Acute hematologic effects of interferon alpha,interferon gamma,tumor necrosis factor alpha and Interleukin 2

Summary This study was designed to investigate acute effects of various doses of the cytokines IFN-alpha, IFN-gamma Interleukin 2 and tumor necrosis factor alpha on white blood cell differential counts. Before initiation of phase II trials, a dose-determination phase was performed, where three different dose levels of each cytokine were applied as a single dose. White blood cell differential counts were assessed immediately before and 2, 12, 24, 48 and 168 h after injection. Patients enrolled suffered from metastatic cancer or chronic active hepatitis. In addition, IFN-alpha was administered to five healthy volunteers. Results indicate that cytokines cause rapid and transient changes in the numbers of leukocyte subsets. Hematologic changes were cell-type- and cytokine-specific: transient lymphopenia was observed after administration of all four cytokines, reaching a nadir 12 to 24 h after subcutaneous injection. Administration of TNF-alpha and IFN-gamma also caused transient monocytopenia. Neutrophilia developed after administration of Interleukin 2, IFN-alpha and TNF-alpha. We conclude that cytokines play a key role in the regulation of peripheral blood cell traffic by their capacity to influence homing patterns of peripheral blood leukocytes.     

Tumor-promoting activity and cytotoxicity of 3,4,3′,4′-tetrachlorobiphenyl on N-nitrosomorpholine-induced murine liver foci

Summary Effects of 3,4,3,4-tetrachlorobiphenyl (TCB) on glucose-6-phosphatase (G6Pase)-altered hepatic foci of N-nitrosomorpholine (NNM)-treated B6C3F1 mice were investigated. TCB was chosen as a selective 3-methylcholanthrene-type inducer and tumor promoter. To initiate hepatocarcinogenesis, mice were treated with NNM (160 mg/l, in drinking water for 7 weeks), as in previous studies with the rat model. After a treatment-free interval of 22 weeks, TCB was administered (5×50 mg/kg, every 3 days), and liver foci were analysed 10 weeks after the start of TCB treatment. Unexpectedly, the number of G6Pase-negative and-positive foci per liver was markedly diminished following TCB treatment (to 32% and 57%, respectively). On the other hand, the mean volume of the remaining G6Pase-altered foci was enhanced, owing to an increase in the percentage of foci of large size (>0.5mm²). Throughout the experimental period of 39 weeks prolonged liver injury due to NNM and TCB treatment was demonstrated by histology and by elevated serum levels of glutamate-oxaloacetate transaminase. The results suggest that (in contrast to the rat system) TCB exhibited opposing effects on liver foci in the mouse model: (a) moderate tumor-promoting effects and (b) cytotoxic effects in NNM-injured liver, leading to decreased numbers of liver foci.Abbreviations used G6Pase glucose-6-phosphatase - NNM N-nitrosomorpholine - TCB 3,4,3,4-tetrachlorobiphenyl     

Gabexate mesilate, a synthetic protease inhibitor, attenuates carbon tetrachloride-induced liver injury in rats

Background Gabexate mesilate, a synthetic protease inhibitor, is used to treat acute pancreatitis and disseminated intravascular coagulation because it inhibits various serine proteases; however, whether gabexate mesilate prevents acute liver failure has not yet been studied. The aim of the present study was to investigate the effect of gabexate mesilate in carbon tetrachloride (CCl₄)-induced liver injury in rats.Methods Acute hepatic failure was induced by administration of CCl₄ intragastrically to male Sprague–Dawley rats. The effects of gabexate mesilate were examined in terms of serum transaminase levels, liver histology, and the prognosis of rats.Results Gabexate mesilate treatment significantly decreased the elevation of serum transaminase levels and improved liver histology 24h after the administration of CCl₄ (0.2ml/100g rat weight). Plasma tumor necrosis factor- (TNF-) and interleukin-1 (IL-1) decreased significantly in the gabexate mesilate-treated rats compared with saline-treated rats. Gabexate mesilate treatment also significantly improved survival rate after a lethal dose of CCl₄ (0.5ml/100g rat weight) from 0% to 20%.Conclusions Gabexate mesilate treatment attenuated CCl₄-induced liver injury via a suppression of proinflammatory cytokine production. In addition, these investigations suggest that gabexate mesilate treatment may provide therapeutic strategies for human acute liver failure.     

Plasma immunoreactive glucagon fractions in four cases of glucagonoma: Increased “Large glucagon — immunoreactivity”

Summary Immunoreactive glucagon (IRG) fractions from plasma of 8 normal subjects and 4 patients with glucagon secreting tumors were studied by gel filtration techniques on Bio Gel P-30 and Sephadex G-50 columns. The pancreatic glucagon specific anti serum (30K) of Unger was utilized to measure IRG. Columns were calibrated with labelled albumin, proinsulin, insulin and glucagon. Four peaks were defined in normal and tumor bearing patients: peak I (>20000 mol. wt.), peak II (primarily 9000 mol. wt.), peak III pancreatic glucagon (3500 mol. wt.) and peak IV small glucagon (<3500 mol. wt.). Glucagonoma patients differed from our normal and reported normal subjects in that peak II contained most of the circulating IRG. The percent of IRG associated with peak II was 9.5–31.5% in normals and 39.1–61.2% in glucagonomas. Glucagon-like biological activity in an isolated hepatocyte system was demonstrated for all peaks. However, relative to immunoreactivity, peak II showed reduced activity (25–33%). Immunoassay of dilutions of all peaks revealed the probability of immuno determinants identical with porcine pancreatic glucagon. The presence of heterogeneous IRG peaks with biological glucagon-like activity suggest that the larger molecules may be prohormones. Further, it is possible that specific elevation of peak II may be a diagnostic feature of glucagonomas.Supported in part by NIH Grant #AM 17431-02.     

Plasma leptin and TNF-alpha levels in chronic hepatitis C patients and their relationship to hepatic fibrosis

The aim of this study was to examine the possible relationship between the plasma levels of leptin and tumor necrosis factor (TNF)- and the stage of hepatic fibrosis in a cohort of patients with chronic hepatitis C. Leptin and TNF levels were measured by RIA in 135 patients and in 75 age- and sex-matched controls. Liver disease was evaluated by the stage of fibrosis and the extent of inflammatory infiltrate in the liver biopsy. Leptin levels correlated with BMI values in healthy controls and in patients with chronic hepatitis C (men, r = 0.61, P = 0.0001; women, r = 0.68, P = 0.003). Leptin levels increased as hepatic fibrosis stage progressed both in male and in female patients (P < 0.001); also, TNF levels were higher in patients with an advanced stage of fibrosis (P = 0.006). In these patients, levels of leptin increased according to the progression of the stage of fibrosis; these data suggest that leptin may play a role in the regulation of hepatic fibrosis.     

Differences of cellular composition and adhesion molecule expression in “leukemic” as compared with “normal” human long-term bone marrow cultures

Summary Human long-term bone marrow cultures (HLTBMCs) were established with bone marrow samples collected from 15 patients with acute myeloid leukemia (AML) and compared with HLTBMCs from eight healthy volunteers. During 6 weeks of culture, the cellular composition of HLTBMCs was quantitatively studied. The cells of the HLTBMCs were divided into three main categories: fibroblasts, macrophages, and other cells (endothelial cells, hematopoietic cells and undefined cells). HLTBMCs derived from healthy volunteers demonstrated a very consistent development. The number of fibroblasts increased during culture and the number of macrophages decreased, resulting in a steady state after 3 weeks of culture. In contrast, HLTBMCs derived from patients with AML showed a strikingly different pattern of irregular development and a steady state was not reached under our conditions. The APAAP technique was used to demonstrate expression of adhesion molecules. VLA2, VLA5, VLA6, LFA1, Mac1, p150/95, ₂-chain, HCAM, ICAM1, NCAM, and VCAM1 were more expressed on normal as compared with leukemic bone marrow stromal cells, although this reached significance only for ₂-chain and NCAM. VLA1, 3, and 4 were expressed in a higher percentage on leukemic stroma (not significant). More expression was seen on normal as opposed to leukemic macrophages for the adhesion molecules tested, except for VLA5. The differences reached significance for the majority of molecules tested. It is concluded that striking differences exist in cellular composition and adhesion molecule expression between HLTBMCs from healthy individuals and those from patients with AML. This may have an impact on the pathogenesis of AML.     

Fluorometrische Bestimmung von “aktiviertem” Cyclophosphamid und Ifosfamid im Blut

Zusammenfassung Aktivierte N-(2-Cloräthyl)-amido-oxazaphosphorine wie 4-Hydroxycyclophosphamid, 4-Hydroperoxycyclophosphamid, 4-Hydroxyifosfamid und 4-Hydroperoxyifosfamid können über die Freisetzung von Acrolein und dessen Kondensation mit m-Aminophenol zu 7-Hydroxychinolin fluorometrisch bestimmt werden. Die Nachweisempfindlichkeit beträgt 1x10^-10 mol; der Test ist spezifisch für aktivierte N-(2-Chloräthyl)-amidooxazaphosphorin-Metabolite mit aldehydogener Funktion am Kohlenstoff 4 des Oxazaphosphorin-Rings, welche unter Testbedingungen Acrolein freisetzen. Weder Cyclophosphamid oder Ifosfamid, noch andere Metabolite dieser Cytostatica stören den Test. Im Mäuseblut wurden die Spiegel von freiem 4-Hydroxycyclophosphamid und 4-Hydroxyifosfamid nach Injektion von Cyclophosphamid bzw. Ifosfamid in einem Zeitraum vom 90 min nach der Injektion bestimmt.Mit Unterstützung durch die Deutsche Forschungsgemeinschaft     

Preventive Effect of FK 506 (Tacrolimus Hydrate) on Experimentally Induced Acute Liver Injury in Rats

The aim of the study was to investigate the effect of the immunosuppressant FK 506 (tacrolimus hydrate) on acute liver injury induced by Propionibacterium acnes and lipopolysaccharide (LPS). Acute liver injury was induced in male Wistar rats by injecting the animals with P. acnes (10 mg/rat), and administering LPS (10 g/rat) seven days later. One group was given FK 506 (1 mg/kg) 24 and 2 hr before administration of LPS, and the other group was given the same dose of saline. The 24-hr survival rate, serum alanine aminotransferase (ALT) concentration, and tumor necrosis factor (TNF) - mRNA and protein concentrations in the liver and spleen were then compared. Hepatic macrophages were also isolated from rats seven days after P. acnes injection, LPS, and FK 506 or saline were added to the culture supernatant, and TNF- production was studied. The 24-hr survival rate was 100% in the FK 506-treated group, in contrast with 16.6% in the saline group. Four hours after LPS injection, the serum ALT concentration was 755 ± 401 in the saline group versus 119 ± 42 units/ml (P < 0.01) in the FK 506-treated group. The serum TNF- concentration was lower in the FK 506-treated group (1419 ± 957 pg/ml) than in the saline group (9205 ± 2215) (P < 0.01). The mRNA and protein concentrations in the liver and spleen in the two groups did not differ significantly 1 hr after LPS injection but were significantly lower in the FK 506-treated group after 4 hr. FK 506 did not directly inhibit TNF- production by isolated cultured hepatic macrophages. FK 506 is unable to inhibit initial TNF- production by hepatic macrophages (or probably that by splenic macrophages either) stimulated by injection of LPS in P. acnes + LPS-induced acute liver injury. However, the immunosuppressant does limit hepatic damage by inhibiting subsequent aggravation of inflammation by the cytokine network.     

Autologous tandem transplantation in patients with primary progressive or relapsed/refractory lymphoma

Patients with primary progressive or refractory Hodgkins disease (HD) or aggressive non-Hodgkins lymphoma (NHL) have a particularly poor prognosis. Here we report the results of autologous tandem transplantation in these patients. Patients aged 18–55 years with primary progressive or refractory relapsed HD and aggressive NHL were included. Patients received high-dose etoposide (2000 mg/m²) followed by peripheral blood stem cell harvest (PBSC). The first high-dose chemotherapy (TMC) consisted of thiotepa (750 mg/m²), mitoxantrone (40 mg/m²), and carboplatin (990 mg/m²). Patients with no change (NC), partial remission (PR), or complete remission (CR) after TMC then received BEAM with carmustine (300 mg/m²), etoposide (1200 mg/m²), cytarabine (1600 mg/m²), and melphalan (140 mg/m²). Patients with bulky disease (>5 cm) or residual lymphoma received involved field radiotherapy. Twenty-five patients were included (HD=10, NHL=15, median age 34 years). Two patients with HD achieved a CR and five patients a PR [response rate (RR) 70%]. Three patients (30%) experienced treatment failure including two deaths due to peritransplant complications. Five patients with aggressive NHL were in CR and two patients in PR (RR 46%). Of the eight patients (56%) with treatment failure, three had progressive disease and five died from peritransplant complications. Freedom from treatment failure (FFTF) and overall survival (OS) for all patients after 12 months was 28% and 40%, respectively. Tandem HDCT followed by autologous stem cell transplantation (ASCT) offers a chance of cure in these poor prognostic patients, but is associated with risks.J.-P. Glossmann and J. O. Staak have contributed equally to this work.     

Correlation Between Stellate Cell Activation and Serum Fibrosis Markers in Choline-Deficient l-Amino Acid-Defined Diet-Induced Rat Liver Fibrosis

The aim of this study was to investigate the association of stellate cell activation with serum fibrosis markers in a rat model of hepatic fibrosis prepared using a choline-deficient l-amino acid (CDAA) defined diet. CDAA diet administration resulted in increased liver hydroxyproline contents in a time-dependent manner with activated stellate cells, expressing -smooth muscle actin (-SMA) as well as increased serum concentrations of amino-terminal procollagen type III peptide (PIIIP) and the 7S fragment of type IV collagen. Hydroxyproline content of the liver showed a closer correlation with the serum 7S (r = 0.75, P < 0.01) concentration than with the serum PIIIP (r = 0.51, P < 0.01) concentration. The percent area of -SMA-positive cells showed stronger correlation with the serum PIIIP concentration (r = 0.85, P < 0.01) than with the 7S concentration (r = 0.50, P < 0.01). These results indicate that the serum PIIIP concentration reflects the activity of fibrogenesis, while the serum 7S concentration reflects the accumulation of collagen fibers in the liver.     

Activation of imidazoline receptors in adrenal gland to lower plasma glucose in streptozotocin-induced diabetic rats

Aims/hypothesis The present study investigated the effect of agmatine, an endogenous ligand of imidazoline receptors, on plasma glucose in streptozotocin-induced diabetic rats (STZ-diabetic rats).Methods Plasma glucose was assessed by the glucose oxidase method. Plasma insulin and -endorphin-like immunoreactivity in plasma or adrenal medulla were measured by enzyme-linked immunosorbent assay. Systolic blood pressure was determined by the tail-cuff method. The mRNA levels of glucose transporter subtype 4 (GLUT4) in soleus muscle and phosphoenolpyruvate carboxykinase (PEPCK) in liver were detected by northern blotting. Protein levels of GLUT4 in soleus muscle and hepatic PEPCK were estimated using western blotting analysis.Results After intravenous injection into fasting STZ-diabetic rats for 30 min, agmatine decreased plasma glucose in a dose-dependent manner without changing systolic blood pressure. At the same time, plasma -endorphin-like immunoreactivity also increased in STZ-diabetic rats receiving the same treatment. Plasma glucose was significantly elevated in STZ-diabetic rats by an intravenous injection of clonidine at a dose sufficient to decrease systolic blood pressure. Involvement of I₁-imidazoline receptors and/or ₂-adrenoceptors in this effect of agmatine was thus unlikely. The lowering of plasma glucose and increase of plasma -endorphin-like immunoreactivity by agmatine were abolished by pretreating the rats with BU-224 at a dose sufficient to block I₂-imidazoline receptors. Both effects of agmatine were also abolished in adrenalectomised STZ-diabetic rats. Moreover, agmatine enhanced -endorphin-like immunoreactivity release from the isolated adrenal medulla of STZ-diabetic rats, an effect also blocked by BU-224. Release of -endorphin from the adrenal glands by I₂-imidazoline receptor activation seems responsible for the plasma glucose-lowering action of agmatine. This was supported by the fact that intravenous injection of naloxone or naloxonazine at doses sufficient to block opioid -receptors inhibited the action of agmatine. In addition to lowering plasma glucose, repeated intravenous injection of agmatine into STZ-diabetic rats for 4 days also increased mRNA and protein levels of GLUT4 in soleus muscle. The same treatment also reversed the higher mRNA and protein levels of PEPCK in liver of STZ-diabetic rats.Conclusions/interpretation Our results suggest that agmatine may activate I₂-imidazoline receptors in the adrenal gland. This enhances secretion of -endorphin, which can activate opioid -receptors to increase GLUT4 gene expression and/or suppress hepatic PEPCK gene expression, resulting in a lowering of plasma glucose in diabetic rats lacking insulin. The results provide a potential new target for intervention in type 1 diabetes.     

A role for chronic hepatitis C virus infection in a patient with cutaneous vasculitis,cryoglobulinemia, and chronic liver disease

Summary A six-year history of repeated attacks of fatigue, fever, arthralgias, skin changes, Raynaud's phenomenon, and neuropathy is reported in a patient with chronic liver disease. The following diagnoses were made: (1) leukocytoclastic vasculitis; (2) acute urticaria; (3) cryoglobulinemia type II with Raynaud's phenomenon and low serum level of C4; (4) peripheral polyneuropathy; (5) sicca syndrome; and (6) chronic hepatitis C virus infection. Despite therapy with corticosteroids symptoms increased gradually over years. In the first PCR of the nested PCR analysis, HCV-RNA was exclusively detected in the cryoglobulin fraction but not in the serum supernatant, suggesting that antibodies bind HCV particles, forming circulating immune complexes. As diagnoses 1-5 are well-known organ manifestations of cryoglobulinemia, we speculated whether treatment of hepatitis C with IFN- (3 million IU IFN-_2b three times a week) would inhibit HCV replication, decrease the cryocrit level and thereby ameliorate organ manifestations such as neuropathy and vasculities. During treatment with IFN- only a very weak or no signal could be detected for HCV-RNA in the cryoglobulin fraction as well as in the serum supernatant. This held true also for the serum supernatant in the second PCR. In parallel, cryoglobulin level, immunoglobulins, and liver enzymes decreased substantially to normal or near normal levels. Clinical symptoms—leukocytoclastic vasculitis and neuropathy—disappeared. We conclude that chronic HCV infection is involved in the pathogenesis of cryoglobulinemia and that IFN- might be an effective treatment in these patients.Dedicated to Gustav Paumgartner on his 60th birthday.     

γ/δ Receptor-expressing T-cell clones from a cutaneous T-cell lymphoma suppress hematopoiesis

Summary Recently we described a cutaneous T-cell lymphoma expressing the / T-cell receptor [5]. The patient suffering from this lymphoma showed low numbers of myeloid and T cells in peripheral blood, while B and NK cells were relatively increased. In vitro culture of the patient's bone marrow (BM) cells revealed a significant suppression of myeloid/monocyte colony formation (GM-CFU) compared with normal controls. This was not due to infiltration of the BM with lymphoma cells. We speculated that a soluble factor either secreted or induced by the lymphoma cells might be responsible for the marked suppression of hematopoiesis in this patient. From a skin biopsy with infiltrating / T-lymphoma cells we established T-cell clones bearing the / T-cell receptor and resembling the phenotype of the lymphoma cells. The supernatant (SN) of these / T-cell clones reduced the number of colonies in a CFU-GM assay (using normal control BM) in comparison to SN of / T-cell clones established from the same biopsy. This suppression was seen mainly on day 7 of culture and was not neutralized by the addition of placenta-CM. The main mediator of this suppression seems to be IFN-,since it was detectable in high amounts in the SN of these / T-cell tumor clones as well as in the serum of the patient. In addition, anti-IFN- antibodies can reverse the T-cell SN-mediated suppression of CFU-GM. We conclude that high serum levels of interferon-, which is secreted in high amounts from / T-cells grown from a biopsy of a cutaneous lymphoma, can suppress hematopoiesis.Abbreviations TCR T-cell receptor - IFN- interferon- - SN supernatant - placenta CM placenta conditioned medium - BM bone marrow - CFU-GM myeloid/monocyte colony formation - NK cells natural killer cells - Ab antibody M. Wilhelm was supported by theDeutsche Forschungsgemeinschaft (DFG Wi 728-2)     

Oxidative stress induces insulin resistance by activating the nuclear factor-κB pathway and disrupting normal subcellular distribution of phosphatidylinositol 3-kinase

Aims/hypothesis Oxidative stress is associated with diabetes, hypertension and atherosclerosis. Insulin resistance is implicated in the development of these disorders. We tested the hypothesis that oxidative stress induces insulin resistance in rats, and endeavoured to identify mechanisms linking the two.Methods Buthionine sulfoximine (BSO), an inhibitor of glutathione synthase, was administered to Sprague-Dawley rats and 3T3-L1 adipocytes. Glucose metabolism and insulin signalling both in vivo and in 3T3-L1 adipocytes were examined. In 3T3-L1 adipocytes, the effects of overexpression of a dominant negative mutant of inhibitory B (IB), one role of which is to block oxidative-stress-induced nuclear factor (NF)-B activation, were investigated.Results In rats given BSO for 2 weeks, the plasma lipid hydroperoxide level doubled, indicating increased oxidative stress. A hyperinsulinaemic-euglycaemic clamp study and a glucose transport assay using isolated muscle and adipocytes revealed insulin resistance in BSO-treated rats. BSO treatment also impaired insulin-induced glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes. In BSO-treated rat muscle, adipose tissue and 3T3-L1 adipocytes, insulin-induced IRS-1 phosphorylation in the low-density microsome (LDM) fraction was specifically decreased, while that in whole cell lysates was not altered, and subsequent translocation of phosphatidylinositol (PI) 3-kinase from the cytosol and the LDM fraction was disrupted. BSO-induced impairments of insulin action and insulin signalling were reversed by overexpressing the dominant negative mutant of IB, thereby suppressing NF-B activation.Conclusions/interpretation Oxidative stress induces insulin resistance by impairing IRS-1 phosphorylation and PI 3-kinase activation in the LDM fraction, and NF-B activation is likely to be involved in this process.Abbreviations BSO buthionine sulfoximine - GMSA gel mobility shift assay - IB inhibitory B - IKK IB kinase - LDM low-density microsome - NF-B nuclear factor-B - PI phosphatidylinositol     

Hepatic iron contents and response to interferon-α in patients with chronic hepatitis C

Recent reports have shown that response to interferon treatment is influenced by hepatic iron contents in patients with chronic hepatitis C. In those reports, however, hepatitis C virus (HCV) genotypes and serum HCV-RNA levels were not examined. The aim of the present study was to investigate whether hepatic iron contents influence the response to interferon in patients with chronic hepatitis C and whether HCV genotypes and serum HCV-RNA levels play a role in this relationship. Among 65 patients with chronic hepatitis C, hepatic iron contents were significantly high in patients with a history of excess drinking of alcohol (more than 80 g/day) compared to those without, and significantly low in female patients before menopause. Having excluded these patients, hepatic iron contents were significantly higher in patients with genotype 1b infection than those with genotype 2a and 2b infection. There was no significant correlation between hepatic iron contents and plasma HCV-RNA levels. Among the patients with genotype 1b infection, hepatic iron contents were significantly lower in the responders to interferon than those in the nonresponders (429 ± 100 vs 875 ± 110 µg/g liver,P<0.05). From these results, it is concluded that response to interferon is mainly influenced by HCV genotypes, while hepatic iron contents may play an important role in response to interferon in patients with genotype 1b infection.     

Characterization of an acute T lymphoblastic leukemia expressing the γ/δ T-cell receptor

Summary Leukemic cells of a 20 year old patient, suffering from acute lymphoblastic leukemia, were characterized by surface marker and functional analysis. A significant cell population within this type of leukemia expresses concomitantly the CD4 and CD8 antigen on the same cell and might represent a new differentiation stage of T-cells with the / receptor. The leukemic cells show a distinct pattern of growth response to mitogens and lymphokines, which might correlate to their differentiation stage. Moreover, a natural killer-like activity can be induced in these cells by IL-2.Abbreviations FITC fluorescein isothiocyanate - PE phycoerythrin - IL-2 interleukin 2; - / TCR gamma/delta T cell receptor - NK natural killer - PBL peripheral blood lymphocytes - T-ALL acute T lymphoblastic leukemia - ConA concanavalin A - PMA phorbol myristate acetate - BM bone marrow - IL-2R IL-2 receptor - TdT terminal deoxynucleotidyl transferase Supported by the Deutsche Forschungsgemeinschaft (DFG Wi-728/3-1)     

Antioxidative effects of fluvastatin on superoxide anion activated by angiotensin II in human aortic smooth muscle cells

We examined the antioxidative effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (statin), on superoxide anion formation activated by angiotensin II (Ang II) in vitro. The effects of fluvastatin were also compared to simvastatin and a water-soluble analog of -tocopherol, trolox. Treatment of human aortic smooth muscle cells (hASMC) with Ang II for 24 hours resulted in a 3.2 ± 0.5-fold increase in intracellular superoxide anion formation as detected by lucigenin assay. hASMC treated with clinical concentrations of fluvastatin (0–100 nM) showed a dose-dependent decrease in Ang II-activated superoxide anion formation. The addition of similar concentrations of trolox to hASMC inhibited Ang II-activated superoxide anion formation in a dose-dependent manner. However, simvastatin at similar doses failed to inhibit Ang II-activated superoxide anion formation by hASMC. Our results indicate that in addition to its hypocholesterolemic effect, fluvastatin may have direct antioxidative effects, suggesting its possible protective effect on atherosclerotic process.