ToF-SIMS imaging shows specific lipophilic vitamin alterations in chronic reprotoxicity caused by the emerging contaminant Pravastatin in Gammarus fossarum.

Blood lipid-lowering agents, such as Pravastatin, are among the most frequently used pharmaceuticals released into the aquatic environment. Although their effects on humans are very well understood, their consequences on freshwater organisms are not well known, especially in chronic exposure conditions. Gammarus fossarum is commonly used as sentinel species in ecotoxicology because of its sensitivity to a wide range of environmental contaminants and the availability of standardized bioassays. Moreover, there is an increased interest in linking molecular changes in sentinel species, such as gammarids, to observed toxic effects. Here, we performed a reproductive toxicity assay on females exposed to different concentrations of pravastatin (30; 300; 3,000 and 30,000 ng L-1) during two successive reproductive cycles and we applied ToF-SIMS imaging to evaluate the effect of pravastatin on lipid homeostasis in gammarids. Reproductive bioassay showed that pravastatin could affect oocyte development in Gammarus fossarum inducing embryotoxicity in the second reproductive cycle. Mass spectrometry imaging highlighted the disruption in vitamin E production in the oocytes of exposed female gammarids at the second reproductive cycle, while limited alterations were observed in other lipid classes, regarding both production and tissue distribution. The results demonstrated the interest of applying spatially resolved lipidomics by mass spectrometry imaging to assess the molecular effects induced by long-term exposure to environmental pharmaceutical residues in sentinel species.

In vivo evaluation of combination therapy targeting the isoprenoid biosynthetic pathway.

Geranylgeranyl diphosphate synthase (GGDPS), an enzyme in the isoprenoid biosynthetic pathway (IBP), produces the isoprenoid (geranylgeranyl pyrophosphate, GGPP) used in protein geranylgeranylation reactions. Our prior studies utilizing triazole bisphosphonate-based GGDPS inhibitors (GGSIs) have revealed that these agents represent a novel strategy by which to induce cancer cell death, including multiple myeloma and pancreatic cancer. Statins inhibit the rate-limiting enzyme in the IBP and potentiate the effects of GGSIs in vitro. The in vivo effects of combination therapy with statins and GGSIs have not been determined. Here we evaluated the effects of combining VSW1198, a novel GGSI, with a statin (lovastatin or pravastatin) in CD-1 mice. Twice-weekly dosing with VSW1198 at the previously established maximally tolerated dose in combination with a statin led to hepatotoxicity, while once-weekly VSW1198-based combinations were feasible. No abnormalities in kidney, spleen, brain or skeletal muscle were observed with combination therapy. Combination therapy disrupted protein geranylgeranylation in vivo. Evaluation of hepatic isoprenoid levels revealed decreased GGPP levels in the single drug groups and undetectable GGPP levels in the combination groups. Additional studies with combinations using 50% dose-reductions of either VSW1198 or lovastatin revealed minimal hepatotoxicity with expected on-target effects of diminished GGPP levels and disruption of protein geranylgeranylation. Combination statin/GGSI therapy significantly slowed tumor growth in a myeloma xenograft model. Collectively, these studies are the first to demonstrate that combination IBP inhibitor therapy alters isoprenoid levels and disrupts protein geranylgeranylation in vivo as well as slows tumor growth in a myeloma xenograft model, thus providing the framework for future clinical exploration.

Atorvastatin and pravastatin stimulate nitric oxide and reactive oxygen species generation, affect mitochondrial network architecture and elevate nicotinamide N-methyltransferase level in endothelial cells.

Statins belong to the most often prescribed medications, which efficiently normalise hyperlipidaemia and prevent cardiovascular complications in obese and diabetic patients. However, beside expected therapeutic results based on the inhibition of 3-hydroxyl-3-methylglutaryl-CoA reductase, these drugs exert multiple side effects of poorly understood characteristic. In this study, side effects of pravastatin and atorvastatin on EA.hy926 endothelial cell line were investigated. It was found that both statins activate proinflammatory response, elevate nitric oxide and reactive oxygen species (ROS) generation and stimulate antioxidative response in these cells. Moreover, only slight stimulation of the mitochondrial biogenesis and significant changes in the mitochondrial network organisation have been noted. Although biochemical bases behind these effects are not clear, they may partially be explained as an elevation of AMP-activated protein kinase (AMPK) activity and an increased activating phosphorylation of sirtuin 1 (Sirt1), which were observed in statins-treated cells. In addition, both statins increased nicotinamide N-methyltransferase (NNMT) protein level that may explain a reduced fraction of methylated histone H3. Interestingly, a substantial reduction of the total level of histone H3 in cells treated with pravastatin but not atorvastatin was also observed. These results indicate a potential additional biochemical target for statins related to reduced histone H3 methylation due to increased NNMT protein level. Thus, NNMT may directly modify gene activity.

Pravastatin as the statin of choice for reducing pre-eclampsia-associated endothelial dysfunction.

OBJECTIVES: There is avid interest in pravastatin as a therapeutic intervention for pre-eclampsia, however little is known on statin action on endothelial dysfunction. This study aimed to evaluate the ability of pravastatin, simvastatin and rosuvastatin to reduce pre-eclampsia-associated markers of endothelial dysfunction in human endothelial cells. STUDY DESIGN: Primary human umbilical vein endothelial cells (HUVECs) and uterine microvascular cells (UtMVs) were isolated and treated with 0.2, 2, 20 and 200 µM pravastatin, simvastatin and rosuvastatin for 24 h, either with or without pre-treatment with TNF-α to induce endothelial dysfunction. MAIN OUTCOME MEASURES: Cell viability (MTS) assays were performed and cells were visually inspected. Expression of endothelial dysfunction markers, endothelin-1 (ET-1) and vascular cell adhesion molecule-1 (VCAM-1) were assessed by qPCR (n=3). Intracellular VCAM-1 protein was examined by Western Blotting (n=5). ET-1 and soluble fms-like tyrosine kinase-1 (sFLT-1) protein secretion was assessed by ELISA in HUVEC conditioned media (n=3). RESULTS: High doses of simvastatin and rosuvastatin significantly compromised HUVEC survival. 200 µM simvastatin significantly reduced UtMV survival. Abnormal cell structure was observed with these doses and thus were excluded from further analysis. The statins did not mitigate TNF-α induced ET-1 or VCAM-1 expression in either HUVECs or UtMVs, nor VCAM-1 protein expression in HUVECs. 0.2 µM pravastatin and simvastatin significantly reduced ET-1 and sFLT-1 protein secretion. CONCLUSIONS: Pravastatin significantly reduced secretion of both ET-1 and sFLT-1, key mediators of endothelial dysfunction. Importantly, pravastatin had no toxic effects, in contrast to rosuvastatin and simvastatin. This further supports selection of pravastatin for clinical applications to combat pre-eclampsia.

Biotechnological Production of Statins: Metabolic Aspects and Genetic Approaches.

Statins are drugs used for people with abnormal lipid levels (hyperlipidemia) and are among the best-selling medications in the United States. Thus, the aspects related to the production of these drugs are of extreme importance for the pharmaceutical industry. Herein, we provide a non-exhaustive review of fungal species used to produce statin and highlighted the major factors affecting the efficacy of this process. The current biotechnological approaches and the advances of a metabolic engineer to improve statins production are also emphasized. The biotechnological production of the main statins (lovastatin, pravastatin and simvastatin) uses different species of filamentous fungi, for example Aspergillus terreus. The statins production is influenced by different types of nutrients available in the medium such as the carbon and nitrogen sources, and several researches have focused their efforts to find the optimal cultivation conditions. Enzymes belonging to Lov class, play essential roles in statin production and have been targeted to genetic manipulations in order to improve the efficiency for Lovastatin and Simvastatin production. For instance, Escherichia coli strains expressing the LovD have been successfully used for lovastatin production. Other examples include the use of iRNA targeting LovF of A. terreus. Therefore, fungi are important allies in the fight against hyperlipidemias. Although many studies have been conducted, investigations on bioprocess optimization (using both native or genetic- modified strains) still necessary.

Atorvastatin affects negatively respiratory function of isolated endothelial mitochondria.

The purpose of this research was to elucidate the direct effects of two popular blood cholesterol-lowering drugs used to treat cardiovascular diseases, atorvastatin and pravastatin, on respiratory function, membrane potential, and reactive oxygen species formation in mitochondria isolated from human umbilical vein endothelial cells (EA.hy926 cell line). Hydrophilic pravastatin did not significantly affect endothelial mitochondria function. In contrast, hydrophobic calcium-containing atorvastatin induced a loss of outer mitochondrial membrane integrity, an increase in hydrogen peroxide formation, and reductions in maximal (phosphorylating or uncoupled) respiratory rate, membrane potential and oxidative phosphorylation efficiency. The atorvastatin-induced changes indicate an impairment of mitochondrial function at the level of ATP synthesis and at the level of the respiratory chain, likely at complex I and complex III. The atorvastatin action on endothelial mitochondria was highly dependent on calcium ions and led to a disturbance in mitochondrial calcium homeostasis. Uptake of calcium ions included in atorvastatin molecule induced mitochondrial uncoupling that enhanced the inhibition of the mitochondrial respiratory chain by atorvastatin. Our results indicate that hydrophobic calcium-containing atorvastatin, widely used as anti-atherosclerotic agent, has a direct negative action on isolated endothelial mitochondria.

Chronic use of pravastatin reduces insulin exocytosis and increases ß-cell death in hypercholesterolemic mice.

We have previously demonstrated that hypercholesterolemic LDL receptor knockout (LDLr(-/-)) mice secrete less insulin than wild-type mice. Removing cholesterol from isolated islets using methyl-beta-cyclodextrin reversed this defect. In this study, we hypothesized that in vivo treatment of LDLr(-/-) mice with the HMGCoA reductase inhibitor pravastatin would improve glucose-stimulated insulin secretion. Female LDLr(-/-) mice were treated with pravastatin (400mg/L) for 1-3 months. Isolated pancreatic islets were assayed for insulin secretion rates, intracellular calcium oscillations, cholesterol levels, NAD(P)H and SNARE protein levels, apoptosis indicators and lipidomic profile. Two months pravastatin treatment reduced cholesterol levels in plasma, liver and islets by 35%, 25% and 50%, respectively. Contrary to our hypothesis, pravastatin treatment increased fasting and fed plasma levels of glucose and decreased markedly (40%) fed plasma levels of insulin. In addition, ex vivo glucose stimulated insulin secretion was significantly reduced after two and three months (36-48%, p<0.05) of pravastatin treatment. Although reducing insulin secretion and insulinemia, two months pravastatin treatment did not affect glucose tolerance because it improved global insulin sensitivity. Pravastatin induced islet dysfunction was associated with marked reductions of exocytosis-related SNARE proteins (SNAP25, Syntaxin 1A, VAMP2) and increased apoptosis markers (Bax/Bcl2 protein ratio, cleaved caspase-3 and lower NAD(P)H production rates) observed in pancreatic islets from treated mice. In addition, several oxidized phospholipids, tri- and diacylglycerols and the proapoptotic lipid molecule ceramide were identified as markers of pravastatin-treated islets. Cell death and oxidative stress (H2O2 production) were confirmed in insulin secreting INS-1E cells treated with pravastatin. These results indicate that chronic treatment with pravastatin impairs the insulin exocytosis machinery and increases ß-cell death. These findings suggest that prolonged use of statins may have a diabetogenic effect.

Different effects of statins on induction of diabetes mellitus: an experimental study.

BACKGROUND: To determine the effect of different statins on the induction of diabetes mellitus. MATERIALS AND METHODS: Four statins (atorvastatin, pravastatin, rosuvastatin, and pitavastatin) were used. Cytotoxicity, insulin secretion, glucose-stimulated insulin secretion, and G0/G1 phase cell cycle arrest were investigated in human pancreas islet ß cells, and glucose uptake and signaling were studied in human skeletal muscle cells (HSkMCs). RESULTS: Human pancreas islet ß cells treated with 100 nM atorvastatin, pravastatin, rosuvastatin, and pitavastatin had reduced cell viability (32.12%, 41.09%, 33.96%, and 29.19%, respectively) compared to controls. Such cytotoxic effect was significantly attenuated by decreasing the dose to 10 and 1 nM, ranged from 1.46% to 17.28%. Cells treated with 100 nM atorvastatin, pravastatin, rosuvastatin, and pitavastatin had a reduction in the rate of insulin secretion rate by 34.07%, 30.06%, 26.78%, and 19.22%, respectively. The inhibitory effect was slightly attenuated by decreasing the dose to 10 and 1 nM, ranging from 10.84% to 29.60%. Insulin secretion stimulated by a high concentration of glucose (28 mmol/L) was significantly higher than a physiologic concentration of glucose (5.6 mmol/L) in all treatment groups. The glucose uptake rates at a concentration of 100 nM were as follows: atorvastatin (58.76%) < pravastatin (60.21%) < rosuvastatin (72.54%) < pitavastatin (89.96%). We also found that atorvastatin and pravastatin decreased glucose transporter (GLUT)-2 expression and induced p-p38 MAPK levels in human pancreas islet ß cells. Atorvastatin, pravastatin, and rosuvastatin inhibited GLUT-4, p-AKT, p-GSK-3ß, and p-p38 MAPK levels in HSkMCs. CONCLUSION: Statins similar but different degree of effects on pancreas islet ß cells damage and induce insulin resistance in HSkMC.

Selected statins produce rapid spinal motor neuron loss in vitro.

BACKGROUND: Hmg-CoA reductase inhibitors (statins) are widely used to prevent disease associated with vascular disease and hyperlipidemia. Although side effects are uncommon, clinical observations suggest statin exposure may exacerbate neuromuscular diseases, including peripheral neuropathy and amyotrophic lateral sclerosis. Although some have postulated class-effects, prior studies of hepatocytes and myocytes indicate that the statins may exhibit differential effects. Studies of neuronal cells have been limited. METHODS: We examined the effects of statins on cultured neurons and Schwann cells. Cultured spinal motor neurons were grown on transwell inserts and assessed for viability using immunochemical staining for SMI-32. Cultured cortical neurons and Schwann cells were assessed using dynamic viability markers. RESULTS: 7 days of exposure to fluvastatin depleted spinal motor neurons in a dose-dependent manner with a KD of < 2 µM. Profound neurite loss was observed after 4 days exposure in culture. Other statins were found to produce toxic effects at much higher concentrations. In contrast, no such toxicity was observed for cultured Schwann cells or cortical neurons. CONCLUSIONS: It is known from pharmacokinetic studies that daily treatment of young adults with fluvastatin can produce serum levels in the single micromolar range. We conclude that specific mechanisms may explain neuromuscular disease worsening with statins and further study is needed.

Possible mechanisms underlying statin-induced skeletal muscle toxicity in L6 fibroblasts and in rats.

3-Hydroxy-3-methylglutaryl CoA reductase inhibitors (statins) are safe and well-tolerated therapeutic drugs. However, they occasionally induce myotoxicity such as myopathy and rhabdomyolysis. Here, we investigated the mechanism of statin-induced myotoxicity in L6 fibroblasts and in rats in vivo. L6 fibroblasts were differentiated and then treated with pravastatin, simvastatin, or fluvastatin for 72 h. Hydrophobic simvastatin and fluvastatin decreased cell viability in a dose-dependent manner via apoptosis characterized by typical nuclear fragmentation and condensation and caspase-3 activation. Both hydrophobic statins transferred RhoA localization from the cell membrane to the cytosol. These changes induced by both hydrophobic statins were completely abolished by the co-application of geranylgeranylpyrophosphate (GGPP). Y27632, a Rho-kinase inhibitor, mimicked the hydrophobic statin-induced apoptosis. Hydrophilic pravastatin did not affect the viability of the cells. Fluvastatin was continuously infused (2.08 mg/kg at an infusion rate of 0.5 mL/h) into the right internal jugular vein of the rats in vivo for 72 h. Fluvastatin infusion significantly elevated the plasma CPK level and transferred RhoA localization in the skeletal muscle from the cell membrane to the cytosol. In conclusion, RhoA dysfunction due to loss of lipid modification with GGPP is involved in the mechanisms of statin-induced skeletal muscle toxicity.

Involvement of organic anion transporting polypeptides in the toxicity of hydrophilic pravastatin and lipophilic fluvastatin in rat skeletal myofibres.

BACKGROUND AND PURPOSE: There is a discrepancy in the adverse effect of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, statins between the clinical reports and the studies using skeletal muscle cell models. In the clinical reports, both hydrophilic and lipophilic statins induce myotoxicity, whereas in in vitro experiments using cell lines of myoblasts, lipophilic, but not hydrophilic, statins exert myotoxicity. We investigated the cause of this discrepancy. EXPERIMENTAL APPROACH: Skeletal myofibres, fibroblasts and satellite cells were isolated from rat flexor digitorum brevis (FDB) muscles. Using these primary cultured cells as well as the L6 myoblast cell line, we compared the toxicity of hydrophilic pravastatin and lipophilic fluvastatin. The mRNA expression levels of possible drug transporters for statins were also examined in these cells using reverse transcriptase-PCR. KEY RESULTS: In the skeletal myofibres, both pravastatin and fluvastatin induced vacuolation and cell death, whereas in the mononuclear cells only fluvastatin, but not pravastatin, was toxic. mRNA of the organic anion transporting polypeptides (Oatp) 1a4 and Oatp2b1 were expressed in the skeletal myofibres, but not in mononucleate cells. Estrone-3-sulphate, a substrate for Oatps, attenuated the effects of pravastatin and fluvastatin in skeletal myofibres; p-aminohippuric acid, a substrate for the organic anion transporters (Oats), but not Oatps, failed to do so. CONCLUSIONS AND IMPLICATIONS: The statin transporters Oatp1a4 and Oatp2b1 are expressed in rat skeletal myofibres, but not in satellite cells, fibroblasts or in L6 myoblasts. This is probably why hydrophilic pravastatin affects skeletal muscle, but not skeletal myoblasts.

Effect of medium pH on the cytotoxicity of hydrophilic statins.

PURPOSE: The aim of this study was to examine the mechanism of pravastatin- and rosuvastatin-induced cytotoxicity and the relationship between pravastatin- and rosuvastatin-induced cytotoxicity and medium pH using human prototypic embryonal rhabdomyosarcoma cell line (RD) and rat myoblast cell line (L6) as a model of in vitro skeletal muscle. METHODS: Statin-induced reduction of cell viability and apoptosis was measured by 3-(4,5-dimethylthiazol-2-yl)2,5 -diphenyl tetrazolium bromide (MTT) assay and caspase assay. Intracellular accumulation of statins was determined using an HPLC system. RESULTS: Rosuvastatin cytotoxicity, reduction of cell viability, morphological changes and caspase activation at acidic pH (pH 6.8) were significantly greater than those at neutral pH (pH 7.4). Rosuvastatin accumulation at acidic pH was greater than that at pH 7.4. On the other hand, medium pH had no effect on pravastatin accumulation. CONCLUSIONS: Rosuvastatin cytotoxicity at acidic pH is associated with increasing intracellular accumulation of rosuvastatin. On the other hand, medium pH had no effect on cytotoxicity of pravastatin.

Toxicity of statins on rat skeletal muscle mitochondria.

We investigated mitochondrial toxicity of four lipophilic stains (cerivastatin, fluvastatin, atorvastatin, simvastatin) and one hydrophilic statin (pravastatin). In L6 cells (rat skeletal muscle cell line), the four lipophilic statins (100 micromol/l) induced death in 27-49% of the cells. Pravastatin was not toxic up to 1 mmol/l. Cerivastatin, fluvastatin and atorvastatin (100 micromol/l) decreased the mitochondrial membrane potential by 49-65%, whereas simvastatin and pravastatin were less toxic. In isolated rat skeletal muscle mitochondria, all statins, except pravastatin, decreased glutamate-driven state 3 respiration and respiratory control ratio. Beta-oxidation was decreased by 88-96% in the presence of 100 micromol/l of the lipophilic statins, but only at higher concentrations by pravastatin. Mitochondrial swelling, cytochrome c release and DNA fragmentation was induced in L6 cells by the four lipophilic statins, but not by pravastatin. Lipophilic statins impair the function of skeletal muscle mitochondria, whereas the hydrophilic pravastatin is significantly less toxic.

Eisai hyperbilirubinemic rat (EHBR) as an animal model affording high drug-exposure in toxicity studies on organic anions.

The Eisai hyperbilirubinemic rat (EHBR) should be a useful animal model for studies on the toxicity of organic anions which are substrates of multidrug resistance-associated protein 2 (Mrp2), since the systemic exposure to these compounds is expected to be increased in EHBR. In this study, we tested the value of EHBR for this purpose, using pravastatin (PV) and methotrexate (MTX) as model compounds. In the case of a single oral dose of PV (200 mg/kg), C(max) in plasma was 4.0-fold higher and AUC(0-infinity) was 3.6-fold larger than those of normal Sprague-Dawley rats (SDR), respectively. When multiple doses of PV were given to EHBR without co-administration of any other compound, drug-induced skeletal muscle toxicity (myopathy/rhabdomyolysis) and increased creatine phosphokinase (CPK) level were observed, whereas a control experiment using SDR did not show any toxic change. When a single dose of MTX (0.6 mg/kg) was given to EHBR orally, C(max) was 1.7-fold higher and AUC(0-infinity) was 1.6-fold larger than those of SDR, respectively. When multiple doses of MTX were given to EHBR, the changes in bone marrow, spleen and intestines were more severe than those in SDR. These findings support the view that EHBR would be a valuable animal model for toxicity studies on organic anion compounds which are substrates of Mrp2.

Chronic hydroxymethylglutaryl coenzyme a reductase inhibition and endothelial function of the normocholesterolemic rat: comparison with angiotensin-converting enzyme inhibition.

Hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors seem to have clinical benefits beyond those predicted by their lipid-lowering action. The objective was to evaluate the vascular effect of long-term treatment with statins on isolated rat aorta and their ability to prevent the acute toxicity of oxidized low-density lipoproteins (oxLDLs) compared with angiotensin-converting enzyme (ACE) inhibitors. Four groups of Wistar rats were treated for 5 weeks. Group 1 received pravastatin 20 mg/kg/d orally; group 2 received atorvastatin 10 mg/kg/d; group 3 received ciprofibrate 25 mg/kg/d; and group 4 served as control. Total cholesterol and triglyceride plasma levels were not altered, except in group 3, in which both parameters were decreased. The inhibitory effect of the endothelium on serotonin-induced contractions was significantly increased in group 1. A significant leftward shift of the concentration-response curves to acetylcholine (1 nM-0.1 mM) was observed in group 1 but the maximal relaxation to acetylcholine was similar in the four groups. In contrast, in the presence of human Cu2+-oxLDL (300 microg/ml, 30 min of preincubation), the maximal relaxation to acetylcholine was markedly decreased (p < 0.02) in groups 3 and 4 versus that of groups 1 and 2. No difference in superoxide accumulation was observed by the chemiluminescence technique. Cyclic guanosine monophosphate (cGMP), measured by enzyme immunoassay in aortic tissues, was increased in group 1 in the presence of superoxide dismutase. Endothelial nitric oxide synthase (eNOS) expression was not altered (Western blot and enzyme-linked immunosorbent assay). In aortas isolated from a fifth group of rats treated with an ACE inhibitor (ramipril 10 mg/kg/d for 6 weeks), similar results to those of group 1 were observed except that the eNOS abundance was significantly enhanced. Thus, long-term statin treatment upregulates the eNOS pathway and attenuates the acute toxicity of human oxLDL. In contrast to chronic ACE inhibition, the eNOS abundance is not increased.

Lovastatin inhibits phenylephrine-induced ERK activation and growth of cardiac.

The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) exert numerous cellular effects through the inhibition of cholesterol synthesis. The objectives of these experiments were to determine the following: (1) whether lovastatin (LOV) inhibits phenylephrine (PE)-induced growth of neonatal rat cardiac myocytes without inducing cytotoxicity and (2) whether growth-inhibiting effects of LOV are associated with reduced PE activation of extracellular signal regulated kinases 1 and 2 (ERK 1/2). After 48 h of exposure, LOV alone (0.1-10 microM) inhibited 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) reduction without significant changes in propidium iodide staining, and 100 microM mevalonic acid prevented the effect of LOV on MTT reduction. PE (50 or 100-microM for 48 h) induced significant increases in protein-to-DNA ratios. PE (100 microM for 5 min) significantly increased the phosphorylated forms of ERK 1 and ERK 2 and activity of ERK. After 24 h pretreatment or 48 h cotreatment, LOV (10 microM) significantly inhibited PE-induced growth. In addition, LOV pretreatment significantly inhibited the stimulatory effect of PE on ERK 2 phosphorylation and ERK activity. These results demonstrate that LOV, at concentrations that do not alter membrane integrity, inhibits PE-induced growth of cardiac myocytes, potentially through reduced activation of ERK 1/2.

Comparative toxicity of high doses of vastatins currently used by clinicians, in CD-1 male mice fed with a hypercholesterolemic diet.

The CD-1 male-mouse model was employed to evaluate comparatively the toxicity of four vastatins (VTS) currently used in clinical medicine: lovastatin (LVT), simvastatin (SVT), pravastatin (PVT) and fluvastatin (FVT). Each vastatin was used orally in doses of 500 mg/Kg body weight/day, in animals with a hypercholesterolemic diet (HD) 5 days, or with a control diet (CD) 30 days. The association of high doses of VTS + HD produced a significant increase in liver weight and liver weight to body weight ratio in animals with SVT and FVT. Cholesterol (Chol) and triacylglycerols (TAG) in the liver increased significantly with FVT but not with the other VTS; Chol increased and TAG decreased in serum very significantly with FVT and SVT. The serum aminotransferases increased quite significantly with FVT but not with other VTS. In the experiment with high doses of VTS + CD, the animals receiving SVT or FVT showed a moderate loss of body weight. Liver weight and liver weight to body weight ratios were similar among all groups. Liver Chol showed a significant decrease with all VTS. Serum Chol decreased moderately with LVT and FVT. TAG in serum and liver showed a moderate decrease with all VTS. The serum aminotransferases were not modified by any vastatin. Our results indicate that high doses of VTS in male mice with a hypercholesterolemic diet result in a decreasing toxicity as follows: FVT>SVT>LVT>PVT.

Evaluation of kidney and liver subacute toxicity induced by Bezalip-Pravastatin-Lopid antihyperlipidaemic compounds in rats.

Renal and hepatic subacute toxicity induced by the antihyperlipidaemic drugs: Bezalip-Pravastatin and Lopid was investigated in rats using serum biochemical parameters. Toxicological evaluation was performed in serum samples following the administration of the therapeutic dose regimens of the compounds that were previously shown to be effective in inhibition of 3-hydroxy-methylglutaryl coenzyme A (HMG CoA) reductase, the enzyme controlling the rate-limiting step in the synthesis of cholesterol, and acyl-CoA cholesterol acyl transferase (ACAT) which converts intracellular free cholesterol to cholesterol ester. Renal and hepatic subacute toxicity was evaluated by measuring enzyme activity or concentrations of: alanine aminotransferace, alkaline phosphatase, aspartate aminotransferase, gamma-glutamyltransferase, glucose, potassium, sodium, blood urea nitrogen, uric acid and creatinine. The use of the above serum biochemical parameters indicated that the overall toxicity impact of antihyperlipidaemic drugs was Bezalip = Pravastatin < Lopid. We have found that the Pravastatin--in contrast to the above antihyperlipidaemic drugs--only transiently affects the biochemical parameters associated with toxicity, but, it affects some of the biochemical parameters associated with hepatic and renal toxicity, up to a significantly lower extent than the antihyperlipidaemic drugs.

Effects of HMG-CoA reductase inhibitors on excitation-contraction coupling of rat skeletal muscle.

3-Hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors currently used as cholesterol-lowering drugs produce side effects in patients, one of which is myopathy. In the present study we compared the effect of a 3-month chronic treatment with two different compounds, simvastatin and pravastatin, on the excitation-contraction coupling of rat skeletal muscle fibers, the mechanism which links membrane depolarization to the movements of cytosolic Ca2+ from intracellular stores. The voltage threshold for mechanical activation of extensor digitorum longus muscle fibers in response to depolarizing pulses of various durations was studied in vitro by the two intracellular microelectrode method in 'point' voltage clamp mode. Simvastatin (5-50 mg/kg) modified the mechanical threshold of striated fibers in a dose-dependent manner. The muscle fibers of rats treated with 10 mg/kg and 50 mg/kg of simvastatin needed significantly less depolarization to contract than did untreated fibers at each pulse duration, suggesting that levels of cytosolic Ca2+ were higher. Consequently, the rheobase voltage for fiber contraction was significantly shifted toward more negative potentials with respect to controls by 2.4 mV and 7.1 mV in the 10 mg/kg and 50 mg/kg simvastatin-treated animals, respectively. Pravastatin treatment at 100 mg/kg did not produce any alteration of excitation-contraction coupling since the rheobase voltage was similar to that of controls. The different physicochemical properties of the two drugs may underlie the different effect observed because lipophilic agents, such as simvastatin, have been shown to affect sterol biosynthesis in many tissues, whereas the hydrophilic pravastatin is hepato-selective.