Mitochondria-targeted antioxidants protect pancreatic β-cells against oxidative stress and improve insulin secretion in glucotoxicity and glucolipotoxicity

Mitochondrial oxidative damage is thought to play a key role in pancreatic β-cell failure in the pathogenesis of type 2 diabetes. Despite this, the potential of mitochondria-targeted antioxidants to protect pancreatic β-cells against oxidative stress has not yet been studied. Therefore, we investigated if mitochondria-targeted antioxidants protect pancreatic β-cells such as RINm5F and HIT-T15 cells against oxidative stress under glucotoxic and glucolipotoxic conditions. When β-cells were incubated under these conditions, the expression levels of mitochondrial electron transport chain complex subunits, mitochondrial antioxidant enzymes (such as MnSOD and Prx3), β-cell apoptosis, lipogenic enzymes (such as ACC, FAS and ABCA1), intracellular lipid accumulation, oxidative stress, ER stress, mitochondrial membrane depolarization, nuclear NF- κB and sterol regulatory element binding protein 1c (SREBP1c) were all increased, in parallel with decreases in intracellular ATP content, citrate synthase enzymatic activity and glucose-stimulated insulin secretion. These changes were consistent with elevated mitochondrial oxidative stress, and incubation with the mitochondria-targeted antioxidants, MitoTempol or Mitoquinone (MitoQ), prevented these effects. In conclusion, mitochondria-targeted antioxidants protect pancreatic β-cells against oxidative stress, promote their survival, and increase insulin secretion in cell models of the glucotoxicity and glucolipotoxicity associated with Type 2 diabetes.     

Myoglobin causes oxidative stress,increase of NO production and dysfunction of kidney's mitochondria

Rhabdomyolysis or crush syndrome is a pathology caused by muscle injury resulting in acute renal failure. The latest data give strong evidence that this syndrome caused by accumulation of muscle breakdown products in the blood stream is associated with oxidative stress with primary role of mitochondria. In order to evaluate the significance of oxidative stress under rhabdomyolysis we explored the direct effect of myoglobin on renal tubules and isolated kidney mitochondria while measuring mitochondrial respiratory control, production of reactive oxygen and nitrogen species and lipid peroxidation. In parallel, we evaluated mitochondrial damage under myoglobinurea in vivo. An increase of lipid peroxidation products in kidney mitochondria and release of cytochrome c was detected on the first day of myoglobinuria. In mitochondria incubated with myoglobin we detected respiratory control drop, uncoupling of oxidative phosphorylation, an increase of lipid peroxidation products and stimulated NO synthesis. Mitochondrial pore inhibitor, cyclosporine A, mitochondria-targeted antioxidant (SkQ1) and deferoxamine (Fe-chelator and ferryl-myoglobin reducer) abrogated these events. Similar effects (oxidative stress and mitochondrial dysfunction) were revealed when myoglobin was added to isolated renal tubules. Thus, rhabdomyolysis can be considered as oxidative stress-mediated pathology with mitochondria to be the primary target and possibly the source of reactive oxygen and nitrogen species. We speculate that rhabdomyolysis-induced kidney damage involves direct interaction of myoglobin with mitochondria possibly resulting in iron ions release from myoglobin's heme, which promotes the peroxidation of mitochondrial membranes. Usage of mitochondrial permeability transition blockers, Fe-chelators or mitochondria-targeted antioxidants, may bring salvage from this pathology.     

Supplementation of endothelial cells with mitochondria-targeted antioxidants inhibit peroxide-induced mitochondrial iron uptake, oxidative damage, and apoptosis

The mitochondria-targeted drugs mitoquinone (Mito-Q) and mitovitamin E (MitoVit-E) are a new class of antioxidants containing the triphenylphosphonium cation moiety that facilitates drug accumulation in mitochondria. In this study, Mito-Q (ubiquinone attached to a triphenylphosphonium cation) and MitoVit-E (vitamin E attached to a triphenylphosphonium cation) were used. The aim of this study was to test the hypothesis that mitochondria-targeted antioxidants inhibit peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC) through enhanced scavenging of mitochondrial reactive oxygen species, thereby blocking reactive oxygen species-induced transferrin receptor (TfR)-mediated iron uptake into mitochondria. Glucose/glucose oxidase-induced oxidative stress in BAECs was monitored by oxidation of dichlorodihydrofluorescein that was catalyzed by both intracellular H(2)O(2) and transferrin iron transported into cells. Pretreatment of BAECs with Mito-Q (1 microM) and MitoVit-E (1 microM) but not untargeted antioxidants (e.g. vitamin E) significantly abrogated H(2)O(2)- and lipid peroxide-induced 2',7'-dichlorofluorescein fluorescence and protein oxidation. Mitochondria-targeted antioxidants inhibit cytochrome c release, caspase-3 activation, and DNA fragmentation. Mito-Q and MitoVit-E inhibited H(2)O(2)- and lipid peroxide-induced inactivation of complex I and aconitase, TfR overexpression, and mitochondrial uptake of (55)Fe, while restoring the mitochondrial membrane potential and proteasomal activity. We conclude that Mito-Q or MitoVit-E supplementation of endothelial cells mitigates peroxide-mediated oxidant stress and maintains proteasomal function, resulting in the overall inhibition of TfR-dependent iron uptake and apoptosis.     

Mitochondria-targeted antioxidants do not prevent tumour necrosis factor-induced necrosis of L929 cells

Mitochondrial production of reactive oxygen species (ROS) is widely reported as a central effector during TNF-induced necrosis. The effect of a family of mitochondria-targeted antioxidants on TNF-induced necrosis of L929 cells was studied. While the commonly used lipid–soluble antioxidant BHA effectively protected cells from TNF-induced necrosis, the mitochondria-targeted antioxidants MitoQ₃, MitoQ₅, MitoQ₁₀ and MitoPBN had no effect on TNF-induced necrosis. Since BHA also acts as an uncoupler of mitochondrial membrane potential, two additional uncouplers were tested. FCCP and CCCP both provided dose-dependent inhibition of TNF-induced necrosis. In conclusion, the generation of mitochondrial ROS may not be necessary for TNF-induced necrosis. Instead, these results suggest alternative mitochondrial functions, such as a respiration-dependent process, are critical for necrotic death.     

Mitochondria-targeted antioxidants do not prevent tumour necrosis factor-induced necrosis of L929 cells

Mitochondrial production of reactive oxygen species (ROS) is widely reported as a central effector during TNF-induced necrosis. The effect of a family of mitochondria-targeted antioxidants on TNF-induced necrosis of L929 cells was studied. While the commonly used lipid-soluble antioxidant BHA effectively protected cells from TNF-induced necrosis, the mitochondria-targeted antioxidants MitoQ₃, MitoQ₅, MitoQ₁₀ and MitoPBN had no effect on TNF-induced necrosis. Since BHA also acts as an uncoupler of mitochondrial membrane potential, two additional uncouplers were tested. FCCP and CCCP both provided dose-dependent inhibition of TNF-induced necrosis. In conclusion, the generation of mitochondrial ROS may not be necessary for TNF-induced necrosis. Instead, these results suggest alternative mitochondrial functions, such as a respiration-dependent process, are critical for necrotic death.     

Consequences of long-term oral administration of the mitochondria-targeted antioxidant MitoQ to wild-type mice

The mitochondria-targeted quinone MitoQ protects mitochondria in animal studies of pathologies in vivo and is being developed as a therapy for humans. However, it is unclear whether the protective action of MitoQ is entirely due to its antioxidant properties, because long-term MitoQ administration may alter whole-body metabolism and gene expression. To address this point, we administered high levels of MitoQ orally to wild-type C57BL/6 mice for up to 28 weeks and investigated the effects on whole-body physiology, metabolism, and gene expression, finding no measurable deleterious effects. In addition, because antioxidants can act as pro-oxidants under certain conditions in vitro, we examined the effects of MitoQ administration on markers of oxidative damage. There were no changes in the expression of mitochondrial or antioxidant genes as assessed by DNA microarray analysis. There were also no increases in oxidative damage to mitochondrial protein, DNA, or cardiolipin, and the activities of mitochondrial enzymes were unchanged. Therefore, MitoQ does not act as a pro-oxidant in vivo. These findings indicate that mitochondria-targeted antioxidants can be safely administered long-term to wild-type mice.     

Mitochondria-targeted antioxidants for treatment of Parkinson's disease: Preclinical and clinical outcomes

Parkinson's disease is a progressive neurodegenerative disease in the elderly, and no cure or disease-modifying therapies exist. Several lines of evidence suggest that mitochondrial dysfunction and oxidative stress have a central role in the dopaminergic neurodegeneration of Parkinson's disease. In this context, mitochondria-targeted therapies that improve mitochondrial function may have great promise in the prevention and treatment of Parkinson's disease. In this review, we discuss the recent developments in mitochondria-targeted antioxidants and their potential beneficial effects as a therapy for ameliorating mitochondrial dysfunction in Parkinson's disease. This article is part of a Special Issue entitled: Misfolded Proteins, Mitochondrial Dysfunction, and Neurodegenerative Diseases.     

A mitochondria-targeted derivative of ascorbate: MitoC

Mitochondrial oxidative damage contributes to a wide range of pathologies. One therapeutic strategy to treat these disorders is targeting antioxidants to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation. To date only hydrophobic antioxidants have been targeted to mitochondria; however, extending this approach to hydrophilic antioxidants offers new therapeutic and research opportunities. Here we report the development and characterization of MitoC, a mitochondria-targeted version of the hydrophilic antioxidant ascorbate. We show that MitoC can be taken up by mitochondria, despite the polarity and acidity of ascorbate, by using a sufficiently hydrophobic link to the TPP moiety. MitoC reacts with a range of reactive species, and within mitochondria is rapidly recycled back to the active ascorbate moiety by the glutathione and thioredoxin systems. Because of this accumulation and recycling MitoC is an effective antioxidant against mitochondrial lipid peroxidation and also decreases aconitase inactivation by superoxide. These findings show that the incorporation of TPP function can be used to target polar and acidic compounds to mitochondria, opening up the delivery of a wide range of bioactive compounds. Furthermore, MitoC has therapeutic potential as a new mitochondria-targeted antioxidant, and is a useful tool to explore the role(s) of ascorbate within mitochondria.     

New Data on Effects of SkQ1 and SkQT1 on Rat Liver Mitochondria and Yeast Cells

Mitochondria are involved in many processes in eukaryotic cells. They play a central role in energy conservation and participate in cell metabolism and signaling pathways. Mitochondria are the main source of reactive oxygen species, excessive generation of which provokes numerous pathologies and cell death. One of the most promising approaches to the attenuation of oxidative stress in mitochondria is the use of targeted (i.e., transported exclusively into mitochondria) lipophilic cationic antioxidants. These compounds offer advantages over conventional water-soluble antioxidants because they induce the so-called “mild uncoupling” and can prevent collapse of the membrane potential in low, nontoxic concentrations. A novel mitochondria-targeted antioxidant, SkQT1, was synthesized and tested within the framework of the research project guided by V. P. Skulachev. The results of these experiments were initially reported in 2013; however, one publication was not able to accommodate all the data on the SkQT1 interactions with isolated mitochondria and cells. Here, we examined comparative effects of SkQT1 and SkQ1 on rat liver mitochondria (with broader spectrum of energy parame- ters being studied) and yeast cells. SkQT1 was found to be less effective uncoupler, depolarizing agent, inhibitor of respiration and ATP synthesis, and “opener” of a nonspecific pore compared to SkQ1. At the same time SkQ1 exhibited higher antioxidant activity. Both SkQT1 and SkQ1 prevented oxidative stress and mitochondria fragmentation in yeast cells exposed to t-butyl hydroperoxide and promoted cell survival, with SkQT1 being more efficient than SkQ1. Together with the results presented in 2013, our data suggest that SkQT1 is the most promising mitochondria-targeted antioxidant that can be used for preventing various pathologies associated with the oxidative stress in mitochondria.     

Role of oxidative stress and mitochondria in onset of urinary bladder dysfunction under acute urine retention

Acute urine retention is a frequent complication in patients with benign hyperplasia of the prostate gland. According to studies made on experimental animals and people, it is accompanied by the deterioration of the bladder blood supply. This study attempts to explore the relationship of intramural blood flow, production of reactive oxygen species, and functional state of the bladder detrusor in modeling of acute urine retention in rats, as well as the impact of mitochondria-targeted antioxidants (which are supposed to alleviate the effects of oxidative stress induced by experimental ischemia) on these parameters. The study showed beneficial effects of mitochondria-targeted antioxidant SkQR1 in preventing damage of the bladder caused by acute urinary retention, which suggests the therapeutic use of this type of compounds for the treatment of ischemic abnormalities of the urinary bladder.     

Cationic antioxidants as a powerful tool against mitochondrial oxidative stress

This review describes evidence that mitochondrial reactive oxygen species (mROS) are of great importance under many physiological and pathological conditions. The most demonstrative indications favoring this conclusion originate from recent discoveries of the in vivo effects of mitochondria-targeted antioxidants (MitoQ and SkQs). The latter compounds look promising in treating several incurable pathologies as well as aging.     

Mitochondria-targeted penetrating cations as carriers of hydrophobic anions through lipid membranes

High negative electric potential inside mitochondria provides a driving force for mitochondria-targeted delivery of cargo molecules linked to hydrophobic penetrating cations. This principle is utilized in construction of mitochondria-targeted antioxidants (MTA) carrying quinone moieties which produce a number of health benefitting effects by protecting cells and organisms from oxidative stress. Here, a series of penetrating cations including MTA were shown to induce the release of the liposome-entrapped carboxyfluorescein anion (CF), but not of glucose or ATP. The ability to induce the leakage of CF from liposomes strongly depended on the number of carbon atoms in alkyl chain (n) of alkyltriphenylphosphonium and alkylrhodamine derivatives. In particular, the leakage of CF was maximal at n about 10-12 and substantially decreased at n = 16. Organic anions (palmitate, oleate, laurylsulfate) competed with CF for the penetrating cation-induced efflux. The reduced activity of alkylrhodamines with n = 16 or n = 18 as compared to that with n = 12 was ascribed to a lower rate of partitioning of the former into liposomal membranes, because electrical current relaxation studies on planar bilayer lipid membranes showed rather close translocation rate constants for alkylrhodamines with n = 18 and n = 12. Changes in the alkylrhodamine absorption spectra upon anion addition confirmed direct interaction between alkylrhodamines and the anion. Thus, mitochondria-targeted penetrating cations can serve as carriers of hydrophobic anions across bilayer lipid membranes.     

Impact of simvastatin,niacin, and/or antioxidants on cholesterol metabolism in CAD patients with low HDL

The HDL Atherosclerosis Treatment Study (HATS) demonstrated a clinical benefit in coronary artery disease patients with low HDL cholesterol (HDL-C) levels treated with simvastatin and niacin (S-N) or S-N plus antioxidants (S-N+A) compared with antioxidants alone or placebo. Angiographically documented stenosis regressed in the S-N group but progressed in all other groups. To assess the mechanism(s) responsible for these observations, surrogate markers of cholesterol absorption and synthesis were measured in a subset of 123 HATS participants at 24 months (on treatment) and at 38 months (off treatment). Treatment with S-N reduced desmosterol and lathosterol levels (cholesterol synthesis indicators) 46% and 36% (P < 0.05), respectively, and elevated campesterol and beta-sitosterol levels (cholesterol absorption indicators) 70% and 59% (P < 0.05), respectively, relative to placebo and antioxidant but not S-N+A. Treatment with antioxidants alone had no significant effect. Combining S-N with antioxidants reduced desmosterol and lathosterol by 37% and 31%, and elevated campesterol and beta-sitosterol levels by 54% and 46%, but differences did not attain significance. Mean change in percent stenosis was positively associated with a percent change in lathosterol (r = 0.26, P < 0.005) and negatively associated with a percent change in beta-sitosterol (r = -0.21, P < 0.01). These data suggest that changes in stenosis were attributable, in part, to changes in cholesterol metabolism.     

Mitochondria-targeted antioxidant SkQR1 ameliorates gentamycin-induced renal failure and hearing loss

The influence of the mitochondria-targeted antioxidant SkQR1 on gentamycin-induced nephrotoxicity and ototoxicity has been analyzed. SkQR1 reduces the death of kidney epithelium cells and decreases the severity of renal failure caused by gentamycin application and also lowers the animals’ mortality. Treatment with SkQR1 also decreases gentamycininduced hearing loss. Mitochondria-targeted antioxidants, such as SkQR1, are new promising agents for preventing negative consequences of therapy with antibiotics.     

A mitochondria-targeted nitroxide is reduced to its hydroxylamine by ubiquinol in mitochondria

Piperidine nitroxides such as TEMPOL act as antioxidants in vivo due to their interconversion among nitroxide, hydroxylamine, and oxoammonium derivatives, but the mechanistic details of these reactions are unclear. As mitochondria are a significant site of piperidine nitroxide metabolism and action, we synthesized a mitochondria-targeted nitroxide, MitoTEMPOL, by conjugating TEMPOL to the lipophilic triphenylphosphonium cation. MitoTEMPOL was accumulated several hundred-fold into energized mitochondria where it was reduced to the hydroxylamine by direct reaction with ubiquinol. This reaction occurred by transfer of H() from ubiquinol to the nitroxide, with the ubisemiquinone radical product predominantly dismutating to ubiquinone and ubiquinol, together with a small amount reacting with oxygen to form superoxide. The piperidine nitroxides TEMPOL, TEMPO, and butylTEMPOL reacted similarly with ubiquinol in organic solvents but in mitochondrial membranes the rates varied in the order: MitoTEMPOL > butylTEMPOL > TEMPO > TEMPOL, which correlated with the extent of access of the nitroxide moiety to ubiquinol within the membrane. These findings suggest ways of using mitochondria-targeted compounds to modulate the coenzyme Q pool within mitochondria in vivo, and indicate that the antioxidant effects of mitochondria-targeted piperidine nitroxides can be ascribed to their corresponding hydroxylamines.     

Rational discovery and development of a mitochondria-targeted antioxidant based on cinnamic acid scaffold

A novel mitochondria-targeted antioxidant (TPP-OH) was synthesized by attaching the natural hydrophilic antioxidant caffeic acid to an aliphatic lipophilic carbon chain containing a triphenylphosphonium (TPP) cation. This compound has similar antioxidant activity to caffeic acid as demonstrated by measurement of DPPH/ABTS radical quenching and redox potentials, but is significantly more hydrophobic than its precursor as indicated by the relative partition coefficients. The antioxidant activity of both compounds was intrinsic related to the ortho-catechol system, as the methoxylation of the phenolic functions, namely in TPP-OCH(3) and dimethoxycinnamic acid, gave compounds with negligible antioxidant action. The incorporation of the lipophilic TPP cation to form TTP-OH and TPP-OCH(3) allowed the cinnamic derivatives to accumulate within mitochondria in a process driven by the membrane potential. However, only TPP-OH was an effective antioxidant: TPP-OH protected cells against H(2)O(2) and linoleic acid hydroperoxide-induced oxidative stress. As mitochondrial oxidative damage is associated with a number of clinical disorders, TPP-OH may be a useful lead that could be added to the family of mitochondria-targeted antioxidants that can decrease mitochondrial oxidative damage.     

Mitochondria as source of reactive oxygen species under oxidative stress. Study with novel mitochondria-targeted antioxidants — the “Skulachev-ion” derivatives

Production of reactive oxygen species (ROS) in mitochondria was studied using the novel mitochondria-targeted antioxidants (SkQ) in cultures of human cells. It was shown that SkQ rapidly (1–2 h) and selectively accumulated in mitochondria and prevented oxidation of mitochondrial components under oxidative stress induced by hydrogen peroxide. At nanomolar concentrations, SkQ inhibited oxidation of glutathione, fragmentation of mitochondria, and translocation of Bax from cytosol into mitochondria. The last effect could be related to prevention of conformational change in the adenine nucleotide transporter, which depends on oxidation of critical thiols. Mitochondria-targeted antioxidants at nanomolar concentrations prevented accumulation of ROS and cell death under oxidative stress. These effects required 24 h or more (depending on the cell type) preincubation, and this was not related to slow induction of endogenous antioxidant systems. It is suggested that SkQ slowly accumulates in a small subpopulation of mitochondria that have decreased membrane potential and produce the major part of ROS under oxidative stress. This population was visualized in the cells using potential-sensitive dye. The possible role of the small fraction of “bad” mitochondria in cell physiology is discussed.     

Mitochondria-targeted plastoquinone derivative SkQ<Subscript>1</Subscript> decreases ischemia-reperfusion injury during liver hypothermic storage for transplantation

The ability of the mitochondria-targeted plastoquinone derivative 10-(6′-plastoquinonyl)decyl triphenylphosphonium (SkQ₁) to decrease ischemia-reperfusion injury in isolated liver during hypothermic storage (HS) was studied. Rat liver was stored for 24 h at 4°C without or in the presence of 1 μM SkQ₁ with following reperfusion for 60 min at 37°C. The presence in the storage medium of SkQ₁ significantly decreased spontaneous production of reactive oxygen species and intensity of lipid peroxidation in the liver during HS and reperfusion. The GSH level after HS in solution with SkQ₁ was reliably higher, but reperfusion leveled this effect. At all stages of experiment the presence of SkQ₁ did not prevent the decrease of antioxidant enzyme activities such as catalase, GSH peroxidase, GSH reductase, and glucose-6-phosphate dehydrogenase. The addition of SkQ₁ to the storage medium improved energetic function of the liver, as was revealed in increased respiratory control index of mitochondria and ATP level. SkQ₁ exhibited positive effect on the liver secretory function and morphology after HS as revealed in enhanced bile flow rate during reperfusion and partial recovery of organ architectonics and state of liver sinusoids and hepatocytes. The data point to promising application of mitochondria-targeted antioxidants for correction of the ischemia-reperfusion injury of isolated liver during long-term cold storage before transplantation.     

Fine-tuning the hydrophobicity of a mitochondria-targeted antioxidant

The mitochondria-targeted antioxidant MitoQ comprises a ubiquinol moiety covalently attached through an aliphatic carbon chain to the lipophilic triphenylphosphonium cation. This cation drives the membrane potential-dependent accumulation of MitoQ into mitochondria, enabling the ubiquinol antioxidant to prevent mitochondrial oxidative damage far more effectively than untargeted antioxidants. We sought to fine-tune the hydrophobicity of MitoQ so as to control the extent of its membrane binding and penetration into the phospholipid bilayer, and thereby regulate its partitioning between the membrane and aqueous phases within mitochondria and cells. To do this, MitoQ variants with 3, 5, 10 and 15 carbon aliphatic chains were synthesised. These molecules had a wide range of hydrophobicities with octan-1-ol/phosphate buffered saline partition coefficients from 2.8 to 20000. All MitoQ variants were accumulated into mitochondria driven by the membrane potential, but their binding to phospholipid bilayers varied from negligible for MitoQ3 to essentially total for MitoQ15. Despite the span of hydrophobicites, all MitoQ variants were effective antioxidants. Therefore, it is possible to fine-tune the degree of membrane association of MitoQ and other mitochondria targeted compounds, without losing antioxidant efficacy. This indicates how the uptake and distribution of mitochondria-targeted compounds within mitochondria and cells can be controlled, thereby facilitating investigations of mitochondrial oxidative damage.     

Risk factors for cholangiocarcinoma in high-risk area of Thailand: role of lifestyle, diet and methylenetetrahydrofolate reductase polymorphisms

Background and aim: Cholangiocarcinoma (CCA) is the most common cancer in Northeast Thailand. Endemicity of Opisthorchis viverrini (OV) – a known carcinogen – is responsible, but although infection is very common, the lifetime risk of CCA is only 5%. Other co-factors must exist, including aspects of lifestyle or diet along with variations in genetic susceptibility to them. Change in methylenetetrahydrofolate reductase (MTHFR) activity may influence both DNA methylation and synthesis. This study investigates risk factors for CCA with a focus on lifestyle, diet and MTHFR polymorphisms. Methods: Nested case–control study within cohort study was conducted. 219 subjects with primary CCA were each matched with two non-cancer controls from the same cohort on sex, age at recruitment and presence/absence of OV eggs in stool. Lifestyle and dietary data were obtained at recruitment. MTHFR polymorphisms were analyzed using PCR with high resolution melting analysis. The associations were assessed using conditional logistic regression. Results: Consumption of alcohol, raw freshwater fish and beef sausage increased the risk of CCA, while fruit and/or vegetables consumption reduced risk. There were interactions between MTHFR and preserved freshwater fish and beef. These dietary items are either a source of OV or of pre-formed nitrosamine, folate and antioxidants that are of possible relevance in OV carcinogenesis. Conclusions: Primary prevention of CCA in high-risk population is based upon efforts to reduce OV infection. Reduced consumption of alcohol and preserved meats, and increased consumption of dietary folate, actions with a wider preventive potential, may also help in the reduction of CCA burden.