Search

L’Organizzazione Mondiale della Sanità (OMS) ha recentemente stigmatizzato l’abitudine al fumo come una delle più gravi emergenze sanitarie globali. I dispositivi elettronici per il rilascio di nicotina (DERN), o sigarette elettroniche (e-cigs) sono state immesse sul mercato proprio con lo scopo di fornire al soggetto fumatore la possibilità di assumere nicotina esalando vapore, da un sistema che non prevede la combustione, e proposti al pubblico come metodi a “ridotto rischio per la salute”. Largamente diffusi, specialmente tra i più giovani, in alcuni paesi sono stati addirittura inclusi nei programmi integrati di lotta al tabagismo. Tuttavia, ad oggi, alcuni prestigiosi enti scientifici come il National Institute for Health and Care Excellence (NICE) ha ribadito come i dati sui possibili effetti tossicologici siano ancora limitati esortando la comunità scientifica a condurre studi indipendenti. Negli ultimi anni il nostro gruppo di ricerca ha studiato alcuni aspetti tossicologici associati all’esposizione dei vapori di e-cigs nel modello animale. Abbiamo rilevato aldeidi tossiche nello svapato e nei polmoni di ratto esposto per 4 settimane ed è stato osservato un marcato incremento degli enzimi del metabolismo degli xenobiotici, elevate concentrazione di radicali liberi (ROS), e una inattivazione dei principali enzimi antiossidanti in linea con i marcatori di danno ossidativo a livello sistemico e d’organo (DNA, lipidi, proteine). Test di mutagenesi effettuati su sangue e urine (comet, micronulcei, Ames) hanno evidenziato la potenzialità genotossica associata all’esposizione di e-cig. E’ interessante notare come i risultati delle analisi emocitometriche mostrino l’andamento tipico di pazienti affetti da brocopneumopatie ostruttive (COPD), con un marcato aumento dei neutrofili. I nostri studi hanno inoltre dimostrato come la scelta dell’atomizzatore, quindi del valore di resistenza applicata al sistema, sia inversamente associato alle concentrazioni di ROS e composti carbonilici rilasciati come formaldeide, acetaldeide e acroleina, indipendentemente dal voltaggio applicato. I dati in vivo confermano l’ipotesi di una tossicità direttamente dipendente dal tipo di atomizzatore scelto dal consumatore a parità di condizioni sperimentali. I risultati evidenziano inoltre come resistenze sub-Ohmiche producano un danno più marcato all’epitelio tracheale, mentre a livello polmonare, le immagini di microscopia elettronica mostrano come passando da un atomizzatore a resistenza di 1,5 Ohm a valori sub-Ohmici, il conseguente aumento del vattaggio determini una riduzione del numero e diametro dei bronchioli, fino ad una marcata disorganizzazione del parenchima polmonare con collasso alveolare, presenza di cellule in apoptosi e necrosi. Nel complesso, i risultati suggeriscono come l’utilizzo di e-cig possa costituire un serio rischio per la salute e che la personalizzazione del dispositivo e le modalità di utilizzo influenzino significativamente il rilascio di composti tossici e l’impatto sul sistema respiratorio.

Numerosi studi sperimentali suggeriscono un ruolo chemiopreventivo della vitamina E (VE) nei confronti di numerose patologie tumorali incluso il cancro alla prostata (CA). Tuttavia, le evidenze epidemiologiche più recenti non confermano tale ipotesi, e lo studio clinico SELECT ha addirittura evidenziato un aumento dell’incidenza di CA alla prostata in pazienti sani al momento dell’arruolamento, ai quali era stata somministrata VE giornalmente alla dose di 400 UI/die. Essendo noto in letteratura come alcune isoforme del citocromo P450 (CYP) siano sovra espresse in lesioni prostatiche maligne, e che la VE, agisca come induttore del CYP in alcuni tessuti, abbiamo ipotizzato che l’aumento dell’incidenza di CA possa coinvolgere un meccanismo co-cancerogenico legato all’induzione CYP. L’esposizione a VE produce una marcata induzione del CYP in cellule epiteliali di prostata RWPE-1, incluse le isoforme responsabili dell’attivazione degli Idrocarburi Policiclici Aromatici (IPA), accompagnata da un significativo aumento delle specie reattive dell’ossigeno (ROS). Inoltre, il pathway pro-infiammatorio COX-2-dipendente risulta incrementato, caratteristica che si osserva in diverse forme tumorali incluso quelle che interessano la prostata. Il fenomeno è stato confermato nel modello animale; è stato osservato un aumento delle monoossigenasi e dell’espressione genica di alcune isoforme CYP a livello prostatico unito a una maggiore concentrazione di ROS, in linea con marcatori di danno ossidativo a livello proteico e lipidico, nella prostata di ratto. L’esposizione di cellule IMR90 a VE ha evidenziato un effetto genotossico riportando un’aumentata frequenza dei micronuclei (MN). Il potenziale co-cancerogeno di VE è stato poi confermato dallo studio della trasformazione cellulare mediate l’uso di cellule BALB/c 3T3. Cellule pre-incubate con VE e successivamente esposte a benzo[a]pirene mostrano un significativo incremento della frequenza di trasformazione rispetto a quello senza della vitamina. Il presente studio dimostra come la VE possa, in determinate condizioni, agire come co-cancerogeno tramite un meccanismo CYP-dipendente, causando danno al DNA e promovendo la trasformazione cellulare.

E-cigarettes induce toxicological effects that can raise the cancer risk. The contribute of EPR radical probe technique Donatella Canistro1, Fabio Vivarelli1, Silvia Cirillo1, Andrea Sapone1, Moreno Paolini1, Paola Franchi2, Marco Lucarini2 1Department of Pharmacy and Biotechnology (University of Bologna, Via Irnerio 48, I-40126 Bologna, Italy) 2Department of Chemistry “G. Ciamician” (University of Bologna, Via San Giacomo 11, I-40126 Bologna, Italy) E-mail: paola.franchi@unibo.it Electronic cigarettes (e-cigs) are devices designed to deliver nicotine in a vaping solution rather than smoke and without tobacco combustion. Perceived as a safer alternative to conventional cigarettes, e-cigs are aggressively marketed as lifestyle-choice consumables, thanks to few restrictions and a lack of regulatory guidelines. Despite the burgeoning worldwide consumption of e-cigs, their safety remains largely unproven and it is unknown whether these devices cause in vivo toxicological effects that could contribute to cancer. Here we illustrate the contribute of EPR radical probe technique in a study where it was possible to demonstrate the co-mutagenic and cancer-initiating effects of e-cig vapour in a rat lung model. It was found that e-cig have a powerful booster effect on phase-I carcinogen-bioactivating enzymes, and increase oxygen free radical production and DNA oxidation. We are able to indirectly evaluate the content of reactive oxygen species (ROS) in lungs tissues of exposed rats, by using an appropriate hydroxylamine that in the presence of transient radical species gives rise to a persistent nitroxide radical. (see Scheme 1) Scheme 1 We found a significant increase of radical species production in samples of lungs tissues from exposed rats compared to samples from non-exposed animals. [1] Canistro, D.; Vivarelli, F.; Cirillo, S.; Babot Marquillas, C.; Buschini, A.; Lazzaretti, M.;Marchi, L.; Cardenia, V.; Rodriguez-Estrada, M.T.; Lodovici, M.; Cipriani, C.; Lorenzini, A.; Croco, E.; Marchionni, S.; Franchi, P.; Lucarini, M.; Longo, V.; Della Croce, C.M.; Vornoli, A.; Colacci, A.; Vaccari, M.; Sapone, A.; Paolini, M. SCIENTIFIC REPORTS. 2017, DOI:10.1038/s41598-017-02317-8 [2] Vivarelli, F.; Canistro, D.; Franchi, P.; Sapone, A.; Vornoli, A.; Della Croce, C.; Lucarini, M.; Paolini, M. Life Sciences, 2016, 145, 166-173. DOI:10.1016/j.lfs.2015.12.033 [3] Fabbri, R.; Sapone, A.; Paolini, M.; Vivarelli, F.; Franchi, P.; Lucarini, M.; Pasquinelli, G.; Vicenti, R.; Macciocca, M.; Venturoli, S.; Canistro, D. Hystology and Hystopatology, 2015, 30, 725-730 DOI: 10.14670/HH-30.725

E-CIGARETTES INDUCE TOXICOLOGICAL EFFECTS THAT CAN RAISE THE CANCER RISK. A FRAME FROM DRUG-METABOLISM AND ANTIOXIDANT HOMEOSTASIS. 1)Canistro D. 2)Vivarelli F. 3)Cirillo S. 4)Cardenia V. 5)Rodriguez-estrada MT. Dept of Pharmacy and Biotechnology, Unibo Electronic cigarettes (e-cigs) are devices designed to deliver nicotine in a vaping solution without tobacco combustion. Perceived as a safer alternative to conventional cigarettes, e-cigs are aggressively marketed as lifestyle-choice consumables, thanks to few restrictions and a lack of regulatory guidelines. Despite the burgeoning worldwide consumption of e-cigs, their safety remains largely unproven and it is unknown whether these devices cause in vivo toxicological effects that could contribute to cancer occurrence. In the present study, we investigated the co-mutagenic and cancer-initiating effects of e-cig vapour in a rat model. To explore whether e-cigs induce toxicological effects, such as those involving cytochrome P450 (CYP) changes, we analyzed the modulation of carcinogen metabolizingenzymes in the lung of rats exposed to e-cig vapour. We observed a significant increase in CYP1A1/2 (activating, for example, polychlorinated biphenyls, aromatic amines, dioxins and PAHs), CYP2B1/2 (activating olefins and halogenated hydrocarbons), 2C11 (activating nitrosamines and mycotoxins) and CYP3A (activating hexamethyl phosphoramide and nitrosamines) documented by the sharp rise in the corresponding probes. Conversely, we observed that the antioxidant enzymes catalase, DT-diaphorase and glutathione peroxidase and the conjugating phase II glutathione S-transferases, mainly involved in xenobiotic detoxification, were noticeable decreased, whereas UDP-glucuronyl-transferase was substantially unchanged. Extrapolated to humans, the corresponding boosted CYP-linked monooxygenases together with reduced activity of antioxidant and detoxifying machinery would predispose a subject to an enhanced cancer risk from the widely bioactivated e-cig vapour procarcinogens associated with an increased risk of lung cancer.

Development of microparticles for oral administration of the non-conventional radical scavenger IAC and testing in an inflammatory rat model 1)Cirillo S.. 2)Paolini M.. 3)Vivarelli F.. 4)Passerini N.. 5)Albertini B.. 6)Di sabatino M. 7)Corace G.. 8)Luppi B.. 9)Canistro D.. University of Bologna The bis (1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)-decandioate (IAC) is an innovative nonconventional radical scavenger used with success in several disease models, such as inflammation, neurological disorders, hepatitis and diabetes. To date, the main limit for the drug use is represented by the intraperitoneal (i.p.) route of administration used in the pharmacological treatments. In order to develop a delivery system that allowed both oral administration and the therapeutic efficacy, Solid Lipid Microparticles (SLMs) containing a theoretical 18% w/w of IAC have been produced. Recently, three formulations (A, B, C) have been tested at different dosages in an inflammation and pain rat model. Inflammatory model was induced by the use of an intraplantar injection of 100ml/paw of Freund's complete adjuvant (FCA). Administered per os at different dosages, IAC B (60% stearic acid-20% Compritol® HD5 ATO) was the most efficient formulation in reducing oedema and alleviating pain, compared to the gold standard Paracetamol. Since the anti-diabetic effects of the i.p. formulation of IAC was already demonstrated in vivo, we are now investigating the therapeutic efficacy of the selected (B) oral IAC formulation (SLMs) in streptozotocin-nicotinamide diabetic mice.

Effects of electronic cigarette vapors exposure on oxidative damage and antioxidant status. 1)Bigagli E. 2)Canistro D. 3)Paolini M. 4)Vivarelli F. 5)Cirillo S. 6)Lodovici M. 1)Neurofarba, University of florence, Florence, Italy 2)Farmacia e biotecnologie, University of bologna, Bologna, Italy 3)Farmacia e biotecnologie, University of bologna, Bologna, Italy 4)Farmacia e biotecnologie, University of bologna, Bologna, Italy 5)F Electronic cigarettes (e-cigs) are devices designed to deliver nicotine without burning tobacco and therefore, they are perceived as a safer alternative to the conventional cigarettes. However, because of the high temperature reached by e-cig solutions (> 200 C°) many toxic substances, including polycyclic aromatic hydrocarbons, formaldehyde, nitrosamines, metals and carbonyls can be generated. Very few in vivo animal studies have been conducted so far, most of them based on short-term e-cig exposure. The aim of this study was therefore to evaluate the influence of e-cigs on oxidative stress related parameters, classical markers associated to conventional cigarette toxicity. Male Sprague Dawley rats (8 weeks of age), were housed in an inhalation chamber and exposed either to e- cig vapors (1 mL/day of e-liquid containing 18 mg/mL of nicotine) or to a saline solution. One cycle of treatment consisted in 17 sec puff, 6 sec on, 5 sec off, 6 sec on, followed by 20 minutes stop. e-cigs voltage was set at 5.5. Animals were submitted to 11 cycles/ day for 5 consecutive days/week, for 4 consecutive weeks. At sacrifice, liver, kidneys, lungs, bladder and plasma were collected for the analysis of protein (carbonyl residues) and DNA oxidative damage (8-hydroxy-2’-deoxyguanosine (8-OHdG)) and of the total antioxidant power (FRAP assay). At lung level, we found that FRAP values of rats exposed to e-cigs vapors were markedly reduced compared to controls. A similar trend was observed in the plasma, even if the statistical significance was not reached. Plasma FRAP levels and carbonyl residues were inversely correlated in e-cig exposed rats but not in controls. We also found that 8-OHdG markedly increased in the lung of e-cig vapor exposed rats compared to controls. An inverse correlation between FRAP levels and 8-OHdG in lung tissue from exposed animals was also observed. These results demonstrate that e-cig vapor causes DNA oxidative damage and impairs antioxidant defenses in rats; given that these toxicological outcomes are typically induced by conventional cigarettes smoking, further investigations to better identify harmful e-cig effects especially in the long term, are of paramount importance.

The digestibility (in vitro), toxicity and metabolic effects of rapeseed (RPH) and sunflower (SPH) protein hydrolysates have been evaluated in a murine animal model. The enzyme AlcalaseVR was employed to obtain a mild enzymatic hydrolysis of rapeseed and sunflower defatted seed meals (DSM) protein isolates. Both hydrolysates showed higher in vitro digestibility than the respective DSM, presumably as a consequence of the hydrolysis process that they had undergone. In vivo, RPH and SPH were well tolerated. Body and organ weights, biochemical blood parameters from treated male mice were comparable to controls. Food intake was regular in RPH and SPH animals, suggesting a good palatability of the hydrolysates. Not relevant perturbations of the principal hepatic and renal drug metabolism enzymes were observed in RPH or SPH mice. In conclusion, protein hydrolysates from sunflower and rapeseed DSM did not determine relevant toxicological effects; therefore, they could be considered as alternative protein sources and/or food ingredients.

Phytochemicals have attracted attention in the oncological field because they are biologically friendly and have relevant pharmacological activities. Thanks to the intense and unique spicy aroma, garlic is one of the most used plants for cooking. Its consumption is correlated to health beneficial effects towards several chronic diseases, such as cancer, mainly attributable to allicin, a bioactive sulfur compound stored in different plant parts in a precursor form. The objective of this review is to present and critically discuss the chemistry and biosynthesis of allicin, its pharmacokinetic profile, its anticancer mechanisms and molecular targets, and its selectivity towards tumor cells. The research carried out so far revealed that allicin suppresses the growth of different types of tumors. In particular, it targets many signaling pathways associated with cancer development. Future research directions are also outlined to further characterize this promising natural product.

Despite the knowledge gap regarding the risk-benefit ratio of the electronic cigarette (e-cig), its use has grown exponentially, even in teenagers. E-cig vapor contains carcinogenic compounds (eg, formaldehyde, acetaldehyde, and acrolein) and free radicals, especially reactive oxygen species (ROS) that cause toxicological effects, including DNA damage. The role of e-cig voltage customization on molecule generation has been reported, but the effects of the resistance on e-cig emissions and toxicity are unknown. Here, we show that the manipulation of e-cig resistance influences the carbonyls production from nonnicotine vapor and the oxidative and inflammatory status in a rat model. Fixing the voltage at the conventional 3.5 V, we observed that the amount of the selected aldehydes increased as the resistance decreased from 1.5 to 0.25 Ω. Under these conditions, we exposed Sprague Dawley rats to e-cig aerosol for 28 days, and we studied the pulmonary inflammation, oxidative stress, tissue damage, and blood homeostasis. We found a perturbation of the antioxidant and phase II enzymes, probably related to the increased ROS levels due to the enhanced xanthine oxidase and P450-linked monooxygenases. Furthermore, frames from scanning electron microscope showed a disorganization of alveolar and bronchial epithelium in 0.25 Ω group. Overall, various toxicological outcomes, widely recognized as smoke-related injuries, can potentially occur in e-cig consumers who use low-voltage and resistance device. Our study suggests that certain “tips for vaping safety” cannot be established, and encourages further independent investigations to help public health agencies in regulating the e-cig use.

Purpose: We previously showed the positive effects of the new antioxidant molecule bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)-decandioate (IAC) in reducing basal hyperglycaemia and relieving glucose intolerance in a diabetes model. However, the chemical properties of IAC did not allow an efficient oral administration, thus representing the main failing of that study. Here, we tested the effect of a new oral delivery system based on solid lipid microparticles (SLMs) in a diabetes mouse model. Methods: The diabetes model was induced in C57B1/6J mice using streptozotocin and nicotinamide. Only the animals that overcame the glycaemic threshold of 180 mg/dL were enrolled in the study. Diabetic animals were then randomly assigned to 4 groups (n = 9) and treated once a day for 5 consecutive weeks with IAC (50, 100, and 150 mg/kg b.w.). The control group was composed of (n = 7) healthy mice that received only the vehicle. Glucose level was weekly monitored during the treatment period and up to 3 weeks after the suspension of the treatment. Glucose tolerance and insulin-resistance test were carried out. Results: Our results showed that SLMs maintained the IAC effect in reducing basal hyperglycaemia as well as improving the insulin sensitivity and glucose tolerance. Conclusion: The present study confirms that SLMs are promising drug carriers, which allow the oral administration of IAC ensuring its therapeutic efficacy. The concrete possibility to administer IAC per os represents a significant breakthrough in the putative consideration of this multi-radical scavenger in the diabetes therapeutic approach.

A large number of basic researches and observational studies suggested the cancer preventive activity of vitamin E, but large-scale human intervention trials have yielded disappointing results and actually showed a higher incidence of prostate cancer although the mechanisms underlying the increased risk remain largely unknown. Here we show through in vitro and in vivo studies that vitamin E produces a marked inductive effect on carcinogen-bioactivating enzymes and a pro-oxidant status promoting both DNA damage and cell transformation frequency. First, we found that vitamin E in the human prostate epithelial RWPE-1 cell line has the remarkable ability to upregulate the expression of various phase-I activating cytochrome P450 (CYP) enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), giving rise to supraphysiological levels of reactive oxygen species. Furthermore, our rat model confirmed that vitamin E in the prostate has a powerful booster effect on CYP enzymes associated with the generation of oxidative stress, thereby favoring lipid-derived electrophile spread that covalently modifies proteins. We show that vitamin E not only causes DNA damage but also promotes cell transformation frequency induced by the PAH-prototype benzo[a]pyrene. Our findings might explain why dietary supplementation with vitamin E increases the prostate cancer risk among healthy men.

Despite the lack of knowledge of the effects of electronic cigarettes (e-cigarettes, e-cigs) on public health, they have been proposed as a part of smoking cessation efforts. Recently, several basic scientific studies have pointed out how e-cigs can generate carcinogens, such as e-cig liquid thermal degradation by-products, and how the exposure can lead to genomic damage through inhibiting DNA repair or disrupting the redox homeostasis. However, scientific studies have pointed out how e-cigs can generate carcinogens and their release could be avoided setting the device to a low-voltage regimen. To test this feasibility, we show the effects of e-cig vapour generated from a low-voltage device filled with a nicotine-free liquid on rat testicular functions. The chemical analysis revealed the presence of carbonyls, such as formaldehyde, acetaldehyde and acrolein. Rats exposed reported a lower relative testis weight and higher levels of lactate dehydrogenase (LDH) as tissue damage marker, along with an impairment of 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD) and glucose-6-phosphate dehydrogenase (G6PDH) as key enzymes in the steroidogenesis pathway. The pro-oxidative environment was confirmed by the higher amount of reactive oxygen species (ROS), the development of lipid peroxidation and protein carbonylation, as well as from the disruption of antioxidant capability. Finally, we observed a higher rate of DNA unwinding in white blood cell line and boosted lipoxygenase (LOX)-linked activity, a tumour promotion marker. Even with the device setting at weak conditions, our results if extrapolated to humans suggest that exposure to e-cig vapours might alter gonads function in male vapers.

The chemopreventive effects of isothiocyanate moringin isolated from Moringa oleifera seeds 1)Vivarelli F. 2)Paolini M. 3)Michl C. 4)Rascle A. 5)Weigl J. 6)De nicola G. 7)Iori R. 8)Cirillo S. 9)Canistro D. University of Bologna Over the past years, there has been a growing interest in the natural constituents of vegetables as a potential means of cancer control. Isothiocyanates (ITCs) are considered as ideal chemopreventive agents, due to their abundance in easily accessible brassica vegetables, their ability to target multiple pathways involved in cancer aetiology coupled with a favourable toxicological profile. Recently it was reported that sulphoraphane (SFN) inhibits STAT5-mediated transcription. STAT5 (Signal Transducer and Activator of transcription 5) belongs to a family of transcription factors (STAT1-6) mainly present in an inactive form in the cytoplasm of numerous cell types and which are activated by phosphorylation in response to specific cytokines hormones and growth factors. STAT5 plays a key role in the control of cell proliferation and survival, via the regulation of genes such as c-Myc, Pim-1, Bcl-x, Osm or Cis and it also contributes to immune cell differentiation and function. Its activity is normally tightly controlled, and is hence frequently found deregulated in cancer where it is often constitutively activated. For these reasons STAT inhibitors are considered as potential candidates for cancer prevention or therapy. Beside SFN, more recently the isothiocyanate moringin (GMG-ITC), isolated from the Moringa oleifera, has caught the interest of scientific community for its anti-inflammatory activity through the inhibition of the NF-κ B pathway and it showed interesting anti cancer effects against mouse multiple myeloma and human astrocytoma cell line. However, till now, very little is known about the activity of GMG-ITC on cell signaling pathways. The present study was conceived to explore the potential inhibitory effect of GMG-ITC on crucial pathways commonly up-regulated in cancer, such as JAK/STAT and NF-κB comparing results with those obtained with SFN. Results showed how SFN and GMG-ITC were able to suppress IL-3- induced expression of STAT5 target genes in mouse pro-B cells, however, GMG-ITC reported a stronger inhibitory activity compared to SFN. Both GMG-ITC and SFN did not inhibit STAT5 phosphorylation, suggesting a downstream inhibitory event. Expression of interferon (IFNα)- stimulated STAT1/STAT2 target genes (G1P3, ISG15, STAT1, IRF9) in HeLa cells were downregulated by GMG-ITC and SFN without altering STAT1 and STAT2 phosphorylation. Also in this case GMG-ITC exhibited a more marked activity then those observed with SFN. Notable, basal expression of c-Myc remained unaffected upon treatment with up to 10 μM SFN or GMG-ITC, indicating that both ITCs inhibit STAT5-induced but not basal c-Myc. GMG-ITC and SFN had a limited effect on IFNα-induced STAT1 and STAT2 activity, indicating that both ITCs differentially target JAK/STAT signaling pathways. Furthermore, we showed that GMG-ITC in the micromolar range is a more potent inhibitor of TNF-induced NF-κB activity than SFN. TNF-induced expression of IL-8 and IL-6 was inhibited by both SFN and GMG-ITC, but this last appeared to be more effective. Interestingly, SFN and GMG-ITC inhibit NF-κ B signaling with a greater potency than that mediated by the well-known NF-κB inhibitor curcumin. As a whole, our data indicate how GMGITC could be considered as a potent inhibitor of STAT5, NF-κ B as well as STAT1/STAT2 signaling pathways, often overcoming the effects obtained with SFN. Given the implication of these pathways in the carcinogenesis, inflammatory diseases and immune disorders, GMG-ITC could represent a newsworthy and attractive chemopreventive agent.

The lack of tobacco combustion, the most attractive feature of electronic cigarettes (e-cigs), has quickly made these new devices become the most sought-after alternative to the traditional cigarettes, contributing also to its widespread safety perception. Contrary to the general belief that the electronic nicotine-delivery systems avoid the release of harmful chemicals, the high temperature reached by e-cig solutions can actually generate dozens of toxic compounds, some of which are listed as known human carcinogens. Male Sprague Dawley rats, at 8 weeks of age, were housed under a 12h-light/12h-dark cycle, 22°C, 60% humidity, and fed ad libitum. After 5 days’ adaptation, the rats were randomly split into two groups of 10 animals each (non-exposed (control group) and exposed). Animals were whole-body exposed to 11 cycles/day, in order to consume 1 mL/day of e-liquid containing 18 mg/mL of nicotine. At the end of each cycle, the animals were transferred to a clean chamber to begin the next one. Animals were treated 5 days/week for 4 consecutive weeks. Rats were fasted 16 h prior to sacrifice. Animals were anesthetized by administering Zoletil 100 (100 mg/kg b.w.) and then sacrificed by decapitation according to the approved Ministerial procedures. The rats’ brains were isolated and their lipid matter was subjected to the analysis of total fatty acids, sterols and oxysterols composition. The e-cig aerosol exposure decreased the total lipid content in rat brain. A slight increase of saturated fatty acids and n-6 long-chain polyunsaturated fatty acids (LC-PUFA) was detected, while n-3 LC-PUFA dropped. A reduction of total cholesterol content and its oxidation products was also observed; in fact, the levels of 7alfa-hydroxycholesterol, alfa/beta-epoxycholesterol isomers and triol were lower compared to those found in the control group. The present research study revealed how e-cigs aerosol could disrupt the lipid and cholesterol homeostasis in rat brain, which could contribute to the new occurrence of some neurodegenerative diseases. A deeper investigation is needed to assess the harmful health impact of e-cigs, particularly after long-term exposure.

Aims A large meta-analysis of randomized clinical trials has seriously questioned chemoprevention based on vitamins including vitamin E (VE), and an increased risk for cancer among long-term users was actually seen. However, the mechanism underlying these findings still remain unknown. To clarify the mechanism, in an in vivo model we studied the putative disruption of redox homeostasis and the perturbation of carcinogen metabolizing enzymes determined by VE. Main methods Male Sprague–Dawley rats were treated ip with either 100 or 200 mg/kg b.w. daily for 7 or 14 consecutive days. Controls received vehicle only. Cytochrome P450 (CYP) content, CYP-reductase, CYP-linked monooxygenases, as well as phase-II and the antioxidant enzymes catalase and NAD(P)H:quinone reductase were investigated in both liver and kidney. Free radical species in tissue subcellular preparations were measured by electronic paramagnetic resonance (EPR) spectroscopy coupled to a radical probe technique. Key findings No substantial changes of hepatic xenobiotic metabolism enzymes were determined by VE. Conversely, a powerful booster effect of various renal phase-I carcinogen bioactivating enzymes at both dosages and observational times was recorded. While no relevant changes of post-oxidative phase-II reactions were found in the liver, a significant inactivating effect was caused by VE in renal tissues. Antioxidant enzymes were found mainly downregulated by the treatment. In the kidney, a marked free radical over-generation linked to CYP induction was observed. Significance This study proved that VE acts as a co-carcinogen and pro-oxidant agent. Such epigenetic mechanisms may contribute to explain the harmful outcomes observed in humans.

Evidence from both epidemiological and experimental observations has fuelled the belief that the high consumption of fruits and vegetables rich in nutrients and phytochemicals may help prevent cancer and heart disease in humans. This concept has been drastically simplified from the dietary approaches to the use of single bioactive components both as a single supplement or in functional foods to manipulate xenobiotic metabolism. These procedures, which aim to induce mutagen/carcinogen detoxification or inhibit their bioactivation, fail to take into account the multiple and paradoxical biological outcomes of enzyme modulators that make their effects unpredictable. Here, we show that the idea that the physiological roles of specific catalysts may be easily manipulated by regular long-term administration of isolated nutrients and other chemicals derived from food plants is not viable. In contrast, we claim that the consumption of healthy diets is most likely to reduce mutagenesis and cancer risk, and that both research endeavours and dietary recommendations should be redirected away from single molecules to dietary patterns as a main strategy for public health policy.

Conflicting data on the anticancer properties of the polyphenolic natural product resveratrol (RSV) have been reported. Since the inhibition of "bioactivating" Phase-I xenobiotic metabolizing enzymes (XMEs) and/or induction of "detoxifying" Phase-II XMEs have long been considered important cancer chemopreventive strategies, in the current study we investigated the effect of RSV treatment on several Cytochrome P450 (CYP)-dependent oxidations and Phase-II markers in liver and lung subcellular preparations from CD1 male mice. These mice were i.p treated with RSV (25 or 50mg/Kg b.w.) daily for one or for seven consecutive days. Using either specific probes for different CYPs, or the regio- and stereo-selective metabolism of testosterone, we found that most of the Phase-I XMEs were significantly suppressed (up to approximately 61% loss for the CYP3A1/2-linked 6 beta-hydroxylation of testosterone in liver and up to approximately 97% loss for 2 alpha-hydroxylase in lung) following RSV treatment for 7 days at 50mg/kg b.w. Glutathione S-transferase was significantly inhibited, particularly in lung (approximately 76% loss of activity) after single administration of 25mg/kg b.w. A different response for the UDP-glucuronosyl transferase was observed, where a significant induction was seen (approximately 83%) in the liver and a significant reduction was observed in the lung (up to approximately 83% loss) following treatment with 25mg/kg b.w. for seven days. These data indicate that murine XMEs are altered by RSV, and that this alteration is dependent on the RSV dose, duration and way of administration. These results could provide mechanistic explanations for the conflicting chemopreventive results reported for RSV.

it was previously found that fenarimol, vinclozolin or acephate, three of the most used pesticides worldwide, provoked a marked perturbation of murine cytochrome P450 (CYP)-linked monooxygenases. Here, to more closely mimic human exposure, it was investigated whether different pesticide combinations administered i.p. in male Swiss Albino CD1 mice in single or repeated fashion (daily, for three consecutive days), affect CYP-dependent oxidations. The four simulated mixtures showed a complex pattern of CYP induction and suppression, especially after repeated injection. For example, while fenarimol alone was the most inducing agent - reaching a 79-fold increase over control in testosterone 2 alpha-hydroxylase - followed by vinclozolin and acephate, coadministration with the former markedly reduced induction. Coadministration with vinclozolin, determined various positive and negative modulations. An increase of CYP2B1/2 and CYP3A1/2-associated oxidases and a decrease of ethoxycoumarin metabolism was observed in the acephate and vinclozolin mixture. An equivalent or reduced CYP expression, if compared to double combinations, was seen using the complete mixture. Taken as a whole, the unpredictability of the recorded effects with simple mixtures, shrinks the misleading extrapolation performed on a single pesticide. If reproduced in human, such changes, altering either endogenous metabolism or biotransformation of ubiquitous toxins, might have public health implications. (c) 2007 Elsevier Ltd. All rights reserved.

Epidemiological evidence suggests a link between consumption of chlorinated drinking water and various cancers. Chlorination of water rich in organic chemicals produces carcinogenic organochlorine by-products (OBPs) such as trihalomethanes and haloacetic acids. Since the discovery of the first OBP in the 1970s, there have been several investigations designed to determine the biological effects of single chemicals or small artificial OBP combinations. However, there is still insufficient information regarding the general biological response to these compounds, and further studies are still needed to evaluate their potential genotoxic effects. In the current study, we evaluated the effect of three drinking water disinfectants on the activity of cytochrome P450 (CYP)-linked metabolizing enzymes and on the generation of oxidative stress in the livers of male and female Cyprinus carpio fish (carp). The fish were exposed in situ for up 20 days to surface water obtained from the Trasmene lake in Italy. The water was treated with 1-2 mg/L of either sodium hypochlorite (NaClO) or chlorine dioxide (ClO2) as traditional disinfectants or with a relatively new disinfectant product, peracetic acid (PAA). Micronucleus (MN) frequencies in circulating erythrocytes from the fish were also analysed as a biomarker of genotoxic effect. In the CYP-linked enzyme assays, a significant induction (up to a 57-fold increase in the deethylation of ethoxyresorufin with PAA treatment) and a notable inactivation (up to almost a 90% loss in hydroxylation of p-nitrophenol with all disinfectants, and of testosterone 2beta-hydroxylation with NaClO) was observed in subcellular liver preparations from exposed fish. Using the electron paramagnetic resonance (EPR) spectroscopy radical-probe technique, we also observed that CYP-modulation was associated with the production of reactive oxygen species (ROS). In addition, we found a significant increase in MN frequency in circulating erythrocytes after 10 days of exposure of fish to water treated with ClO2, while a non-significant six-fold increase in MN frequency was observed with NaClO, but not with PAA. Our data suggest that the use of ClO2 and NaClO to disinfect drinking water could generate harmful OBP mixtures that are able to perturb CYP-mediated reactions, generate oxidative stress and induce genetic damage. These data may provide a mechanistic explanation for epidemiological studies linking consumption of chlorinated drinking water to increased risk of urinary, gastrointestinal and bladder cancers.

Sanitation is of crucial importance for the microbiological safety of drinking water. However, chlorination of water rich in organic material produces disinfection by-products (DBPs), many of which have been reported to be mutagenic and/or carcinogenic compounds such as haloacetic acids and trihalomethanes. Epidemiological studies have suggested a link between drinking water consumption and cancer. We previously observed that Cyprinus carpio fish exposed to DBPs, may be subject to epigenetic effects such as those referable to the up-regulation of cytochrome P450 (CYP) superfamily (ex. co-mutagenesis/co-carcinogenesis and oxidative stress) that has been associated to non-genotoxic carcinogenesis. Our goal was to study the xenobiotic metabolism in mollusks exposed in situ to surface water of Lake Trasimene (Central Italy) treated with several disinfectants such as the traditional chlorine dioxide (ClO2), sodium hypochlorite (NaClO) or the relatively new one peracetic acid (PAA). The freshwater bivalves (Dreissena polymorpha) being selected as biomarker, have the unique ability to accumulate pollutants. Freshwater bivalves were maintained in surface water containing each disinfectant individually (1-2 mg/L). Following an exposure period up to 20 days during the fall period, microsomes were collected from the mussels, then tested for various monooxygenases. Strong CYP inductions were observed. These data indicate that drinking water disinfection generates harmful DBP mixtures capable of determining a marked perturbation of CYP-supported reactions. This phenomenon, being associated to an increased pro-carcinogen bioactivation and persistent oxidative stress, could provide an explanation for the observational studies connecting the regular consumption of drinking water to increased risk of various cancers in humans.

BACKGROUND: Obesity is recognized as a leading global health problem, correlated with an increased risk for several chronic diseases. One strategy for weight control management includes the use of vegetables rich in bioactive compounds to counteract weight gain, improve the antioxidant status and stimulate lipid catabolism. AIM OF THE STUDY: The aim of this study was to investigate the role of Raphanus sativus Sango sprout juice (SSJ), a Brassica extraordinarily rich in anthocyanins (AC) and isothiocyanates (ITCs), in a non-genetic model of obesity (high fat diet-HFD induced). METHODS: Control groups were fed with HFD or regular diet (RD). After a 10-week period, animals were assigned to experimental units and treated by gavage for 28 days as follows: HFD and RD control groups (rats fed HFD or RD and treated with vehicle only) and HFD-treated groups (rats fed HFD and treated with 15, 75 or 150 mg/kg b.w. of SSJ). Body weight and food consumption were recorded and serum lipid profile was measured (total cholesterol, triglycerides, and non-esterified fatty acids). Hepatic phase-I, phase-II as well as antioxidant enzymatic activities were assessed. RESULTS: SSJ lowered total cholesterol level, food intake and liver weight compared with HFD rodents. SSJ at medium dose proved effective in reducing body-weight (~19 g reduction). SSJ was effective in up-regulating the antioxidant enzymes catalase, NAD(P)H: quinone reductase, oxidised glutathione reductase and superoxide dismutase, which reached or exceeded RD levels, as well as the phase II metabolic enzyme UDP-glucuronosyl transferase (up to about 43%). HFD up-regulated almost every cytochrome P450 isoform tested, and a mild down-regulation to baseline was observed after SSJ intervention. CONCLUSION: This work reveals, for the first time, the antioxidant, hypolipidemic and antiobesity potential of SSJ, suggesting its use as an efficient new functional food/nutraceutical product.

Ameliorative potential of Raphanus sativus cv. Sango sprout juice in obese rats maintained with high fat diet or switched to regular diet F. Vivarelli1, D. Canistro1, A. Sapone1, G.R. De Nicola2, C. Babot Marquillas1, R. Iori2, F. Gentilini3, M. Paolini1 1Dept. of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Via Irnerio 48, 40126, Bologna, Italy 2Industrial Crop Research Centre, Agricultural Research Council, (CRA-CIN), Via di Corticella, 133, 40128, Bologna, Italy 3Dept. of Veterinary Medical Sciences, Alma Mater Studiorum-University of Bologna, via Tolara di Sopra 50, 40064, Ozzano dell'Emilia, Bologna, Italy Obesity is a worldwide epidemic characterized not only by excessive fat deposition but also by systemic low grade of inflammation and high oxidative stress. Despite much progress has been achieved in recent years, current pharmacological approaches for long-term therapy offer modest benefits for most patients. The present study evaluated the ameliorative effect of Raphanus sativus Sango sprout juice (SSJ), a Brassica extraordinarily rich in anthocyanins (AC) and isothiocyanates (ITCs), in a non-genetic model of obesity (high fat diet-HFD induced). Rats were fed with HFD or regular diet (RD); after 10-weeks, the HFD animals were assigned to four experimental units and the interventional period was 28 days long: 1) HFD-RD, the diet switched from HFD to RD and received the vehicle only; 2) HFD+SSJ and 3) HFD-RD+SSJ, treated with 75 mg/kg b.w. of SSJ; 4) HFD and 5) RD vehicle only, controls. Our model of obesity showed hyperlipidaemia as well as a marked increase of body and liver weight. The transition from HFD to RD, as expected, led to a significant loss of excess weight (-9.83 g; p

Effetti del germoglio di Raphanus Sativus CV Sango in un modello animale di obesità F. Vivarelli1, D. Canistro1, A. Sapone1, C. Babot Marquillas1, G.R. De Nicola2, R. Iori2, I.C. Antonazzo1, F. Gentilini3 e M. Paolini1. 1 Unità di Tossicologia Molecolare, Dipartimento di Farmacia e Biotecnologie, Alma Mater Studiorum, Università di Bologna, 40127 Bologna, Italia. 2 Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Colture Industriali (CRA-CIN), 40128 Bologna, Italia. 3 Dipartimento di Scienze Mediche Veterinarie, Alma Mater Studiorum, Università di Bologna, 40064 Ozzano dell’Emilia, Bologna, Italia. L’Organizzazione Mondiale della Sanità (OMS) ha riconosciuto l’obesità come un’epidemia globale, che costituisce uno dei principali problemi concernenti la salute pubblica, essendo un importante fattore di rischio per varie malattie croniche, come il diabete mellito di tipo 2, malattie cardiovascolari, patologie endocrine e tumori. Evidenze scientifiche sostengono l’ipotesi che alcuni alimenti o derivati abbiano effetti benefici sulla salute e, ad oggi, la corretta nutrizione è considerata un valido strumento di prevenzione per numerose patologiche. E’ noto, come l’obesità influenzi lo status ossidativo, determinando un’anomala produzione di specie reattive dell’ossigeno (ROS), condizione che frequentemente viene esacerbata da una deplezione delle comuni difese antiossidanti a livello cellulare. Diversi vegetali appartenenti alla famiglia delle Brassicaceae, contenenti elevate concentrazioni di glucosinolati (GLs) e isotiocianati (ITCs), hanno attirato l’interesse della comunità scientifica per le loro proprietà ipolipemizzanti e antiossidanti, come evidenziato dal crescente numero di studi preclinici ed epidemiologici. Scopo di questo lavoro è stato quello di indagare gli effetti del succo di germoglio di Raphanus Sativus Sango (SSJ), una Brassicacea straordinariamente ricca in antocianine (AC) e GLs, in un modello animale non genetico di obesità. La patologia è stata indotta sottoponendo ratti del ceppo Sprague Dawley a una dieta iperlipidica (HFD) per 10 settimane consecutive; al gruppo di controllo è stata assegnata una dieta standard (RD). Tre differenti dosaggi (15, 75 o 150 mg/kg p.c.) di SSJ sono stati somministrati per os per 28 giorni consecutivi; durante la fase di intervento, ai relativi gruppi di controllo (HFD e RD) è stato somministrato un volume equivalente del veicolo (acqua). I risultati mostrano una significativa riduzione del peso corporeo nei gruppi trattati con il dosaggio più basso (body weight gain = -0.29 g, p

This work aimed to investigate whether the insecticide acephate (125 or 250 mg/kg b.w.) or diflubenzuron (752 or 1075 mg/kg b.w.), two of the most widely used pesticides worldwide, impairs CYP-linked murine metabolism in liver, kidney and lung microsomes after repeated (daily, for three consecutive days) i.p. administration. The regio- and stereo-selective hydroxylation of testosterone was used as multibiomarker of different CYP isoforms. Both gender and tissue specific effects were observed. Lung was the most responsive tissue to induction by lower diflubenzuron dose, as exemplified by the marked increase of testosterone 7alpha-hydroxylation (CYP2A) (up to 13-fold) in males. Higher dose produced a generalized inactivation. At the lower dose acephate induced 6beta- (CYP3A1/2, liver) as well as 2beta- (CYP2B1/2, kidney) hydroxylase activities ( approximately 5 and approximately 4-fold increase, respectively) in males. In females, a marked suppression of the various hydroxylations was observed. At 250 mg/kg of acephate, animals did not survive. Induction of the most affected isoforms was sustained by immunoblotting analysis. Corresponding human CYP modulations might disrupt normal physiological functions related to these enzymes. Furthermore, the co-mutagenic and promoting potential of these pesticides, phenomena linked to CYP upregulation (e.g. increased bioactivation of ubiquitous pollutants and generation of oxygen free radicals) are of concern for a more complete definition of their overall toxicological potential.

Effect of Vitamin E supplementation on xenobiotic-metabolizing and antioxidant enzymes in rat. F. Vivarelli1 D. Canistro1, A. Sapone1, S. Filippi2, I.C. Antonazzo1, C. Babot Marquillas1 M. Paolini1 1 Molecular Toxicology Unit, Department of Pharmacy and Biotechnology, Alma-Mater Studiorum, University of Bologna, 40127 Bologna, Italy. 2 Interdepartmental Laboratory of Functional and Cellular Pharmacology of Reproduction, Department of NEUROFARBA and Department of Experimental, Clinical and Biomedical Sciences, University of Florence, 50139 Florence, Italy. Since its discovery and isolation Vitamin E (VE) has been considered a “safe” agent. VE has a number of biological functions being the antioxidant one, as peroxyl radical scavenger, the best known. The relationship between VE and cancer risk has been investigated in epidemiological studies and clinical trials with conflicting results. Recently, Selenium and Vitamin E Cancer Prevention Trial (SELECT) unexpectedly showed an increased risk of all primary cancers among healthy man supplemented with VE. As the literature reports that VE induce the P450 in human hepatoma cell line (HepG2), and being also known that such induction is always linked to an increased free radical and metabolite generation (cocarcinogenesis), we hypothesized that a generalized CYP upregulation migth have a role in the SELECT outcomes. Sprague Dawley male rats were intraperitoneally daily treated (for 7 or 14 consecutive days) with VE (α-tocopherol) at 100 or 200 mg/kg b.w. doses. The putative effects of VE on hepatic and renal microsomal CYP-linked monooxygenases as well as on antioxidant and post-oxidative enzymes were investigated. The almost neutral effect exerted by VE on hepatic phase I and II xenobiotic metabolizing enzymes, and the appreciable increase of catalase (up to 73% after 7 day treatment at lowest dose, p