ToxSci Advance Access originally published online on March 30, 2007
Toxicological Sciences 2007 98(1):57-62; doi:10.1093/toxsci/kfm073
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Elevation of 8-Hydroxydeoxyguanosine in DNA from Isolated Mouse Lung Cells Following In Vivo Treatment with Aflatoxin B1
Department of Pharmacology and Toxicology, Queen's University, Kingston, Ontario, Canada, K7L 3N6
1 To whom correspondence should be addressed. Fax: (613) 533-6412. E-mail: masseyt{at}post.queensu.ca.
Received December 21, 2006; accepted March 22, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Aflatoxin B1 (AFB1) is a mycotoxin produced by some strains of Aspergillus and is a recognized pulmonary and hepatic carcinogen. The most widely accepted mechanism of AFB1 carcinogenicity involves bioactivation to AFB1-8,9-exo-epoxide and binding to DNA to form AFB1-N7-guanine. Another potential cause of DNA damage is AFB1-mediated stimulation of reactive oxygen species formation, leading to oxidation of DNA bases. The objective of this study was to determine the ability of AFB1 to cause oxidative DNA damage in lung cell types of the A/J mouse. The formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in freshly isolated mouse lung alveolar macrophages, alveolar type II cells, and nonciliated bronchial epithelial (Clara) cells was assessed by high-performance liquid chromatography with electrochemical detection. An approximately 3-fold increase in 8-OHdG formation occurred in both alveolar macrophage and Clara cell preparations isolated from A/J mice 2 h following treatment with a single tumorigenic dose of 50 mg/kg AFB1 ip (n = 3, p < 0.05). Prior treatment with 300 kU/kg polyethylene glycol–conjugated catalase prevented the AFB1-induced increase in 8-OHdG levels in all mouse lung cell preparations (n = 3, p < 0.05). These results support the possibility that oxidative DNA damage in mouse lung cells contributes to AFB1 carcinogenicity.
Key Words: aflatoxin B1; mouse lung; 8-hydroxy-2'-deoxyguanosine; catalase.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Aflatoxin B1 (AFB1) is a mycotoxin produced by some strains of Aspergillus and is a recognized pulmonary and hepatic carcinogen (Donnelly et al., 1996a
; Massey et al., 2000). It is a group 1 carcinogen according to the International Agency for Research on Cancer, being consistently genotoxic and producing DNA adducts in humans and animals in vivo as well as chromosomal anomalies in rodents (IARC, 1993). Exposure of the lungs to AFB1 can occur both through the diet and by inhalation, with the toxin reaching the lungs either directly or via the circulatory system (Donnelly et al., 1996a). AFB1 is bioactivated in human lung primarily by prostaglandin H synthase and/or lipoxygenase-catalyzed cooxidation (Donnelly et al., 1996b) and in rodents and rabbits by cytochromes P450 (Imaoka et al., 1992). Bioactivation of AFB1 leads to formation of the highly reactive AFB1-8,9-exo-epoxide which, if not detoxified, binds preferentially to the N7 position of guanine residues in DNA (Massey et al., 2000). Susceptibility to the toxicity and carcinogenicity of AFB1 varies markedly between species, although mice are resistant to AFB1-induced hepatocarcinogenesis, they are susceptible to pulmonary tumorigenesis (Massey et al., 2000).
It is widely accepted that epoxide formation and subsequent AFB1-N7-guanine formation results in specific K-ras mutations, initiating mouse lung tumorigenesis (Massey et al., 2000). However, it is also possible that AFB1-induced formation of ROS, leading to 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation, can result in the same K-ras mutation pattern (i.e., predominantly G to T transversion mutations) as DNA alkylation, thus contributing to pulmonary tumorigenesis.
In the present study, we investigated the ability of AFB1 to cause oxidative DNA damage in different mouse lung cell types, as well as the potential protective effect of catalase (CAT) against AFB1-induced oxidative DNA damage in mouse lung.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials.
Chemicals were obtained as follows: AFB1, polyethylene glycol–conjugated CAT (PEG-CAT), DNAse I, alkaline phosphatase, 8-OHdG, and 2'-dG standards from Sigma (St Louis, MO); nuclease S1 from VWR (Mississauga, ON); phosphodiesterases I and II and proteinase K from Amersham Biosciences (Baie D'Urfe, QC); [3H]AFB1 from Moravek (Brea, CA) and Vitrax (Placentia, CA); Biorad Protein Assay dye reagent from Biorad Laboratories, Inc. (Hercules, CA); Ultrafree-MC 10,000 NMWL filter units from Millipore (Bedford, MA); DNeasy Tissue Kit from Qiagen (Mississauga, ON); CAT Assay Kit from Cayman Chemicals (Ann Arbor, MI). All other chemicals were reagent grade and were obtained from common commercial suppliers.
Animal treatments.
Female A/J mice (16–19 g, Jackson Laboratories, Bar Harbor, ME) were housed with a 12-h light/dark cycle and provided food and water ad libitum. Mice were treated with vehicle (40 µl dimethyl sulfoxide [DMSO], ip) or with 50 mg/kg AFB1, a dose that has been shown to result in pulmonary tumorigenesis, but not hepatocarcinogenesis, in AC3F1 mice (Donnelly et al., 1996b). Mice were killed by cervical dislocation 2, 12, 24, or 48 h after treatment. In an independent experiment, mice were treated with either saline (100 µl, ip) or PEG-CAT (300 kU/kg), with one unit being defined as able to decompose 1.0 µmol of hydrogen peroxide (H2O2) per minute at pH 7.0 and 25°C, followed by vehicle (40 µl DMSO, ip) or 50 mg/kg AFB1 12 h later. Mice were then killed by cervical dislocation 2 h after the second treatment. Each animal's trachea was cannulated, and the lungs and livers were perfused with HEPES phosphate-buffered saline (HPBS) (pH 7.4) and excised.
Mouse lung cell isolations.
Alveolar macrophages were isolated by lavage with cold HPBS (pH 7.4) (Donnelly and Massey, 1999). Cell digest (which consists of isolated unseparated cells), alveolar type II cells, and Clara cells were enriched by centrifugal elutriation as described by Donnelly and Massey (1999). Each elutriation employed 20–30 mice per treatment group. The cell fractions were frozen in liquid nitrogen and stored at – 80°C until DNA isolation.
Isolation of DNA from freshly isolated mouse lung cells.
Utilization of a phenol-free method of DNA isolation and complete DNA digestion are considered to be necessary to avoid artifactual production of 8-OHdG (Ravanat et al., 1998). Therefore, calf thymus DNA (CTDNA) was used in optimization of DNA isolation and digestion methods to ensure accuracy in the measurement of oxidative DNA damage. Liver tissue and lung cell preparations were thawed on ice, and DNA was isolated from groups of 5 x 106 cells or 25–50 mg tissue using a phenol-free Qiagen DNeasy Tissue Kit, according to the manufacturer's recommendations. DNA digestion was performed using the method described by Huang et al. (2001), except nuclease S1 (10 units/µl) was used rather than nuclease P1 (1 unit/µl). The digest was filtered through a Millipore Ultra-free MC 10,000 NMWL filter unit.
Determination of 8-OHdG levels by high-performance liquid chromatography with electrochemical detection.
The levels of 8-OHdG and 2'-dG were assessed with a Coularray high-performance liquid chromatography with multichannel electrochemical detector (ESA Inc., Chelmsford, MA). Compounds were separated on a Waters S-3 4.6 x 150 mm column with 5% methanol/95% 100mM sodium acetate buffer (pH 5.2) at a flow rate of 1.0 ml/min (Bolin et al., 2004). The four electrochemical detector channels were set at –100, 250, 475, and 875 mV.
Determination of CAT activity in mouse lung.
Isolated cells were homogenized with a glass 2 ml Dounce homogenizer (Kontes, Vineland, NJ) using 50mM potassium phosphate buffer (pH 7.0) with 1.0mM EDTA, the homogenate centrifuged at 10,000 x g for 15 min, and the supernatant collected. CAT activity of the supernatant was determined using the Cayman Catalase Assay Kit, according to the manufacturer's recommendations. Formaldehyde formation was measured using a Synergy HT multidetection microplate reader (Biotek, Winooski, VT) at 540 nm. Protein content of supernatant was measured using the Biorad Protein Assay.
Determination of [3H]AFB1-DNA binding in mouse lung.
Groups of three female A/J mice were treated with either [3H]AFB1 (40 µl of 100 µCi [3H]AFB1 at 50 mg/kg, ip) or PEG-CAT (300 kU/kg in 100 µl saline, ip) and [3H]AFB1, using the treatment regimen described above. Mice were killed 2 h after treatment by cervical dislocation. The lungs and livers were perfused with HPBS (pH 7.4) and excised. Mouse lung and liver tissue (50–100g) was subjected to proteinase K digestion (10 mg/ml), and DNA was isolated by standard phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation (Devereux et al., 1993). Radioactivity was quantitated by liquid scintillation spectroscopy in a Beckman LS 3800 spectrometer to determine the amount of [3H]AFB1-adduct formed, expressed as the amount of [3H]AFB1-DNA binding per gram of DNA.
Data analysis.
All data are reported as mean ± SD. Statistical analysis was performed using Student's t-test to compare differences in the amount of oxidative DNA damage present in whole organs between the vehicle and AFB1 treatment groups and to compare [3H]AFB1-DNA binding between mice treated with saline or PEG-CAT and between [3H]AFB1-DNA binding in lung versus liver (GraphPad Prism 4 software). A two-way ANOVA combined with the Newman-Keuls post hoc test was used to assess differences among the various cell types. Two-way ANOVA was also used to compare oxidative DNA damage between the PEG-CAT and/or AFB1 treatment groups. p < 0.05 was considered significant in all cases.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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8-OHdG Formation in Freshly Isolated Mouse Lung Cells Following In Vivo Treatment of Mice with AFB1
Consistent with previous studies (Belinsky et al., 1995
), enrichment of the cell preparations was approximately 95% for macrophages and alveolar type II cells and approximately 77% for Clara cells. Background levels of 8-OHdG in CTDNA averaged 1.4 8-OHdG per 105 dG. 8-OHdG levels in control isolated cell fractions ranged from 1.49 8-OHdG per 104 dG to 3.19 8-OHdG per 104 dG and were somewhat more variable between experiments for alveolar type II cells than for the other cell fractions (Figs. 1, 2, and 4). Increases of approximately 60, 180, and 200% in 8-OHdG formation occurred in cell digest, enriched alveolar macrophages, and enriched Clara cells, respectively, (compared to vehicle-treated controls) from mice treated with 50 mg/kg AFB1 ip and killed 2 h after treatment (n = 4 pools of lungs from 20 mice per pool for each treatment group, p < 0.05) (Fig. 1). Although an apparent increase was observed in alveolar type II cells, it was not statistically significant (p > 0.05). No increase in 8-OHdG formation was seen in any cell preparation at 12 h following AFB1 treatment (n = 3, p > 0.05) compared to controls (Fig. 2). This remained true at both the 24 h (n = 1) and 48 h (n = 1) time points (data not shown). No increase in 8-OHdG formation was seen in the liver tissue following AFB1 treatment (4.81 ± 0.54 8-OHdG/104 dG) as compared to controls (4.92 ± 0.69 8-OHdG/104 dG, n = 3, p > 0.05).
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significantly different (p < 0.05) from AFB1 group.Effects of PEG-CAT on AFB1-Induced 8-OHdG Formation in Freshly Isolated Mouse Lung Cells
An increase in CAT activity was observed in cell digest, and in preparations enriched in macrophages, alveolar type II cells, and Clara cells isolated from mice treated with 300 kU/kg PEG-CAT ip and killed 12 h after treatment (n = 3 pools of lungs from 30 mice per pool for each treatment group, p < 0.05) (Fig. 3). AFB1-DNA–binding levels in mouse lung were not altered by treatment with PEG-CAT (n = 5, p > 0.05), with the amount of [3H]AFB1-DNA binding per gram of DNA being 84.5 ± 12.6 pmol [3H]AFB1/g DNA and 104 ± 30.9 pmol [3H]AFB1/g DNA for the [3H]AFB1 and PEG-CAT + [3H]AFB1 groups, respectively. The AFB1-DNA and binding levels in liver were approximately 1341% higher being 1218 ± 324.4 pmol [3H]AFB1/g DNA and 84.5 ± 12.6 pmol [3H]AFB1/g DNA in liver and lung, respectively, ([1218 pmol – 84.5 pmol/84.5 pmol] x 100).
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significantly different (p < 0.05) from AFB1 group.Administration of AFB1 alone resulted in a 128% increase in CAT activity in mouse lung cell digest (p < 0.05). PEG-CAT treatment prevented the AFB1-induced increase in 8-OHdG levels in all four of the cell preparations examined (p < 0.05), with 8-OHdG levels being indistinguishable from those of control animals (p < 0.05, Fig. 4). Treatment of mice with 300 kU/kg PEG-CAT 12 h prior to administration of the AFB1 vehicle (i.e., DMSO) resulted in a decrease in endogenous 8-OHdG levels in cell digest and macrophages (p < 0.05, Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The most commonly accepted mechanism of AFB1-induced carcinogenesis involves DNA alkylation via the reaction of AFB1-8,9-exo-epoxide with guanine residues in DNA. In mouse lung, this process is consistent with the observed G to T transversion mutations in K-ras, a potential initiating event in AFB1-induced tumorigenesis (Donnelly et al., 1996a
). However, in addition to alkylating DNA, in vivo treatment with AFB1 can induce ROS formation in some systems (Shen et al., 1995), but ROS formation in lung from AFB1 treatment has not been described previously, and the relevance of 8-OHdG formation to AFB1 carcinogenicity has not been examined. 8-OHdG is often used as a marker for oxidative DNA damage and is formed by reaction of hydroxyl radical (•OH) with guanine residues in DNA (Shen et al., 1995). The presence of 8-OHdG causes alpha polymerase to misincorporate nucleotides during DNA replication, often leading to G to T transversion mutations (Shen et al., 1995). Hence, our demonstration of elevated 8-OHdG in mouse lung cells following AFB1 suggests that ROS may contribute to AFB1 carcinogenesis in this model.
There is considerable inconsistency in the literature concerning endogenous 8-OHdG levels in both in vitro and in vivo systems, with values ranging from approximately four 8-OHdG per 106 dG in HeLa cells (ESCODD, 2003) to four 8-OHdG per 104 dG in mouse liver (Zhang et al., 2004). Therefore, it remains important to ensure proper tissue handling, DNA isolation, and digestion to avoid artifactual production of 8-OHdG. The background CTDNA 8-OHdG levels obtained in the present study (1.4 8-OHdG per 105 dG) were comparable to those published by the European Standards Committee on Oxidative DNA Damage (ESCODD) (Lunec, 1998), which ranged from 1.9–21.3 8-OHdG per 105 dG, suggesting that the method developed avoided the artifactual production of oxidative DNA damage. Although the background levels of 8-OHdG in mouse lung are higher than those cited in HeLa cells by ESCODD (ESCODD, 2003), they are within the range cited above in various tissues and cell types. The levels measured in mouse liver are slightly higher than those commonly reported in literature but are not highly variable and similar to those previously published for mouse liver (Zhang et al., 2004). As well, the levels of 8-OHdG measured in liver were not vastly different from those in the lung, in which we clearly saw an effect of AFB1 (i.e., 4.92 8-OHdG per 104 dG versus approximately two 8-OHdG per 104 dG).
Of particular significance in the present study is that AFB1 induced ROS formation in Clara cells and alveolar type II cells. These cell types are not only sites of AFB1 metabolism, but they are also potential progenitor cells for tumorigenesis in mouse lung (Kannan et al., 2006, Massey et al., 2000). We have found Clara cells and alveolar type II cells to be targets for AFB1-induced K-ras mutations (Donnelly and Massey, 1999), which coincides with the AFB1-induced increases in 8-OHdG formation seen here. The frequency of K-ras mutations between the different cell types is also consistent with the oxidative DNA damage results, with the mutant allele frequency and levels of 8-OHdG being higher in the Clara cell fraction than in the alveolar type II cell fraction (Donnelly and Massey, 1999). This supports the hypothesis that AFB1-induced oxidative DNA damage in these cells contributes to the resulting carcinogenicity of AFB1 in mouse lung.
The mechanism of ROS formation resulting from AFB1 exposure is not clearly understood. However, AFB1 clastogenicity in human lymphocytes has been linked to stimulation of arachidonic acid release, which may induce chromosomal damage by increasing formation of hydroperoxyarachidonic acid metabolites (Amstad et al., 1984). These hydroperoxides could undergo metal-catalyzed degradation to yield •OH, resulting in 8-OHdG production (Halliwell et al., 1999). Furthermore, both AFB1 and aflatoxin M1, a hydroxylated metabolite of AFB1, stimulate formation of free radicals in rat liver (Kodama et al., 1990; Towner et al., 2003). Alternatively, activity of cytochromes P450 may be important in AFB1-stimulated radical formation, since a temporal correlation was found between AFB1-increased ROS levels and expression of P450 activity in cultured rat hepatocytes (Shen et al., 1995). Also, P450 inhibition decreased AFB1-induced hepatic •OH formation (Towner et al., 2003). In the same system, iron chelation also decreased AFB1-stimulated •OH formation, presumably by inhibiting its production from H2O2 by the Fenton reaction (Towner et al., 2003). AFB1-induced H2O2 formation from cytochromes P450 in rat liver microsomes is hypothesized to result from dismutation of superoxide radical anion (O2•–) and from the breakdown of the peroxygenated p450 complex (White, 1991; White et al., 1980). Furthermore, Kupffer cells, which are resident hepatic macrophages, are involved in the metabolism of AFB1. Gadolinium chloride, a Kupffer cell inactivator, decreased AFB1-induced free radical formation (Towner et al., 2003). Therefore, it was proposed that AFB1 metabolism–stimulated free radical formation in rat liver requires initial involvement of cytochromes P450 and subsequent involvement of both iron (for •OH formation by the Fenton reaction) and Kupffer cells (Towner et al., 2003). Corresponding mechanisms have yet to be examined in the lung.
Loss of AFB1-induced oxidative DNA damage after 2 h following AFB1 was likely due to the induction of repair mechanisms, particularly base excision repair (BER), the primary repair pathway of 8-OHdG. In fact, translocation of BER proteins to the nucleus can occur rapidly after exposure to oxidative stress (Mitra et al., 2001). The loss of AFB1-induced elevation of 8-OHdG after 2 h does not preclude its involvement in mutagenesis, since mutations resulting from 8-OHdG formation can occur via errors in BER (Matsuda et al., 2003) and low fidelity bypass polymerases, which can incorrectly insert bases across from 8-OHdG. In fact, the bypass DNA polymerase 
frequently inserts adenine rather than cytosine across from 8-OHdG, leading to a G to T transversion (Jaloszynski et al., 2005).
The potential contribution of oxidative DNA damage to AFB1 mouse lung tumorigenicity is supported by the fact that the levels of AFB1 adducted guanine residues (1.25 x 10–5% of guanines adducted, calculated assuming 1 g of DNA is equal to 3.3 mmol total nucleotides, with 20% being dG) and AFB1-induced 8-OHdG (0.017% in the cell digest of treated mice, [(3.5 8-OHdG – 1.8 8-OHdG)/104 dG] x 100, Fig. 1) were similar. Furthermore, in mouse liver, which is not susceptible to AFB1 carcinogenesis, treatment with the mycotoxin did not elevate 8-OHdG despite the fact that AFB1-induced DNA alkylation, represented by [3H]AFB1 DNA binding, was 14.4-fold higher in liver than in lung ([1218 pmol [3H]AFB1/g DNA]/[84.5 pmol [3H]AFB1/g DNA]).
The fact that ip treatment of mice with PEG-CAT resulted in an increase in lung cellular CAT activity suggested that it might protect against prooxidant damage. CAT was shown previously to prevent AFB1-stimulated ROS production in cultured rat hepatocytes (Shen et al., 1996) but that study did not address effects on DNA damage. Indeed, we found that treatment with PEG-CAT protected mouse lung against AFB1-induced oxidative DNA damage, without affecting binding of AFB1-derived radioactivity to DNA. CAT detoxifies H2O2, thereby preventing formation of •OH by the Fenton reaction, which can lead to production of 8-OHdG (Djordjevic, 2004). Conjugating CAT to PEG blocks renal clearance of the enzyme and thus increases its circulatory half-life. The antigenicity of the native protein is also reduced, preventing its hydrolysis by proteases and increasing cell-associated specific activities (Beckman, 1988). Our results therefore suggest that oxidative DNA damage induced by AFB1 involves H2O2. However, they do not preclude a central role for O2•–, since superoxide dismutase (SOD) converts O2•– to H2O2 and molecular oxygen. Furthermore, there is evidence that CAT itself has some activity for detoxification of O2•– (Lardinois, 1995; Shimizu et al., 1984). We did not examine the potential protective effect of administering exogenous SOD, since it is toxic to mice (Winn et al., 1999), possibly because of accelerating superoxide dismutation to H2O2, with subsequent formation of •OH (Freeman et al., 1986).
The failure of PEG-CAT treatment to significantly alter CAT activity in alveolar macrophages may have been due to the high yet variable levels of endogenous CAT present in this cell type (Coursin et al., 1992).
Although the effect was only statistically significant in the cell digest, the fact that AFB1 itself elevated CAT activity is consistent with a pro-oxidant activity for AFB1, since ROS increase CAT activity by stimulating translocation of the transcriptional regulators NFB, AP-1, and NF-Y into the nucleus (Mantovani, 1999; Zhou et al., 2001).
The explanation for the failure in vivo treatment with a combination of AFB1 and PEG-CAT to significantly alter lung CAT activity is not obvious, but conceivably could be due to the subcellular localization of the administered PEG-CAT compared to endogenous CAT. Endogenous CAT is present primarily in peroxisomes (Ho et al., 2004), while PEG-CAT concentrates in the cytoplasm after entering the cell via membrane protein binding or endocytosis (Beckman et al., 1988). AFB1 metabolites are known to bind to lysine residues of cytoplasmic proteins, potentially inhibiting their activity (McLean et al., 1995; Sujatha et al., 2001). For example, a lysine residue fourth from the COOH terminus constitutes a critical component of the human CAT peroxisomal targeting signal, with substitution of this residue leading to abolition of peroxisomal targeting (Purdue et al., 1996). It is possible that the ROS formed by AFB1 are being eliminated by the elevated CAT activity present initially, but the binding of the AFB1 metabolites to cytosolic PEG-CAT returns the CAT activity to constitutive levels. In any case, the prevention of the increase in 8-OHdG formation by PEG-CAT suggests a potential chemopreventative strategy against the tumorigenicity of AFB1 and warrants further study.
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ACKNOWLEDGMENTS |
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The authors wish to thank Ms Sandra Graham and Ms Pamela Brown for assistance with experimental protocols. This work was supported by Canadian Institutes of Health Research grant no. MT10382.
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