhythm at 1-month follow-up (OR 18.28, 95% CI: 3.3-100, P = 0.001) were associated with the probability of maintaining sinus rhythm at 12 months. CONCLUSION: In unselected patients with atrial fibrillation undergoing elective electrical cardioversion, only 45.7% were in sinus rhythm at 12 months. The presence of sinus rhythm at 1-month follow-up emerged as an independent predictor of maintenance of sinus rhythm. This highlights that early re-evaluation of these patients appears useful for assessing longer term outcomes also from the perspective of a possible selective approach to ablation strategies.

[Diagnostic work-up and clinical management of cardiomyopathies: the operative protocol from the Cardiothoracovascular Department of Trieste, Italy]. / Inquadramento e gestione delle cardiomiopatie: il protocollo della Cardiologia di Trieste.

Cardiomyopathies are primary myocardial disorders, genetically determined, with clinical onset between the third and the fifth decade of life. They represent the main causes of sudden cardiac death and heart failure in the youth. The more common myocardial diseases in clinical practice are dilated cardiomyopathy, arrhythmogenic cardiomyopathy and hypertrophic cardiomyopathy. Next generation sequencing techniques, recently available for genetics researches, together with the diffusion of advanced imaging techniques, permitted in the last years a deeper knowledge of these pathologies. Nevertheless, diagnosis, etiology and several aspects of patients' clinical management remain complex and controversial. This review paper aims to propose some operative flow-charts, derived from scientific evidences and the internal protocol of the Cardiothoracovascular Department of Trieste Hospital, Italian referral Center for cardiomyopathies and heart failure, with more than 30 years of experience in diagnosis and management of patients who suffer from primary myocardial disorders.

Cardiac Tumors: Diagnosis, Prognosis, and Treatment.

PURPOSE OF REVIEW: Cardiac masses frequently present significant diagnostic and therapeutic clinical challenges and encompass a broad set of lesions that can be either neoplastic or non-neoplastic. We sought to provide an overview of cardiac tumors using a cardiac chamber prevalence approach and providing epidemiology, imaging, histopathology, diagnostic workup, treatment, and prognoses of cardiac tumors. RECENT FINDINGS: Cardiac tumors are rare but remain an important component of cardio-oncology practice. Over the past decade, the advances in imaging techniques have enabled a noninvasive diagnosis in many cases. Indeed, imaging modalities such as cardiac magnetic resonance, computed tomography, and positron emission tomography are important tools for diagnosing and characterizing the lesions. Although an epidemiological and multimodality imaging approach is useful, the definite diagnosis requires histologic examination in challenging scenarios, and histopathological characterization remains the diagnostic gold standard. A comprehensive clinical and multimodality imaging evaluation of cardiac tumors is fundamental to obtain a proper differential diagnosis, but histopathology is necessary to reach the final diagnosis and subsequent clinical management.

[Cardiological counseling and perioperative management of heart disease patients. Protocol of the University of Trieste - Year 2019]. / Consulenza cardiologica e gestione perioperatoria del paziente cardiopatico. Protocollo dell'Azienda Sanitaria Universitaria Integrata di Trieste ­ Anno 2019.

The management of patients with heart disease or suspected heart disease, who are hospitalized and/or who should undergo surgery or an invasive procedure, is very complex for the comorbidities often present, the multiple therapies taken and the frequent presence of advanced cardiac devices.The purpose of this document is to provide indications and standardize the behavior of different clinicians in the management of heart disease patients or those with suspected heart disease in order (i) to manage acute cardiac conditions with appropriate timing and accuracy, and (ii) to define the cardiovascular risk in the individual patient with appropriate timing and indications, allowing patients to face any surgery or invasive procedure with the lowest risk correlated to his heart disease.

Quality control of regional wall motion analysis in stress Echo 2020.

BACKGROUND: The trial "Stress Echo (SE) 2020" evaluates novel applications of SE beyond coronary artery disease. The aim of the study was control quality and harmonize reading criteria. METHODS: One reader from 78 centers of the SE 2020 network asked for credentials to read a set of 20 SE video-clips selected by the core lab. All aspiring centers met the pre-requisite of high-volume and the years of experience in SE ranged from 5 to 31years (mean value 18years). The diagnostic gold standard was a reading by the core lab. The a priori determined pass threshold was 18/20 (≥90%). RESULTS: Of the initial 78 who started, 57 completed the first attempt: individual readers' score on first attempt ranged from 07/20 to 20/20 (accuracy from 35% to 100%, mean 78.7±13%) and 44 readers passed it. There was a very poor correlation between years of experience and the reader's score on first attempt (r=-0.161, p=0.231). Of the 13 readers who failed the first attempt, 12 took it again after the web-based session and their accuracy improved (74% vs. 96%, p<0.001). The kappa inter-observer agreement before and after web-based training was 0.59 on first attempt and rose to 0.91 on the last attempt. CONCLUSIONS: In SE reading, the volume of activity or years of experience is not synonymous with diagnostic quality. Qualitative analysis and operator-dependence can become a limiting weakness in clinical practice, in the absence of strict pathways of learning, credentialing and audit.

Stress echo 2020: the international stress echo study in ischemic and non-ischemic heart disease.

BACKGROUND: Stress echocardiography (SE) has an established role in evidence-based guidelines, but recently its breadth and variety of applications have extended well beyond coronary artery disease (CAD). We lack a prospective research study of SE applications, in and beyond CAD, also considering a variety of signs in addition to regional wall motion abnormalities. METHODS: In a prospective, multicenter, international, observational study design, > 100 certified high-volume SE labs (initially from Italy, Brazil, Hungary, and Serbia) will be networked with an organized system of clinical, laboratory and imaging data collection at the time of physical or pharmacological SE, with structured follow-up information. The study is endorsed by the Italian Society of Cardiovascular Echography and organized in 10 subprojects focusing on: contractile reserve for prediction of cardiac resynchronization or medical therapy response; stress B-lines in heart failure; hypertrophic cardiomyopathy; heart failure with preserved ejection fraction; mitral regurgitation after either transcatheter or surgical aortic valve replacement; outdoor SE in extreme physiology; right ventricular contractile reserve in repaired Tetralogy of Fallot; suspected or initial pulmonary arterial hypertension; coronary flow velocity, left ventricular elastance reserve and B-lines in known or suspected CAD; identification of subclinical familial disease in genotype-positive, phenotype- negative healthy relatives of inherited disease (such as hypertrophic cardiomyopathy). RESULTS: We expect to recruit about 10,000 patients over a 5-year period (2016-2020), with sample sizes ranging from 5,000 for coronary flow velocity/ left ventricular elastance/ B-lines in CAD to around 250 for hypertrophic cardiomyopathy or repaired Tetralogy of Fallot. This data-base will allow to investigate technical questions such as feasibility and reproducibility of various SE parameters and to assess their prognostic value in different clinical scenarios. CONCLUSIONS: The study will create the cultural, informatic and scientific infrastructure connecting high-volume, accredited SE labs, sharing common criteria of indication, execution, reporting and image storage of SE to obtain original safety, feasibility, and outcome data in evidence-poor diagnostic fields, also outside the established core application of SE in CAD based on regional wall motion abnormalities. The study will standardize procedures, validate emerging signs, and integrate the new information with established knowledge, helping to build a next-generation SE lab without inner walls.

Comparison of Patient Characteristics and Course of Hypertensive Hypokinetic Cardiomyopathy Versus Idiopathic Dilated Cardiomyopathy.

Hypertensive hypokinetic cardiomyopathy (HHC) is defined by left ventricular (LV) systolic dysfunction with a history of systemic hypertension as the only possible cause. Although commonly encountered in clinical practice, its characterization and differences with true idiopathic dilated cardiomyopathy (IDC) are lacking. The aim of this study was to characterize the clinical instrumental features and the natural history of HHC. We analyzed the data of 4,191 patients referred to our center for newly diagnosed LV systolic dysfunction from 2005 to 2010. Of them, 310 presented idiopathic LV systolic dysfunction (LV ejection fraction <50%): 136 (44%) had a history of systemic hypertension and were defined HHC. The remaining 174 patients were considered IDC. Compared with patients with IDC, those with HHC were older (63 ± 11 vs 47 ± 14 years, p <0.001), with worse comorbidity profile, higher blood pressure, and increased LV mass. During follow-up, patients with HHC showed earlier and higher proportion of LV reverse remodeling (46% vs 21% at 6 months' follow-up). Moreover, they had a better long-term survival free from cardiovascular death/ventricular assist device/heart transplant/malignant ventricular arrhythmias (5.1 vs 12.6 in HHC and IDC, p = 0.03). Indeed, their mortality was mainly driven by noncardiovascular causes (at 10 years 9.6% vs 1.7% in HHC and IDC, p <0.001). In conclusion, HHC has a high prevalence among patients with "idiopathic" LV dysfunction. The natural history of patients with HHC is characterized by a rapid response to optimal therapy for heart failure, a favorable cardiovascular outcome, and a relevant incidence of noncardiovascular events.

Usefulness of high-dose intravenous human immunoglobulins treatment for refractory recurrent pericarditis.

The management of refractory recurrent pericarditis is challenging. Previous clinical reports have noted a beneficial effect of high-dose intravenous human immunoglobulins (IvIgs) in isolated and systemic inflammatory disease-related forms. In this article, we analyzed retrospectively our clinical experience with IvIg therapy in a series of clinical cases of pericarditis refractory to conventional treatment. We retrospectively analyzed 9 patients (1994 to 2010) with refractory recurrent pericarditis, who received high-dose IvIg as a part of their medical treatment. Nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, or colchicine treatment was not discontinued during IvIg treatment. No patients had a history of autoimmune or connective tissue diseases. During an average period of 11 months from the first recurrence, patients had experienced a mean of 5 relapses before the first IvIg treatment. In 4 cases, patients showed complete clinical remission with no further relapse after the first IvIg cycle. Two patients experienced a single minor relapse, responsive to short-term nonsteroidal anti-inflammatory drugs. In 2 patients, we performed a second cycle of IvIg after a recurrence of pericarditis, with subsequent complete remission. One patient did not respond to 3 cycles of IvIg and subsequently underwent pericardial window and long-term immunosuppressive treatment. No major adverse effect was observed in consequence of IvIg administration in all the cases. In conclusion, although IvIg mode of action is still poorly understood in this setting, this treatment can be considered as an option in patients with recurrent pericarditis refractory to conventional medical treatment and, in our small series, has proved to be effective in 8 of 9 cases.

[Indications for cardiology consultation and management of cardiac patients who will undergo surgical or endoscopic procedures: the proposal of the University Hospital of Trieste, Italy]. / Indicazioni per la consulenza cardiologica e gestione del paziente cardiopatico da operare o sottoporre a procedure endoscopiche: la proposta dell'Azienda Ospedaliero-Universitaria di Trieste.

The number of patients affected by cardiovascular disease admitted to internal medicine and geriatric wards is expanding due to the increasing prevalence of cardiovascular disease in the ageing population. This contributes to a growing demand for cardiology consult visits, with requests for perioperative risk stratification for non-cardiac surgery or endoscopy, and general clinical management. This document was jointly drafted by the Cardiology and Anesthesiology departments, medical and surgical departments, and endoscopy services of the Azienda Ospedaliero-Universitaria "Ospedali Riuniti" in Trieste (Italy). It addresses critical issues such as antiplatelet and anticoagulant therapy in non-cardiac surgery, electric device management, and prophylaxis of bacterial endocarditis. It provides general guidelines and appropriateness criteria, prompted by the Joint Commission International and approved by the Hospital Guidelines Committee. It provides a basis for periodic educational meetings, and will be periodically updated. Periodic audits will monitor its application, and critical and controversial points, in order to promote quality of health care, organizational efficiency, and appropriateness.

[If heart failure or cardiac arrest complicate pregnancy and delivery]. / Quando lo scompenso o l'arresto cardiaco complicano la gravidanza ed il parto.

Physiological adaptation to pregnancy exposes mother's cardiovascular system to relevant hemodynamic overload. These changes and other specific conditions of pregnancy, such as amniotic embolism, can point out unrecognized preexisting heart disease or, in the presence of some cofactors, be burdensome even for healthy hearts. Thus, tragic cases of heart failure or cardiac arrest may occur, whose management requires several considerations with respect of trying to save two lives at the same time, the need for drugs potentially harmful to the fetus, and assessment of emergent cesarean section.

Improved metabolic stimulus for glucose-induced insulin secretion through GK and PFK-2/FBPase-2 coexpression in insulin-producing RINm5F cells.

The glucose sensor enzyme glucokinase plays a pivotal role in the regulation of glucose-induced insulin secretion in pancreatic beta-cells. Activation of glucokinase represents a promising concept for the treatment of type 2 diabetes. Therefore, we analyzed the glucokinase activation through its physiological interaction partner, the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) and the resulting effect on glucose metabolism in insulin-producing cells. In RINm5F-GK-PFK-2/FBPase-2 cells stably overexpressing glucokinase plus islet PFK-2/FBPase-2, colocalization between both enzymes as well as elevation of glucokinase activity were significantly increased at a stimulatory glucose concentration of 10 mmol/liter. RINm5F-GK-PFK-2/FBPase-2 cells showed under this culture condition a significant increase in glucose utilization and in the ATP/ADP ratio compared with RINm5F-GK cells, which only overexpress glucokinase. Also glucose-induced insulin secretion was elevated in RINm5F-GK-PFK-2/FBPase-2 cells in comparison to RINm5F-GK cells. Furthermore, pyruvate accumulation and lactate production in RINm5F-GK-PFK-2/FBPase-2 cells were significantly lower at both 10 and 30 mmol/liter glucose than in RINm5F-GK and RINm5F cells. The significant improvement of glucose metabolism after PFK-2/FBPase-2 overexpression is apparently not exclusively the result of high glucokinase enzyme activity. Stabilization of the closed glucokinase conformation by PFK-2/FBPase-2 may not only activate the enzyme but also improve metabolic channeling in beta-cells.

Interaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) with glucokinase activates glucose phosphorylation and glucose metabolism in insulin-producing cells.

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) was recently identified as a new intracellular binding partner for glucokinase (GK). Therefore, we studied the importance of this interaction for the activity status of GK and glucose metabolism in insulin-producing cells by overexpression of the rat liver and pancreatic islet isoforms of PFK-2/FBPase-2. PFK-2/FBPase-2 overexpression in RINm5F-GK cells significantly increased the GK activity by 78% in cells expressing the islet isoform, by 130% in cells expressing the liver isoform, and by 116% in cells expressing a cAMP-insensitive liver S32A/H258A double mutant isoform. Only in cells overexpressing the wild-type liver PFK-2/FBPase-2 isoform was the increase of GK activity abolished by forskolin, apparently due to the regulatory site for phosphorylation by a cAMP-dependent protein kinase. In cells overexpressing any isoform of the PFK-2/FBPase-2, the increase of the GK enzyme activity was antagonized by treatment with anti-FBPase-2 antibody. Increasing the glucose concentration from 2 to 10 mmol/l had a significant stimulatory effect on the GK activity in RINm5F-GK cells overexpressing any isoform of PFK-2/FBPase-2. The interaction of GK with PFK-2/FBPase-2 takes place at glucose concentrations that are physiologically relevant for the activation of GK and the regulation of glucose-induced insulin secretion. This new mechanism of posttranslational GK regulation may also represent a new site for pharmacotherapeutic intervention in type 2 diabetes treatment.

Effects of gamma-hexachlorocyclohexane and L-3,3',5-triiodothyronine on rat liver cytochrome P4502E1-dependent activity and content in relation to microsomal superoxide radical generation.

Liver microsomal cytochrome P4502E1-dependent p-nitrophenol (PNP) hydroxylation and expression of cytochrome P4502E1 were studied in rats subjected to gamma-hexachlorocyclohexane (HCCH) or L-3,3,5-triiodothyronine (T3) administration as a possible mechanism contributing to superoxide radical (O2.-) generation. HCCH treatment (a single dose of 40 mg/kg body wt) produced a 43% increase in the content of total cytochrome P450, whereas T3 (daily doses of 0.1 mg/kg body wt for two consecutive days) led to a 37% decrease. NADPH-dependent O2.- generation was elevated by HCCH and T3, expressed as either per mg of protein or per nmol of cytochrome P450, with a 135% enhancement in the O2.- production/superoxide dismutase (SOD) activity ratios being observed in both conditions. This was partly due to depression of SOD activity. Concomitantly, the molecular activity of NADPH-cytochrome p450 reductase was enhanced by 90 and 69% by HCCH and T3, respectively. In these conditions, microsomal PNP hydroxylation showed increases of 58 and 45% in HCCH- and T3-treated rats over control values, respectively, with a parallel 31% (HCCH) and 41% (T3) enhancement in the content of cytochrome P4502E1 assessed by western immunoblotting. We conclude that HCCH and T3 enhance the expression and activity of cytochrome P4502E1 and that of NADPH-cytochrome P450 reductase in rat liver, regardless of the changes in total cytochrome P450 content, representing major contributory mechanisms to microsomal NADPH-dependent O2.- generation.

Effects of gamma-hexachlorocyclohexane and L-3, 3', 5-triiodothyronine on rat liver cytochrome P4502E1-dependent activity and content in relation to microsomal superoxide radical generation

Liver microsomal cytochrome P4502E1-dependent p-nitrophenol (PNP) hydroxylation and expression of cytochrome P4502E1 were studied in rats subjected to gamma-hexachlorocyclohexane (HCCH) or L-3,3,5-triiodothyronine (T3) administration as a possible mechanism contributing to superoxide radical (O2.-) generation. HCCH treatment (a single dose of 40 mg/kg body wt) produced a 43 per cent increase in the content of total cytochrome P450, whereas T3 (daily doses of 0.1 mg/kg body wt for two consecutive days) led to a 37 per cent decrease. NADPH-dependent O2.- generation was elevated by HCCH and T3, expressed as either per mg of protein or per nmol of cytochrome P450, with a 135 per cent enhancement in the O2.- production/superoxide dismutase (SOD) activity ratios being observed in both conditions. This was partly due to depression of SOD activity. Concomitantly, the molecular activity of NADPH-cytochrome p450 reductase was enhanced by 90 and 69 per cent by HCCH and T3, respectively. In these conditions, microsomal PNP hydroxylation showed increases of 58 and 45 per cent in HCCH- and T3-treated rats over control values, respectively, with a parallel 31 per cent (HCCH) and 41 per cent (T3) enhancement in the content of cytochrome P4502E1 assessed by western immunoblotting. We conclude that HCCH and T3 enhance the expression and activity of cytochrome P4502E1 and that of NADPH-cytochrome P450 reductase in rat liver, regardless of the changes in total cytochrome P450 content, representing major contributory mechanisms to microsomal NADPH-dependent O2.- generation.