Giorgio Cantelli-Forti

Comparison between Chinese medical herb Pueraria lobata crude extract and its main isoflavone puerarin: Antioxidant properties and effects on rat liver CYP-catalysed drug metabolism

Ge-gen (Radix Puerariae; RP) is used in traditional oriental medicine for various medicinal purposes. The drug is the root of a wild leguminous creeper, Pueraria lobata (Willd) Ohwi. It possesses a high content of flavonoid derivatives, the most abundant of which is puerarin (PU). Here, using the enhanced chemiluminescence technique based on horseradish peroxidase and a luminol-oxidant-enhancer reagent, we evaluated in vitro the antioxidant activity of PU and RP crude extract. Both biological samples inhibited the steady-state chemiluminescent reaction in a dose-dependent fashion. However, different inhibition mechanism were postulated, since only RP behaved like conventional antioxidants. This activity was supposed to be due the presence of compounds other than PU in the crude extract. Using each of the specific substrates to different cytochrome P450 (CYP) isoforms or the regio- and stereo-selective hydroxylation of testosterone as polyfunctional probe we found that when intragastrically administered in male Wistar rats, PU (100 or 200 mg/kg b.w.) and RP (700 or 1,400 mg/kg b.w.) significantly altered hepatic CYP-linked monooxygenases. While both CYP content and NADPH-(CYP)-c-reductase activity were significantly increased in all situations, a complex pattern of CYP modulation was observed, including both induction (PU: CYP2A1, 1A1/2, 3A1, 2C11; RP: CYP1A2, 3A1, 2B1) and inactivation (PU and RP: CYP3A, 2E1, 2B1), the latter being due to either parental agents or metabolites, as demonstrated by in vitro studies. Overall, these findings indicate that RP contains compounds with potent antioxidant activity and that both PU and RP impairs CYP-catalysed drug metabolism.

Neuroprotective effects of anthocyanins and their in vivo metabolites in SH-SY5Y cells

Recent in vivo studies have highlighted an important role for the neuroprotective actions of dietary anthocyanins. However, one consistent result of these studies is that the systemic bioavailability of anthocyanins, including cyanidin 3-O-glucopyranoside (Cy-3G), is very poor. Cy-3G has been demonstrated to be highly instable at physiological pH, so its in vivo metabolites, such as the aglycon cyanidin (Cy) and protocatechuic acid (PA), may be responsible for both the antioxidant activitiy and the neuroprotective effects observed in vivo. Therefore, we investigated the protective effects of Cy-3G, Cy and PA against H(2)O(2)-induced oxidative stress in a human neuronal cell line (SH-SY5Y). We determined their ability to counteract reactive oxygen species (ROS) formation and to inhibit apoptosis in terms of mitochondrial functioning loss and DNA fragmentation induced by H(2)O(2). We demonstrated that pretreatment of SH-SY5Y cells with Cy-3G, Cy and PA inhibits H(2)O(2)-induced ROS formation at different cellular levels: Cy-3G at membrane level, PA at cytosolic level and Cy at both membrane and cytosolic levels. In addition, Cy showed a higher antioxidant activity at membrane and cytosolic level than Cy-3G and PA, respectively. Interestingly, both Cy and PA, but not Cy-3G, could inhibit H(2)O(2)-induced apoptotic events, such as mitochondrial functioning loss and DNA fragmentation. These results suggest that Cy and PA may be considered as neuroprotective molecules and may play an important role in brain health promotion. These in vitro findings should encourage further research in animal models of neurological diseases to explore the potential neuroprotective effects of compounds generated during in vivo metabolism of anthocyanins.

β-Carotene: a cancer chemopreventive agent or a co-carcinogen?

Evidence from both epidemiological and experimental observations have fueled the belief that the high consumption of fruits and vegetables rich in carotenoids may help prevent cancer and heart disease in humans. Because of its well-documented antioxidant and antigenotoxic properties, the carotenoid beta-carotene (betaCT) gained most of the attention in the early 1980s and became one of the most extensively studied cancer chemopreventive agents in population-based trials supported by the National Cancer Institute. However, the results of three randomized lung cancer chemoprevention trials on betaCT supplementation unexpectedly contradicted the large body of epidemiological evidence relating to the potential benefits of dietary carotenoids. Not only did betaCT show no benefit, it was associated with significant increases in lung cancer incidence, cardiovascular diseases, and total mortality. These findings aroused widespread scientific debate that is still ongoing. It also raised the suspicion that betaCT may even possess co-carcinogenic properties. In this review, we summarize the current data on the co-carcinogenic properties of betaCT that is attributed to its role in the induction of carcinogen metabolizing enzymes and the over-generation of oxidative stress. The data presented provide convincing evidence of the harmful properties of this compound if given alone to smokers, or to individuals exposed to environmental carcinogens, as a micronutrient supplement. This has now been directly verified in a medium-term cancer transformation bioassay. In the context of public health policies, while the benefits of a diet rich in a variety of fruits and vegetables should continue to be emphasized, the data presented here point to the need for consideration of the possible detrimental effects of certain isolated dietary supplements, before mass cancer chemoprevention clinical trials are conducted on human subjects. This is especially important for genetically predisposed individuals who are environmentally or occupationally exposed to mutagens and carcinogens, such as those found in tobacco smoke and in industrial settings.

Inflammation-Induced Alteration of Astrocyte Mitochondrial Dynamics Requires Autophagy for Mitochondrial Network Maintenance

Accumulating evidence suggests that changes in the metabolic signature of astrocytes underlie their response to neuroinflammation, but how proinflammatory stimuli induce these changes is poorly understood. By monitoring astrocytes following acute cortical injury, we identified a differential and region-specific remodeling of their mitochondrial network: while astrocytes within the penumbra of the lesion undergo mitochondrial elongation, those located in the core-the area invaded by proinflammatory cells-experience transient mitochondrial fragmentation. In brain slices, proinflammatory stimuli reproduced localized changes in mitochondrial dynamics, favoring fission over fusion. This effect was triggered by Drp1 phosphorylation and ultimately resulted in reduced respiratory capacity. Furthermore, maintenance of the mitochondrial architecture critically depended on the induction of autophagy. Deletion of Atg7, required for autophagosome formation, prevented the reestablishment of tubular mitochondria, leading to marked reactive oxygen species accumulation and cell death. Thus, our data reveal autophagy to be essential for regenerating astrocyte mitochondrial networks during inflammation.

Neuroprotective effect of sulforaphane in 6-hydroxydopamine-lesioned mouse model of Parkinson's disease

Parkinsons disease (PD) is characterized by the selective loss of dopaminergic nigrostriatal neurons, which leads to disabling motor disturbances. Sulforaphane (SFN), found in cruciferous vegetables, is a potent indirect antioxidant and recent advances have shown its neuroprotective activity in various experimental models of neurodegeneration. This study was undertaken to examine the effects of SFN on behavioral changes and dopaminergic neurotoxicity in mice exposed to 6-hydroxydopamine (6-OHDA). For this purpose, mice were treated with SFN (5mg/kg twice a week) for four weeks after the unilateral intrastriatal injection of 6-OHDA. The increase in 6-OHDA-induced rotations and deficits in motor coordination were ameliorated significantly by SFN treatment. In addition, SFN protected 6-OHDA-induced apoptosis via blocking DNA fragmentation and caspase-3 activation. These results were further supported by immunohistochemical findings in the substantia nigra that showed that SFN protected neurons from neurotoxic effects of 6-OHDA. The neuroprotective effect of SFN may be attributed to its ability to enhance glutathione levels and its dependent enzymes (glutathione-S-transferase and glutathione reductase) and to modulate neuronal survival pathways, such as ERK1/2, in the brain of mice. These results suggest that SFN may potentially be effective in slowing down the progression of idiopathic PD by the modulation of oxidative stress and apoptotic machinery.

The nature of prooxidant activity of vitamin C.

It was recently reported that vitamin C (500 mg/day for 6 weeks) administered as a dietary supplement to healthy humans exhibits a prooxidant, as well as an antioxidant effect in vivo. Here we show that high intakes of vitamin C (500 mg/kg b.w. for 4 days) in the rat are able to markedly induce hepatic cytochrome P4502E1-linked monooxygenases, measured as p-nitrophenol hydroxylase activity and corroborated by means of Western blot analyses. Furthermore, using Electron Paramagnetic Resonance Spectroscopy (EPR) coupled to a spin-trapping technique, we have also found that this induction generates large amounts of the anion radical superoxide (O2-). Therefore we can conclude that the adverse prooxidant outcomes (i.e. oxidative DNA damage) associated to vitamin C supplementation, being associated to a typical reversible boosting effect (i.e. enzymatic induction), may be easily controlled by a discontinuous supply. However, since the induced P4502E1 isoforms by vitamin C are responsible for ethanol metabolism to highly reactive radicals, care should be taken even in moderate drinkers.

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.

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 H2O2 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.

Effect of licorice and glycyrrhizin on murine liver CYP-dependent monooxygenases

This study is aimed to investigate the effect of the prolonged intake of conspicuous amounts of licorice (LE), or its natural constituent glycyrrhizin (G) on murine liver CYP-catalyzed drug metabolism. For this purpose the modulation of the regio- and stereo-selective hydroxylation of testosterone, together with the use of highly specific substrates as probes for different CYP isoforms such as ethoxyresorufin (CYP1A1), methoxyresorufin (1A2), pentoxyresorufin (2B1), p-nitrophenol (2E1) and aminopyrine (3A), were investigated. Daily doses of licorice root extract (3,138 or 6,276 mg/kg b.w. per os), or G (240 or 480 mg/kg b.w. per os), were administered to different groups of Swiss Albino CD1 mice of both sexes for 1, 4 or 10 consecutive days. While a single LE or G dose was unable to affect the multienzymatic CYP-system, using both schedules of repeated treatment, either LE or G were able to significantly induce hepatic CYP3A- and, to a lesser extent, 2B1- and 1A2-dependent microsomal monooxygenase activities, as well as 6beta- (mainly associated to CYP3A), 2alpha-, 6alpha- (CYP2A1, 2B1), 7alpha-, 16alpha- (CYP2B9) and 16beta-testosterone hydroxylase (TH) activities in male and female mice. Data on CYP3A modulation, the major isoform present in human liver, was confirmed by using Western immunoblotting with anti-CYP3A1/2 rabbit polyclonal antibodies raised against purified rat CYP3A. Northern blotting analysis using CYP3A cDNA biotinylated probe showed that the expression of such isozyme is regulated at the mRNA level. These results suggest that the induction of cytochrome P450-dependent activities by the prolonged intake of high LE or G doses, may result in accelerated metabolism of coadministered drugs with important implications for their disposition. The adverse effects associated with CYP changes such as toxicity/cotoxicity and comutagenicity may also have clinical consequences.

Neuroprotective effects of cyanidin 3-O-glucopyranoside on amyloid beta (25-35) oligomer-induced toxicity.

Recent studies suggest that the oligomers of short amyloid beta (Abeta) peptides such as Abeta(25-35) as well as full-length Abeta peptides (i.e. Abeta(1-40) and Abeta(1-42) peptides) are responsible for synaptic dysfunction and/or neuronal loss in Alzheimers disease (AD). Among antioxidant phytochemicals derived from fruit and vegetables, cyanidin 3-O-glucoside (Cy-3G) has recently gained attention for its neuroprotective properties. In this in vitro study, we demonstrated that Cy-3G can inhibit Abeta(25-35) spontaneous aggregation into oligomers and their neurotoxicity in human neuronal SH-SY5Y cells. In particular, the pre- and co-treatment of SH-SY5Y cells with Cy-3G reduced the neuronal death, in terms of apoptosis and necrosis, elicited by Abeta(25-35) oligomers. Cy-3G also shows the interesting ability to prevent the early events leading to neuronal death such as the Abeta(25-35) oligomer binding to plasma membrane and the subsequent membrane integrity loss. Taken together, these findings suggest that Cy-3G may be considered a phytochemical with neuroprotective properties useful in finding potential drug or food supplements for the therapy of AD.