Genotoxic effects of the cyanobacterial pentapeptide nodularin in HepG2 cells.

The cyanobacterial pentapeptide nodularin (NOD), mainly produced by genus Nodularia, is a potent inhibitor of protein phosphatases PP1 and PP2A, and causes animal mortality. The few studies available indicate that NOD is a potential non-genotoxic carcinogen. In the present study we evaluated NOD (0.01, 0.1 and 1 µg/ml) genotoxic activity in human hepatoma (HepG2) cells with the comet, γH2AX and cytokinesis block micronucleus cytome assays. In addition, induction of oxidative stress was studied. Moreover changes in the expression of selected genes from the P53 pathway, involved in the response to DNA damage (P53, GADD45α, CDKN1A, MDM2), apoptosis (BAX, BCL2) and oxidative stress (GPX1, GSR, GCLC, CAT, SOD1) were determined using qPCR. Non-cytotoxic concentrations induced time and dose dependant increase in reactive oxygen species (ROS) production and substantially increased the formation of oxidative DNA damage. In addition, elevated formation of micronuclei was detected. For the first time it has been shown that NOD deregulated the mRNA level of DNA damage (CDKN1A, GADD45α) and oxidative stress (GPX1, GSR, GCLC, CAT and SOD1) responsive genes and anti-apoptotic gene BCL2. Our results provide new evidence that NOD genotoxic effects are mediated through ROS production, already at low environmentally relevant concentrations.

Muscle gene electrotransfer is increased by the antioxidant tempol in mice.

Electropermeabilization (EP) is an effective method of gene transfer into different tissues. During EP, reactive oxygen species (ROS) are formed, which could affect transfection efficiency. The role of generated ROS and the role of antioxidants in electrotransfer in myoblasts in vitro and in Musculus tibialis cranialis in mice were, therefore, investigated. We demonstrate in the study that during EP of C2C12 myoblasts, ROS are generated on the surface of the cells, which do not induce long-term genomic DNA damage. Plasmid DNA for transfection (pEGFP-N1), which is present outside the cells during EP, neutralizes the generated ROS. The ROS generation is proportional to the amplitude of the electric pulses and can be scavenged by antioxidants, such as vitamin C or tempol. When antioxidants were used during gene electrotransfer, the transfection efficiency of C2C12 myoblasts was statistically significantly increased 1.6-fold with tempol. Also in vivo, the transfection efficiency of M. tibialis cranialis in mice was statistically significantly increased 1.4-fold by tempol. The study indicates that ROS are generated on cells during EP and can be scavenged by antioxidants. Specifically, tempol can be used to improve gene electrotransfer into the muscle and possibly also to other tissues.

Microcystin-LR induced DNA damage in human peripheral blood lymphocytes.

Human exposure to microcystins, which are produced by freshwater cyanobacterial species, is of growing concern due to increasing appearance of cyanobacterial blooms as a consequence of global warming and increasing water eutrophication. Although microcystins are considered to be liver-specific, there is evidence that they may also affect other tissues. These substances have been shown to induce DNA damage in vitro and in vivo, but the mechanisms of their genotoxic activity remain unclear. In human peripheral blood lymphocytes (HPBLs) exposure to non-cytotoxic concentrations (0, 0.1, 1 and 10µg/ml) of microcystin-LR (MCLR) induced a dose- and time-dependent increase in DNA damage, as measured with the comet assay. Digestion of DNA from MCLR-treated HPBLs with purified formamidopyrimidine-DNA glycosylase (Fpg) displayed a greater number of DNA strand-breaks than non-digested DNA, confirming the evidence that MCLR induces oxidative DNA damage. With the cytokinesis-block micronucleus assay no statistically significant induction of micronuclei, nucleoplasmic bridges and nuclear buds was observed after a 24-h exposure to MCLR. At the molecular level, no changes in the expression of selected genes involved in the cellular response to DNA damage and oxidative stress were observed after a 4-h exposure to MCLR (1µg/ml). After 24h, DNA damage-responsive genes (p53, mdm2, gadd45a, cdkn1a), a gene involved in apoptosis (bax) and oxidative stress-responsive genes (cat, gpx1, sod1, gsr, gclc) were up-regulated. These results provide strong support that MCLR is an indirectly genotoxic agent, acting via induction of oxidative stress, and that lymphocytes are also the target of microcystin-induced toxicity.

Cylindrospermopsin induced DNA damage and alteration in the expression of genes involved in the response to DNA damage, apoptosis and oxidative stress.

Cylindrospermopsin (CYN), a potent cyanobacterial cytototoxin produced by certain freshwater cyanobacteria, is regularly found in water supplies in many parts of the world, and has been associated with the intoxication of humans and livestock. The few genotoxicity studies available indicate that CYN is genotoxic, generally implying that it is pro-genotoxic. In human peripheral blood lymphocytes (HPBLs) CYN (0, 0.05, 0.1 and 0.5 µg/ml) induced the formation of DNA single strand breaks, applying the comet assay. Time and dose dependent significant increase in the frequency of micronuclei and nuclear buds was observed after the exposure of HPBLs to CYN, while there was only slight increase in the number of nucleoplasmic bridges. For the first time the modulation of gene expression in HPBLs was studied after the exposure to CYN (0.5 µg/ml), using the quantitative real-time PCR. The genes presumably involved in CYN metabolism (CYP1A1 and CYP1A2) were up-regulated after the exposure. CYN induced changes in the mRNA expression of P53 and its downstream regulated DNA damage responsive genes MDM2, GADD45α and apoptosis genes, BCL-2 and BAX, as well as oxidative stress responsive genes (GPX1, SOD1, GSR, GCLC), while no changes in the expression of genes CDKN1A and CAT were observed. These results provide strong evidence that CYN should be considered as genotoxic and that lymphocytes can also be a target of cylindrospermopsin induced genotoxicity.

Protective effect of linalool, myrcene and eucalyptol against t-butyl hydroperoxide induced genotoxicity in bacteria and cultured human cells.

We studied the protective effect of monoterpenes myrcene, eucalyptol and linalool against t-butyl hydroperoxide (t-BOOH) induced genotoxicity in reverse mutation assay with Escherichia coli WP2 IC185 strain and its oxyR mutant IC202, and with the comet assay in human hepatoma HepG2 and human B lymphoid NC-NC cells. The monoterpenes were tested in concentration ranges 0.05-1.5 mg/plate and 0.01-1.0 microg/ml in bacteria and mammalian cells, respectively. Suppression of t-BOOH induced mutagenesis was detected only in IC202 strain, and correlated with the observed inhibition of lipid peroxidation by the three monoterpenes. Linalool and myrcene strongly suppressed t-BOOH induced mutagenesis. Eucalyptol, in addition to moderate suppression of t-BOOH induced mutagenesis, suppressed also spontaneous mutagenesis. In NC-NC cells linalool and myrcene reduced t-BOOH induced DNA damage by about 50% at 0.01 microg/ml, while eucalyptol was less efficient (about 50% reduction at 1.0 microg/ml). In HepG2 cells linalool and eucalyptol reduced DNA damage by 30% and 40%, respectively, while myrcene was ineffective. The repair of t-BOOH induced DNA damage, studied in HepG2 cells, was not affected by monoterpenes. The results indicate that linalool, eucalyptol and myrcene have substantial protective effect against oxidant induced genotoxicity, which is predominately mediated by their radical scavenging activity.

Both cerivastatin and fenofibrate improve arterial vasoreactivity in patients with combined hyperlipidaemia.

OBJECTIVES: The aim of this study was to compare the effects of cerivastatin and fenofibrate on endothelium dependent and independent arterial dilation. DESIGN: In a prospective, double blind study, 38 overweight, nonsmoking, males aged between 40 and 60 years with combined hyperlipidaemia were randomized and, after 6 weeks run-in phase with American Heart Association step I diet treatment, submitted to 12 weeks' treatment either with fenofibrate (250 mg daily) or cerivastatin. Cerivastatin was given in a daily dose of 0.2 mg for 6 weeks and was increased to 0.4 mg daily, if the LDL-C did not decrease below 3.0 mmol x L(-1). Flow-mediated (endothelium-dependent) dilation (FMD) and nitroglycerin-induced (endothelium-independent) [gliceryltrinitrate (GTN)] dilation of brachial artery were measured using high resolution ultrasound. RESULTS: The FMD increased from 3.4 +/- 3.3 to 9.3 +/- 2.4% (P < 0.001) in the cerivastatin group, and from 3.3 +/- 2.8 to 6.5 +/- 3.1% (P < 0.001) in the fenofibrate group, the improvement being significantly better after cerivastatin (P=0.006). GTN increased from 11.5 +/- 4.1 to 16.2 +/- 3.5% (P < 0.01) and from 11.1 +/- 2.5 to 16.0 +/- 2.9% (P < 0.01), respectively, with no difference between the groups. Cerivastatin reduced total cholesterol by 24%, LDL-cholesterol by 31%, triglycerides by 24%, ox-LDL by 29% and increased HDL-cholesterol by 5%, whilst, after fenofibrate, these changes were -15, -13, -41, -17 and 18%, respectively. Only the decrease of LDL-C turned out to be an independent predictor the FMD improvement. The improvement in GTN-induced dilation did not correlate with the changes in blood lipids. CONCLUSIONS: Both cerivastatin and fenofibrate lead to an improvement of endothelium-dependent and endothelium-independent dilation of brachial artery in overweight patients with combined hyperlipidaemia and no other atherosclerotic risk factors. The effects on FMD were greater in subjects receiving cerivastatin than in subjects receiving fenofibrate, but the effects on GTN were equal in both groups.

Fibrinolytic parameters and insulin resistance in young survivors of myocardial infarction with heterozygous familial hypercholesterolemia.

A characteristic feature of patients with heterozygous familial hypercholesterolemia (FH) is the premature occurrence of coronary artery disease because of elevated LDL cholesterol levels. Hyperinsulinemia and insulin resistance, important characteristics of the cardiovascular dysmetabolic syndrome (CDS), were found to be associated with coronary artery disease in FH subjects, as in the general population. We investigated whether hypofibrinolysis, as part of CDS, is independently associated with symptomatic coronary artery disease in these high-risk patients. Clinical examination (body mass index, waist circumference, blood pressure) and blood analysis (plasma tissue plasminogen activator (t-PA) antigen, plasminogen activator inhibitor (PAI-1) antigen and activity, fibrinogen, serum lipids and lipoproteins, fasting glucose and insulin) were carried out in 39 male patients with heterozygous FH (aged 46.6 +/- 8.8 years). Insulin resistance was calculated using the homeostasis model assessment (HOMA) mathematical model. Thirteen of the patients had suffered a myocardial infarction (MI) 5 to 8 years ago (aged 47.8 +/- 6.1 years) and 26 were free of coronary artery disease (aged 45.9 +/- 9.9 years). There was no difference in total and LDL cholesterol between the two groups. Patients with previous myocardial infarction had significantly higher levels of insulin, insulin resistance, triglycerides, t-PA antigen, PAI-1 antigen and activity, and significantly lower values of HDL cholesterol. Other widely recognised risk factors for coronary artery disease, such as smoking, systolic and diastolic blood pressure, obesity and age, did not differ significantly between the groups. In the logistic regression model, PAI-1 antigen, as a marker of hypofibrinolysis, emerged as an independent risk factor for the occurrence of myocardial infarction (odds ratio 1.55; p = 0.02). In summary our results suggest that the impairment of fibrinolytic activity resulting from elevated levels of PAI-1 antigen and activity and t-PA antigen is an independent variable in CDS associated with the premature occurrence of myocardial infarction in male patients with FH.

Primary breast cancer of the vulva: a case report and review of the literature.

Since 1872, 40 cases of ectopic mammary gland tissue in the vulva have been reported in the literature. Out of these, 12 had a primary cancer in the ectopic breast tissue. Seven metastases of an orthotopic breast cancer have been found in this location. We are presenting the 20th case of cancerous breast tissue in the vulva whom we classified as the 13th case of primary cancer based on clinical and histopathological criteria of primary and metastatic malignant disease. Because of the advanced age of the patient, wide local excision followed by adjuvant hormonal therapy was opted for. Nineteen months after surgery, there is no evidence of recurrent disease. Due to the rarity of this entity, its management presents therapeutic dilemmas, and variable treatment strategies are being found in the literature. In our opinion, the same basic principles used for treatment of cancers of the orthotopic breast should be applied in ectopic breast carcinoma.