Ageing Research Reviews 9 (2010) 424430

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Ageing Research Reviews

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Review

Commonly adopted caloric restriction protocols often involve malnutrition

Fernanda M. Cerqueira , Alicia J. Kowaltowski
Departamento de Bioqumica, Instituto de Qumica, Universidade de So Paulo, So Paulo, Brazil

a r t i c l e

i n f o

a b s t r a c t
Undernutrition without malnutrition is an intervention that enhances laboratory animal life span, and is widely studied to uncover factors limiting longevity. In a search of the literature over a course of four years, we found that most protocols currently adopted as caloric restriction do not meet micronutrient standards set by the National Research Council for laboratory rats and mice. We provide evidence that the most commonly adopted caloric restriction protocol, a 40% restriction of the AIN-93 diet without vitamin or mineral supplementation, leads to malnutrition in both mice and rats. Furthermore, others and we nd that every other day feeding, another dietary intervention often referred to as caloric restriction, does not limit the total amount of calories consumed. Altogether, we propose that the term caloric restriction should be used specically to describe diets that decrease calorie intake but not micronutrient availability, and that protocols adopted should be described in detail in order to allow for comparisons and better understanding of the effects of these diets. 2010 Elsevier B.V. All rights reserved.

Article history: Received 13 April 2010 Received in revised form 10 May 2010 Accepted 10 May 2010

Keywords: Ageing Diet Energy restriction Calorie restriction Micronutrients Vitamins

1. Caloric restriction: early studies McCay et al. rst demonstrated in 1935 that caloric restriction (CR) increases maximum lifespan (McCay, 1935), offering restricted rats 20% less food than the ad libitum controls ingested. The authors of this groundbreaking study were aware that micronutrient malnutrition could be associated with the restricted diet, and the CR rats received additional daily supplements of cod liver oil and yeast to prevent vitamin and mineral deciencies. Most CR studies within the next decades kept micronutrient supplementation: Ross and colleagues used semi-puried diets in a series of studies on longevity and cancer in rats, enriching the diets of CR rodents in contents of salts and vitamins (Ross, 1961). Following Ross protocol, Weindruch and Walford developed a diet to study the inuences of CR and ageing on mitochondrial respiration (Weindruch et al., 1980), immune function (Weindruch et al., 1982), lifespan, and cancer incidence (Weindruch and Walford, 1982a; Weindruch et al., 1986) in mice. The diets fed to the restricted

mice were also enriched, and solidied the term CR as a label for a dietary intervention involving a decrease in calorie intake without micronutrient limitation, which is associated with lifespan extension in many animal models. 2. AIN diets and increased interest in CR: what happened to micronutrient supplementation? In 1977, the American Institute of Nutrition (AIN) Ad Hoc Committee on Standards for Nutritional Studies released a report with recommendations on nutritional methodology which would serve as a guideline for scientists who work with animal models. Fixedformula diets, in which kinds and amounts of ingredients do not vary from batch to batch, are often called open-formula diets when the formula is openly declared. A xed-formula diet may contain multiple sources of protein, fat and carbohydrates, thereby reducing the importance of variation in the composition of any particular ingredient from batch to batch (Knapka et al., 1974). The AIN-76 xed-formula diet supported growth, reproduction and lactation comparable to those observed using the best cereal-based diets. This diet was modied over the years, resulting nally in the AIN93 diet, in two formulations: AIN-93G, for growth, pregnancy and lactation, and AIN-93M for adult maintenance (Reeves et al., 1993). The criteria used for the AIN-93 formulations were (a) diets can be prepared from puried ingredients; (b) they conform to or exceed nutrient requirements suggested by the National Research Council (NRC) (National Research Council, 1995); (c) they can be prepared with readily available ingredients at a reasonable cost; (d) compositions are consistent and reproducible; and (e) diets can be used for a wide range of applications (Reeves, 1997). The AIN-93 diet is

Disclaimer: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. All animal experiments follow international standards and were approved by the Comit de tica em Cuidados e Uso Animal, an NIH accredited ethics committee which oversees animal research within the Instituto de Qumica of the Universidade de So Paulo, and follows standards set by the Colgio Brasileiro de Experimentaco Animal. Rats and mice were bread and housed in the Biotrio de Produco e Experimentaco da Faculdade de Cincias Farmacuticas e Instituto de Qumica, Universidade de So Paulo. Corresponding author at: Av. Prof. Lineu Prestes, 748, Cidade Universitria, So Paulo, 05508-900, SP, Brazil. Tel.: +55 11 30913810; fax: +55 11 38155579. E-mail address: fmeneses@iq.usp.br (F.M. Cerqueira). 1568-1637/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.arr.2010.05.002

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Table 1 NRC micronutrient recommendations for mice and rats versus the micronutrient contents provided by AIN-93M and NIH-31 used in 40% DR. 1 NRC recommendation for rats and mice (National Research Council, 1995). 2 AIN-93M (Reeves, 1997): a lower than recommended for rats; b lower than recommended for mice. 3 NIH-31 (Pugh et al., 1999): a lower than recommended for rats; b lower than recommended for mice. Micronutrients Growth recommendations1 unit/100 g rat Minerals Calcium Chlorine Magnesium Phosphorus Potassium Sodium Copper Iron Manganese Zinc Molybdenium Selenium Iodine Vitamins 3-Hydroxy retinol Cholecalciferol dl- -Tocopheryl acetate Menadione Biotin Choline Folic acid Niacin Pantothenic acid Riboavin Thiamin Pyridoxine Cobalamin g g g g g g mg mg mg mg g g g mg g mg mg mg mg mg mg mg mg mg mg g (unit/100 g mouse) 0.50 0.05 0.05 0.30 0.05 (0.20) 0.05 0.50 (0.60) 3.50 1.00 1.50 (1.00) 15.00 15.00 15.00 0.07 2.00 1.80 (2.20) 0.10 0.02 75.00 (200.00) 0.10 (0.05) 1.50 1.00 (1.60) 0.30 (0.70) 0.40 (0.50) 0.60 (0.80) 5.00 (1.00) AIN-93M2 unit/100 g 0.50 0.16 0.05 0.30 0.60 0.10 0.60 3.50 1.00 3.00 15.00 15.00 20.00 0.40 2.50 5.00 0.08a 0.20 250.00 0.20 3.00 1.50 0.60 0.50 0.60 2.50a unit/60 g 0.30ab 0.09 0.03ab 0.18ab 0.31 0.06 0.36ab 2.10ab 0.60ab 1.80 9.00ab 9.00ab 12.00ab 0.24 1.50ab 3.00 0.06ab 0.12 150.00b 0.12 1.80 0.96ab 0.36b 0.30ab 0.42ab 1.50a NIH-313 unit/100g 1.01 0.42 0.20 0.92 0.59 0.26 1.32 30.02 15.28 5.04 ND ND 19.40 1.06 1.05ab 3.83 0.22 0.01ab 196 0.17 9.22 3.95 0.78 7.73 1.02 5.3 unit/60 g 0.61 0.25 0.12 0.55 0.35 0.16 0.79 18.01 9.17 3.02 ND ND 11.64ab 1.06 1.05ab 3.83 0.22 0.01ab 196 0.17 9.22 3.95 0.78 7.73 1.02 5.3

Abbreviations: American Institute of Nutrition (AIN); caloric restriction (CR); dietary restriction (DR); every other day feeding (EOD); National Institute of Aging (NIA); National Research Council (NRC).

enriched with a vitamin and mineral mix that supports the nutrient requirements suggested by the NRC (National Research Council, 1995), and exceeds those in many, but not all, cases (compare the recommended vitamin and mineral amounts and content per 100 g AIN-93 diet in Table 1). The increased implementation of AIN diets was paralleled by an enhanced interest in CR studies. Unfortunately, the protocols began to diverge with respect to mineral and/or vitamin supplementation. Table 2 lists papers added to the PubMed database between January 1st, 2006 and December 31st, 2009 with the widely adopted term caloric restriction in the title, as well as the dietary intervention used. Surprisingly, 60% of all articles uncovered in our search do not specify the use of micronutrient supplementation. Note that the papers uncovered are a fraction of all publications involving some sort of dietary restriction during this period, since not all publications include this exact term in their title. However, we believe this fraction is representative of a general tendency to adopt diets that do not supplement micronutrients. Indeed, a preliminary search for papers using energy restriction or calorie restriction (a common variation of the term) reveals a similar pattern (not shown). The widespread use of diets without micronutrient supplementation may be due to the high content of vitamins and minerals in AIN formulations, which could lead researchers to believe supplementation was unnecessary. The use of ready-made commercial diets may also have hampered the ease to obtain manipulated enriched diets for CR studies. Furthermore, studies with animals on non-supplemented dietary restricted diets presented some results expected for CR animals (Keenan et al., 1997; Yu et al., 1982). Unfortunately, no direct comparisons were made between dietary restriction (DR, a general limitation of diet, not necessarily associated with enhanced lifespan) and CR, and it is therefore not possible to conclude that all effects of CR are reproduced in DR. Indeed, many effects attributed to CR uncovered in DR models could, in fact, be

the result of decreased levels of micronutrients, even if these are still above recommended daily needs. As protocols changed, the terms (and acronyms) that describe them also diverged. Terms such as malnutrition, protein-energy (calorie) malnutrition, starvation, food restriction, DR, undernutrition without malnutrition (Weindruch and Walford, 1982b), and, more recently, every other day feeding (EOD) are often used interchangeably with CR. It is clear today that there is no consensus regarding the use of micronutrient supplementation in these studies, that protocols vary widely and that the results of these different protocols are not necessarily comparable. Furthermore, the extent of time in which animals are maintained on the diet also varies, as described in Table 2. Although short-term CR has a similar impact on gene expression as long-term CR (Cao et al., 2001), the age at which CR is introduced affects changes in metabolic parameters (Colman et al., 2007), and the extent of time in which the animals are kept on the diet most probably also affects these results. In an example of one of the few well-controlled and detailed protocols available that includes supplementation, the National Institute on Aging formulated a specic diet, the NIH-31 OpenFormula Diet (see Table 1 for micronutrient contents), and a diet implementation protocol (Pugh et al., 1999). The restricted NIH-31 diet contains supplemental vitamins (but not minerals) to provide approximately the same intake as that of the ad libitum mice (Pugh et al., 1999) (see Table 2). 3. Micronutrient contents of diets versus NRC recommendations A nutritionally balanced diet is important both for the welfare of laboratory animals and to ensure that experimental results are not biased by unintended nutritional factors (National Research Council, 1995). The NRC established diet contents based on maxi-

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Table 2 Dietary protocols used in rat and mouse studies. Original articles that were added to the PubMed database between January 1st, 2006 and December 31st, 2009 with caloric restriction in the title and using rodents as models are listed divided by type of protocol used. Feeding strategies 4050% CR with vitamin and mineral supplementation Articles, n 9 References (time of study, wk) (Cleary et al., 2007) (3); (Hagopian et al., 2004) (112); (Hamadeh and Tarnopolsky, 2006) (0.5); (Hepple et al., 2008) (128); (Resuehr et al., 2006) (2); (Westerbeek et al., 2008) (wk 14 until natural death); (Yoshida et al., 2006) (wk 4 until natural death); (Zangarelli et al., 2006) (20); (Bonorden et al., 2009) (2) (Yoshida et al., 2006) (wk 4 until natural death); (Hagopian et al., 2008) (4); (Shinmura et al., 2007) (2) (Kim et al., 2008) (18-96) (Pearson et al., 2008a)a (wk 20 until natural death); (Adams et al., 2008)a (31119); (Chang et al., 2007)a (8-84); (Ferguson et al., 2007)a (5672); (Hepple et al., 2006)a (19143); (Jamieson et al., 2007)a (779); (Kalani et al., 2006)a (8 or 104); (Linford et al., 2007)a (96); (Newton et al., 2008)a (40116); (Selesniemi et al., 2008)a (48); (Shi et al., 2007)a (40116); (Wang et al., 2007)a (72); (Yang et al., 2007)a (3032); (Chen et al., 2007)a (96); (Selman et al., 2006) (2); (Ugochukwu and Figgers, 2006) (9); (Ugochukwu and Figgers, 2007a) (9); (Ugochukwu and Figgers, 2007b) (9); (Yang et al., 2009)a (23); (Csiszar et al., 2009)a (112); (Kalmar et al., 2009)a (28) (Tatsumi et al., 2008) (48) (Boily et al., 2008) (44); (Colom et al., 2007) (12); (Crescenzo et al., 2006) (2); (Donati et al., 2008) (4896); (Duarte et al., 2008) (8); (Halagappa et al., 2007) (28 or 56); (Hannan et al., 2007) (10); (Knight et al., 2006) (2); (Mager et al., 2006) (16); (Mulligan et al., 2008) (116); (Niemann et al., 2008) (24); (Rohrbach et al., 2008) (24); (Summermatter et al., 2008) (2); (Thoms-Moy et al., 2006) (48); (Valle et al., 2007a) (14); (Valle et al., 2007b) (12); (Valle et al., 2008a) (48); (Valle et al., 2008b) (12); (Deng et al., 2009) (4); (Mouton et al., 2009) (16); (Yang et al., 2009) (4694); (Stranahan et al., 2009) (12); (Podkowka-Sieczka et al., 2009) (48) (Niemann et al., 2008) (24); (Al-Regaiey et al., 2007) (76); (Asami et al., 2008) (120); (Barger et al., 2008a) (14); (Barger et al., 2008b) (64); (Denny et al., 2006) (3.6); (Erdos et al., 2007) (2496); (Gmez et al., 2007) (67); (Hambly et al., 2007) (7); (Masternak et al., 2006) (84); (Rohrbach et al., 2006) (12); (Kawamura et al., 2009 Oct) (during pregnancy); (Ribeiro et al., 2009) (12); (Kondo et al., 2009) (4); (Hagopian et al., 2009) (112); (Teske and Kotz, 2009) (4) (Zha et al., 2008) (wk 6 until natural death); (Halagappa et al., 2007) (28 or 56); (Mager et al., 2006) (16); (Bonelli et al., 2008) (wk 12 until natural death); (Chiba et al., 2008) (2226); (Facchetti et al., 2007) (2496); (Fontn-Lozano et al., 2007) (2436); (Higami et al., 2006) (18); (Muscari et al., 2006) (48); (Yamaza et al., 2007) (2230); (Yamamoto et al., 2009) (2); (Ranhotra, 2009) (13) (Bonelli et al., 2008) (wk 12 until natural death); (Facchetti et al., 2007) (2496)

2035% CR with vitamin and mineral supplementation <20% CR with vitamin and mineral supplementation 40% DR with vitamin but not mineral supplementation

3 1 21

3 d/wk feeding with calcium and phosphorus supplementation 4060% DR without supplementation

1 23

835% DR without supplementation

16

Every other day feeding (EOD) without supplementation

12

Food deprivation 1 d/wk without supplementation

a

NIA protocol (Pugh et al., 1999).

mal growth and reproduction during the rst months of life, but the improvement of life span, which involves long-term maintenance of adult and elderly animals, is pursued in CR studies. Therefore, even NRC recommendations may not be adequate for these studies. Furthermore, in non-supplemented DR there is a reduction in the amount of food offered to the animals. In general, the food consumption of control littermates is used to calculate how much of the same diet should be offered to restricted animals. In this sense, the nutrient kg1 ratio is not altered in non-supplemented food restriction, but the nal intake obviously is. To allow for easier comparisons between supplemented and non-supplemented dietary restriction (see Table 1), we calculated micronutrient contents provided by 100 g and 60 g AIN or NIH diets, corresponding to the same reduction in nutrient intake observed in 40% non-supplemented dietary restriction, the most commonly employed restriction range. Clear differences between micronutrient consumption of the ad libitum and dietary restriction protocols can be observed. In addition, quantities of micronutrients vary widely between the AIN and NIH diets. AIN-93 diets certainly do not supply all micronutrients in excess: vitamin B12 and vitamin K

levels are below the NRC recommendations even for ad libitum rats and mice, and many micronutrients, such as vitamin B6, calcium, manganese, iron, molybdenum and selenium, are at the lower limit. When this diet is used in non-supplemented DR protocols, these nutrients are available at levels lower than NRC recommendations. Other micronutrients provided by AIN-93 are in excess, sometimes more than 5 times the recommended levels, such as retinol, choline and biotin. In NIH-31 diets, micronutrients are increased compared to NCR recommendations and, in general, the excess is higher than that observed in the AIN-93 diet, reaching 10, 20 or 30 times the recommended level in many cases. The NIH-31 diet used in CR studies, as well as Altromin 1340/1500 (which presents similar micronutrient levels), is enriched with a vitamin mix, but not minerals, keeping the high vitamin level excess and reducing minerals to lower levels, closer to NRC recommendations. 4. What denes a CR protocol? McCays (1935) concept of CR implied a limitation of calories, not micronutrients. Over the years many protocols ceased to adopt

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micronutrient supplementation (Pugh et al., 1999), although the perception that CR diets should account for this lack is reected by NIA protocols. When we analyzed four years of original articles using the phrase caloric restriction (Table 2), we found that the amount of calories/food restricted was most commonly 40% (but varied from 8 to 60%), mostly without mentioning micronutrient supplements any micronutrient (60% of articles), even in long-term studies (Table 2). Most DR studies do not follow lifespans, so the effect of a non-supplemented restricted diet on animal longevity is still poorly known. Although Keenan et al. (1996) saw a 25% increase in the mean survival age for male rats eating 23% less of a unsupplemented diet relative to ad libitum controls, Duffy et al. (2002) noted that DR of rats on AIN-93 diets did not result in the lifespan extension observed on NIH-31 diets, suggesting that micronutrient supplementation is important for the long-term effects of CR. To add to the confusion regarding the denition of CR, recent studies have adopted other restriction strategies. An increasingly popular strategy is EOD feeding, where animals are fed on alternate days. Thirteen percent of feeding strategies employed in the fouryear period analyzed are EOD (Table 2). Some EOD studies provide ad libitum food on the feeding day (Anson et al., 2003; Pearson et al., 2008a; Caro et al., 2008), while others give a dened amount of food, limiting total consumption to less than that of control animals (Shimokawa et al., 2003; Zha et al., 2008). The diet is not vitamin and mineral enriched in any EOD protocol uncovered in our search. Furthermore, in our animal facilities, EOD feedings resulted in total food ingestion levels equal to those in control animals. This is in agreement with previous studies demonstrating that food ingestion in EOD protocols varies very slightly from that of controls (Anson et al., 2003; Hipkiss, 2007), although at least two groups report a 3040% decrease in food intake in EOD feedings (Caro et al., 2008; Nisoli et al., 2005). Most EOD studies involve short-term interventions, and we found only two which follow full lifespans: in one study (Goodrick et al., 1990), both mean and maximal lifespan were enhanced, while the second paper (Pearson et al., 2008b) shows no increment in lifespan associated with EOD feedings. In all, EOD is an interesting protocol in many aspects, although direct comparisons between the effects of this regimen and traditional CR must be made with caution, since it is not clear that this regimen is associated with prolonged life. More complexity is added by the nding that some authors refer to EOD as intermediate feeding, while other authors use intermediate feeding to describe other feeding strategies such as offering food on specic days (Mondays, Wednesdays and Fridays (Tatsumi et al., 2008)). The effects of such rarer protocols on animal longevity are unknown.

Fig. 1. Rodent growth on supplemented versus non-supplemented diets. (A) Female Swiss outbred albino mice, purchased originally from Taconic Farms, were bred (5 mice/cage) and lodged at the Biotrio de Produco e Experimentaco da Faculdade de Cincias Farmacuticas e Instituto de Qumica, under controlled temperature (22 C), humidity, light (12 h light/dark cycles) and pressure conditions. Starting at 9 weeks of age, the Control group was kept on ad libitum AIN-93M diets (produced by Rhoster Indstria e Comrcio, Aracoiaba da Serra, So Paulo, Brazil), while 40% of ad libitum dietary restriction (DR) was initiated for DR mice, initially without micronutrient supplementation. At 15 weeks, where indicated by the arrow, vitamin and mineral supplementation were added to the restricted diet, in order to reach micronutrient levels equal to controls, but maintaining 40% food restriction. Food quantities were adjusted weekly by weight, based on ad libitum food consumption of the Control group, and animals were weighted weekly. Restricted diets were offered daily at 18:00. Values are mean SEM, n = 10. (B) Male Sprague-Dawley albino rats purchased originally from Taconic Farms were bred (3 rats/cage) and lodged under the same conditions as described for Panel A, on ad libitum AIN-93M diets. Starting at week 12, the Control group was kept on the ad libitum AIN-93M diet, while 40% dietary restriction (DR) without supplementation (calculated based on weekly measurements of Control animal ingestion), 40% dietary restriction with vitamin and mineral supplementation (CR) or non-supplemented ad libitum every other day (EOD) feedings were introduced to the other experimental groups. These dietary interventions were maintained during the following 15 weeks, and the weight gain over this experimental period was recorded. DR and CR food quantities were adjusted weekly by weight, based on ad libitum food consumption of the Control group. Restricted diets were offered daily at 18:00, the same time in which the diet was removed or placed for the EOD group. Results are expressed as mean SEM, n = 6.

In our animal facilities, as well as in others (Anson et al., 2003; Hipkiss, 2007), ad libitum EOD intake did not signicantly decrease total food ingestion (22.8 2.4 g day1 animal1 versus 20.15 0.9 g day1 animal1 for ad libitum versus EOD), although the weight gain of EOD rats was smaller (Fig. 1B), indicating a lower efciency of energy conversion. Together, these results suggest that, although it may present some metabolic changes observed in CR, EOD is not necessarily a form of calorie restriction, and the terms should not be used interchangeably. 6. A proposal for nomenclature and protocols Our data mining indicates clearly that a large diversity of protocols is adopted as caloric restriction in the literature (Table 2). In addition to differing widely, these protocols are often implemented with little knowledge regarding their long-term effects. The consequence of this variability in the literature is that experimental results cannot be compared adequately, and often cannot be directly related to a positive lifespan effect. Other researchers have proposed care in respect to CR nomenclature. Piper et al. (2005) proposed the use of dietary restriction as a term of choice, arguing that it is difcult to quantify useable energy (calories) in a diet. Masoro (2009) suggests the use of food restriction instead, because dietary can refer to anything, including the restriction of a single nutrient. We would like to propose that the terms calorie restriction and caloric restriction retain their original meaning of energy, but

5. Potential impact of DR on animal development It rst became clear to us that non-supplemented DR protocols could be nutritionally decient when we submitted adult Swiss mice on AIN-93M diets to 40% food restriction (Fig. 1A) and observed pronounced (20%) weight loss. A signicant weight gain (p < 0.001) was observed in these animals after the supplementation of micronutrients. Weight loss was less than 10% during the same period with the same level of restriction of the diet supplemented with vitamins and minerals, to equal micronutrient ingestion in controls (CR, results not shown). Furthermore, we found that Sprague-Dawley rats on long-term dietary restriction presented signicantly larger weight gains over time when they received micronutrient supplementation (p = 0.04, Fig. 1B). Our results demonstrate that non-supplemented DR in young rodents does not only limit calories, but also involves micronutrient malnutrition.

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not micronutrient limitation, and be used to refer to diets in which total calories are limited, with vitamin and mineral supplementation. Fully supplemented diets should be the standard intervention studied in longevity research due to their well-established lifespan effects in rodents. Diets such as non-supplemented chow restriction, EOD and other variants should perhaps more precisely be referred to as dietary restriction or food restriction, and their lifespan effects should be closely evaluated. Most importantly, all work in the area should include detailed methodology describing the animal model, genotype, type of diet adopted, restriction levels and how these were calculated, the age in which the diet was introduced, length of time in which the animals were maintained on the diet, supplements included, housing conditions and methods of feeding. With such standards and information, research in the area will certainly gain more signicant insight into the mechanistic effects of diets on longevity. Contributors FMC designed, conducted and analyzed the data presented. FMC and AJK reviewed the literature and prepared the manuscript. Conict of interest None. Acknowledgements The authors are in debt to Camille C. Caldeira-da-Silva for expert technical assistance and Silvania M.P. Neves, Renata S. Fontes, Flavia M.P. Ong and Maria de Ftima Rodrigues for excellent animal care. This work is supported by grants from the Fundaco de Amparo Pesquisa do Estado de So Paulo (FAPESP), Instituto Nacional de Cincia e Tecnologia de Processos Redox em Biomedicina (Redoxoma), Conselho Nacional de Desenvolvimento Cientco e Tecnolgico (CNPq) and the John Simon Guggenheim Memorial Foundation. References
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