Affiliations of authors: J.-S. Wang, X. Shen, X. He, A.Zarba, P. A. Egner, J. D. Groopman, T. W. Kensler (Department of Environmental Health Sciences), L. P. Jacobson, A. Muñoz, K. J. Helzlsouer (Department of Epidemiology), The Johns Hopkins University, Baltimore, MD; Y.-R. Zhu, B.-C. Zhang, J.-B. Wang, Qidong Liver Cancer Institute, Qidong, Jiangsu Province, People's Republic of China; G.-S. Qian, S.-Y. Kuang, Shanghai Cancer Institute, Shanghai, People's Republic of China.
Correspondence to: Thomas W. Kensler, Ph.D., Department of Environmental Health Sciences, The Johns Hopkins School of Hygiene and Public Health, 615 N. Wolfe St., Baltimore, MD 21205 (e-mail: tkensler{at}jhsph.edu).
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ABSTRACT |
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INTRODUCTION |
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The extent of aflatoxin contamination in foods is a function of ecology
and is not completely preventable. Secondary prevention programs, such
as chemoprevention, may be useful in this setting.
Experimentally, aflatoxin-induced hepatocarcinogenesis can be inhibited
by more than a score of different chemopreventive agents
(7,8). One of the most potent and effective agents in these
animal models is the antischistosomal drug oltipraz (9).
Dietary administration of oltipraz to rats afforded complete protection
against aflatoxin-induced hepatocarcinogenesis when administered before
and during the period of carcinogen exposure. Chemoprevention in these
animals is reflected in lowered levels of several aflatoxin biomarkers
(9-11). The protective actions of oltipraz in this model are
thought to result primarily from an altered balance between the
activation and detoxification of aflatoxin B1
(AFB1) in the hepatocyte. As shown in Fig.
1, anticarcinogenic concentrations of oltipraz in
the diet can markedly induce activities of detoxifying phase 2 enzymes
such as glutathione S-transferases (GSTs) in rat tissues. This
induction facilitates conjugation of glutathione to the ultimate
carcinogenic species, aflatoxin-8,9-oxide, thereby enhancing its
elimination as a mercapturic acid (AFB-NAC) and coordinately
diminishing DNA adduct formation (12,13). Formation of these
DNA adducts is an essential but insufficient component of
aflatoxin-induced hepatocarcinogenesis. Induction of GSTs by oltipraz
in primary cultures of human hepatocytes has been observed
(14), although the catalytic activity of human GSTs toward the
8,9-epoxide appears to be lower than that of rodent GSTs (15).
Oltipraz can also influence phase 1 enzymes, particularly cytochrome
P450 activities. Enzyme kinetic studies on heterologously expressed
human CYP1A2 indicate that oltipraz is a competitive inhibitor, with an
apparent inhibition constant (Ki) of 10
µM (16), a pharmacologically achievable
concentration in rats and humans (17). CYP3A4 can also be
inhibited but with an eightfold higher Ki value
(16). Inhibition of CYP1A2 by oltipraz results in diminished
metabolism of aflatoxin to the genotoxic 8,9-epoxide and the
hydroxylated metabolite aflatoxin M1 (AFM1) in
primary cultures of rat and human hepatocytes (16). Urinary
excretion of AFM1 also drops dramatically immediately after
oltipraz administration to aflatoxin-treated rats (18). Thus, both
inhibition of cytochrome P450s and induction of electrophile detoxification
enzymes are likely to contribute to chemoprevention by oltipraz.
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SUBJECTS AND METHODS |
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The phase IIa chemoprevention trial with oltipraz was a randomized, placebo-controlled, double-blind study. Signed informed consent was obtained from all participants in accordance with institutional and federal guidelines of the People's Republic of China and of the United States. Two hundred forty adults in good general health without any history of major chronic illnesses and with detectable serum aflatoxin-albumin adduct levels at baseline were randomly assigned into one of three intervention arms: A) placebo, B) 125 mg oltipraz administered daily, or C) 500 mg oltipraz administered weekly. The trial included men and women and did not exclude individuals positive for hepatitis B virus surface antigen who had evidence of normal liver function. The rationale, methods, participant characteristics, compliance, adverse events, and initial results on modulation of biomarkers from this trial have been reported (21-23).
Study participants were recruited from Daxin Township, Qidong. Daxin is a rural farming community of approximately 40 000 residents and is located at the mouth of the Yangtze River, 15 km southeast of Qidong. After an initial screening of 1006 residents, 233 eligible participants actually reported to the Daxin Medical Clinic on the first day of the study (July 9, 1995), where they completed another physical examination and provided blood and urine samples. One additional participant, included in the randomization scheme, missed this initial visit but was allowed to participate starting at week 3. All study participants remained eligible as determined on-site and were given their randomized identification number and their first dose of study drug at the clinic. Thereafter, two identical capsules containing either placebo (arm A) or active drug (125 [arm B] or 250 [arm C] mg oltipraz) were administered daily for 8 weeks. In practice, each daily administration in intervention arm B contained one capsule with 125 mg oltipraz and one placebo capsule. In intervention arm C, individuals received 500 mg oltipraz (two 250-mg capsules of oltipraz) on the first day of each weekly cycle, followed by two placebo capsules per day for the next 6 days. Blood and urine samples, collected throughout the intervention and follow-up periods, provided the basis for measuring aflatoxin biomarkers. Overnight urine samples were collected on the second, third, and fourth mornings of weekly cycles 1, 3, 5, 9, 13, 15, and 17. Urine samples were collected at the participants' homes by village doctors and delivered by motorbike courier to the Qidong Liver Cancer Institute by mid-morning of each collection day at which time they were logged in, distributed into several tubes, and frozen at -20 °C. At the conclusion of the clinical trial, samples were shipped by air to Baltimore, MD, and stored at -80 °C before assay.
Analysis of Urinary Levels of AFM1 and AFB-NAC
Five milliliters of urine was adjusted to an acidic pH with 0.5 mL of 1 M ammonium formate (pH 4.5), and the volume was increased to 10 mL with water. The sample was then applied to a 1-mL preparative monoclonal antibody column at a flow rate of 0.3 mL/minute as described previously (24,25). The affinity column was then washed twice with 5 mL of phosphate-buffered saline (pH 7.4) and once with 10 mL of water to remove nonspecifically bound materials. Aflatoxin derivatives were eluted from the immunoaffinity column with 2 mL of 80% methanol in water. The eluate was reduced to about 100 µL with an argon stream and mixed with an equal amount of 5 mM triethylammonium formate (pH 3.0) before analysis by high-performance liquid chromatography (HPLC).
AFM1 and AFB-NAC were analyzed by reversed-phase HPLC on a Hewlett-Packard model 1040A diode-array detector connected in series with a Dynmax FL-2 fluorescence detector (366-nm excitation wavelength and 436-nm emission wavelength) to quantify aflatoxin metabolites. The HPLC column used was a C18 5-µm (4 x 250 mm) Microsorb analytical column (Rainin Inst. Co., Woburn, MA), and chromatographic separation was obtained by a 5%-25% ethanol linear gradient in water generated over a 25-minute period followed by isocratic elution with 25% ethanol in water, all at a flow rate of 1 mL/minute. The mobile phase was buffered with 5-mM triethylammonium formate (pH 3.0), and the column temperature was maintained at 45 °C. The eluted peaks were integrated and AFM1 and AFB-NAC were quantitated with the regression formulae obtained from standard curves for each metabolite. Authentic AFB-NAC (25) was eluted at 27.1 minutes and AFM1 was eluted at 34.4 minutes. The limit of detection of this fluorescence HPLC method was 0.5 pg for AFM1 and 2 pg for AFB-NAC.
The experimental conditions for metabolite analyses were optimized during method development for several parameters, including volume of urine sample, size of immunoaffinity column, and constitutents of the immunoaffinity resin. An equal admixture of two monoclonal antibodies (2B11 and 2F5) was used. The capacity of the column to bind aflatoxin derivatives was assessed with AFM1 and [3H]AFB1. Up to 500 ng of AFM1 applied to a 1-mL affinity column could be recovered at rates of 90%-98%. To ensure quality control of the analysis, all measurements were conducted on blinded samples and 5% of the samples were analyzed pairwise with "spiked" (0.1 ng of AFM1 added) and "unspiked" sample for the same individuals. Cross-analysis with a separate HPLC-fluorescence system was also carried out on all outlier samples. The variances between the pairwise and cross-analyses were less than 5%.
Characterization of Urinary AFM1 and AFB-NAC
A Finnigan LCQ liquid chromatography mass spectrometry system was used to perform electrospray ionization mass spectrometry in positive-ion mode to confirm the identity of AFM1 and AFB-NAC. The elution fractions of either AFM1 or AFB-NAC from the HPLC-fluorescence system were collected and loaded onto a Waters Oasis column (3 mL) to remove salts. The column eluate was then reduced to about 100 µL with an ultra-high-purity argon stream before analysis by liquid chromatography-mass spectrometry. A Thermal Systems Products HPLC was used to provide a constant flow of 200 µL/minute to an ODS J-sphere M-80 column (2 x 250 mm) (YMC, Inc., Wilmington, NC). A gradient starting at 4% acetonitrile/2% methanol and finishing at 13% acetonitrile/12% methanol in 25 minutes was used for separating aflatoxins. A buffer containing 0.1% formic acid (pH 2.5) was used throughout the run. The HPLC column was maintained at 55 °C and the column effluent was directed through a UV detector (365 nm) and into the electrospray ionization interface on the mass spectrometer. The sample was scanned from 200 to 600 atomic mass units at 1 second per scan. A collision energy of 22% was used for collision reaction monitoring.
Statistical Analyses
The distributions of the levels of both urinary metabolites were highly skewed. Therefore, nonparametric Wilcoxon rank sum tests were used to compare urinary metabolite levels in each treatment arm to the results in the placebo arm. For this analysis, values of L/2 (where L is the limit of detection) for AFM1 and AFB-NAC were inserted for all nondetect values, as described by Hornung and Reed (26). To test whether lower levels of AFM1 excretion at week 5 predicted the overall change in aflatoxin-albumin adducts from baseline through week 13, we used the slope of adducts in each individual as the outcome and, by linear regression methods, determined whether the individuals with the lowest levels of urinary AFM1 at week 5 had the fastest declining adducts. Because a decline in aflatoxin-albumin adducts had only been observed in those individuals randomly assigned to receive the weekly dose of 500 mg oltipraz (22), this latter analysis was restricted to that group. All reported P values are two-sided.
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RESULTS |
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Fig. 2, A, shows a representative chromatogram of
AFM1 and AFB-NAC immunopurified from 5 mL of urine collected
from a study participant and detected by the HPLC-fluorescence system.
Peaks corresponding to AFM1 and AFB-NAC by retention time
and fluorescence characteristics were then isolated from multiple urine
samples and subjected to liquid chromatography-mass spectrometry
analysis. Fig. 2
, B, shows the mass spectrum of 200 pg of
AFM1 isolated from these urine samples. The positive
molecular ion (MH+) at m/z = 329 is identical to
that observed with authentic standard (18). Fig. 2
,
C, shows
the data obtained by collision reaction monitoring of the m/z
329 ion. The major fragmentation ions for the m/z 329 ion are
m/z 301, 273, and 259, and they reflect the loss of
CO+, loss of another CO+ from the m/z 301
ion, and loss of CH2 from the m/z 287 ion,
respectively. Fig. 2
, D, shows the mass spectrum of AFB-NAC isolated
from urine. The molecular ion m/z = 492 is identical to that
observed with authentic standard and the mercapturic acid characterized
in the urine of rats receiving AFB1 (25). A
monosodium adduct at m/z 514 is also observed. Collision
reaction monitoring of the m/z 492 ion is shown in Fig. 2
, E.
The major fragmentation ion is m/z 329, which reflects the
loss of the N-acetylcysteine group. Characteristic aflatoxin
fragments at m/z 311 and 271 are also seen.
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Analyses were conducted on 189 urine samples collected during week
5, the midpoint of the active intervention phase of the study. Because
of the short half-life of urinary aflatoxin metabolites, this period
was judged a priori as most likely to reveal possible
treatment-related effects on biomarker levels. Samples collected at
other time points have not been analyzed for urinary aflatoxin
biomarkers. As shown in Table 1, 197 individuals
remained active participants in the intervention trial at this time.
One hundred ninety-five of these participants provided an overnight
urine sample on the morning of the second day of weekly cycle 5. Six of
these samples had abnormally low levels of creatinine (<12 mg/dL)
and were excluded from further analysis. Review of study compliance, as
judged by pill counts, indicated that all but four of these 189
individuals took their assigned capsules during the 48 hours before
this urine collection. Thus, in practice, urine was collected 36-48
hours after administration of the weekly dose of 500 mg oltipraz and
12-24 hours after administration of the most recent of the daily doses
of 125 mg of oltipraz.
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Elevation of AFB-NAC (Phase 2) Excretion
AFB-NAC could be detected in 124 (65.6%) of the 189 urine
samples, with the samples in which AFB-NAC was not detected
distributed as 43.1% (31 of 72 samples), 21.1% (12 of 57
samples), and 35.0% (21 of 60 samples) of the placebo arm and arms
receiving 125 mg oltipraz daily and 500 mg oltipraz weekly,
respectively. Fig. 3 (right) shows the distributions of the levels of
AFB-NAC in the three intervention arms. The median level of AFB-NAC
in the urine of participants receiving placebo was 7.1 pg/mg of
creatinine, with a range of nondetectable to 156.6 pg/mg.
Administration of 125 mg oltipraz daily for 4 weeks before collection
led to a statistically significant (P = .017) elevation in the
amount of AFB-NAC excreted (median, 18.6 pg/mg; range, nondetectable
to 245.5 pg/mg). This increase was primarily driven by the diminished
number of nondetectable values within this treatment group. By
contrast, administration of 500 mg oltipraz once a week for 4 weeks had
no statistically significant (P = .871) effect on urinary excretion
of AFB-NAC (median, 8.3 pg/mL; range, nondetectable to 189.4 pg/mL).
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DISCUSSION |
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Because AFM1 is formed by the same cytochrome P450 that yields the 8,9-epoxide, AFM1 may serve as a reasonable surrogate for the genotoxic potential of aflatoxin exposures in individuals. Such a possibility has been examined in residents of Fusui County, Guangxi Autonomous Region, People's Republic of China, where a high incidence of HCC has also been reported. Zhu et al. (34) analyzed AFM1 concentration in urine samples by enzyme-linked immunoabsorbent assay and noted correlations between levels of AFM1 excretion and levels of AFB1 in corn and peanut oil samples collected from different households. Using immunoaffinity and HPLC methods, Groopman et al. (24) have observed that measurements of urinary excretion of AFM1 and the labile DNA adduct aflatoxin-N7-guanine showed strong and highly statistically significant correlations with aflatoxin intake in this region. Moreover, direct associations between excretion of AFM1 and levels of aflatoxin-albumin adducts in individuals were noted in this ecologic survey (35). In a prospective, nested, case-control study, Qian et al. (4) reported that the relative risk of HCC for individuals whose urine contained AFM1 was 4.4 (95% confidence interval = 2.1-9.6) compared with those not excreting this biomarker. Similarly, Yu et al. (36) reported a statistically significant dose-response relationship between urinary AFM1 levels and HCC. The odds ratio encompassing the highest with the lowest tertile of AFM1 levels was 6.0 (95% confidence interval = 1.2-29.0). Thus, urinary levels of AFM1 may provide some index of altered risk for use in chemopreventive interventions. Indeed, Scholl et al. (18) have reported that excretion levels of AFM1 fell precipitously when oltipraz was administered to rats continuously exposed to AFB1. Diminished AFM1 excretion persisted for the duration of the intervention but rebounded rapidly when the intervention was discontinued. This transient nature of the inhibition in vivo reflects the competitive inhibition of CYP1A2 by oltipraz and the relatively short half-life (~24 hours) of the target enzyme (16,37).
There is a moderate association between downward modulation of the slopes of curves for serum aflatoxin-albumin adducts as a function of time and diminished excretion of AFM1 in the urine of study participants randomly assigned to the arm receiving 500 mg oltipraz weekly. This association suggests that inhibition of cytochrome P450 activity leads to the observed decline in serum aflatoxin-albumin adducts. By contrast, enhanced glutathione conjugation of the 8,9-epoxide to yield AFB-NAC does not appear to be associated with altered aflatoxin-albumin adduct levels. A comprehensive analysis of the predictive value of the aflatoxin-albumin biomarker has been conducted in rats. This analysis indicated a reasonable association between the level of the aflatoxin-albumin biomarker and risk of HCC at the population level but no association between biomarker levels and individual risks of HCC (11). Moreover, measurements of adduct levels, in both DNA and protein, consistently underestimate the chemopreventive efficacy of oltipraz in rodent aflatoxin hepatocarcinogenesis models (9-11). Thus, adduct biomarkers, particularly the aflatoxin-albumin adduct, do not provide the full picture. Reduction in AFM1 formation may have direct consequences in addition to those reflected in its role as a surrogate marker for diminished aflatoxin genotoxicity. Although AFM1 is considerably less active as a hepatocarcinogen in the rat (38), it is equipotent to AFB1 as a hepatotoxin (39). Perhaps reduction in AFM1 production by oltipraz and the attenuation of the contribution of AFM1 to the cytotoxic autopromoting component of aflatoxin hepatocarcinogenesis are important elements of the overall chemopreventive outcome. Other oxidative metabolites of aflatoxin (e.g., aflatoxin P1 and aflatoxin Q1) are much less toxic than AFB1 or AFM1; however, it is not known whether inhibition of CYP1A2 by oltipraz appreciably shunts aflatoxin metabolism to other cytochrome P450 enzymes. Finally, additional chemopreventive mechanisms unrelated to effects on the metabolism of aflatoxin have also been identified for oltipraz (40-42).
Measurement of urinary levels of AFM1 can serve as a biomarker for aflatoxin exposure. Cheng et al. (43) using similar analytic methodologies have recently reported on excretion rates of urinary AFM1 in 69 counties throughout rural China. They reported the mean and highest levels of AFM1 excretion to be 3.2 and 108 ng per 12 hours, respectively. If an excretion rate of 0.7 g of creatinine per 12 hours (normal range, 0.3-1.0 g per 12 hours) is assumed, then the mean level of excretion in the placebo arm of this trial would be 12 ng per 12 hours. The nearly fourfold higher excretion rate in the present study reflects the selection of Qidong as a study site because of the known high prevalence of aflatoxin-contaminated foods in the diet and the elevated risk of HCC in this region. In the study by Cheng et al. (43), investigators intentionally dosed themselves with 1.0 µg of pure AFB1 and determined that 5%-6% of the administered dose was excreted over the subsequent week as AFM1. Other reports from field studies have estimated that 1%-2% of ingested AFB1 is eliminated as AFM1 in humans (34). By extrapolation, it can be inferred that the AFB1 exposure of the residents of Daxin Township in Qidong in the summer of 1995 was approximately 1-2 µg/day. However, because of the heterogeneity of aflatoxin contamination of foodstuffs, it is likely that there is large inter- and intra-individual variation in this estimate. Nonetheless, this estimate is less than half that reported for the region 15 years earlier (44) and may reflect a switch from corn to rice as the primary dietary staple within the past decade. After rising steadily during the 1970s and 1980s, age-adjusted incidence rates for HCC in Qidong have plateaued and perhaps begun a modest decline over the past few years. Whether such trends reflect reduced aflatoxin exposures, the implementation of hepatitis B virus vaccination programs, or additional combinations of factors remains unclear. Nonetheless, incidence rates for HCC remain untenably high. If sustainable over the long term, statistically significant alterations in the formation and fate of the ultimate carcinogenic metabolite aflatoxin-8,9-epoxide (as brought about through multiple mechanisms with oltipraz at one point in this intervention) could provide substantive protective effects against the adverse actions of aflatoxin in this population.
A follow-up 12-month phase IIb intervention with oltipraz will be conducted in this region from 1999 through 2000. The primary goal will be to assess the full extent and persistence of the initial modulation of aflatoxin biomarkers seen in the phase IIa trial. Participants will be randomly assigned to receive placebo or 250 or 500 mg oltipraz once a week. The exclusive selection of a weekly schedule is driven in part by the effects of 500 mg oltipraz weekly on the urinary and serum aflatoxin biomarkers, as well as some practical considerations. The weekly schedule is likely to improve compliance, both by attenuating the intense monitoring required with daily administration of study drug and by reducing the occurrence of adverse events. In this phase IIa trial, fewer adverse events were reported by individuals in the weekly arm than in the daily arm (21), perhaps reflecting the lower cumulative dose. Drug costs are also substantially reduced in a weekly intervention, rendering the widespread use of oltipraz in high-risk populations more feasible. The upcoming phase IIb trial is also an intermediate step in the development of chemopreventive strategies in that phase III studies with a duration of 5 years or more will ultimately be required to establish the extent to which a decrease in the concentration of aflatoxin biomarkers translates into a reduction or delay in the development of HCC.
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NOTES |
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We thank Drs. Gary Kelloff, James Crowell, Earnest Hawk, Charles Boone, Kenneth Olden, and Gary Gordon for their helpful discussions and Drs. Nancy Davidson, Mary Gorman, and Hans Prochaska for their medical oversight in Qidong. We also thank the staff of the Daxin Medical Clinic, the village doctors, and the residents of Daxin Township for their participation.
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Manuscript received July 20, 1998; revised December 3, 1998; accepted December 21, 1998.
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