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1Aflatoxins
Aflatoxins are mycotoxins that contaminate feed and
foods, considered by the Food and Drug Administration (FDA) as being inevitable contaminants (Kensler et al., 2004). There are more than 20 aflatoxins derivatives produced by different species of fungi. For example, Aspergillus flavus synthesize AFB1 and AFB2
, while
A. parasiticus could synthesize AFB1, AFB2, AFG1, and
AFG2 (D’Mello & McDonald, 1997; Bennett & Klich, 2003) (Figure 1).
Aflatoxin B1 is a very potent carcinogen in many
species, including humans, birds, swine, fish, and rodents (Bennett & Klich, 2003; Kensler et al., 2004). AFB1 induces chromosomal aberrations, micronuclei, sister chromatid exchange, chromosomal strand breaks, and forms adducts in fish, birds and mammalian cells (IARC, 1993). On the basis of numerous evidence concerning its toxicity, AFB1 was classified in the group 1 of toxicity as a human carcinogen (IARC, 1993).
In each species, the liver is the primary target organ
of acute injury, and the aflatoxins have been extensively studied in relation to liver cancer (Wang & Groopman, 1999). However, other adverse effects, including alterations of productive parameters, immune impair –
ment, or toxic effects in other organs than liver have been widely reported (Marin et al., 2002; Meissonnier et al., 2008).
The AFB1 metabolism plays a major role in determin-
ing the toxicity of the toxin. The liver biotransformation of AFB1 is related to its toxic and carcinogenic effects (Bailey et al., 1996). After an initial oxidation, AFB1 is transformed into an AFB1-exo-8, 9-epoxide (AFBO), which is later detoxified through a variety of metabolic processes (Wild et al., 2002). Epoxidation of AFB1 to the exo-8, 9-epoxide is a critical step in the genotoxic pathway of this carcinogen. The exo-epoxide is highly unstable, and binds with DNA to form the predomi-nant trans-8,9- dihydro-8-(N7-guanyl)-9-hydroxy-AFB1 (AFB1-N7-Gua) adduct, a promutagenic DNA lesion (Raney et al., 1993; Wild et al., 2002) – (Figure 2). In addi-tion to formation of the 8, 9-oxide, stable, less toxic, uri-nary metabolites (AFM1, AFQ1 and AFP1) result from the AFB1 metabolism (Wild et al., 2002). This review present an overview on aflatoxins and oxidative stress based on the literature found in PubMed and Science Direct using the following key terms: aflatoxin, oxidative stress, lipid Review
Overview on aflatoxins and oxidative stress
Daniela E. Marin and Ionelia Taranu
Laboratory of Animal Biology, National Institute for Research and Development for Biology and Animal
Nutrition, Calea Bucuresti no. 1, Balotesti, Ilfov, 077015, Romania
Abstract
Aflatoxins are naturally occurring mycotoxins that are produced by many species of Aspergillus, a fungus, mainly by Aspergillus flavus and Aspergillus parasiticus. Aflatoxins, especially aflatoxin B1 (AFB1)
, are very potent carcinogens
in many species, including humans, birds, swine, fish, and rodents. The oxidative stress caused by AFB1 may be one of the underlining mechanisms for AFB1-induced cell injury and DNA, protein and lipid damages, which lead to tumorigenesis. This review presents an overview on aflatoxins and oxidative stress, with an emphasis on the protective role of the antioxidants.
Keywords: aflatoxins, oxidative stress, antioxidants, vitamins, adducts
Address for Correspondence: Laboratory of Animal Biology, National Institute for Research and Development for Biology and Animal
Nutrition, Calea Bucuresti no. 1, Balotesti, Ilfov, 077015, Romania. Tel: 004 021 351 20 82. Fax: 004 021 351 20 80.
E-mail: daniela.marin@ibna.ro
(Received 31 July 2012; revised 20 August 2012; accepted 20 August 2012)Toxin Reviews, 2012; Early Online: 1–12
© 2012 Informa Healthcare USA, Inc.ISSN 1556-9543 print/ISSN 1556-9551 onlineDOI: 10.3109/15569543.2012.730092
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10.3109/15569543.2012.7300922012Aflatoxins and oxidative stress
D. E. Marin and I. Taranu
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2 D. E. Marin and I. Taranu
Toxin Reviewsperoxydation, protein carbonyl, DNA oxidation, antioxy-
dants, polyphenols.
Free radicals and oxidative stress
Reactive oxygen species (ROS) are the result of the nor –
mal cellular metabolism, but could result also from the ingestion/inhalation of the drugs, or environment pol-lutants (Halliwell & Cross, 1994; Ray et al., 2012). ROS induce alterations of the cellular components, leading to changes in cell functions and viability; these changes include DNA lesions, protein cross-links, and lipid oxida-tion (Sies, 1991; Halliwell & Cross, 1994).The well-known ROS are anion superoxide (O2
•−),
hydrogen peroxide (H2O2), peroxyl radical (OH•), and
the reactive nitrogen species are nitric oxide (NO) and peroxynitrite (ONOO
−). The main sites of ROS produced
in living organisms are mitochondrial electron transport system, peroxisomal fatty acid, cytochrome P-450, and phagocytic cells (Bayir, 2005; Ray et al., 2012).
Cells are naturally provided with protective enzy-
matic and non-enzymatic antioxidants that counteract these potentially injurious oxidizing agents (Sies 1991; McCord, 1993; Halliwell & Cross, 1994).
Thus, animal organisms could synthesize antioxidant
enzymes that efficiently disprove the ROS. Superoxide dismutase, catalase and glutathione peroxidase are intracellular antioxidant enzymes that convert potential substrates (superoxide anion radicals and hydrogen per –
oxide) to less reactive forms in the body (Remacle, 1992; Finaud et al., 2006; Afonso et al., 2007).
Several extracellular antioxidants such as proteins
(transferrin, lactoferrin, albumin, ceruloplasmin) and urate prevent free radical reactions in the body by seques –
tering transition metal ions by chelation. Albumin, bili-rubin and urate may also scavenge free radicals directly (Slater, 1984; Gutteridge, 1995).
Even this multifunctional protective system cannot
completely counteract the deleterious effects of ROS, however, oxidatively damaged molecules can accumulate in cell generating the oxidative stress. Oxidative stress has been defined as a disturbance in the balance between anti-oxidants and prooxidants (free radicals and other reactive species), with increased levels of pro-oxidants leading to potential damage (Slater, 1984; Betteridge, 2000).
Figure 2. Aflatoxin B1 biotransformation pathways [adapted with permission from Bammler et al. (1994)].
Figure 1. Chemical structure of aflatoxins.
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Aflatoxins and oxidative stress 3
© 2012 Informa Healthcare USA, Inc. This imbalance can be due to the decrease of endog-
enous antioxidants, low intake of dietary antioxidants,
and/or increased formation of free radicals and other reactive species.
The clinical implications of these alterations can
be severe; in fact, the accumulation of ROS in several cellular components is thought to be a major cause of molecular injury leading to cell aging and to age-related degenerative diseases such as cancer, immune system decline, brain dysfunction, cataracts, and coronary heart disease (Halliwell, 1999).
effects of aflatoxin on the oxidative stress
Recent studies have shown that AFB1 enhances ROS formation and causes oxidative damage. The oxidative stress caused by AFB1 may be one of the underlining mechanisms for AFB1-induced cell injury and DNA damage, which eventually lead to tumorigenesis (Shen
et al., 1994).
It has been noted that there is free radical generation
during AFB1 metabolism (Kodama et al., 1990), and oxidative damage is one type of damage caused by AFB1 in human lymphocytes (Amstad et al., 1984). Luminol-dependent CL activity, which reflects the production of reactive oxygen species from human polymorpho-nuclear leukocytes, was up-regulated to approximately 150% when PMNs were treated with 0.05 ng/mL of AFB1
upon stimulation with N-formyl-methionine-leucine-phenylalanine or zymosan (Ubagai et al., 2008).
AFB1 can cause an increase in ROS formation in ani-
mals’ target organs, including rat liver, duck liver, and mouse lung (Shen et al., 1995a; Barraud et al., 2001; Guindon et al., 2007). AFB1 induced an important liver cell injury, as shown by the significant increase in serum transaminases, phosphatases (acid and alka-line), dehydrogenases (sorbitol, lactate and glutamate), cholesterol, triglycerides, total lipids, bilirubin, creati-nine, uric acid and nitric oxide, but also a strong lipid peroxidation in liver and kidney, accompanied with a significant decrease in total antioxidant capacity in rats (El-Nekeety et al., 2011; Hathout et al., 2011; Rastogi et al., 2011), mice (Adedara et al., 2010; Kanbur et al., 2011), and chicken (Sirajudeen, 2011). Administration of AFB1 to rats (2 mg/kg intraperitoneally) caused also
significant increase in the activities of gamma-glutamyl transpeptidase (GGT), 5’-nucleotidase, acid phos –
phatase, acid ribonuclease, as well as content of lipid peroxides in liver after six weeks (Rastogi et al., 2001a). Also, a strong iNOS and nitrotyrosine immunoreactiv –
ity was observed in the livers of chicks given 300 ppb of aflatoxin (Karaman et al., 2010).
Recently, it is accepted that oxidative stress is an apopto-
sis inducer (Chandra et al., 2000). Many agents that induce apoptosis are either oxidants or stimulators of cellular oxi-dative metabolism. It was shown that AFB1 increased the expression of pro apoptotic proteins p53 and bax (Duan et al., 2005; Brahmi et al., 2011) and bcl2 (Duan et al., 2005). Also, the levels of caspase-3 activities, a cysteine protease with a major role in the apoptotic pathway and of the heat shock protein-70 (HSP70 level) in AFB1 intoxicated rats were significantly higher than in control group, being associated with high levels of LPO and NO, as indicators of the oxidative stress (Meki et al., 2004). Similarly, in Ross chicks given 150–300 ppb AFB1 for 21 days, many apop-totic cells were detected in the livers, together with a high level of malondialdehyde (MDA) in the liver and kidney of intoxicated animals (Ozen et al., 2009).
Conversely, many inhibitors of apoptosis have antiox –
idant activities or enhance cellular antioxidant defences (Freeman & Grapo, 1982).
AFB1 was linked with increase in ROS, which surpass
the capacity of antioxidant mechanisms of defence, leav –
ing cells vulnerable to nucleic acids, proteins or lipid oxidation (Clayson et al., 1994; Shen et al., 1995a; Bedard & Massey, 2006).
Aflatoxins have been implicated as risk factors in the
pathogenesis of liver cancer, and the oxidative stress is considered to be a key player in the development and the progression of liver cirrhosis which is known to be a pre-cursor of human hepatocellular carcinoma (HCC) (Choi et al., 2001; Liu et al., 2008).). The activation of the phos –
phoinositide 3-kinase /Akt pathway could represent one of the mechanisms by which ROS modulate cell survival during carcinogenesis (Halliwell, 2007).
Aflatoxins and oxidative lipid damage
Polyunsaturated lipids are essential for the cells, being important constituents of cell membranes, endoplasmic reticulum, and mitochondria. Thus, the disruption of their structural properties could have consequences for cellular function. Lipid peroxidation is the most exten-sively investigated free radical induced process, and is one of the main factors responsible for the structural and functional alterations of the cell membrane following oxidative stress (Halliwell & Chirico, 1993), and initiation of carcinogenesis (Banakar et al., 2004).
It is not very clear if the mycotoxins stimulate the
lipid peroxidation directly through the increase of the ROS synthesis, or the increase of the tissue susceptibil-ity to the peroxydation is the result of the compromised antioxidant defence, but it seems that both processes are involved. Initiation of lipid peroxidation is caused by attack of any species that has sufficient reactivity to abstract a hydrogen atom from a methylene group upon a PUFA (Gutteridge, 1995; Moore & Roberts, 1998; De Zwart et al., 1999; Halliwell, 1999). The peroxidation of PUFAs can be realized not only through non-enzymatic free radical-induced pathways, but also through pro-cesses that are catalyzed by enzymes as cyclooxygenase and lipoxygenase (Halliwell & Chirico, 1993; Gutteridge, 1995). It was shown that also 8, 9-epoxide increases lipid peroxidation, followed by loss of membrane stability and the blockage of the membrane bound enzyme activity (Toskulkao & Glinsukon, 1988).
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4 D. E. Marin and I. Taranu
Toxin ReviewsTwo of the most commonly assays used for the evalu-
ation of the lipid damage are based on the measurement
of thiobarbituric acid reactive substances (TBARSs) or MDA by the thiobarbituric acid (TBA) test and conju-gated dienes.
AFB1 induced an increase of the TBARS concentration
in liver (Naaz et al., 2007) or in human hepatoma cells (Lee et al., 2005). The increase of the lipid peroxides (LPO) syn-thesis was observed not only in liver (Madhusudhanan et al., 2004; Rastogi et al., 2006, 2007), but also in kidney and brain (Madhusudhanan et al., 2004), cutaneous tissue (Rastogi et al., 2006, 2007). This alteration was associated with a significant increase in conjugated diene formation (Madhusudhanan et al., 2004).
Exposure to AFB1 for up to 72 h resulted in signifi-
cantly elevated levels of lactate dehydrogenase (LDH) being released into the medium, as well as the MDA generation in cultured hepatocytes (Shen et al., 1995a). The same group of authors (Shen et al., 1994) showed that AFB1 induced an increase of the synthesis of MDA and conjugated dienes in liver homogenate after 1 day of administration. It reached the peak level 3 days after dosing, and remained at an elevated level up to 14 days. Measurements of lipid peroxidation in the subcellu-lar fractions revealed that microsomes had the highest concentration of MDA, followed by those of the nuclear fraction and mitochondria. MDA concentration was not detectable in the cytosolic fraction. Concentrations of MDA + 4-hydroxyalkenals (4-HDA) -, as an index of LPO, were increased by AFB1 in the liver, lung, brain and tes –
tis, but not the kidney of male Wistar rats (Gesing et al., 2008). 4-hydroxynonenal (4-HNE), a major electrophilic by-product of lipid peroxidation caused by oxidative stress, interacts with DNA to form exocyclic guanine products, which have been shown to be increased in a rat model of hepatocarcinogenesis (Marquez et al., 2007). AFB1 induces lipid peroxidation in rat liver, and this may be an underlying mechanism of carcinogenesis (Shen et al., 1996). Thus, AFB1 administered HCC-bearing rats showed increased levels of LPOs, TBARs, and decreased levels of enzymic and nonenzymic antioxidants when compared to control animals (Ravinayagam et al., 2012). The effect of lipid peroxidation is amplified in the condi-tion of iron overload, as shown by massive deposits of 4-HNE observed in liver sections, as well as by the additive effect between LPO and ALT in the serum of male Wistar rats treated with both Fe and AFB1 (Asare et al., 2007).
Aflatoxins and oxidative proteins damage
ROS can lead also to oxidation of amino acid residue side chains, formation of protein-protein cross-linkages and oxidation of the protein backbone resulting in pro-tein fragmentation, and the modified forms of proteins will accumulate in organism (Berlett & Stadtman, 1997). By its capacity to generate ROS, AFB1 can promote the ROS-mediated oxidative damages in proteins (Amici
et al., 2007; Peng et al., 2007a, 2007b; Ubagai et al., 2008). At the same time, AFB1 could inhibit some (serine) pro-teolytic enzymes (Clausen et al., 2002) responsible for the degradation of damaged proteins, with consequent relevant implications in hepatocarcinogenesis (Peng et al., 2007a, 2007b). It was suggested that many actions of the aflatoxins may be mediated by their interactions with the proteasome, the main structure responsible for the degradation of most of the cytosolic and nuclear proteins in eukaryotic cells. Indeed, AFB1 mediates an inhibition of cellular 20S proteasomes, affecting the cellular defence against oxidative stress (Amici et al., 2007). Because the 20S proteasome is the proteolytic machinery responsible for removing oxidized proteins (Saeki & Tanaka, 2012), its inhibition could contribute to a higher protein carbon-yls (PCC) content observed in cultured hepatoma cells lysates (Amici et al., 2007).
AFB1 was able also to induce a significant increase
in PCC in liver, kidney and brain of different species (Madhusudhanan et al., 2004; Peng et al., 2007a; Liu et al., 2008). In human populations with a high exposure to AFB1 and with a high incidence of hepatocellular car –
cinoma (HCC), PCC have long been used for assessing aflatoxin exposure and oxidative stress to proteins (Peng et al., 2007b). In these populations, the plasma PCC level was associated with the amino acid adducts level, and with the levels of alanine aminotransferase and aspartate aminotransferase in hepatitis B virus-infected subjects, suggesting the roles of aflatoxin exposure, oxidative stress and hepatitis B virus infection in the development of HCC (Peng et al., 2007b).
Aflatoxins and oxidative DNA damage
Oxidative DNA damage is a general definition for all types of changes (structural or functional) of DNA, due to the interaction of ROS with DNA.
The addition of hydroxyl radical to the C8-position of
DNA guanine produces C8–OH adduct radical (Steenken, 1989), which is subsequently converted to 8-OH-guanine (8-OH-Gua) by one-electron oxidation (Kasai, 1992).
While damaged lipids and proteins can be removed
by metabolic turnover of these molecules, impaired DNA has to be repaired in situ, or destroyed by apoptotic processes, conversely, mutations result in the absence of these processes (Luo et al., 2006).
In humans, the 8-OH-Gua glycosylase is the pri-
mary enzyme for the repair of 8-OH-Gua in short-patch base-excision repair (Evans, 2004). The excised form of 8-OH-Gua is a promutagenic adduct: 8-hydroxydeoxy-guanosine (8-OHdG), which is excreted into urine with-out further metabolism and is stable for a significant time (Moriwaki, 2000; Pilger, 2001).
8-OHdG is widely considered as a key biomarker of
oxidative DNA damage (Shen et al., 1995a; Valavanidis
et al., 2009). 8-OHdG formation were observed after AFB1 administration in liver or in HepG2 cells (Shen et al., 1994; Liu et al., 1999; Lee et al., 2005), lung (Guindon et al., 2007, 2008), with time- and dose dependent responses of
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Aflatoxins and oxidative stress 5
© 2012 Informa Healthcare USA, Inc. 8-OHdG. The urinary 8-OhdG was correlated with both
the level of DNA 8-OHdG and the expression of human OGG1 gene (hOGG1) in peripheral leukocytes in AFB1 intoxicated patients with a high risk for HCC (Peng et al., 2007).
The widely accepted mechanism of AFB1-induced
carcinogenesis involves the metabolisation into AFBO. This compound interacts with DNA, resulting in forma-tion of AFB1-N7-Gua, mutations of K-ras gene (in the mouse lung), and tumorigenesis (Massey et al., 2000; Guindon et al., 2008). K-ras activation is an early, critical event in AFB1 responsible for pulmonary carcinogenesis in AC3F1 mice (Donnelly et al., 1996), and it is also pos –
sible that AFB1-induced formation of ROS, leading to 8-OHdG formation, can result in the same K-ras muta-tion pattern (i.e., predominantly G to T transversion mutations) similar to that due to AFBO, thus contributing to tumorigenesis.
It was shown that AFB1-induced 8-OHdG was pre-
vented by prior treatment with polyethylene glycol-con-jugated catalase (PEG-CAT) in isolated mouse lung cells following in vivo treatment with the toxin (Guindon et al., 2007), but PEG-CAT was not protective against AFB1 carcinogenicity in mouse lung despite preventing DNA oxidation (Guindon et al., 2008). Luo et al. (2006) have shown that chemoprevention with green tea poliphe-nols is effective in diminishing oxidative DNA damage, through the reduction of the urinary 8-OHdG levels.
Aflatoxins and the antioxidant defence
The marked increase in the lipid peroxide levels and a concomitant alteration of the enzymic (superoxide dis –
mutase, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione-S-transferase) and nonenzymic (reduced glutathione, vitamin C and vitamin E) antioxidants in the hepatic tissue were observed after aflatoxin treatment. Depending on the experimental conditions (species, dose, time and way of administration, other antioxidant concentration etc.), the activities of other enzymes with antioxidant properties could increase as a response to the oxidative stress or could decrease through the direct or indirect action of the mycotoxins.
A part of the oxidative metabolism products of AFB1
constitute a substrate for the phase II detoxification enzymes. In the majority of animal species, the primary way to detoxify the AFB1 is through the conjugation of AFBO with the reduced glutathione (GSH) (Eaton & Gallagher, 1994). This way of detoxification is the principal way of AFB1 excretion in many animal species. The reac –
tion is catalyzed by the glutathione S transpherase (GST) (Eaton & Gallagher, 1994). It was observed that in mice, the reduced sensibility to aflatoxins is correlated with the constitutive increase of the GST iso enzyme (Raney
et al., 1992). The resistance of adult mice to AFB1 has been suggested to be due to constitutive of the A3 subunit of GST (mGSTA3; also known as Yc or Ya3) expression in liver, which exhibits high catalytic activity toward AFB1 (Buetler & Eaton, 1992; Hayes et al., 1992; McDonagh et al., 1999). The role of mGSTA3 in the mice protection from AFB1 toxicity was studied in mGSTA3 knockout mice following a single AFB1 administration (Ilic et al., 2010). After AFB1 administration the KO mice showed more than a 100-fold increase in AFB1-N7-DNA adduct levels in their livers, relative to the levels in wild-type control mice, demonstrating that the high expression of mGSTA3 subunit in mouse liver confers intrinsic resistance to AFB1 hepatocarcinogenesis (Hayes et al., 1992).
It was also shown that mGSTA3 may have a function
in an antioxidant defence mechanism (Hayes et al., 1992), and that the expression of the mGSTA3 subunit is regulated by the Nrf2 transcription factor through an antioxidant response element (Jowsey et al., 2003). Nrf2 transcription factor binds to this promoter and, at least in part, controls its expression.
Due to mGSTA3, mice, which are resistant to the car –
cinogenic properties of aflatoxin, present a three to five times higher activity of GST comparing with susceptible species as the rat. The studies shown that the detoxifica-tion mechanism is relatively weak in humans comparing with rats, mice or rabbits (Eaton & Gallagher, 1994).
The glutathione concentration could increase as a
result of the AFB1 treatment. Thus, the GSH concentra-tion in kidney was increased after 10 days of treatment with aflatoxin (Beers et al., 1992a). Similarly, the liver concentration of GSH was increased after 2 and 8 hours following a single dose of AFB1, and continue to increase after 5 daily doses of AFB1 (Beers et al., 1992b). Also, AFB1 intoxication led to a significant increase in SOD activ –
ity that was observed in Peking ducks treated with AFB
1
(Barraud et al., 2001). Taking into consideration the fact that the main way of AFB1 detoxification is through the AFBO conjugation GSH, these results could be explained as an attempt of the organism to defend itself against the AFB1 toxicity.
However, in many cases, the capacity of the organ-
ism to defend is surpassed, and some studies showed a decreased concentration of GSH. Indeed, intraperito-neal administration of AFB1 in rats (2mg/kg) resulted in decrease of the level of glutathione (GSH) in liver (Nili-Ahmadabadi et al., 2011), or in the culture of hepa-tocytes (Liu et al., 1999). Also, AFB1 intoxication resulted in significant decrease of activity of enzymes involved in the antioxidant defence: succinate dehydrogenase, glu-cose-6-phosphatase, glutathione peroxidase (GSPx) and glutathione reductase (GR), catalase (CAT) superoxide dismutase (SOD) and GST in mice (Adedara et al., 2010) and rats (Rastogi et al., 2001b; El-Agamy et al., 2010). The decrease of the GSH was accompanied by an increase of the MDA and NO concentrations in the liver and kidney of AF-treated chicks (Karaman et al., 2010) or rats (Meki et al., 2001). It was shown that in rats AFB
1 can lead to
direct or indirect caspase-3 activation, and consequently to apoptosis in rat liver, and caspase-3 activity was posi-tively correlated with MDA while negatively correlated
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6 D. E. Marin and I. Taranu
Toxin Reviewswith GSH, GSPx and GR in rat livers treated with AFB1
(Meki et al., 2001).
Aflatoxins and dietary antioxidants
Antioxidants can stabilize the ROS, maintaining in this way the structural and functional integrity of the cells, and also increasing the capacity of the immune system to respond to the antigens (Bendich, 1990). Among the antioxidants, selenium, carotenoids, vitamins A, C and E are the most important. These antioxidants could be administered as diet supplements, because it was shown that their dietary deficiency could lead to alterations of different organs functions, tumor progression, infec –
tions, and inflammation and alteration of the general health status (Dror & Allen, 2011; Tanumihardjo, 2011; Traber & Stevens, 2011).
The balance between the antioxidants and the pro-
oxidants is responsible for the regulation of the different metabolic pathways that allow the maintaining of immu-nocompetence, and the protection in stress conditions (Surai & Dvorska, 2001). Nutritional stress factors have a negative impact on this balance antioxidant/pro-oxidant. Considering this aspect, aflatoxins could be considered to belong to the most important dietary stress factors.
Effect of AFB1 on the antioxidant concentration
In many situations, the lipid peroxidation caused by AFB1 was associated with the decrease of the antioxidant concentration. AFB1 interferes with the accumulation of carotenoids in chicken tissues (Schaeffer et al., 1988). In fact, AFB1 determine a significant depression of lutheins in liver, serum and mucosa (Schaeffer et al., 1988). In young birds, AFB1 reduced the content of the lutheine in the jejunum mucosa at 35% and of the serum lutheine at 70% (Tyczkowski & Hamilton, 1987a), suggesting that AFB1 interfere with the absorption, transport and carot –
enoids storage.
The dietary carotenoids inhibited the negative effects
exerted by AFB1 at the liver level (Gradelet et al., 1997). The authors claim that beta-apo-8’carotenal, cantaxan-tine and astaxantine exert their protective effects through the deviation of the AFB1 metabolism toward the detoxi-fication ways, leading to the aflatoxin M1. Certain apo-carotenoids, which are precursors of vitamin A, were found to be very efficient in inhibiting the adduct for –
mation (Gowami et al., 1989). Also, dietary extracts rich in carotenoids were responsible for an inhibition of the biochemical and cellular processes considered to forego the hepatocarcinogenesys (He et al., 1997).
The barrows intoxicated with AFB1 have serum simi-
lar levels of tocopherol and retinol, and have a decrease of the concentration of tocopherol in the heart tissue (Harvey et al., 1994). Also, other animal studies indicate that exposure to aflatoxin may reduce plasma and tissue vitamin A in rabbits (Abdelhamid et al., 1990), chickens (Reddy et al., 1989; Pimpukee et al., 2004), camels (Abbas & Ali, 2001), and humans (Obuseh et al., 2010, 2011).Effects on vitamin absorption
Aflatoxicosis is associated with the lipid malabsorption syndrome (Hamilton, 1977). It was suggested that the decrease of the concentration of the antioxidants could be due to a malabsorption mechanism that require later investigations. One possible explanation for the malab-sorption is that the aflatoxins stimulate the lipid peroxi-dation in enterocytes, leading to alterations that favor this alteration. No data are available concerning this aspect, but it was shown that AFB1 could affect the permeability of the epithelial cell monolayer in human epithelial cell line Caco2 (Mata et al., 2004), or cell viability and IL-8 synthesis in porcine epithelial cell line IPEC-1 (del Río García et al., 2007). All these alterations at the intestinal level could result in antioxidants malabsorbtion. Indeed, Tyczkowski and Hamilton (1987b) have shown that AFB
1 affect the absorption of the lutheine in chickens.
Generally speaking, the syndrome of malabsorption is considered a result of the mycotoxicosis. For example, low concentrations of aflatoxins administered to broilers from hatching to the age of 3 weeks induced a syndrome of malabsorption characterized by steatoreea, hipoca-rotenoidemia and decrease of the gallbladder salts and pancreatic enzymes (Osborne et al., 1982).
Effect of AFB1 in cases of hipovitaminoses
It seems that the vitamin A status of animals can influence AFB1 genotoxic activity. Indeed, Decoudu et al. (1992) showed that the activities of metabolizing enzymes which specifically activate or deactivate AFB1 were found to be significantly decreased in vitamin A-deficient animals and weakly modified in vitamin A-supplemented ani-mals. Also, Webster et al. (1996) have shown that the vita-min A status is correlated with the hepatocarcinogenicity of AFB
1 in rats. The authors have observed an increase of
the negative effects of AFB1 correlated with the vitamin
A deficiency, effects that were annulled by the vitamin A dietary supplement. Two compounds of the vitamin A (3-dehydroretinol and 3 dehydroretinil palmitate) present in the fish meat exerted an efficient action in the inhibition of the formation of high adduct concentration (AFB-AD) (Aboobaker et al., 1997). This inhibition could be the result of the modulation of the expression of the microsomal enzymes, suggesting a protective effect of these compounds to the AFB
1 induced carcinogenesis.
The vitamin A status was found to play a role in regulating glutathione S-transferase activity of the liver cytosol frac –
tion, the activity being low in deficiency, but increased progressively with increasing supplementation of vita-min A (Bhattacharya et al., 1989).
In humans, the serum vitamin A and E deficiency was
associated with high adduct concentration (AFB-AD) (Tang et al., 2009), and dietary vitamin supplements reduced AFB1-induced oxidative stress (Alpsoy et al., 2009, 2011). However, the association of AFB1 con-tamination and the HCC risk with plasma levels of various antioxidant vitamins remains to be elucidated because other studies have shown that vitamin E and
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Aflatoxins and oxidative stress 7
© 2012 Informa Healthcare USA, Inc. beta-carotene were observed to enhance AFB1–DNA
adduct formation in cultured woodchuck hepatocytes or in the urine of humans from a Taiwan population with a high risk of HCC (Yu et al., 1994, 1997).
Effects of dietary antioxidant supplementation in
aflatoxicosis
There are many studies concerning the protective effects
of some antioxidants in alleviating the toxic effect of afla-toxins, when they are administered before or at the same time as the carcinogen (Figure 3). Alpsoy and Yalvac (2011) analyzed the roles of antioxidants in AFB1 intoxi-cations with a focus on the vitamins A, C, and E. Some plant extracts with antioxidant properties were also tested with the aim to reduce the oxidative stress induced by the aflatoxins. A short overview of the principal find-ings concerning the role of antioxidants in AFB1-induced oxidative stress will be presented in this chapter.
Among the vitamins, vitamin E rapidly reacts with
the peroxide radicals in order to form tocopheroxyl, a form of stable radicals, capable to generate α-tocopherol
through the reaction with the ascorbate (Atroshi et al., 2002). A study realized in AFB1 intoxicated rats, Cassand et al. (1991), showed that dietary vitamin E protects directly the membrane against damage induced by lipid peroxidation, and indirectly the hepatic microsomal monooxygenase activities. Dietary vitamin E decreases the genotoxic effects of AFB1 through the alteration of the activities of hepatic microsomal cytochrome P-450 activities, Cassand et al. (1993).
Also, in mice testis, vitamin E pretreatment sig-
nificantly ameliorates the aflatoxin-induced lipid per –
oxidation, which could be due to higher enzymatic and non-enzymatic antioxidants in the testis as compared with those given aflatoxin alone (Verma & Nair, 2001). The capacity of α-tocopherol to reduce the concentration of ROS (Ibeh & Saxena, 1998) following AFB1 exposure was associated with a reduction in the AFB1 metabo-lism (Ibeh & Saxena, 1998). In rats, vitamin E, but also other antioxidants (ascorbic acid, selenium, etc.) could have a protective effect in the AFB1 liver induced cancer (Nyandieka et al., 1990). On the other hand, vitamin E increases the activity of biomarkers associated with the oxidative stress (Alpsoy & Yalvac, 2011) and was not able to reduce AFB-AD formation (Yu et al., 1994). Also, Harvey et al. (1994) showed that vitamin E has no benefi-cial effects on the toxicity associated with the AF intoxi-cation, but AF was able to reduce the concentrations of serum retinol and tocopherol.
In some cases, supplementation of vitamins A and E
ameliorated aflatoxin-induced changes and inhibited aflatoxin-induced carcinogenesis through anti-muta-genic effect (Nyandieka & Wakhisi, 1993; Gradelet et al., 1997; Verma & Nair, 2001).
Lycopene and beta-carotene are effective in inhibiting
the in vitro toxicity induced by AFB1 on human hepato-cytes by decreasing apoptosis and the level of AFB-AD (Alpsoy & Yalvac, 2011). These carotenoids also inhibited AFB1-induced mutations in p53 tumor suppressor gene and inhibited the metabolism of AFB1 (Reddy et al., 2006).
In cultured human lymphocytes, vitamins A, C, and E
could effectively inhibit AFB1-induced sister chromatid exchange (Alpsoy et al., 2009a) and exhibited protective effects by inhibiting AFB(1)-induced ROS generation (Alpsoy et al., 2009a). In woodchuck hepatocytes, vita-min C decreases the AFB1-related lipid peroxidation and inhibits the AFB-AD formation (Yu et al., 1994). It was suggested that vitamin C protects the animals from acute toxicity of AFB1 by activating AFB1-epoxide hydroxylase, aldehyde reductase, and CYP3A enzymes located in the enterocytes (Netke et al., 1997).
Figure 3. Effect of aflatoxins on the oxidative stress; the alleviating role of antioxidants.
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8 D. E. Marin and I. Taranu
Toxin ReviewsIn a study realized in rats, Shi et al. (1994) have shown
that selenium inhibit the liaison between AFB1-ADN
and the adduct formation. The same authors (1995) have shown in a study on hamster cells that sodium selenite and the yeast extract enriched in selenium protect the cells from the AFB1 cytotoxicity, but not from the muta-genic effect. Selenium protects the hepatic cells from the hepatotoxic effects of the AFB1 in intoxicated swine, (Davila et al., 1993). On the other side, McLeod et al. (1997) showed that rats fed a selenium deficient diet were more resistant to AFB1 as comparing with the animals fed a diet enriched in selenium. It was suggested that the protection conferred by the selenium deficiency in AFB1 intoxicated animals was associated with the liver expres –
sion of an aldo-Keto reductase and of a GST subunit that efficiently metabolize the mycotoxin.
effects of plant extracts on the oxidative
stress induced by AFB1
It has been reported that the diets rich in vegetables and fruits provide, beside the contents in vitamins and pro-vitamins, a great amount of other antioxidant phy-tochemicals, such as polyphenolic acids, terpenoids, flavonoids and vegetable pigments, which offer protec –
tion against cellular damage due to their ability to bind oxygen-derived free radicals by donating electron, che-late to redox-active metals and inhibit lipooxygenases (Łuczaj and Skrzydlewska, 2005; Singh et al., 2009).
Extracts from different plants as: Allii fistulosi;
Salvia miltiorrhiza; Rosmarinus officinalis; Acacia sal-icina or plant preparations: Kalpaamruthaa (mixtures of Semecarpus anacardium, Emblica officinalis and
honey); Tridham (mixtures of Terminalia chebula seed
coat, Elaeocarpus ganitrus fruits, and Prosopis cineraria
leaves) were tested with the aim to counteract the oxida-tive stress induced by AFB1 (Liu et al., 1999; Lee et al., 2005; Costa et al., 2007; Umarani et al., 2008; Bouhlel
et al., 2010). The extracts were able to protect cells against the consequences of oxidative stress by inhibiting the formation of intracellular reactive oxygen species, reduc –
ing the levels of TBARS, decreasing the LPO; increasing the level of reduced glutathione and the PHGPx gene expression; decrease of PCC level (Liu et al., 1999; Lee et al., 2005; Costa et al., 2007; Umarani et al., 2008; Bouhlel et al., 2010; Ravinayagam et al., 2012).
The mechanism involved in this protective effect is
not very clear. It was mentioned that the phenolic com-pounds (flavonoids and phenolic acids) have the capac –
ity to scavenge ROS (Gülçin, 2012; Zambonin, 2012), the antioxidant activity depending on the number and positions of hydroxyl groups and glycosylation of flavo-noid molecules (Cai et al., 2006). Furthermore, it was suggested that the binding of phenolic compounds to plasma membranes may result in inhibition of lipid per –
oxide generation (El-Sharaky et al., 2009). Also, the devel-opment of lipid peroxidation could be impaired by the chelation of the metal ions by the phenolic compounds (Wang et al., 2009). Another mechanism that might be taken into consideration is the binding of aflatoxin to phenolic compounds found in the plant extracts (San & Chan, 1987). However, more studies are still needed, in order to explain the mechanisms involved in the pro-tection of plant extracts against the oxidative damage induced by AFB1.
Conclusions
The oxidative stress caused by AFB1 may be one of the underlining mechanisms for AFB1-induced cell injury and DNA, protein and lipid damages, which lead to tumorigenesis. Antioxidants (vitamins, polyphenolic acids, terpenoids, flavonoids and vegetable pigments) are a good alternative for the reduction of the toxic effects of aflatoxins, but more studies are needed in order to understand the protection mechanisms.
Acknowledgment
The authors want to thank to Dr. Olga Starodub for her help with the English corrections.
Declaration of interest
This work was financed through the project Research and Development Project from the Nucleus Programme no 09-38 0104.
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