Effects of Dietary Oltipraz and Ethoxyquin on Aflatoxin B1 Biotransformation in Non-Human Primates

Theo K. Bammler, Dennis H. Slone and David L. Eaton1

Department of Environmental Health, University of Washington, Seattle, Washington 98195

Received August 5, 1999; accepted October 27, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Following aflatoxin B1 (AFB) exposure, rats readily develop liver tumors. However, treatment of rats with a variety of compounds, including the synthetic dithiolthione oltipraz and the antioxidant ethoxyquin, protects these rodents from AFB-induced hepatocarcinogenesis. Several epidemiological studies strongly suggest that AFB is also a causative agent of liver cancer in humans. However, relatively little is known about the efficacy of cancer chemoprevention in human and non-human primates. To this end, we examined the effects of chemopreventive agents on AFB metabolism in non-human primates. Hepatic aflatoxin B1 metabolism profiles of macaque (Macaca nemestrina) and marmoset (Callithrix jacchus) monkeys were determined and compared to humans. Quantitatively, the oxidative metabolism of this mycotoxin was similar in the three primate species. In contrast to macaques, both humans and marmosets lacked AFB-glutathione conjugating activity. It was concluded that marmosets resembled human AFB metabolism more closely than the macaques, and therefore, marmoset monkeys were chosen for this study. Eleven adult male marmosets were randomly assigned to three groups. Animals received the synthetic dithiolthione oltipraz, the antioxidant ethoxyquin, or vehicle only. In addition, two single doses of AFB were also administered orally before and after animals were treated with aforementioned compounds. Both oltipraz and ethoxyquin induced aflatoxin B1-glutathione conjugating activity in the livers of some but not all marmosets. In addition, 10 µM oltipraz inhibited cytochrome P450-mediated activation of AFB to the ultimate carcinogenic metabolite, aflatoxin B1-8,9-epoxide,in vitro, up to 51%. Furthermore, animals treated in vivo with oltipraz, but not ethoxyquin, exhibited a significant reduction (53% average) in AFB-DNA adduct formation relative to the control animals (p < 0.05). Together, our data suggest that chemoprevention is also effective in primates; however, most likely to a lesser degree than in rodents.

Key Words: aflatoxin B1; chemoprevention; glutathione S-transferase; cytochrome P450; oltipraz; ethoxyquin; primates.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The potent hepatocarcinogen aflatoxin B1 (AFB), produced by the common molds Aspergillus flavus and Aspergillus parasiticus, is a widely encountered contaminant of cereal crops and other foodstuffs in regions of the world with hot and humid climates (Austwick and Ayerst, 1963Go; Eaton and Gallagher, 1994Go). Many species, including rat, turkey, duck, trout, and primates, are susceptible to the carcinogenic effects of AFB (Newberne and Butler, 1969Go). In addition, epidemiological studies strongly suggest that AFB exposure alone and in combination with hepatitis B infection are causative agents in hepatocarcinogenesis (Qian et al., 1994Go). Furthermore, there is convincing evidence that this mycotoxin produces mutations in the p53 tumor suppressor gene, particularly at codon 249 (Bressac et al., 1991Go; Eaton and Gallagher, 1994Go; Hsu et al., 1991Go; Ozturk et al., 1991Go).

AFB is metabolized in mammalian liver through cytochromes P450 to the ultimate carcinogen aflatoxin B1-8,9-exo-epoxide (exo-AFBO) or to the much less mutagenic polar metabolites aflatoxin M1 (AFM1), aflatoxin Q1 (AFQ1) and aflatoxin P1 (AFP1) (for recent reviews see Eaton and Gallagher, 1994; Massey et al., 1995; McLean and Dutton, 1995) (Fig. 1Go). exo-AFBO is highly unstable, with a half life of approximately 1 s in aqueous solution (Johnson et al., 1996Go), and reacts with cellular macromolecules including DNA, or undergoes rapid non-enzymatic hydrolysis to aflatoxin B1-8,9-dihydrodiol (Raney et al., 1992cGo) (Fig. 1Go). At physiological pH, aflatoxin B1-8,9-dihydrodiol yields a dialdehydic phenolate ion, which forms Schiff bases with primary amine groups in proteins (Sabbioni et al., 1987Go; Sabbioni and Wild, 1991Go). Raney and co-workers have shown that both the exo- and endo-stereoisomers of AFBO are formed by mammalian hepatic microsomes (Raney et al., 1992aGo). However, it is exclusively the exo-epoxide that reacts with DNA, resulting mostly in N7-guanine adducts (Iyer et al., 1994Go).



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FIG. 1. Overview of biotransformation pathways for aflatoxin B1.

 
Interestingly, the susceptibility of mouse and rat to the carcinogenic effects of AFB differs greatly; the mouse is resistant, whereas the rat is highly sensitive (Degen and Neumann, 1981Go; Newberne and Butler, 1969Go). Mice constitutively express the alpha class glutathione S-transferase (GST) A3 gene (also known as Yc) and the encoded protein efficiently detoxifies exo-AFBO by conjugation with glutathione (Buetler and Eaton, 1992Go; Buetler et al., 1992Go; Hayes et al., 1992Go). In contrast, rats lack the constitutive expression of such a GST-mediated detoxification pathway and readily develop liver tumors following exposure to AFB. However, dietary treatment with a variety of compounds, including the antioxidant ethoxyquin (Cabral and Neal, 1983Go; Kensler et al., 1986Go) and the synthetic dithiolthione oltipraz (Kensler et al., 1987Go), protect rats from AFB-induced hepatocarcinogenesis by inducing the alpha class rGSTA5 gene (also known as Yc2). The corresponding gene product also detoxifies exo-AFBO (Hayes et al., 1994Go). In addition, treatment of mice with glutathione-depleting agents dramatically potentiates AFB genotoxicity (Monroe and Eaton, 1988Go). Both rodent species activate AFB, with the mouse generating the exo-AFBO even more efficiently (Monroe and Eaton, 1987Go). Together, these data strongly suggest that GST-mediated aflatoxin B1-glutathione conjugating (AFB-SG) activity is a major determinant of species susceptibility to the carcinogenic effects of AFB.

It is not known whether chemoprevention strategies based on GST induction that are effective in rodents are also effective in primates. Although there is some evidence that diets high in cruciferous vegetables can induce alpha class GSTs in humans (Bogaards et al., 1994Go; Nijhoff et al., 1995Go), we and others have failed to detect significant AFB-SG activity in human liver cytosol (Slone et al., 1995Go, Moss and Neal, 1985aGo). Furthermore, purified recombinant hGSTA1-1 has no detectable activity toward AFBO (Buetler et al., 1996Go), although human mu class GSTs do have some measurable activity toward AFB-epoxide (Raney et al., 1992bGo). The question arises whether chemointervention affords protection against AFB-induced hepatocarcinogenesis in human subjects that are exposed to high levels of AFB in their diet, as is the case in large parts of Africa and China. Recently, Kensler and colleagues (1998) examined the ability of oltipraz, an FDA-approved antischistosomal drug, to modulate AFB metabolism in a human population naturally exposed to AFB in the diet, by measuring established biomarkers such as AFB-albumin adducts. Although there was some suggestion of a protective effect, for obvious reasons it was not possible to determine the extent of effects directly in the liver. Importantly, the liver is not only the major organ of metabolism for AFB, it is also the target organ of genotoxicity for this mycotoxin. Therefore, this study was designed to complement the human chemointervention trial and examine the effects of dietary treatment on AFB biotransformation and DNA adduct formation directly in the liver of non-human primates. Our objective was to determine whether dietary oltipraz and/or ethoxyquin could modulate hepatic AFB metabolism in vivo in a way that was protective against its genotoxic and potentially carcinogenic effects. The antioxidant ethoxyquin was chosen for this study, because it is an even more potent inducer of hepatic AFB-glutahtione conjugating activity than oltipraz in rat liver (Hayes et al., 1994Go), but little is known about its chemoprotective effects in primates.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Oltipraz was a kind gift from Dr. Thomas Kensler (Department of Environmental Health Sciences, The Johns Hopkins University, Baltimore, MD). Aflatoxins B1, G1, and ethoxyquin were obtained from Sigma Chemical Co. (St. Louis, MO). [3H]-AFB1 was purchased from Moravek Biochemicals (Brea, CA). {Delta}5-Androstene-3,17-dione was purchased from Steraloids, Inc. (Wilton, NH). HPLC-grade reagents were supplied by J. T. Baker (Phillipsburg, NJ). Other reagents were of analytical grade and obtained from various commercial sources.

Animals and treatments.
The 3 adult male marmosets (Callithrix jacchus) used for the pilot study were purchased from New England Regional Primate Center (Southbridge, MA). These 3 animals received the same treatment as the animals in the control group (see below). The other 11 adult male marmosets were obtained from the Biomedical Resources Foundation, Houston, TX. The ages of the marmosets ranged from 3 to 9 years and the weights from 250 to 406 g. All animals were housed at the University of Washington Regional Primate Research Center. Marmoset monkeys were fed a standard primate chow and fruit diet, and were given guava/orange juice and water ad libitum. Oltipraz (18 mg/kg/day) was mixed in applesauce and fed directly to the appropriate animals. Ethoxyquin (30 mg/kg/day) was mixed with a small amount of propylene glycol and Karo syrup and injected into mealworms (which were a normal part of the daily diet) that were fed to the appropriate animals. The time between injection of the mealworms with ethoxyquin and ingestion of these worms by the marmosets was approximately 1 min.

In preliminary studies, it was found that ethoxyquin mixed in fruit juice or applesauce was unpalatable and directly irritating to mucous membranes. Therefore, an alternative vehicle was necessary. All animals received all vehicles (applesauce, propylene glycol, Karo syrup, and mealworms) regardless of treatment regimen. Commercial Primate Chow contains small amounts of synthetic antioxidants. The total amount of synthetic antioxidants contained in the different components of the standard diet was less than 3% of that fed to the animals in the ethoxyquin group. Animals were assigned randomly to one of 3 groups; control (n = 4), oltipraz (n = 4) and ethoxyquin (n = 3). At time point 0, each animal received a single dose of [3H]-AFB (100 µg/kg, 0.5-mCi/kg) by gavage, and blood samples were drawn at 0, 2, 24, and 48 h. On days 16 through 28, animals in the treatment groups received 18 mg/kg oltipraz or 30 mg/kg ethoxyquin daily in their diet, whereas the control animals received vehicles only. On day 26, each animal received a second dose of [3H]-AFB (100-µg/kg, 0.5-mCi/kg) by gavage, and blood samples were drawn at the same time intervals as described above. On day 28, animals were euthanized by a veterinarian and livers were excised, flash frozen in liquid nitrogen, and stored at –80°C.

For comparative purposes, livers from adult males of the monkey species Macaca nemestrina were obtained through the University of Washington's Primate Center tissue program. All experimental procedures were approved by the University of Washington Animal Care Committee.

Human liver samples.
Human liver samples H5, H8, and H123 were obtained from organ donors through the University of Washington Hospital (Seattle, WA) and stored at –80°C until microsomal and cytosolic fractions were prepared. The age, gender and drug histories of the donors are as follows: H5, male, age 21, no drug history; H8, male, age 46, inderal; H123, male, age 33, phenytoin and dexamethasone. For the oltipraz inhibition study (see Table 3Go), hepatic microsomal fractions were prepared from 9 individual human liver samples that were obtained through the University of Washington Liver Bank. Microsomal fractions were pooled. The age of the 6 male and 3 female donors ranged from 15 to 67 years; no other identifying information was available. Approval for human subjects was obtained under exemption 4 of NIH subjects guidelines.


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TABLE 3 In Vitro Inhibition of the Formation of Oxidative Metabolites of Aflatoxin B1 at a Concentration of 16 µM by Oltipraz, Expressed as Percentage of Control
 
Preparation of microsomal and cytosolic fractions.
All of the following steps were carried out at 4°C. Frozen tissue was thawed in 0.9% NaCl, blot dried, and weighed. Tissue was minced with scissors in 1.5 volumes (v/w) of homogenization buffer (0.25 M sucrose, 0.2 mM DTE, 1 mM EDTA, 10 mM Tris–HCl, pH 7.4), and homogenized in a Waring blender for 10 s. Each sample was centrifuged at 10,000 x g for 10 min, the pellet was discarded, and supernatant was centrifuged at 15,000 x g for 20 min. This supernatant was centrifuged at 105,000 x g for 60 min, the resulting supernatant was filtered through gauze to remove lipids, and the pellet was rinsed carefully 3 times with wash buffer (0.25 M sucrose, 5 mM MgCl, 25 mM KCl, 1 mM EDTA, and 80 mM Tris–HCl, pH 7.4). Using a hand-held Teflon-glass homogenizer, the pellet was resuspended and centrifuged at 105,000 x g for 60 min. The microsomal pellet was again rinsed, resuspended, and centrifuged as before. The final pellet was resuspended in an equal volume (compared to the original tissue weight) of resuspension buffer (0.25 M sucrose, 1 mM EDTA, 20 mM Tris–HCl, pH 7.4), divided into appropriate aliquots, flash frozen in liquid nitrogen, and stored at –80°C.

Aflatoxin B1 oxidation assay.
Microsomal oxidation of AFB was carried out using the method by Ramsdell et al. (1991) with the following modifications. Approximately 500 µg of microsomes, 500 µg of a 2:1 (v:v) mixture of rat:mouse hepatic cytosol, 1 mM NADP+, 0.125 units glucose-6-phosphate dehydrogenase, 5 mM glucose 6-phosphate, 5 mM reduced glutathione in 0.1 M potassium phosphate, pH 7.2, were mixed in a total volume of 240 µL. Following a 5-min preincubation at 37°C, the reaction was started by the addition of 10 µL of 16 or 126 µM AFB dissolved in dimethyl sulfoxide (DMSO). To ensure that high levels of AFB-SG activity was present, mouse liver cytosol was isolated from adult male animals that received a diet containing 0.75% butylated hydroxyanisole for 10 days prior to sacrifice. The final concentration of DMSO was 4% (v/v). AFB concentrations of stock solutions were determined spectrophotometrically. The reactions proceeded for 5 or 10 min before termination by addition of 250 µL ice-cold methanol containing 1% trifluoro-acetic acid and AFG1 (10 µM) as HPLC internal standard. Samples were vortexed, precipitated overnight at –20°C, centrifuged at 10,000 x g for 3 min and analyzed. The aflatoxin B1-8,9-epoxide (trapped as the glutathione conjugate via the high activities of rat:mouse cytosolic GSTs), AFQ1, AFM1, and AFP1 were quantified by reverse-phased, high-performance liquid chromatography analysis as described by Monroe and Eaton (1987). Reaction times were chosen to ensure linearity of metabolite formation.

Oltipraz or ethoxyquin inhibitions of AFB oxidation were performed as described above, with the following modification. Oltipraz or ethoxyquin dissolved in DMSO was added to the pre-incubation mixture five min before reaction was started, to a final concentration of 10, 50, 150, or 250 µM. Concentrations of stock solutions of oltipraz/ethoxyquin and AFB were chosen such that the final concentration of DMSO in the incubation mixtures was 4%.

Glutathione S-transferase assay.
Protein concentrations were estimated using the Bradford colorimetric assay from Bio-Rad (Hercules, CA), using bovine serum albumin as a standard. GST activity toward aflatoxin B1-8,9-epoxide (AFBO) was measured using a modified method by Monroe and Eaton (1987). AFBO is highly unstable in aqueous solution; therefore, this substrate was generated in situ using hepatic microsomes isolated from male mice (Swiss Webster) fed for 10 days with a diet containing 0.75% butylated hydroxyanisole. Approximately 250 µg of mouse microsomes, 126 µM AFB, and hepatic cytosolic fractions in a total volume of 250 µL of 0.1 M potassium phosphate, pH 7.2 were pre-incubated at 37°C for 5 min. The reaction was started by the addition of 1 mM NADPH. Following a 10-min incubation, the reaction was stopped with 250 µL of 1% trifluoro-acetic acid in methanol with 10 µM aflatoxin G1 as an HPLC internal standard.

Measurement of DNA adducts.
Total genomic DNA was isolated from approximately 1g of liver tissue by chloroform:phenol extraction as described by Beland et al. (1979). DNA concentration was estimated spectrophotometrically and purity was determined by measuring the 260/280 nm absorption ratio. [3H]-AFB-derived radioactivity in DNA fractions was quantified in Westcom EcoLite scintillation fluid (Scientific Resource Associates, Inc., Redmond, WA) using a Beckmann Model 3800 scintillation counter with automated quench correction, and using a set of 3H-standards. Results are expressed as pmol of AFB equivalents bound per mg DNA.

Measurement of albumin adducts.
Blood samples were collected in heparinized tubes to prevent clotting, mixed and centrifuged in serum separator tubes at 2000 x g for 5 min, to separate blood cells from serum. Serum samples were stored at –80°C and shipped on dry ice to the Johns Hopkins University for analysis of [3H]-AFB albumin in the laboratory of Dr. Thomas Kensler. [3H]-AFB albumin adducts were determined as described by Egner et al. (1995) with the modification that the protein pellet was washed and re-precipitated 4 times to remove all of the non-covalently-bound material.

Statistical analysis.
Statistical analyses were performed using Dunnett's modified t-test for multiple comparisons to a control, and linear-regression analysis. Analyses were performed using the program InStat for the Macintosh computer.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatic AFB Metabolism in Vitro in 3 Primate Species: Homo sapiens, Macaca nemestrina, and Callithrix jacchus
To identify a non-human primate species with an hepatic aflatoxin B1 (AFB) metabolism most similar to that of humans, we analyzed hepatic microsomal and cytosolic fractions prepared from untreated adult male macaques (Macaca nemestrina), male marmosets (Callithrix jacchus), and humans (Homo sapiens). Previous studies from this laboratory have demonstrated that human liver microsomal activation of AFB occurs primarily by a combination of CYP1A2 and CYP3A4 activity, and follows non-linear kinetics at concentrations above 10 µM due to non-linear, Hill-type kinetics of CYP3A4 toward AFB (Gallagher et al., 1996Go). A concentration of 16 µM AFB was chosen for this in vitro experiment as the best practical compromise between the relatively low hepatic concentrations of AFB possibly encountered naturally in the diet (likely to be less than 0.1 µM), and a higher AFB concentration necessary to accurately measure the products of AFB oxidation. Microsomes from all 3 species metabolized AFB at this concentration to varying degrees to AFM1, AFQ1, and also to the extremely reactive aflatoxin B1-8,9-epoxide (AFBO). With the exception of one human sample, the predominant oxidative metabolite in all primate species was AFQ1, ranging in the human, macaque, and marmoset samples from 45 to 361, 452 to 549, and 391 to 521 pmol/min/mg, respectively (Table 1Go). The humans and macaques exhibited somewhat higher rates of AFBO formation than the marmosets. However, a 3-fold variation was observed among the human samples, and the lowest human sample displayed a rate of AFBO production very similar to the highest marmoset sample. AFM1 was, in all species, a relatively minor metabolite at this AFB concentration. AFM1 and AFBO formations of the non-human primate samples did not differ significantly from the human samples (p > 0.05). However, in contrast to the marmoset samples, AFQ1 formation by macaque microsomes was significantly different from the human samples (p < 0.05). AFP1 formation was not detected in any of the samples.


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TABLE 1 Hepatic Aflatoxin B1 Metabolites in Human, Macaque and Marmoset Monkey
 
In contrast to the oxidative metabolism, the 3 species showed clear differences in their capacity to utilize AFBO as a substrate for GST-mediated conjugation with glutathione (GSH). GST activity toward AFBO in both marmoset and human liver cytosol was below the detection limit of the assay (0.7 pmol/min/mg), whereas liver cytosol from macaques exhibited clearly detectable activity (Table 1Go). These results suggest that marmosets mimicked human hepatic AFB metabolism more closely than the macaques and, therefore, marmosets were chosen over the macaques for the in vivo chemointervention study.

Effect of Oltipraz or Ethoxyquin Treatment of Marmosets on in Vivo AFB Metabolism as Determined by AFB-lysine Albumin Adduct Formation
Once AFB has been activated by cytochromes P450 to AFBO, it readily binds to cellular macromolecules including DNA and proteins. Sabbioni et al. (1987) identified the serum AFB-lysine albumin adduct as one of the major AFB adducts formed. Thus, effective chemoprevention strategies would be expected to reduce AFB-albumin adduction formation as well as AFB-DNA adducts. To monitor the effect of oltipraz (OPZ) and ethoxyquin (EQ) on the net AFBO production in vivo, we determined serum [3H]-AFB-albumin adducts.

All animals received two doses of [3H]-AFB, the first 16 days before OPZ or EQ was administered, and for the second 10 days after the animals had been treated with these compounds on a daily basis. To ensure that the peak time of AFB-albumin adduct formation was covered, blood samples were drawn at time points 0, 2, 24, and 48 h following [3H]-AFB administration. Adduct levels reached their maximum at 2 h, and therefore, data analysis was performed using this time point (data for time points 24 and 48 h are not shown). Adduct levels at the 2 h time point following the 2nd [3H]-AFB administration were, in all animals, expressed as percentages of the corresponding time point following the first [3H]-AFB administration (e.g., prior to EQ, OPZ, or vehicle treatment), for each animal (Fig. 2Go).



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FIG. 2. Aflatoxin B1-albumin adducts of individual marmosets. All animals received a single dose of [3H]-AFB1 (100 µg/kg, 0.5 mCi/kg) at time point zero. On days 16 through 28, animals in the treatment groups received 18 mg/kg/day oltipraz or 30 mg/kg/day ethoxyquin daily in their diet, whereas the control animals received vehicle only. On day 26, each animal received a second dose of [3H]-AFB (100 µg/kg, 0.5 mCi/kg); 2 h following each [3H]-AFB1 administration, blood samples were drawn and AFB-albumin adducts were determined. Bars represent adduct levels of individual animals measured at the 2 h time point following the 2nd [3H]-AFB administration, expressed as percentages of the corresponding time point following the first [3H]-AFB administration. Bars from left to right represent data from individual animals in the following order: control, #105, #34, #50, #38; oltipraz-treated, #162, #243, #579, #106; ethoxyquin-treated, #231, #219, #70.

 
No significant differences (p > 0.05) in AFB-albumin adduct levels were found between the different treatment groups. One animal in the control group showed a marginal 6% increase, whereas 3 marmosets in the same group displayed 20, 25, and 33% reductions in adduct levels. Changes in albumin adduct levels ranged from a 19% increase to a 36% decrease in animals treated with oltipraz. A 48% reduction in adduct levels was found in one ethoxyquin-treated animal, whereas the remaining 2 animals in this group showed slight decreases of 4 and 9%. It is noteworthy that 0.1 to 5.2% of adduct levels measured 2 h after the first [3H]-AFB administration were still detectable, in animals of all treatment groups, when the second dose of [3H]-AFB was administered 26 days later.

Effect of Oltipraz or Ethoxyquin Treatment of Marmosets on in Vivo AFB Metabolism as Determined by Hepatic AFB-DNA Adduct Formation
To estimate the effects of OPZ and EQ on the overall genotoxicity of AFB in marmosets, we determined that AFB-DNA adducts were present 2 days after the second and final dose of [3H]-AFB had been administered. Total DNA was isolated from marmoset livers, and radioactivity, due to [3H]-AFB equivalents bound to DNA, was measured. AFB-DNA adducts ranged from 0.48 to 1.44, 0.23 to 0.45, and 0.18 to 0.84 pmol [3H]-AFB equivalents bound per mg of DNA in the control, OPZ, and EQ groups, respectively. The difference between the control and the OPZ groups was marginally statistically significant (p = 0.047). In contrast, a comparison between the EQ group with either the control (p = 0.151) or the OPZ (p = 0.760) groups lacked significance, although inspection of the data clearly suggested a lack of any effect in one animal, with possibly significant effects in the other 2 EQ-treated marmosets (Fig. 3Go).



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FIG. 3. AFB-DNA adduct levels measured in the liver of individual animals. Total genomic DNA was isolated from liver tissue and covalent binding of [3H]-AFB to DNA was quantified using a scintillation counter; triplicate measurements were carried out for each sample. Bars represent pmol of [3H]-AFB equivalents bound per mg DNA for each individual animal; error bars represent SE. Bars from left to right represent data from individual animals in the following order: control, #105, #34, #50, #38; oltipraz-treated, #162, #243, #579, #106; ethoxyquin-treated, #231, #219, #70. In contrast to the ethoxyquin group, the difference between the oltipraz and control groups was significant (p < 0.05).

 
Effect of Oltipraz and Ethoxyquin on Hepatic Aflatoxin-Glutathione Conjugating Activity
To determine whether dietary OPZ or EQ effectively induced AFB-glutathione conjugating (AFB-SG) activity in marmoset liver, cytosolic fractions of all animals were analyzed. In contrast to the 3 marmosets in our pilot study, one of the 4 control animals exhibited detectable AFB-SG activity (2.5 pmol/min/mg), whereas 2 out of 4 OPZ (2.0 and 5.5 pmol/min/mg) and 2 out of 3 EQ (9.7 and 3.2 pmol/min/mg)-treated animals displayed this activity (Fig. 4Go). Thus, when the 3 untreated animals from our pilot study (labeled pre-control in Fig. 4Go) are included in the control group, 1 of 7 (14%) control animals was able to conjugate AFBO with glutathione, whereas 2 of 4 (50%) OPZ- and 2 of 3 (67%) EQ-treated animals possessed GST activity toward AFBO. Only animals 231 and 243 produced sufficient AFB-SG activities to allow determination of the relative amounts of exo-and endo-stereoisomers used as substrates; approximately 70% of total cytosolic AFB-SG activity was directed toward exo-AFBO.



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FIG. 4. Aflatoxin B1-glutathione conjugating activity (AFB-SG) in the livers of individual marmosets. AFB-SG activities of hepatic cytosolic fractions were determined as described in Materials and Methods. Bars represent triplicate measurements of AFB-SG activities of individual animals; error bars represent SE. AFB-SG activities of the oltipraz or ethoxyquin groups were not significantly different from the control group (p > 0.05). AFB-SG activities were also determined for animals in the pilot study. These animals are referred to as pre-control. Activities are expressed as pmol/min/mg. BDL, below the detection limit (0.7 pmol/min/mg). Bars from left to right represent data from individual animals in the following order: pre-control, #66, #67, #68; control, #105, #34, #50, #38; oltipraz-treated, #162, #243, #579, #106; ethoxyquin-treated, #231, #219, #70.

 
Effects of Oltipraz and Ethoxyquin on in Vitro Hepatic Microsomal Oxidation of AFB
Independent of treatment and consistent with the results from our pilot study, at an AFB concentration of 16 µM, AFQ1 accounted for more than 90% of total oxidative metabolites formed in vitro by hepatic microsomes from all animals, followed by less than 10% of AFBO and less than 1% of AFM1 (Table 2Go). AFBO and AFM1 formation were significantly decreased in both OPZ- and EQ-treated animals, relative to the concurrent controls (22–41% decrease). However, if data from the animals that did not receive chemoprotectors or AFB in the pilot study (Table 1Go) are included in the control group, differences in AFBO production between the treatment and control groups are not statistically significant (p > 0.05). In contrast, rates of AFM1 production between the non-treated (n = 7) and the OPZ group remain statistically significant (p = 0.0275), whereas this is not the case for the EQ group (p > 0.05).


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TABLE 2 Hepatic Oxidative Aflatoxin B1 Metabolism in Marmosets at 16 µM Aflatoxin B1
 
In Vitro Inhibition of Oxidative AFB Metabolism by Oltipraz
To determine whether OPZ treatment of marmosets might inhibit oxidative AFB metabolism in vivo, we determined the rates of in vitro hepatic microsomal oxidation of AFB in the presence of 10, 50, 150 and 250 µM OPZ. To ensure that hepatic microsomes isolated from animals that had received dietary OPZ did not contain residual oltipraz that might mask an inhibitory effect, we included microsomal preparations from control animals. OPZ or a metabolite thereof inhibited AFM1 and AFQ1 production in a dose dependent fashion (Table 3Go). The lowest in vitro concentration of OPZ used in this study (10 µM) decreased AFM1 formation by an average of 61 and 52% in the control and OPZ groups respectively, but had little effect on AFQ1 formation. In a similar manner, in human liver microsomes (prepared and pooled from 9 individual livers) 10 µM OPZ reduced initial rates of AFM1 production by 59%, and also had no effect on AFQ1 formation. In addition, while OPZ, or a metabolite thereof, inhibited AFM1 and AFQ1 formations, the inhibition was not strictly dose dependent. This phenomenon applied particularly to AFM1 formation. This is most likely due to the low levels of AFM1 formed, making the measurement particularly susceptible to measurement error.

In contrast to the oxidative metabolites AFM1 and AFQ1, AFBO cannot be measured directly, due to its extremely short half-life (Johnson et al., 1996Go). However, it can be measured indirectly as the AFB-SG conjugate (see Fig. 1Go). The assay conditions have been optimized such that virtually all of the AFBO formed (both the endo and exo isomers) by the marmoset microsomes is conjugated with glutathione by an excess of glutathione S-transferase activity present in the assay in the form of a mixture of mouse and rat cytosol (see Materials and Methods for details). Using this assay system, 10 µM OPZ decreased AFBO formation (measured as AFB-SG) by an average of 42 and 27% in the control and OPZ-treated animals, respectively. For comparison, 10 µM OPZ inhibited human microsomal oxidation of AFB to AFBO by 69% (Table 3Go). The inhibition was dose dependent, as higher concentrations of oltipraz increased inhibition of AFBO formation in both marmoset and humans.

To rule out the possibility that oltipraz was inhibiting the GST-mediated conjugation of AFBO rather than the cytochrome P450-mediated oxidation of AFB to AFBO, we carried out an additional assay that measured AFBO as AFB-8,9-dihydrodiol, as described previously (Monroe and Eaton, 1987Go); no glutathione S-transferases were present in the assay. The results were virtually identical, clearly showing that OPZ, at concentrations up to 50 µM, did indeed inhibit the oxidative formation of AFBO (results not shown).

Regardless of the nature of metabolite formation and concentration of inhibitor, differences between control and OPZ groups were not significant (p > 0.05). This suggests that there were no significant remnants of oltipraz present in the microsomal samples isolated from the oltipraz-treated marmosets.

In Vitro Inhibition of Oxidative AFB Metabolism by Ethoxyquin
The spectral properties of EQ or a metabolite thereof interfered with the HPLC-based method that was used to measure the various oxidative AFB metabolites. Therefore, the potential inhibitory properties of ethoxyquin on P450-mediated metabolism of AFB could not be assessed. Currently, we are developing an assay that circumvents this problem.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemoprotection against AFB has been proposed as an attractive strategy to combat the high incidence of hepatocarcinogenesis in those parts of the world where individuals are exposed to relatively high doses of this mycotoxin in their diet. The Qidong region in the Republic of China is such an area, and was recently chosen for a Phase IIa chemointervention trial in which 234 adults participated (Kensler et al., 1998Go; Wang et al., 1999Go). The FDA-approved antischistosomal drug, oltipraz (4-methyl-5-[N-2-pyrazynil]-1,2-dithiole-3-thione), which has been shown to protect rats from the carcinogenic effects of AFB (Kensler et al.,1987Go), was used in that study. While this trial is crucial in assessing the efficacy of this drug in vivo in humans, it was limited to measuring AFB-albumin adducts, urinary AFM1, and AFB-mercapturic acid. For obvious reasons, this trial is not able to measure effects directly in the target organ (liver). To complement this trial, we designed an in vivo study using non-human primates, which would allow us to examine directly whether dietary oltipraz and/or ethoxyquin (a) would afford protection from AFB-induced hepatic DNA damage, and (b) could induce aflatoxin B1 glutathione conjugating activity (AFB-SG) in liver. This is the first report to assess directly the chemoprotective effects of oltipraz in primate liver.

Initially, we conducted a pilot study to identify an appropriate non-human primate model for human hepatic AFB metabolism. While an in vitro comparison of marmosets, macaques, and humans showed similar oxidative metabolic profiles, their capacity to conjugate AFBO with glutathione was markedly different. Both humans and marmosets lacked any constitutive AFBO-SG activity, whereas the macaques expressed measurable constitutive GST activity toward AFBO (Table 1Go). As one of our main objectives was to assess whether a non-constitutively expressed, GST-mediated AFBO detoxification pathway could be induced in primates, as had been shown in rats (Kensler et al., 1986Go, 1987Go), we chose marmosets for our chemointervention study.

In contrast to most laboratory animals, the marmoset monkeys used in our experiments were not derived from an inbred strain, and therefore, considerable inter-animal variability was expected. To facilitate the interpretation of our results, we chose an experimental design that allowed each animal to serve as its own control. This was thought to be feasible by measuring aflatoxin-lysine albumin adducts before and after the animals received oltipraz, ethoxyquin, or vehicle only. Any treatment-related effects of the chemoprotectors on AFB metabolism in vivo was expected to be reflected by differences in serum albumin adducts in the blood samples drawn before and after treatment.

However, for reasons that are unclear and difficult to interpret, the AFB-lysine-albumin adduct data were highly variable. Values among control animals ranged from a 6% increase to a 33% decrease relative to adduct levels measured following the first AFB administration. A similar variability in response was observed for both the oltipraz- and ethoxyquin-treated marmosets, with the exception of animal 231 that exhibited a slightly larger reduction (48%) in adducts (Fig. 2Go). While the reasons for the variation among animals in the control group are not clear, it is possible that it is a reflection of the variability of the experimental procedure and/or the assay itself. Alternatively, the initial administration of [3H]-AFB itself could have altered its own metabolism by modulating gene expression. However, it is questionable whether such an effect would still be noticeable 26 days past the initial exposure to AFB. In any case, neither oltipraz nor ethoxyquin treatments affected AFB-albumin adduct formation significantly (p > 0.05).

It is informative to compare our results in non-human primates to the preliminary phase IIa chemointervention trial conducted by Kensler and colleagues (Kensler et al., 1998Go) in an aflatoxin-exposed Chinese population. The participants of that trial received a placebo, a daily dose of 125-mg oltipraz, or a once-weekly dose of 500-mg oltipraz. Assuming an average body weight of 70 kg, these doses translate into a daily dose of 1.8 mg/kg or a weekly dose of 7.1 mg/kg compared to the daily dose of 18 mg/kg used in our study. In addition, the human subjects received oltipraz for 8 weeks, whereas the marmosets received this compound for a total of 12 days. Furthermore, the study subjects were exposed to AFB daily, due to contamination of their diet; however, the marmosets received only 2 doses of AFB, separated by a 26-day period. Despite the aforementioned differences, results obtained from the human intervention trial and our study show a similar data spread of the control groups, i.e., albumin adducts also showed a random decline in the human subjects over a given time period. In the Kensler et al. study, the random median change in aflatoxin-albumin adduct levels in the placebo group ranged from a 0.6% increase to a 5.6% decrease. While individual values for this group were not provided in the report by Kensler et al. (1998), they are likely to be larger and similar to the albumin-adduct variations we have observed among the control marmosets. Interestingly, Kensler and colleagues found that only the weekly dosing regimen resulted in a reduction of aflatoxin-albumin adduct levels and only after 5 weeks of treatment. This finding raises the possibility that the 12-day treatment period may not have been long enough to see the maximum protective effect in the marmosets as judged by this biomarker.

Although no treatment-related effects were seen in the AFB-lysine biomarker, a treatment-related trend was observed with respect to AFB-DNA adduct formation in vivo in the liver, in both treatment groups (Fig. 3Go). Two ethoxyquin and all 4 oltipraz-treated animals exhibited less [3H]-AFB equivalents bound to DNA than any of the control marmosets. While two ethoxyquin-treated animals displayed the lowest levels of adducts, the remaining animal in this group had an adduct load comparable to those found in control animals. Statistical analysis revealed that the differences between the control and the OPZ groups reached significance (p = 0.047), whereas this was not the case for the EQ group (p = 0.151).

Currently, we have no clear explanation as to the different trends observed for albumin and DNA adducts measured in individual animals. However, it has to be appreciated that the only major metabolite that reacts with DNA is aflatoxin B1-8,9-exo-epoxide (exo-AFBO) (Iyer et al., 1994Go). In contrast, both the endo- and exo-stereoisomers of the epoxide readily hydrolyze in aqueous solution and form aflatoxin B1-8,9-dihydrodiol, which yields a dialdehydic phenolate ion that in turn can form Schiff bases with primary amines in proteins (Sabbioni et al., 1987Go) (Fig. 1Go). In addition, microsomal epoxide hydrolase (mEH) has been implicated in catalyzing the hydrolysis of AFBO (Decad et al., 1979Go), although the role of mEH is controversial (Johnson et al., 1997Go). Such a reaction would result in attenuating genotoxicity but not cytotoxicity. There is some limited epidemiological data to support the hypothesis that human mEH might protect against AFB-induced hepatocarcinogenesis (McGlynn et al., 1995Go). Recent work from our laboratory has provided evidence that human mEH attenuates the genotoxic effects of AFB in intact eukaryotic cells (Kelly et al., 1999Go). To complicate the picture further, Hayes et al. (1993) have identified an inducible enzyme, rat aflatoxin B1 aldehyde reductase (AFAR) that is capable of detoxifying the dialdehydic phenolate ion via conversion to the dialcohol. Recently, a human form of this enzyme has been cloned (Ireland et al., 1998Go, Knight et al., 1999Go). It is also important to note that, in contrast to protein adducts, AFB-DNA adducts can be repaired. In addition, O'Dwyer and colleagues (1997) have provided evidence that oltipraz treatment of HT-29 adenocarcinoma cells stimulates the repair of DNA adducts. Thus, it becomes clear that several potential mechanisms exist, each of which could modulate DNA and albumin-adduct formation independently. Therefore, the different trends found for DNA and albumin adduct formations are not necessarily contradictory. Our results also suggest that monitoring of AFB-lysine albumin adduct formation may underestimate the anti-carcinogenic effect provided by chemointervention in primates.

It has been demonstrated that hepatic tumor incidence induced by AFB is proportional to hepatic AFB-DNA adducts in rats and rainbow trout (Bechtel, 1989Go). As mentioned previously, aflatoxin B1-8,9-exo-epoxide (exo-AFBO) is by far the single most potent AFB metabolite that reacts with DNA (Iyer et al., 1994Go). Therefore, the steady-state level of exo-AFBO will be a major factor in determining an organism's susceptibility toward the genotoxic effects of AFB. Furthermore, several lines of evidence strongly suggest that a glutathione S-transferase-mediated pathway that detoxifies AFBO efficiently is the single most important mechanism that protects rodents from AFB0-induced hepatocarcinogenicity (for recent reviews see Eaton and Gallagher, 1994; Hayes et al., 1991).

Our data suggest that OPZ and EQ induce modest AFB-SG activity in the liver of some, but not all marmosets (Fig. 4Go). In addition, the majority of this activity (approximately 70%) was directed against the exo isomer of AFBO. While this study provides no direct evidence that the AFB-SG activity measured by us in vitro also occurs in vivo, Moss and coworkers (1985b) identified AFB-mercapturic acid in urine samples collected from marmosets that had been exposed to the mycotoxin. This finding strongly suggests that GST-mediated AFB-SG activity does occur in vivo. Furthermore, our results are consistent with the chemointervention trial carried out by Kensler and colleagues (Wang et al., 1999Go), who reported a statistically significant increase in urinary AFB-mercapturic acid in subjects exposed to a daily dose of 125 mg of oltipraz.

It should be noted that the hepatic GST activities toward AFBO exhibited by the non-human primates in this study were approximately 2 orders of magnitude lower than those in mice (Borroz et al., 1991Go), a species resistant to the carcinogenic effects of AFB. This finding suggests that, in contrast to rodents, a GST-mediated detoxification pathway is likely to play a lesser role in primates. The question arises, what alternative mechanism(s) could explain the DNA-adduct profile of the 2 treatment groups?

Langouet and co-workers (1995) have demonstrated that OPZ not only induces certain GST isoforms in human primary hepatocytes, but also inhibits both CYPs 1A2 (Ki = 10 µM) and 3A4 (Ki = 80 µM), the two major enzymes activating AFB in humans (Gallagher et al., 1994Go; Raney et al., 1992cGo); AFM1 is a marker substrate for CYP1A2 and AFQ1 for CYP3A4. In addition, Wang et al. (1999) provided evidence that 500 mg of oltipraz, administered once weekly to Chinese subjects participating in a chemointervention trial, decreased urinary AFM1 significantly. Consistent with these reports, we found that OPZ inhibited the formation of AFBO by marmoset hepatic microsomes at concentrations as low as 10 µM (Table 3Go). Furthermore, based on the report by Gupta et al. (1995), we estimated that the transient OPZ concentration in vivo in the plasma of the marmosets treated with this compound was approximately 50 µM. Based on our in vitro studies, such a concentration would provide substantial inhibition of CYP-mediated AFB oxidation if the hepatic concentration equaled or exceeded that of plasma. Thus, the decrease in DNA adducts found in the animals treated with OPZ may, at least in part, be rationally explained by inhibition of CYP-mediated AFBO formation.

We have identified 2 potential mechanisms that contribute to a decrease in steady state of exo-AFBO: GST-mediated detoxification of exo-AFBO and inhibition of cytochrome(s) P450 that form it. It is difficult to assess whether and to what degree these mechanisms alter DNA-adduct formation in vivo. However, if they affect the steady state level of exo-AFBO, then they should also affect AFB-DNA adduct formation. To test this hypothesis, we analyzed the correlation between the AFBO production measured in vitro and DNA adducts formed in vivo. When oltipraz inhibition and AFB-SG activities in the individual animals are not taken into account, linear-regression analysis reveals an R2 value of 0.395 with a p value of 0.038. When OPZ inhibition (i.e., AFBO data of OPZ-treated animals are substituted with data obtained using 50 µM OPZ; see Table 3Go), or AFB-SG activities (i.e., rates of AFB-SG activities of animals are simply subtracted from AFBO rates) were taken into account separately, the correlation improved, with R2 values of 0.475 (p = 0.019) and 0.416 (p = 0.032) respectively. When in vitro AFBO production was adjusted for both GST activity and CYP inhibition, the correlation improved further: R2 = 0.505 (p = 0.014) (Table 4Go). While the results from these linear-regression analyses have to be interpreted with caution, they suggest that both mechanisms are likely to play some role in decreasing AFB-induced genotoxicty in vivo. However, it should be emphasized that no definitive conclusions can be drawn as to the extent to which these mechanisms afford protection against AFB-induced hepatocarcinogenicity.


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TABLE 4 Correlation between Aflatoxin B1-DNA Adducts and Total Aflatoxin B1-8,9-Epoxide Formation Alone or Adjusted to Inhibitory Effects of Oltipraz and/or Aflatoxin B1-Glutathione Conjugating Activity of Individual Marmosets
 
In summary, our results indicate that dietary OPZ decreases AFB-DNA adduct formation in marmoset monkeys in vivo. In addition, OPZ and EQ induce modest AFB-SG activity in some but not all marmosets. Furthermore, OPZ is an effective inhibitor of cytochrome P450-mediated AFBO formation. Both induction of AFB-SG activity and inhibition of AFB activation by OPZ are likely to afford some protection against AFB-induced genotoxicity. Consistent with data obtained from a chemointervention trial in a human population, our results suggest that such an approach is likely to attenuate the adverse effects of AFB in primates. Currently, we are in the process of identifying the specific GST isoform(s) in marmoset liver that is/are responsible for AFB-SG activity.


    ACKNOWLEDGMENTS
 
The authors gratefully acknowledge Ms. Pat Egner and Dr. Thomas Kensler for AFB-albumin analysis of serum samples, Dr. Bill Griffith for advice on statistical analysis, and Dr. Ed Kelly for critical reading of this manuscript. This work was supported by NIH grants RO1 ES-05780, NIEHS Center grant ES-07033, and NIH grant RR00166 to the Regional Primate Research Center at the University of Washington. The help provided by the staff of the Regional Primate Research Center is gratefully appreciated.


    NOTES
 
1 To whom correspondence should be addressed at the Department of Environmental Health, University of Washington, 4225 Roosevelt Way NE, Suite 100, Seattle, WA 98195. Fax: (206) 685-4696. E-mail: deaton{at}u.washington.edu. Back


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Articles by Bammler, T. K.
Articles by Eaton, D. L.