Perfluoroheptanoic acid induces Leydig cell hyperplasia but inhibits spermatogenesis in rats after pubertal exposure.

Perfluoroheptanoic acid (PFHpA) is a short-chain alternative to long-chain perfluoroalkyl substances, which have been reported to possess reproductive toxicity. However, it is unclear whether PFHpA affects Leydig cell development during puberty. The 35-day-old Sprague Dawley male rats were exposed to PFHpA by gavage with 0 (corn oil), 10, 50, and 100 mg/kg/day for 21 days. PFHpA did not affect the body weight of rats, but it reduced testis weight, relative testis weight, and epididymis weight at 100 mg/kg. It significantly increased serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels at a dose of 100 mg/kg without affecting serum estradiol levels. PFHpA suppressed sperm production at a dose of 100 mg/kg. PFHpA induced Leydig cell hyperplasia (increased number of CYP11A1-positive Leydig cells) at a dose of 100 mg/kg, but down-regulated the expression of Cyp11a1, Hsd3b1, and Cyp17a1 in individual Leydig cell pe se and up-regulated the expression of Fshr in the Sertoli cell pe se. PFHpA did not affect the number of HSD11B1 (a biomarker for more mature Leydig cells) positive Leydig cells and SOX9 positive Sertoli cells. PFHpA increased BCL2, and the phosphorylation of AKT1, AKT2, ERK1/2, and JNK, but decreased BAX levels. However, it had no effect on SIRT1 and PGC-1α levels. In conclusion, PFHpA induces Leydig cell hyperplasia due to the increase in the secretion of luteinizing hormone through negative feedback after down-regulating the expression of steroidogenic enzymes and inhibiting testosterone production in individual Leydig cells. This proliferation may be mediated by increasing BCL2 and phosphorylation of AKT, ERK1/2, and JNK, and decreasing BAX level.

The suitability of reconstructed human epidermis models for medical device irritation assessment: A comparison of In Vitro and In Vivo testing results.

The ISO 10993 standards on biocompatibility assessment of medical devices discourage the use of animal tests when reliable and validated in vitro methods are available. A round robin validation study of in vitro reconstructed human epidermis (RhE) assays was performed as potential replacements for rabbit skin irritation testing. The RhE assays were able to accurately identify strong irritants in dilute medical device extracts. However, there was some uncertainty about whether RhE tissues accurately predicted the results of the rabbit skin patch or intracutaneous irritation test. To address that question, this paper presents in vivo data from the round robin and subsequent follow-up studies. The follow-up studies included simultaneous in vitro RhE model and in vivo testing of round robin polymer samples and the results of dual in vitro/in vivo testing of currently marketed medical device components/materials. Our results show for the first time that for both pure chemicals and medical device extracts the intracutaneous rabbit test is more sensitive to detect irritant activity than the rabbit skin patch test. The studies showed that the RhE models produced results that were essentially equivalent to those from the intracutaneous rabbit skin irritation test. Therefore, it is concluded that RhE in vitro models are acceptable replacements for the in vivo rabbit intracutaneous irritation test for evaluating the irritant potential of medical devices.

Subacute dermal toxicity of perfluoroalkyl carboxylic acids: comparison with different carbon-chain lengths in human skin equivalents and systemic effects of perfluoroheptanoic acid in Sprague Dawley rats.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are used in various fields but raise concerns regarding human health and environmental consequences. Among PFASs, perfluorooctanoic acid (PFOA) and short-chain perfluoroalkyl carboxylic acids (SC PFCAs) are detectable in skin-contact consumer products and have dermal absorption potential. Here, we investigated the effects of dermal exposure to PFOA and SC PFCAs using in vitro and in vivo models. Human skin equivalents were topically treated with 0.25 mM and 2.5 mM PFOA and SC PFCAs (perfluoropentanoic acid, PFPeA; perfluorohexanoic acid, PFHxA; and perfluoroheptanoic acid, PFHpA) for 6 days, and cell viability, interleukin (IL)-1α, oxidative stress markers (malondialdehyde, MDA; and 8-hydroxydeoxyguanosine, 8-OHdG), and histopathology were examined. MDA levels were significantly higher in the PFASs groups than in controls. Compared with SC PFCAs, 2.5 mM PFOA caused more IL-1α (p < 0.001) release, decreased skin thickness and microscopic abnormalities. To evaluate systemic effects, Sprague Dawley (SD) rats were dermally treated with 250 and 1000 mg/kg PFHpA for 2 weeks and clinical and anatomic pathology were assessed. At 1000 mg/kg, 83% of the rats died, with severe ulcerative dermatitis at the application site. Adverse PFHpA-treated systemic changes were observed in the kidney, liver and testes, and histopathologic lesions such as renal tubular necrosis, hepatocellular necrosis, and germ cell degeneration were seen at 250 and 1000 mg/kg. Our study suggests that SC PFCAs have fewer effects on the skin than PFOA, but SC PFCAs can have adverse effects on major organs with systemic exposure at high concentrations.

Persistent organic pollutants and gestational diabetes: A multi-center prospective cohort study of healthy US women.

BACKGROUND: Persistent organic pollutants (POPs) are linked with insulin resistance and type-2 diabetes (T2D) in the general population. However, their associations with gestational diabetes (GDM) are inconsistent. OBJECTIVE: We prospectively evaluated the associations of POPs measured in early pregnancy with GDM risk. We also assessed whether pre-pregnancy BMI (ppBMI) and family history of T2D modify this risk. METHODS: In NICHD Fetal Growth Study, Singletons, we measured plasma concentration of 76 POPs, including 11 organochlorine pesticides (OCPs), 9 polybrominated diphenylethers (PBDEs), 44 polychlorinated biphenyls (PCBs), and 11 per-and polyfluoroalkyl substances (PFAS) among 2334 healthy non-obese women at 8-13 weeks of gestation. GDM was diagnosed by Carpenter and Coustan criteria. We constructed chemical networks using a weighted-correlation algorithm and examined the associations of individual chemical and chemical networks with GDM using multivariate Poisson regression with robust variance. RESULTS: Higher concentrations of PCBs with six or more chlorine atoms were associated with increased risk of GDM in the overall cohort (risk ratios [RRs] range: 1.08-1.13 per 1-standard deviation [SD] increment) and among women with a family history of T2D (RRs range: 1.08-1.48 per 1-SD increment) or normal ppBMI (RRs range: 1.08-1.22 per 1-SD increment). Similar associations were observed for the chemical network comprised of PCBs with ≥6 chlorine atoms and the summary measure of total PCBs and non-dioxin like PCBs (138, 153, 170, 180). Furthermore, four PFAS congeners - perfluorononanoic acid (PFNA), perfluorooctanoic acid (PFOA), perfluoroheptanoic acid (PFHpA), and perfluorododecanoic acid (PFDoDA) - showed significant positive associations with GDM among women with a family history of T2D (RRs range:1.22-3.18 per 1-SD increment), whereas BDE47 and BDE153 showed significant positive associations among women without a family history of T2D. CONCLUSIONS: Environmentally relevant levels of heavily chlorinated PCBs and some PFAS and PBDEs were positively associated with GDM with suggestive effect modifications by family history of T2D and body adiposity status.

Peak AAA fatty acid homolog contaminants present in the dietary supplement l-Tryptophan associated with the onset of eosinophilia-myalgia syndrome.

The eosinophilia-myalgia syndrome (EMS) outbreak that occurred in the USA and elsewhere in 1989 was caused by the ingestion of Showa Denko K.K. (SD) L-tryptophan (L-Trp). "Six compounds" detected in the L-Trp were reported as case-associated contaminants. Recently the final and most statistically significant contaminant, "Peak AAA" was structurally characterized. The "compound" was actually shown to be two structural isomers resulting from condensation reactions of L-Trp with fatty acids derived from the bacterial cell membrane. They were identified as the indole C-2 anteiso (AAA1-343) and linear (AAA2-343) aliphatic chain isomers. Based on those findings, we utilized a combination of on-line HPLC-electrospray ionization mass spectrometry (LC-MS), as well as both precursor and product ion tandem mass spectrometry (MS/MS) to facilitate identification of a homologous family of condensation products related to AAA1-343 and AAA2-343. We structurally characterized eight new AAA1-XXX/AAA2-XXX contaminants, where XXX represents the integer molecular ions of all the related homologs, differing by aliphatic chain length and isomer configuration. The contaminants were derived from the following fatty acids of the bacterial cell membrane, 5-methylheptanoic acid (anteiso-C8:0) for AAA1-315; n-octanoic acid (n-C8:0) for AAA2-315; 6-methyloctanoic acid (anteiso-C9:0) for AAA1-329; n-nonanoic acid (n-C9:0) for AAA2-329; 10-methyldodecanoic acid (anteiso-C13:0) for AAA1-385; n-tridecanoic acid (n-C13:0) for AAA2-385; 11-methyltridecanoic acid (anteiso-C14:0) for AAA1-399; and n-tetradecanoic acid (n-C14:0) for AAA2-399. The concentration levels for these contaminants were estimated to be 0.1-7.9 µg / 500 mg of an individual SD L-Trp tablet or capsule The structural similarity of these homologs to case-related contaminants of Spanish Toxic Oil Syndrome (TOS) is discussed.

Evaluation of the medical devices benchmark materials in the controlled human patch testing and in the RhE in vitro skin irritation protocol.

Several irritants were used in the in vitro irritation medical device round robin. The objective of this study was to verify their irritation potential using the human patch test (HPT), an in vitro assay, and in vivo data. The irritants were lactic acid (LA), heptanoic acid (HA), sodium dodecyl sulfate (SDS), Genapol® X-80 (GP), and Y-4 polymer. Dilute saline and sesame seed oil (SSO) solutions of each were evaluated using a 4 and 18 h HPT and the EpiDerm™ SIT-MD RhE assay; results were then compared to existing rabbit skin irritation test data. Results from the 4 h HPT were negative in most cases except for GP and SDS, while the 18 h HPT also identified some LA, HA, and GP samples as irritants. EpiDerm™ SIT-MD correctly identified all irritants except GP in SSO due to limited solubility. Data from cutaneous rabbit irritation tests were negative, while all intracutaneous results were strongly or weakly positive except for the most dilute GP solutions. These findings indicate that EpiDerm™ SIT-MD results correlate with those from the rabbit intracutaneous test and confirm that RhE assays are suitable replacements for animals in evaluating the tissue irritation potential of medical devices.

Correlation between mast cell-mediated allergic inflammation and length of perfluorinated compounds.

Perfluorinated compounds (PFC) have widely been used in numerous applications including clothing, food packaging, and nonstick coating. With the widespread use of PFC, concerns regarding potential adverse health effects in humans and wildlife have increased. In spite of the known PFC-mediated immunotoxiciy, correlation with PFC and allergic inflammation still requires elucidation. The aim of this study was to examine the effect of four types of PFC (perfluoroheptanoic acid [PFHpA], perfluorononanoic acid [PFNA], perfluorodecanoic acid [PFDA], and perfluoroundecanoic acid [PFUnA]) on mast cell-mediated allergic inflammation in the presence of high-affinity immunoglobulin (Ig) E receptor (FcεRI) cross-linking. Among PFC family, long-chain PFDA and PFUnA increased release of histamine and ß-hexosaminidase by up-regulation of intracellular calcium levels in IgE-stimulated mast cells. In addition, PFDA and PFUnA enhanced gene expression of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and IL-8 by activation of nuclear factor-κB in IgE-stimulated mast cells. In ovalbumin (OVA)-induced model of systemic anaphylaxis in the presence of hypothermia, PFNA, PFDA, and PFUnA exacerbated allergic symptoms accompanied by elevation in serum histamine, TNF-α, IgE, and IgG1. Our data indicate that some PFC aggravated high-affinity IgE receptor (FcεRI)-mediated mast cell degranulation and allergic symptoms. Consequently, the results demonstrated that carbon-chain length of PFC may serve as a factor in allergic inflammation.

Exposure to perfluoroalkyl substances during pregnancy and child behaviour at 5 to 9years of age.

We examined associations between prenatal exposure to perfluorohexane sulfonic acid (PFHxS), perfluoroheptanoic acid (PFHpA), perfluorononanoic acid (PFNA), and perfluorodecanic acid (PFDA) - and child behaviour (SDQ-total) and hyperactivity (sub-scale) at 5-9years of age in birth cohorts from Greenland and Ukraine. Pregnancy serum samples (N=1023) were analysed for perfluoroalkyl substances (PFASs) and categorised into tertiles and also used as continuous exposure variables. Problem behaviour and hyperactivity were assessed, using the Strength and Difficulties Questionnaire (SDQ) and categorised as normal/borderline and abnormal. Associations were analysed using multiple logistic and linear regression. High compared to low prenatal PFHxS exposure was associated with 1.16 (95% confidence interval (CI): 0.08; 2.25) point higher SDQ-total (more problem behaviour) in Greenland and 0.80 (CI: 0.06; 1.54) point higher SDQ-total in the combined analyses, whereas no association was present in Ukraine alone. One natural log-unit increase in prenatal PFNA exposure was associated with 0.90 (CI: 0.10; 1.71) points higher SDQ-total in Greenland and 0.72 (CI: 0.13; 1.31) points higher in the combined analysis and no association in Ukraine. Prenatal PFAS exposure was unrelated to problem behaviour (abnormal SDQ-total). In the combined analysis, odds ratio (OR) (CI) for hyperactivity was 1.8 (1.0; 3.2) for one natural log-unit increase in prenatal PFNA and 1.7 (1.0; 3.1) for one natural log-unit increase in prenatal PFDA exposure. Findings are compatible with weak effects on child behaviour of prenatal exposure to some PFASs although spurious results are not entirely unlikely. The associations were strongest in Greenland.

Perfluoroheptanoic acid affects amphibian embryogenesis by inducing the phosphorylation of ERK and JNK.

Perfluoroalkyl compounds (PFCs) are globally distributed synthetic compounds that are known to adversely affect human health. Developmental toxicity assessment of PFCs is important to facilitate the evaluation of their environmental impact. In the present study, we assessed the developmental toxicity and teratogenicity of PFCs with different numbers of carbon atoms on Xenopus embryogenesis. An initial frog embryo teratogenicity assay-Xenopus (FETAX) assay was performed that identified perfluorohexanoic (PFHxA) and perfluoroheptanoic (PFHpA) acids as potential teratogens and developmental toxicants. The mechanism underlying this teratogenicity was also investigated by measuring the expression of tissue-specific biomarkers such as phosphotyrosine­binding protein, xPTB (liver); NKX2.5 (heart); and Cyl18 (intestine). Whole­mount in situ hybridization, reverse transcriptase­polymerase chain reaction (RT-PCR), and histologic analyses detected severe defects in the liver and heart following exposure to PFHxA or PFHpA. In addition, immunoblotting revealed that PFHpA significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), while PFHxA slightly increased these, as compared with the control. These results suggest that PFHxA and PFHpA are developmental toxicants and teratogens, with PFHpA producing more severe effects on liver and heart development through the induction of ERK and JNK phosphorylation.

Does coenzyme-Q have a protective effect against atorvastatin induced myopathy? A histopathological and immunohistochemical study in albino rats.

INTRODUCTION: In addition to their lipid-lowering effect, statins have pleiotropic effects that may extend their use to the treatment and prevention of various other diseases such as cancer, osteoporosis, multiple sclerosis, rheumatoid arthritis, type 2 diabetes, and Alzheimer's disease. Consequently, the number of patients taking statins is expected to increase. A side effect of statins, statin-induced myopathy, which may result from reduced muscular coenzyme Q10 levels, limits their use. The current study investigates if supplementing with CoQ10 could ameliorate statin induced myopathy. MATERIALS AND METHODS: Forty adult male albino rats were randomized into 4 groups, with 10 rats per group. The following was administered to the rats using oral gavage for 4 weeks: Group 1: 2 ml of 0.5% carboxymethyl cellulose once daily. Group 2: 100 mg/kg/ day coenzyme Q10 dissolved in 2 ml of cotton seed oil. Group 3: 10 mg/kg once daily atorvastatin dissolved in 0.5% carboxymethyl cellulose. Group 4: concomitantly received CoQ10 and atorvastatin similar to groups 2 and 3 respectively. Plasma creatine kinase levels were measured by using spectrophotometer. The right extensor digitorum longus muscle sections were stained for histological (Haematoxylin & Eosin, Masson trichrome and Phosphotungstic acid haematoxylin) and immunohistochemical (cytochrome C and Bax) examinations. Quantitative measures of cytochrome C and Bax were carried out using image analyzer. RESULTS: Atorvastatin induced increased total creatine kinase, skeletal muscle variations in the sizes and shapes, necrosis, disorganization, nuclear pyknosis, karyorrhexis, karyolysis, dismantled plasma membrane, excess collagen fibers and lipid deposition in addition to loss of cross striation. Atorvastatin increased the intensity of the immune-positive reactions of cytochrome C and Bax. These changes were ameliorated by concomitantly giving coenzyme Q10. CONCLUSION: CoQ10 may ameliorate atorvastatin induced skeletal muscle injury.

Evaluation of the effect of andrographolide on atherosclerotic rabbits induced by Porphyromonas gingivalis.

Epidemiologic evidence has demonstrated significant associations between atherosclerosis and Porphyromonas gingivalis (Pg). We had investigated the effect of andrographolide (AND) on atherosclerosis induced by Pg in rabbits. For experimental purpose, we separated thirty male white New Zealand rabbits into 5 groups. Group 1 received standard food pellets; Groups 2-5 were orally challenged with Pg; Group 3 received atorvastatin (AV, 5 mg/kg), and Groups 4-5 received 10 and 20 mg/kg of AND, respectively, over 12 weeks. Groups treated with AND showed significant decrease in TC, TG, and LDL levels (P<0.05) and significant increase in HDL level in the serum of rabbits. Furthermore, the treated groups (G3-G5) exhibited reductions in interleukins (IL-1ß and IL-6) and C-reactive protein (CRP) as compared to atherogenicgroup (G2). The histological results showed that the thickening of atherosclerotic plaques were less significant in treated groups (G3-G5) compared with atherogenicgroup (G2). Also, alpha-smooth muscle actin (α-SMA) staining decreased within the plaques of atherogenicgroup (G2), while it was increased in treated groups (G3-G5). Lastly, groups treated with AV and AND (G3-G5) showed significant reduction of CD36 expression (P<0.05) compared to atherogenicgroup (G2). These results substantially proved that AND contain antiatherogenic activity.

Suppression of memory acquisition following co-administration of lithium and atorvastatin through nitric oxide pathway in mice.

PURPOSE: The aim of this study was to investigate the interactive effect of lithium and atorvastatin on cognitive performance and the role of NO as a potential mechanism involved in this interaction. MATERIALS AND METHODS: Memory performance was evaluated in a two-trial recognition Y-maze test and a step-through passive avoidance task in mice. Lithium (5, 10, 20 or 40 mg/kg, i.p.) and atorvastatin (1 mg/kg, p.o.) were administered 1 h before each trial, L-NAME, a non-specific NO synthase inhibitor (3, 10 mg/kg, i.p.); aminoguanidine, a specific inducible NO synthase (iNOS) inhibitor (100 mg/kg); and L-arginine, a NO precursor (750 mg/kg) were administered 30 min before training sessions. The level of plasma NO end-products (NOx) was determined using Griess reagent protocol. RESULTS: 1) Lithium (40 mg/kg) impaired the acquisition of spatial recognition memory; 2) lithium did not affect the retrieval phase of spatial memory; 3) atorvastatin (1 mg/kg) significantly impaired the memory performance, when co-administered with the sub-effective dose of lithium (10 mg/kg), but did not affect the status when administered with lithium (5 mg/kg); 4) L-NAME (10 mg/kg) and aminoguanidine (100 mg/kg) dramatically decreased memory performance in mice received sub-effective doses of both lithium (5 mg/kg) and atorvastatin (1 mg/kg); 5) L-arginine (750 mg/kg) improved the memory acquisition in mice administered lithium (10 mg/kg) and atorvastatin (1 mg/kg); 6) lithium did not affect the cognitive performance in the passive avoidance test. All results were compatible and confirmed with in vitro determination of plasma NOx levels. CONCLUSIONS: Lithium, dose dependently, impaired acquisition phase of spatial recognition memory. Lithium and atorvastatin co-administration impaired spatial recognition memory mediating by nitrergic pathway. In addition to L-arginine, our data from L-NAME and aminoguanidine also support the involvement of NO pathway in this interaction.

Mechanisms of the statins cytotoxicity in freshly isolated rat hepatocytes.

Statins are potent drugs, used as lipid-lowering agents in cardiovascular diseases. Hepatotoxicity is one of the serious adverse effects of statins, and the exact mechanism of hepatotoxicity is not yet clear. In this study, the cytotoxic effects of the most commonly used statins, that is, atorvastatin, lovastatin, and simvastatin toward isolated rat hepatocytes, were evaluated. Markers, such as cell death, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential, and the amount of reduced and oxidized glutathione in the statin-treated hepatocytes, were investigated. It was found that the statins caused cytotoxicity toward rat hepatocytes dose dependently. An elevation in ROS formation, accompanied by a significant amount of lipid peroxidation and mitochondrial depolarization, was observed. Cellular glutathione reservoirs were decreased, and a significant amount of oxidized glutathione was formed. This study suggests that the adverse effect of statins toward hepatocytes is mediated through oxidative stress and the hepatocytes mitochondria play an important role in the statin-induced toxicity.

Prophylactic effect of egualen sodium, a stable azulene derivative, on gastrointestinal damage induced by ischemia/reperfusion, double antiplatelet therapy and loxoprofen in rats.

We examined the effect of egualen, a stable azulene derivative, against gastric damage induced by ischemia/reperfusion (I/R), gastric bleeding induced by double antiplatelet therapy with aspirin (ASA) plus clopidogrel, and small intestinal damage generated by loxoprofen, and investigated the possible mechanisms involved in its protective action. Male C57BL/6 mice or SD rats were used under urethane anesthesia (gastric lesions) or in a conscious (intestinal lesions) state. I/R-induced gastric injury was produced in mice by clamping the celiac artery for 30 min, followed by reperfusion for 60 min. Gastric bleeding was induced in rats by luminal perfusion with 25 mM ASA+50 mM HCl for 2 hours in the presence of clopidogrel (30 mg/kg). To produce small intestinal lesions the rats were given loxoprofen (60 mg/kg) p.o. and killed 24 hours later. Egualen was given i.d. 60 min before I/R or ASA perfusion, while given p.o. twice 30 min before and 6 hours after loxoprofen. Egualen significantly prevented the I/R-induced gastric damage, and the effect was equivalent to that of seratrodast (TXA2 antagonist). This agent also significantly suppressed gastric bleeding induced by ASA plus clopidogrel, similar to PGE2. Likewise, egualen significantly prevented loxoprofen-induced damage in the small intestine, accompanied by an increase in the secretion of mucus and suppression of bacterial invasion as well as iNOS expression. These results suggest that egualen has a prophylactic effect against various lesions in the gastrointestinal mucosa, probably through its characteristic pharmacological properties, such as TXA2 antagonistic action, local mucosal protection, and stimulation of mucus secretion.

HMG-CoA reductase inhibitors induce apoptosis of lymphoma cells by promoting ROS generation and regulating Akt, Erk and p38 signals via suppression of mevalonate pathway.

Statins, the inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are widely used cholesterol-lowering drugs. Convincing evidence indicates that statins stimulate apoptotic cell death in several types of proliferating tumor cells in a cholesterol-lowering-independent manner. The objective here was to elucidate the molecular mechanism by which statins induce lymphoma cells death. Statins (atorvastatin, fluvastatin and simvastatin) treatment enhanced the DNA fragmentation and the activation of proapoptotic members such as caspase-3, PARP and Bax, but suppressed the activation of anti-apoptotic molecule Bcl-2 in lymphoma cells including A20 and EL4 cells, which was accompanied by inhibition of cell survival. Both increase in levels of reactive oxygen species (ROS) and activation of p38 MAPK and decrease in mitochondrial membrane potential and activation of Akt and Erk pathways were observed in statin-treated lymphoma cells. Statin-induced cytotoxic effects, DNA fragmentation and changes of activation of caspase-3, Akt, Erk and p38 were blocked by antioxidant (N-acetylcysteine) and metabolic products of the HMG-CoA reductase reaction, such as mevalonate, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). These results suggests that HMG-CoA reductase inhibitors induce lymphoma cells apoptosis by increasing intracellular ROS generation and p38 activation and suppressing activation of Akt and Erk pathways, through inhibition of metabolic products of the HMG-CoA reductase reaction including mevalonate, FPP and GGPP.

Prooxidative toxicity and selenoprotein suppression by cerivastatin in muscle cells.

Statins are the most widely used drugs for the treatment of hypercholesterolemia. In spite of their overall favorable safety profile, they do possess serious myotoxic potential, whose molecular origin has remained equivocal. Here, we demonstrate in cultivated myoblasts and skeletal muscle cells that cerivastatin at nanomolar concentrations interferes with selenoprotein synthesis and evokes a heightened vulnerability of the cells toward oxidative stressors. A correspondingly increased vulnerability was found with atorvastatin, albeit at higher concentrations than with cerivastatin. In selenium-saturated cells, cerivastatin caused a largely indiscriminate suppression of selenoprotein biosynthesis and reduced the steady state-levels of glutathione peroxidase 1 (GPx1) and selenoprotein N (SelN). Selenite, ebselen, and ubiquinone were unable to prevent the devitalizing effect of statin treatment, despite the fact that the cellular baseline resistance against tert-butyl hydroperoxide was significantly increased by picomolar sodium selenite. Mevalonic acid, in contrast, entirely prevented the statin-induced decrease in peroxide resistance. These results indicate that muscle cells may be particularly susceptible to a statin-induced suppression of essential antioxidant selenoproteins, which provides an explanation for the disposition of these drugs to evoke adverse muscular side-effects.

Atorvastatin up-regulate toxicologically relevant genes in rainbow trout gills.

There are large and increasing discharges of statins into the aquatic environment. Statins are cholesterol-lowering pharmaceuticals, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, an enzyme in the cholesterol synthesis pathway. Earlier studies have shown that statins will affect the expression of a range of genes in mammalian tissues and this group of pharmaceuticals has also been shown to affect membrane transporters. Changes in gene expression and ion transport in aquatic organisms may have dramatic consequences for the individual. The aim of the present study was to clarify whether waterborne exposure to a selected statin, atorvastatin, would affect gene expression in rainbow trout (Oncorhynchus mykiss) gill or liver or ion regulation in gills. Juvenile rainbow trout were exposed to two atorvastatin acid and atorvastatin lactone concentrations for 7 days (nominal concentrations 200 ng L(-1) and 10 µg L(-1)). The exposures caused up-regulated gene expression in gill, not liver, and only at the lowest concentration. Genes involved in membrane transport (pgp, mrp1), oxidative stress response (sod, mt), apoptosis (bax) and biotransformation (sult2b) were differentially expressed whereas the expression of genes involved in cholesterol biosynthesis (hmgr, fdps) or peroxisomal proliferation (ppar) were not affected. There were no significant changes in gill Na(+)/K(+) ATPase activity following exposure to atorvastatin. The pattern of differentially expressed genes in rainbow trout gills differ from responses previously observed in mammalian tissues following statin exposure.

Genotoxic effects in fish induced by pharmacological agents present in the sewage of some Italian water-treatment plants.

The presence of pharmaceutical substances in the municipal effluents is currently considered the principal source of bio-active molecule emissions into aquatic environments. This study analyzes the genotoxic damage caused by gemfibrozil and atorvastatin, two regulators of the hematic level of lipids, and sildenafil citrate, a vasodilator, on the teleost Danio rerio. The genotoxicity of these three compounds was evaluated using the comet assay, diffusion assay, and RAPD-PCR. The alkaline version (pH 12.1) of the comet assay was used for the erythrocytes of the zebrafish to evaluate the presence of single strand DNA breaks. Furthermore, the diffusion assay was used to estimate the number of apoptotic cells. The fish were treated with the three pharmacological agents at the average concentrations previously found at some Italian treatment plants and were then sacrificed from 5 to 35 days after exposure. The data of the comet assay showed a statistically significant loss of DNA integrity after 5 days of exposure to atorvastatin and after one week of exposure to gemfibrozil. This damage was, however, repaired after 14 days. Sildenafil citrate produced, instead, a statistically significant loss of DNA integrity at the concentrations found only after 35 days of exposure. The genotoxicity at the molecular level was tested by RAPD-PCR. The results from this investigation are in agreement with those from two other tests, confirming the efficacy of the use of the three experimental approaches for the complete evaluation of genotoxic damage.

Species-dependent sensitivity to contaminants: an approach using primary hepatocyte cultures with three marine fish species.

There is limited knowledge about the sensitivity of different fish species to environmental pollutants. Such information is pivotal in risk assessment and to understand why some species appear to be more tolerant to contaminants than others. The aim of the current study was to evaluate whether primary hepatocyte cultures of three marine fish species could be established in the field and whether their sensitivity to selected contaminants would differ. Primary hepatocyte cultures of three marine fish species (plaice, long rough dab, Atlantic cod) were established and exposed for 24 h to copper (20-2500 mg L⁻¹) and statins (1-200 mg L⁻¹). Endpoints were esterase activity, metabolic activity and reduced glutathione (GSH) content, all using fluorescent probes. Flatfish hepatocytes were more susceptible to copper and statin exposure than hepatocytes from cod. This study has shown that species-dependent differences in contaminant sensitivity can be investigated using primary hepatocyte cultures.