ANMCO/ISS/AMD/ANCE/ARCA/FADOI/GICR-IACPR/SICI-GISE/SIBioC/SIC/SICOA/SID/SIF/SIMEU/SIMG/SIMI/SISA Joint Consensus Document on cholesterol and cardiovascular risk: diagnostic-therapeutic pathway in Italy.

Atherosclerotic cardiovascular disease still represents the leading cause of death in Western countries. A wealth of scientific evidence demonstrates that increased blood cholesterol levels have a major impact on the outbreak and progression of atherosclerotic plaques. Moreover, several cholesterol-lowering pharmacological agents, including statins and ezetimibe, have proved effective in improving clinical outcomes. This document focuses on the clinical management of hypercholesterolaemia and has been conceived by 16 Italian medical associations with the support of the Italian National Institute of Health. The authors discuss in detail the role of hypercholesterolaemia in the genesis of atherosclerotic cardiovascular disease. In addition, the implications for high cholesterol levels in the definition of the individual cardiovascular risk profile have been carefully analysed, while all available therapeutic options for blood cholesterol reduction and cardiovascular risk mitigation have been explored. Finally, this document outlines the diagnostic and therapeutic pathways for the clinical management of patients with hypercholesterolaemia.

Relationship between Lipid Phenotypes, Overweight, Lipid Lowering Drug Response and KIF6 and HMG-CoA Genotypes in a Subset of the Brisighella Heart Study Population.

The existence of genetic traits might explain the susceptibility to develop hypercholesterolemia and the inter-individual differences in statin response. This study was performed to evaluate whether individuals' polymorphisms in HMG-CoA and KIF6 genes are independently associated with hypercholesterolemia, other lipid-associated traits, and statin response in unselected individuals enrolled in the Brisighella heart study (Survey 2012). A total of 1622 individuals, of which 183 under statin medication, were genotyped for a total of five polymorphisms (KIF6 rs20455, rs9471077, rs9462535; HMG-CoA rs3761740, rs3846662). The relationships between the five loci and clinical characteristics were analyzed. The principal basic parameters calculated on 12 h fasting blood included total cholesterol (TC), High Density Lipoprotein Cholesterol (HDL-C), Low-Density Lipoprotein Cholesterol (LDL-C), and triglycerides (TG). Hypercholesterolemia was defined as a TC >200 mg/dL or use of lipid-lowering medication. 965 individuals were characterized by hypercholesterolemia; these subjects were significantly older (p < 0.001), with body mass index (BMI) and waist circumference significantly higher (p < 0.001) compared to the others. HMG-CoA rs3846662 GG genotype was significantly over-represented in the hypercholesterolemic group (p = 0.030). HMG-CoA rs3846662 genotype was associated with the level of TC and LDL-C. Furthermore, in the same subset of untreated subjects, we observed a significant correlation between the KIF6 rs20455 and HDL-C. KIF6 variants were associated with a significantly lower (rs20455) or higher (rs9471077 and rs9462535) risk of obesity, in males only. No association between responsiveness to statins and the polymorphisms under investigation were observed. Our results showed associations between HMG-CoA rs3846662 and KIF6 rs20455 and lipid phenotypes, which may have an influence on dyslipidemia-related events. Moreover, this represents the first study implicating KIF6 variants with obesity in men, and point to the possible involvement of this genetic locus in the known gender-related differences in coronary artery disease.

The Water to Water Cycles in Microalgae.

In oxygenic photosynthesis, light produces ATP plus NADPH via linear electron transfer, i.e. the in-series activity of the two photosystems: PSI and PSII. This process, however, is thought not to be sufficient to provide enough ATP per NADPH for carbon assimilation in the Calvin-Benson-Bassham cycle. Thus, it is assumed that additional ATP can be generated by alternative electron pathways. These circuits produce an electrochemical proton gradient without NADPH synthesis, and, although they often represent a small proportion of the linear electron flow, they could have a huge importance in optimizing CO2 assimilation. In Viridiplantae, there is a consensus that alternative electron flow comprises cyclic electron flow around PSI and the water to water cycles. The latter processes include photosynthetic O2 reduction via the Mehler reaction at PSI, the plastoquinone terminal oxidase downstream of PSII, photorespiration (the oxygenase activity of Rubisco) and the export of reducing equivalents towards the mitochondrial oxidases, through the malate shuttle. In this review, we summarize current knowledge about the role of the water to water cycles in photosynthesis, with a special focus on their occurrence and physiological roles in microalgae.

Micronucleus frequency in human peripheral blood lymphocytes as a biomarker for the early detection of colorectal cancer risk.

The early detection of colorectal cancer (CRC) can significantly improve the prognosis of affected patients. The loss of genomic stability and the resulting gene alteration play an important role in the molecular pathological steps that occur early in tumorigenesis of CRC. Thus, the identification of non-invasive biomarkers, whose function may provide useful insights into critical early events in the CRC process, is of great interest. In this regard, micronucleus (MN) frequency in peripheral blood lymphocytes (PBL) has become one of the most established biomarkers for studying DNA damage in the human population. This study investigated the MN frequency in the PBL of 82 subjects (30 females and 52 males; aged 50-70 years) who were participating in a screening programme for CRC prevention. All 82 patients were positive in fecal occult blood tests and they were subsequently classified, according to colonoscopy and histological findings, as patients with CRC, patients with colon polyps or subjects without intestinal lesion, referred to as study controls. This study also examined the relationship between the plasma clastogenic activity and the frequency of micronuclei of the study population. The MN frequency was significantly higher in CRC patients than in both colon polyp patients (16.82±6.56 versus 12.23±1.88; P = 0.002) and controls (16.82±6.56 versus 8.00±1.77; P < 0.001). An increased MN frequency was detected in the lymphocytes of the polyp group in comparison to the control group, although this was lower than that observed in CRC patients (12.23±1.88 versus 8.00±1.77; P < 0.001). In the overall study population, the increase of MN frequency, which was observed in the lymphocytes of the subjects involved, was significantly associated with the clastogenic activity detected in their plasma (r = 0.594, P < 0.001). Overall, the results suggest that the MN test can become a promising biomarker for the early detection of CRC.

Association between imatinib transporters and metabolizing enzymes genotype and response in newly diagnosed chronic myeloid leukemia patients receiving imatinib therapy.

Imatinib has so far been the first-choice treatment in chronic myeloid leukemia with excellent results. However, only a proportion of patients achieve major molecular response - hence the need to find biological predictors of outcome to select the optimal therapeutic strategy now that more potent inhibitors are available. We investigated a panel of 20 polymorphisms in seven genes, potentially associated with the pharmacogenetics of imatinib, in a subset of 189 patients with newly diagnosed chronic myeloid leukemia enrolled in the TOPS trial. The analysis included polymorphisms in the transporters hOCT1, MDR1, ABCG2, OCTN1, and OATP1A2, and in the metabolizing genes CYP3A4 and CYP3A5. In the overall population, the OCTN1 C allele (rs1050152), a simple combination of polymorphisms in the hOCT1 gene and another combination in the genes involved in imatinib uptake were significantly associated with major molecular response. The combination of polymorphisms in imatinib uptake was also significantly associated with complete molecular response. Analyses restricted to Caucasians highlighted the significant association of MDR1 CC (rs60023214) genotype with complete molecular response. We demonstrate the usefulness of a pharmacogenetic approach for stratifying patients with chronic myeloid leukemia according to their likelihood of achieving a major or complete molecular response to imatinib. This represents an attractive opportunity for therapy optimization, worth testing in clinical trials.

Polymorphisms in OCTN1 and OCTN2 transporters genes are associated with prolonged time to progression in unresectable gastrointestinal stromal tumours treated with imatinib therapy.

The two basic mainstays of gastrointestinal stromal tumours (GIST) treatment are surgery and imatinib, a selective tyrosine kinase inhibitor that allows achieving a stable or responding disease in about 80% of patients with unresectable/metastatic GIST. Response to imatinib mainly depends from KIT and PDGFRα mutational status. Nevertheless, some patients with a potentially responsive genotype do not respond, and others develop a pattern of resistance to imatinib which is not associated with secondary mutations. This emphasizes the presence of mechanisms of resistance other than the receptor-related genotype, and the need of biological predictors to select the optimal therapeutic strategy, particularly now that other potent inhibitors are available. We investigated a panel of 31 polymorphisms in 11 genes, potentially associated with the pharmacogenetics of imatinib, in a group of 54 unresectable/metastatic GISTs treated with imatinib 400mg daily as first line therapy. Included in this analysis were polymorphisms in the transporters' family SLC22, SLCO, ABC, and in the metabolizing genes CYP-3A4 and -3A5. Time to progression was significantly improved in presence of the C allele in SLC22A4 (OCTN1 rs1050152), and the two minor alleles (G) in SLC22A5 (OCTN2 rs2631367 and rs2631372). Importantly, multivariate analysis, adjusting for age, gender, KIT/PDGFRα mutational status, and tumour size, revealed that all the three genotypes maintained independent predictive significance. In conclusion, in this study we showed that SLC22A4 and SLC22A5 genotypes may be an important predictor of time to progression in GIST patients receiving imatinib therapy. Further investigations are required in an attempt to further personalize GIST therapy.

Regulation of electron transport in microalgae.

Unicellular algae are characterized by an extreme flexibility with respect to their responses to environmental constraints. This flexibility probably explains why microalgae show a very high biomass yield, constitute one of the major contributors to primary productivity in the oceans and are considered a promising choice for biotechnological applications. Flexibility results from a combination of several factors including fast changes in the light-harvesting apparatus and a strong interaction between different metabolic processes (e.g. respiration and photosynthesis), which all take place within the same cell. Microalgae are also capable of modifying their photosynthetic electron flow capacity, by changing its maximum rate and/or by diverting photogenerated electrons towards different sinks depending on their growth status. In this review, we will focus on the occurrence and regulation of alternative electron flows in unicellular algae and compare data obtained in these systems with those available in vascular plants. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.

Environmental exposure to benzene, micronucleus formation and polymorphisms in DNA-repair genes: a pilot study.

This report is part of a biomarker study conducted in an Italian population with exposure to environmental benzene ranging from 1.43 to 31.41 µg/m³ (values from personal sampling). DNA damage induced by benzene is the crucial mechanism of its genotoxicity, which leads to chronic benzene poisoning, haematotoxicity and leukaemia. Therefore, genetic variation in DNA-repair genes may modulate susceptibility to benzene-induced DNA damage. In light of this, the effects of polymorphisms in DNA-repair genes (APEX1, hOGG1, NBS1, XPD, XRCC1, and XRCC3) on micronucleus (MN) formation as a biomarker of early biological effects were evaluated. A significantly higher median MN frequency was recorded in traffic wardens than in controls. However, none of the analysed polymorphisms was significantly associated with the median MN frequency. A gene-gender interaction was observed for the APEX1 genotype. The APEX1 variant genotype was associated with significantly lower median MN frequency in men, not in women. Statistical analysis did not reveal any association between the score of the protective alleles - hypothetically pushing the pathway towards optimal DNA-damage repair - and MN. Even though there are some limitations in the study, our results indicate that the general population may be exposed to benzene concentrations higher than the threshold level for air-quality standards in the European Union of 10 µg/m³. Furthermore, urban traffic wardens are exposed to significantly higher levels of benzene than individuals spending most of the time indoors. This higher exposure may contribute to DNA damage, suggesting that benzene might be implicated both as an environmental and occupational risk factor in leukaemia and other haematological diseases. In conclusion, this study suggest the need for (i) regular monitoring of traffic wardens for possible exposure to benzene, as a precautionary step to reduce the associated health risks, and (ii) more comprehensive studies in order to better elucidate the involvement of APEX1 genotypes in benzene genotoxicity.

Neuroprotective effects of erucin against 6-hydroxydopamine-induced oxidative damage in a dopaminergic-like neuroblastoma cell line.

Oxidative stress (OS) contributes to the cascade leading to the dysfunction or death of dopaminergic neurons during Parkinson's disease (PD). A strategy to prevent the OS of dopaminergic neurons may be the use of phytochemicals as inducers of endogenous antioxidants and phase 2 enzymes. In this study, we demonstrated that treatment of the dopaminergic-like neuroblastoma SH-SY5Y cell line with isothiocyanate erucin (ER), a compound of cruciferous vegetables, resulted in significant increases of both total glutathione (GSH) levels and total antioxidant capacity at the cytosolic level. The increase of GSH levels was associated with an increase in the resistance of SH-SY5Y cells to neuronal death, in terms of apoptosis, induced by 6-hydroxydopamine (6-OHDA). The pretreatment of SH-SY5Y cells with ER was also shown to prevent the redox status impairment, in terms of intracellular ROS and O(2) (•-) formation, and loss of mitochondrial membrane potential, early events that are initiators of the apoptotic process, induced by 6-OHDA. Last, the antiapoptotic and antioxidant effects of ER were abolished by buthionine sulfoximine, supporting the main role of GSH in the neuroprotective effects recorded by ER. These results suggest that ER may prevent the oxidative damage induced by 6-OHDA.

The onset of NPQ and Deltamu(H)+ upon illumination of tobacco plants studied through the influence of mitochondrial electron transport.

The relationship between the development of photoprotective mechanisms (non-photochemical quenching, NPQ), the generation of the electrochemical proton gradient in the chloroplast and the capacity to assimilate CO(2) was studied in tobacco dark-adapted leaves at the onset of illumination with low light. These conditions induce the generation of a transient NPQ, which relaxes in the light in parallel with the activation of the Calvin cycle. Wild-type plants were compared with a CMSII mitochondrial mutant, which lacks the respiratory complex I and shows a delayed activation of photosynthesis. In the mutant, a slower onset of photosynthesis was mirrored by a decreased capacity to develop NPQ. This correlates with a reduced efficiency to reroute electrons at the PSI reducing side towards cyclic electron flow around PSI and/or other alternative acceptor pools, and with a smaller ability to generate a proton motive force in the light. Altogether, these data illustrate the tight relationship existing between the capacity to evacuate excess electrons accumulated in the intersystem carriers and the capacity to dissipate excess photons during a dark to light transition. These data also underline the essential role of respiration in modulating the photoprotective response in dark-adapted leaves, by poising the cellular redox state.

Exposure to low environmental levels of benzene: evaluation of micronucleus frequencies and S-phenylmercapturic acid excretion in relation to polymorphisms in genes encoding metabolic enzymes.

An integrated approach based on environmental and biological monitoring, including the analysis of biomarkers of exposure [excretion of S-phenylmercapturic acid (S-PMA)], early biological effects [micronucleus (MN) frequency] and susceptibility (genetic polymorphisms), was applied to characterize benzene exposure in a group of 70 traffic policemen and 40 employees of the city of Bologna, Italy. Median personal benzene exposure was 6.55-fold higher for traffic policemen than for controls (P<0.0001). This higher exposure was confirmed by a significant, 2.53-fold higher S-PMA excretion in traffic policemen compared with that observed for indoor workers (P<0.0001). Median MN frequency was also significantly higher in policemen compared with indoor workers (P=0.001), emphasizing the genotoxic effect potentially associated with benzene exposure. With regard to biomarkers of susceptibility, the analysis revealed that high epoxide hydrolase (mEH) (predicted) enzyme activity was significantly correlated with a lower median MN frequency (P=0.003). A gene-gender interaction was observed for the glutathione-S-transferase M1 (GSTM1) genotype. The GSTM1-null genotype was associated with a significantly higher median MN frequency in men, not in women. Statistical analysis did not reveal any association between the presence of the protective allele, pushing the pathway towards benzene detoxification, and MN frequency or S-PMA excretion. Even though there are some limitations in the study, our results indicate that policemen are exposed to higher levels of benzene than individuals spending most of the time indoors. This higher exposure may contribute to DNA damage, suggesting an increase health risk from traffic benzene emission. Finally, a more comprehensive study is warranted in order to better elucidate the involvement of EPHX1 genotypes combination in benzene genotoxicity.

The role of respiration in the activation of photosynthesis upon illumination of dark adapted Chlamydomonas reinhardtii.

It is reported that O(2) is required for the activation of photosynthesis in dark adapted Chlamydomonas reinhardtii in State 1, under low light intensity. The concentration of dissolved O(2) of ca. 9 microM is sufficient to saturate the requirement. When the concentration of O(2) is 3 muM or below, the activation of photosynthesis is strongly inhibited by myxothiazol, a specific inhibitor of the mitochondrial cytochrome bc(1). The effect of this inhibitor decreases as the O(2) concentration is raised, to disappear completely above 50 muM. Low concentrations of uncouplers delay the activation of photosynthesis, but do not inhibit it when steady state is reached. It is concluded that in State 1 C. reinhardtii mitochondrial respiration is required for the activation of photosynthesis upon illumination of dark adapted cells only when the concentration of O(2) is too low (less than 5 muM) to allow an appreciable activity of the Mehler reaction. The role of respiration does not seem to be due to the synthesis of ATP by oxidative phosphorylation, because photosynthesis activation is not sensitive to oligomycin.

Sulforaphane as an inducer of glutathione prevents oxidative stress-induced cell death in a dopaminergic-like neuroblastoma cell line.

The total GSH depletion observed in the substantia nigra (SN) appears to be responsible for subsequent oxidative stress (OS), mitochondrial dysfunction, and dopaminergic cell loss in patients with Parkinson's disease. A strategy to prevent the OS of dopaminergic cells in the SN may be the use of chemopreventive agents as inducers of endogenous GSH, antioxidant and phase 2 enzymes. In this study, we demonstrated that treatment of the dopaminergic-like neuroblastoma SH-SY5Y cell line with sulforaphane (SF), a cruciferous vegetables inducer, resulted in significant increases of total GSH level, NAD(P)H : quinone oxidoreductase-1, GSH-transferase and -reductase, but not GSH-peroxidase, catalase and superoxide dismutase activities. Further, the elevation of GSH levels, GSH-transferase and NAD(P)H:quinone oxidoreductase-1 activities was correlated to an increase of the resistance of SH-SY5Y cells to toxicity induced by H(2)O(2) or 6-hydroxydopamine (6-OHDA). The pre-treatment of SH-SY5Y cells with SF was also shown to prevent various apoptotic events (mitochondrial depolarization, caspase 9 and 3 activation and DNA fragmentation) and necrosis elicited by 6-OHDA. Further, the impairment of antioxidant capacity and reactive oxygen species formation at intracellular level after exposure to 6-OHDA was effectively counteracted by pre-treatment with SF. Last, both the cytoprotective and antioxidant effects of SF were abolished by the addition of buthionine sulfoximine supporting the main role of GSH in the neuroprotective effects displayed by SF. These findings show that SF may play a role in preventing Parkinson's disease.

Protective effect of creatine against RNA damage.

It is well documented that damage to DNA could be very harmful for all cells and is the source of several consequences such as cancer development, apoptosis or genetic diseases. In contrast, RNA damage is a poorly examined field in biomedical research, despite its potential to affect cell physiology. For example, a significant loss of RNA integrity has been demonstrated in advanced human atherosclerotic plaques as compared with non-atherosclerotic mammary arteries, and oxidative RNA damage has been described in several neurodegenerative diseases including Alzheimer disease. In the present study, we investigated whether RNA damage could be related to the exposure of particular xenobiotics and then we studied the potential protective activity of creatine against RNA-damaging activity of a series of chemicals with different mechanisms of action [ethyl methanesulfonate (EMS), H(2)O(2), doxorubicin, spermine NONOate, S-nitroso-N-acetylpenicillamine (SNAP)]. Since the protective effect against RNA damage can be mediated by different mechanisms, such as alterations of the rates of toxic agent absorption and uptake, trapping of electrophiles as well as free radicals, and protection of nucleophilic sites in RNA, we used two different treatment protocols (pre- and co-treatment) for understanding the mechanism of the inhibitory activity of creatine. We demonstrated that total RNA is susceptible to chemical attack by doxorubicin, H(2)O(2), spermine and SNAP. Creatine significantly reduced the RNA-damaging activity of only two of the toxic tested agents (H(2)O(2) and doxorubicin), while it lacked activity in counterstaining the RNA damage induced by the NO donors spermine and SNAP. Its inhibitory activity could be at least partially dependent on its capacity to directly scavenge free radicals and/or to maintain phosphocreatine store and ATP regeneration.

Micronuclei frequency induced by bleomycin in human peripheral lymphocytes: correlating BLHX polymorphism with mutagen sensitivity.

Mutagen sensitivity assay, by measuring chromosome damage induced by an in vitro treatment of peripheral lymphocytes with bleomycin, has been proposed as a biomarker for assessing cancer susceptibility. Recently, a single nucleotide polymorphism (SNP A1450G) of the gene for bleomycin hydrolase (BLHX), a specific neutral cysteine protease able to metabolise bleomycin, was proposed as a plausible candidate to variation in mutagen sensitivity. To shed more light on the effect of BLHX genotype on the expression of chromosome damage induced in vitro by bleomycin, we determined mutagen sensitivity for 45 non-smoker healthy volunteers. The level of bleomycin-induced chromosome damage was assessed as frequencies of micronuclei (MN) in cytokinesis-blocked lymphocytes. The subjects were genotyped for the BLHX gene, to determine the possible effect of this polymorphism on mutagen sensitivity. No difference in the spontaneous value of MN was detected between the homozygotes wild-type (A/A) and the carriers of variant alleles A/G heterozygotes or G/G homozygotes (MN/1000 binucleated (BN) cells: 6.69+/-2.53 and 6.37+/-4.87, respectively). A substantial effect of BLHX polymorphism in predetermining individual mutagen sensitivity status was observed: subjects with the BLHX A/A genotype displayed significantly lower mean levels of bleomycin-induced MN frequency than the carriers of A/G or G/G variant alleles combined (12.00+/-3.76 MN/1000 BN vs. 16.37+/-8.86 MN/1000 BN, respectively; P=0.029). The multiple regression analysis, including BLHX genotype and age, confirmed the significant effect of BLHX variant alleles (A/G, G/G) on the chromosome damage induced by bleomycin (P=0.01), whereas age correlated only with the spontaneous MN frequency.

RNA as a new target for toxic and protective agents.

In contrast to damage of genomic DNA and despite its potential to affect cell physiology, RNA damage is a poorly examined field in biomedical research. Potential triggers of RNA damage as well as its pathophysiological implications remain largely unknown. While less lethal than mutations in genome, such non-acutely lethal insults to cells have been recently associated with underlying mechanisms of several human chronic diseases. We investigated whether RNA damage could be related to the exposure of particular xenobiotics by testing the RNA-damaging activity of a series of chemicals with different mechanisms of action. Cultured human T-lymphoblastoid cells were treated with ethyl methanesulfonate (EMS), H(2)O(2), doxorubicin, spermine, or S-nitroso-N-acetylpenicillamine (SNAP). Furthermore, we studied the potential protective activity of a pomegranate extract against RNA damage induced by different chemicals. Special attention has been paid to the protective mechanisms of the extract. The protective effect of pomegranate can be mediated by alterations of the rates of toxic agent absorption and uptake, by trapping of electrophiles as well as free radicals, and protection of nucleophilic sites in RNA. We used two different treatment protocols (pre- and co-treatment) for understanding the mechanism of the inhibitory activity of pomegranate. We demonstrated that total RNA is susceptible to chemical attack. A degradation of total RNA could be accomplished with doxorubicin, H(2)O(2), spermine and SNAP. However, EMS, a well-known DNA-damaging agent, was devoid of RNA-damaging properties, while spermine and SNAP, although lacking of DNA-damaging properties, were able to damage RNA. Pomegranate reduced the RNA-damaging effect of doxorubicin, H(2)O(2), and spermine. Its inhibitory activity could be related with its ability to forms complexes with doxorubicin and H(2)O(2), or interacts with the intracellular formation of reactive species mediating their toxicity. For spermine, an alteration of the rates of spermine absorption and uptake can also be involved.

Inherited susceptibility to bleomycin-induced micronuclei: correlating polymorphisms in GSTT1, GSTM1 and DNA repair genes with mutagen sensitivity.

Susceptibility to DNA damage varies among individuals and sensitivity to bleomycin (BLM) may reflect the inter-individual differences. BLM sensitivity in part may be explained by inherited differences in DNA repair genes. We investigated the association between genetic polymorphisms in the GSTT1, GSTM1, XPD, XRCC1 and XRCC3 genes and the levels of spontaneous and BLM-induced DNA damage in peripheral blood lymphocytes from 200 healthy, unexposed individuals. The investigation of BLM sensitivity on cancer- or disease-free subjects and not occupationally exposed to known mutagen represents the strengths of the present study, as the detection of genetic damage is not biased by any disease- and occupational-related factor. The micronucleus (MN) assay was used to detect the spontaneous and BLM-induced genetic damage whereas, genotype analysis was carried out using methods based on polymerase chain reaction. Poisson regression analysis showed that subject's age, gender and smoking status had no effect on the spontaneous and BLM-induced MN frequencies. Genotype analysis revealed a clear association between GSTT1-null and XPD polymorphisms and both spontaneous and BLM-induced MN frequencies, whereas the effect of the XRCC1 polymorphism was marginally significant only with regard to spontaneous MN frequency. Genotype analysis did not reveal a clear association between the other studied SNPs (GSTM1 and XRCC3) and MN frequencies. Poisson regression analysis revealed no association between the score of protective alleles and the frequency of spontaneous MN. However, an increased number of protective alleles was significantly associated with a lower frequency of BLM-induced MN (P=0.0003). This finding highlights the genetic basis for BLM sensitivity, which could be a valid and useful surrogate for identifying genotypes that might increase susceptibility in population exposed to carcinogens. Further investigations in a large sample size and including more SNPs, reflecting the complexity of DNA repair machinery, might lead to the identification of a genetic profile responsible for the susceptibility to genotoxicants, with a far-reaching long-term impact on primary prevention and early detection of disease associated genes.

Induction of differentiation in human promyelocytic cells by the isothiocyanate sulforaphane.

BACKGROUND: The consumption of cruciferous vegetables has long been associated with a reduced risk for the occurrence of cancer at various sites. This protective effect is associated with their isothiocyanate content. Sulforaphane (SFN) is by far the isothiocyanate most extensively studied to uncover the mechanisms behind this chemoprotection. In the present study, the ability of SFN to induce cytodifferentiation and apoptosis in a leukemia cell line was investigated. MATERIALS AND METHODS: Cells were treated with different concentrations of SFN (0-100 microM). Analysis of cell differentiation was performed by nonspecific/specific acid esterase activity. Apoptosis induction was performed by flow cytometry. RESULTS: SFN induced cytodifferentiation toward both granulocytic and macrophagic lineage, mediated by the involvement of phosphatidylinositol 3-kinase/protein kinase C. It also caused a significant increase in the apoptotic cell fraction. CONCLUSION: These findings suggest that SFN may be a promising antileukemic agent and should encourage further investigation as regards its chemotherapeutic potential.

Comparison of Adaptive Neuroprotective Mechanisms of Sulforaphane and its Interconversion Product Erucin in in Vitro and in Vivo Models of Parkinson's Disease.

Several studies suggest that an increase of glutathione (GSH) through activation of the transcriptional nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the dopaminergic neurons may be a promising neuroprotective strategy in Parkinson's disease (PD). Among Nrf2 activators, isothiocyanate sulforaphane (SFN), derived from precursor glucosinolate present in Brassica vegetables, has gained attention as a potential neuroprotective compound. Bioavailability studies also suggest the contribution of SFN metabolites, including erucin (ERN), to the neuroprotective effects of SFN. Therefore, we compared the in vitro neuroprotective effects of SFN and ERN at the same dose level (5 µM) and oxidative treatment with 6-hydroxydopamine (6-OHDA) in SH-SY5Y cells. The pretreatment of SH-SY5Y cells with SFN recorded a higher (p < 0.05) active nuclear Nrf2 protein (12.0 ± 0.4 vs 8.0 ± 0.2 fold increase), mRNA Nrf2 (2.0 ± 0.3 vs 1.4 ± 0.1 fold increase), total GSH (384.0 ± 9.0 vs 256.0 ± 8.0 µM) levels, and resistance to neuronal apoptosis elicited by 6-OHDA compared to ERN. By contrast, the simultaneous treatment of SH-SY5Y cells with either SFN or ERN and 6-OHDA recorded similar neuroprotective effects with both the isothiocyanates (Nrf2 protein 2.2 ± 0.2 vs 2.1 ± 0.1 and mRNA Nrf2 2.1 ± 0.3 vs 1.9 ± 0.2 fold increase; total GSH 384.0 ± 4.8 vs 352.0 ± 6.4 µM). Finally, in vitro finding was confirmed in a 6-OHDA-PD mouse model. The metabolic oxidation of ERN to SFN could account for their similar neuroprotective effects in vivo, raising the possibility of using vegetables containing a precursor of ERN for systemic antioxidant benefits in a similar manner to SFN.

Combination of doxorubicin and sulforaphane for reversing doxorubicin-resistant phenotype in mouse fibroblasts with p53Ser220 mutation.

Chemoresistance in cancer therapy is a multifactorial process, which includes alterations in drug accumulation, increased activity of gluthatione S-transferases, loss of function, and mutations of p53, etc. One strategy for reversing chemoresistance is the use of chemopreventive agents alongside standard chemotherapeutic protocols. Sulforaphane is one of the most promising chemopreventive agents. Sulforaphane inhibits cell proliferation and induces apoptosis in different tumor cell lines. Its proapoptotic potential could make it effective either alone or in combination with other therapeutic strategies in reversing chemoresistance. We investigated the effects of sulforaphane on mouse fibroblasts bearing a different p53 status (wild-type, knockout, mutated) for understanding whether its activity is prevented by a mutated p53 status. p53-knockout fibroblasts from newborn mice transfected with the p53(Ser220) mutation, observed in different human cancers, were used as a model of mutated p53 status. Moreover, since p53(Ser220) mutation fibroblasts showed a doxorubicin-resistant phenotype, we treated the cells with a combination of doxorubicin plus sulforaphane. Taken together, our results suggest that a mutated p53 status did not prevent the induction of apoptosis by sulforaphane and that sulforaphane was able to reverse the resistance to doxorubicin. The association of sulforaphane-doxorubicin may therefore allow doxorubicin to be administered at lower doses, thereby reducing its potential toxicity.