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Identification of Aflatoxin M1-N7-Guanine in Liver and Urine of Tree Shrews and Rats Following Administration of Aflatoxin B1 Patricia A. Egner,*,† Xiang Yu,† Jesse K. Johnson,† Christopher K. Nathasingh,† John D. Groopman,† Thomas W. Kensler,† and Bill D. Roebuck‡ Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205, and Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755 Received May 28, 2003
Epidemiological studies have shown that exposure to aflatoxin B1 (AFB1) and concurrent infection with hepatitis B lead to a multiplicative risk of developing liver cancer. This chemicalviral interaction can be recapitulated in the tree shrew (Tupia belangeri chinensis). As an initial characterization of this model, the metabolism of AFB1 in tree shrews has been examined and compared to a sensitive bioassay species, the rat. Utilizing LC/MS/MS, an unreported product, aflatoxin M1-N7-guanine (AFM1-N7-guanine), was detected in urine and hepatic DNA samples 24 h after administration of 400 µg/kg AFB1. In hepatic DNA isolated from tree shrews, AFM1-N7-guanine was the predominant adduct, 0.74 ( 0.14 pmol/mg DNA, as compared to 0.37 ( 0.07 pmol/mg DNA of AFB1-N7-guanine. Conversely, in rat liver, 6.56 ( 2.41 pmol/mg DNA of AFB1-N7-guanine and 0.42 ( 0.13 pmol/mg DNA of AFM1-N7-guanine were detected. Rats excreted 1.00 ( 0.21 pmol AFB1-N7-guanine/mg creatinine and 0.29 ( 0.10 pmol AFM1N7-guanine/mg creatinine as compared to 0.60 ( 0.12 pmol AFB1-N7-guanine/mg creatinine and 0.69 ( 0.16 pmol AFM1-N7-guanine/mg creatinine excreted by the tree shrew. Furthermore, tree shrew urine contained 40 times more of the hydroxylated metabolite, AFM1, than was excreted by rats. In vitro experiments confirmed this difference in oxidative metabolism. Hepatic microsomes isolated from tree shrews failed to produce aflatoxin Q1 or aflatoxin P1 but formed a significantly greater amount of AFM1 than rat microsomes. Bioassays indicated that the tree shrew was considerably more resistant than the rat to AFB1 hepatocarcinogenesis, which may reflect the significant differences in metabolic profiles of the two species.
Introduction HCC1 is one of the most common cancers worldwide with incidence rates highest in geographical regions of Africa and Asia exhibiting climatic similarities of high heat, humidity, and poor food storage conditions. In the People’s Republic of China alone, 250 000 deaths annually have been attributed to HCC with 100 cases per 100 000 individuals per year reported in some affected areas (1). Development of HCC is strongly influenced by chemical and viral interactions, especially dietary exposure to aflatoxins and infection with HBV (2). Aflatoxins, naturally occurring mycotoxins found in contaminated foods including maize, peanuts, soy sauce, and locally fermented legumes, have been classified as human carcinogens by the International Agency for Research on Cancer (3). The relationship between dietary aflatoxin exposure and HBV was initially reported in nested case * To whom correspondence should be addressed. E-mail: pegner@ jhsph.edu. † Johns Hopkins Bloomberg School of Public Health. ‡ Dartmouth Medical School. 1 Abbreviations: AFB , aflatoxin B ; AFM , aflatoxin M ; AFP , 1 1 1 1 1 aflatoxin P1; AFQ1, aflatoxin Q1; AFB2, aflatoxin B2; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; oltipraz, 4-methyl-5-(pyrazin2-yl)-1,2-dithiole-3-thione; LC/MS/MS, liquid chromatography/tandem mass spectrometry; GGT + foci, gamma glutamyltranspeptidasepositive foci; GST-P + foci, glutathione-S-transferase-placental form positive foci.
control studies conducted in Shanghai. While a significant risk in developing HCC was seen unilaterally with exposure to aflatoxin or with chronic infection with HBV, a dramatic 60-fold increase in the risk of HCC occurred when aflatoxin exposure was concomitant with HBV infection (4). The presence of the metabolites of AFB1 in urine, either as AFM1 or as the depurinated nucleic acid adduct, AFB1-N7-guanine, was associated with 3- and 4-fold increases in HCC risk, respectively, after adjustment for the presence of the hepatitis B surface antigen (5). Subsequent studies in Taiwan (6) and Qidong (7) have confirmed these findings. Although aflatoxins are extremely potent hepatocarcinogens in animal models such as rat (8), rainbow trout (9), and duck (10), these species have proven to be resistant to infection with human HBV. Therefore, attempts to study the interaction between human HBV and aflatoxin exposure have been hindered. Recently, investigators at the Guangxi Cancer Institute in Nanning, P. R. C., have shown that a close phylogenetic relative of primates, tree shrews (Tupia belangeri chinensis), develop HCC after chronic infection with human HBV and/ or administration of AFB1 (11). Similar to the observations in the Shanghai cohort studies, the incidence of HCC was significantly increased in tree shrews infected with human HBV and concomitantly exposed to AFB1 (12). These findings indicate that the tree shrew may
10.1021/tx034106u CCC: $25.00 © 2003 American Chemical Society Published on Web 08/28/2003
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Figure 1. Biotransformation of AFB1 to urinary excretion products.
mimic the development of HCC seen in some populations and prove to be a valuable animal model for studying the molecular, clinical, and preventive aspects of HCC. The tree shrew appears to be more resistant to AFB1 hepatocarcinogenesis than the F344 rat, which in turn may be more sensitive than humans. Yan et al. (12) fed 400 µg/kg/day of AFB1 dissolved in milk to tree shrews, 6 days per week for 60-80 weeks (total dose, 22-26 mg), and reported a 12% incidence of HCC. We have produced a similar incidence of HCC in rats in a 2 year bioassay in which animals were gavaged with 250 µg/kg/day, 5 days per week for 2 weeks (total dose, 0.25 mg) (13). Pathways of metabolism are important determinants of interspecies susceptibility to the toxicity of xenobiotics such as AFB1. While AFB1 metabolism has been partially characterized in humans and well-studied in the rat, little is known about its metabolism in the tree shrew. As depicted in Figure 1, AFB1 undergoes an initial oxidation by the cytochrome P450 family members CYP1A2 and CYP3A4, yielding two aflatoxin-8,9-epoxide stereoisomers (14-16). The exo epoxide reacts with the N7 atom of guanine to form a promutagenic DNA adduct, AFB1-N7guanine (17, 18). This aflatoxin-DNA adduct is unstable and undergoes depurination leading to its excretion in urine (19). CYP1A2 also catalyzes the hydroxylation of AFB1 in the 10-position to yield AFM1, which is a major aflatoxin metabolite in humans (20). Other oxidation products such as AFP1 and AFQ1 are formed as well. AFQ1 appears to be preferentially formed by CYP3A4 (21). These oxidation products of aflatoxin can be excreted without further biotransformation or, in some cases, can be conjugated by UDP-glucuronosyl transferases. AFM1, a tertiary alcohol, is not a substrate for glucuronidation
(22). The aflatoxin-8,9-epoxide is a substrate for glutathione-S-transferases, which produces a stable, nontoxic, polar product excreted in the bile (23). The aflatoxin-glutathione product also undergoes sequential metabolism in the liver and kidneys to be excreted as a mercapturic acid (aflatoxin-N-acetylcysteine) in the urine (24). Initial studies to examine the metabolism of AFB1 in the tree shrew began with the radiometric detection of two epoxide-derived biomarkers, serum aflatoxinalbumin adducts and urinary AFB1-N7-guanine levels (25). Levels of these AFB1 biomarkers in the tree shrew were significantly lower than levels reported for comparably dosed male F344 rats (13). However, as seen in humans and rats, administration of oltipraz, a chemopreventive agent used to modulate AFB1 metabolism, significantly reduced the levels of these biomarkers in tree shrews (25). In the present investigation, we have directly compared the metabolic disposition of a single, oral dose of AFB1 in tree shrews and F344 rats. Using sensitive LC/MS/ MS techniques, we found that the tree shrew predominantly formed the hydroxylated metabolite of AFB1, namely AFM1. AFM1 is reported to be less carcinogenic than AFB1 on a molar comparison but is considered to be equitoxic (26, 27). In addition, although the wellcharacterized DNA adduct AFB1-N7-guanine was found in both species, AFM1-N7-guanine was found to be the predominant DNA adduct formed in the liver and excreted in the urine of the tree shrew. Characterization of AFM1-N7-guanine in vivo has not been previously reported. While the contribution of AFM1-N7-guanine to the oncogenicity of AFB1 is unknown at this time, the distinctive metabolic disposition of AFB1 in the tree
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shrew as compared to the rat may contribute in part to the reduced susceptibility of the tree shrew to hepatocarcinogenesis.
Experimental Procedures Animal Protocols. Adult tree shrews (T. belangeri chinensis) were purchased from High Water Farms (Kipling, NC) to establish a breeding colony. Offspring from successful pairings were fed a daily diet consisting of equal parts of cottage cheese, celery, apple, green grapes, and AIN 76A diet for all experiments (28). To examine the sensitivity of tree shrews to AFB1, two separate experiments were conducted. In the first, animals received 400 µg/kg/day of AFB in tricaprylin by gavage for five doses per week for 2 weeks. The average total of AFB1 administered to each animal was 375 µg. In the second regimen, tree shrews received the same daily dose of AFB1 by gavage but were dosed for 10 consecutive days and then rested for 10 days throughout six cycles of treatment. The cumulative dose administered was 3.6 mg of AFB1 per tree shrew. All animals were maintained for 2 years. At autopsy, grossly abnormal tissue was examined histologically and standardized sections of liver were cut from the two largest lobes. Visible tumors as well as half of the standardized sections were fixed in formalin, processed by routine methods, embedded in paraffin, sectioned, and stained with H&E. The remaining standardized sections were fixed in acetone, processed similarly, and stained for GGT+ foci and GST-P+ foci (29). As compared to the rat, few foci were encountered in the tree shrew and firm GGT identification was hampered by the extensive background staining found in the bile duct region. In H&E stained sections, foci of at least 10 cells were identified by altered tinctorial characteristics of hepatic parenchymal cells as described by Eustis et al. (30) However, these few foci found in the tree shrew did not react to GST-P antibodies of human, rat, or mouse origin. To directly compare the metabolic disposition of AFB1 in rats and tree shrews, four male tree shrews (100-160 g) and three male F344 rats (100-125 g) (Charles River, Boston, MA) received 400 µg/kg body weight of [3H]AFB1 (specific activity ) 12 µC/µmol) in DMSO by gavage. Animals were housed in glass metabolic cages; rats were fed AIN-76A and water ad libitium. Urine was collected on dry ice, and 24 h after AFB1 administration, animals were killed, and their livers were removed and immediately freeze-clamped. Tissues and urine were stored at -80 °C prior to analyses. Livers were also obtained from untreated animals for in vitro metabolic analyses. Caution: Solid aflatoxins are hazardous and some have been demonstrated to be human carcinogens. Extreme care needs to be exercised when handling aflatoxins including gloves, respiratory masks, and well-ventilated fume hoods. Aflatoxin residues can be destroyed using 3% bleach. Chemicals. Aflatoxins (AFB1, AFM1, AFP1, AFQ1, and AFB2), calf-thymus DNA, and Diazald were purchased from Aldrich/ Sigma Chemical Company (St. Louis, MO). [3H]AFB1 was purchased from Moravek Biochemicals (Brea, CA). All other chemicals and solvents were of analytical/reagent grade or higher. Urine samples were normalized using a spectrophotometic creatinine kit (Sigma). Instrumentation. Aflatoxin metabolites isolated following in vivo administration of AFB1 were analyzed by LC/MS/MS using a ThermoFinnigan Deca Electrospray-Mass Spectrometer (ThermoFinnigan, San Jose, CA) equipped with a 5 µm, 1 mm × 15 mm Luna column (Phenomenex, Torrence CA) eluted at 50 µL/min using an isocratic 60/38/2 mix of 1% glacial acetic acid, methanol, and acetonitrile. MS/MS spectra were generated from the appropriate singly charged parent ion using helium as a collision gas. Standard curves were constructed by measuring the mass abundance of the unique fragment ions generated from the parent aflatoxin or adduct (31). In Vitro Metabolism of [3H]AFB1. Liver microsomes were prepared from male animals, four F344 rats and four tree
Figure 2. Comparison of the oxidation of AFB1 in vitro and in vivo in tree shrews and rats. Left: Microsomes prepared from the liver of rats and tree shrews were incubated with 130 µM AFB1 and analyzed for the transformation of AFQ1, AFP1, and AFM1. Only AFM1 was formed from microsomes isolated from tree shrews. Right: The preferential formation of AFM1 by the tree shrew was confirmed by examining the urinary excretion of AFM1 in vivo. Values are means ( SE; n ) 4 in each group. shrews, according to standard procedures. Washed microsomes were resuspended on ice in 50 mM Tris buffer, pH 7.4, containing a NADPH generating system. Samples were incubated for 5 min at 37 °C prior to the addition of 0.13 µmol of [3H]AFB1 (specific activity ) 5.8 nCi/nmol) to a final concentration of 130 µM (32). Incubations were terminated at 10 min by the addition of an equal volume of ice-cold methanol, and samples were precipitated at -20 °C for 30 min prior to centrifugation at 12 000g. The supernatant was removed from the pellet, and the aflatoxin metabolites were separated by HPLC using a 5 µm C18 Prodigy column (Phenomenex) as described previously (33). Absorbance at 365 nm was measured using a Waters 996 photodiode array detector and compared to authentic standards. Following HPLC separation, 15 s fractions were collected from the column eluant and the radioactivity associated with each metabolite was measured using a Beckman liquid scintillation counter. Preparation of AFB1-N7-Guanine and AFM1-N7-Guanine. The 8,9-epoxy derivatives of both AFB1 and AFM1 were prepared by reacting the specific aflatoxin with dimethyldioxirane as previously described (34). The formation of the reactive epoxides was monitored by spectrophotometry. These epoxides were subsequently reacted with 1 mg/mL solutions of calf thymus DNA dissolved in water for 10 min and hydrolyzed with 0.1 N HCl for 15 min at 90 °C (35). Aliquots were concentrated on Oasis-HLB solid phase extraction columns (Waters Corporation, Cambridge, MA), and the resulting mixture was purified by reverse phase HPLC. Characterization and purity of the AFB1-N7-guanine and AFM1-N7-guanine isolated following the HPLC separation were confirmed using LC/MS/MS. In Vivo Metabolic Analyses. Aliquots (0.25 mL) of urine from each animal were concentrated by solid phase extraction and immunoaffinity chromatography as described in Walton et al. (31). AFB2 (1 ng) was added as an internal standard. DNA adducts were isolated from 1 g of liver and hydrolyzed to the guanine adducts as previously described (35). Total DNA content was measured spectrophotometrically using diphenylamine.
Results Figure 2 shows the pattern of metabolism of AFB1 by hepatic microsomes isolated from rat and tree shrew. Rat liver microsomes formed approximately 21 pmol/mg/min each of the oxidative products AFM1, AFP1, and AFQ1. Conversely, tree shrew microsomes did not form any detectable amounts of AFP1 or AFQ1 but a large amount of AFM1 (63.4 ( 13.0 pmol/mg/min). For microsomal incubations from both species, approximately 2-3% of the AFB1 was metabolized over 10 min. This microsomecatalyzed conversion of AFB1 by the tree shrew to AFM1 was evaluated in vivo by examining the urinary excretion of this hydroxylated metabolite following administration
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Figure 3. Liquid chromatography electrospray mass spectrometry of metabolites of AFB1 excreted in urine of tree shrews. Following enrichment by immunoaffinity chromatography, panels A, B, and C show AFM1-N7-guanine, AFB1-N7-guanine, and AFM1 eluting at 3.6, 5.6, and 11.3 min, respectively. Using MS/MS, the guanine fragment m/z 152.1 was used to measure the amount of AFM1-N7guanine and AFB1-N7-guanine. For AFM1, the amount of the carbonyl ion m/z 301.1 resulting from the fragmentation of the parent ion was used for quantitative determinations. The relative abundance of the fragment ions recorded for panels A, B and C is 74 024, 78 078, and 527 414 026, respectively. The lower panels D-F represent the MS/MS spectra of the protonated molecular ion. For AFM1-N7-guanine and AFB1-N7-guanine, only the guanine fragment (m/z 152.1) and the aflatoxin moiety (m/z 345.1 and m/z 329.1) were acquired. Panel F shows the complete AFM1 fragmentation pattern.
of 400 µg/kg AFB1. Also shown in Figure 2, the tree shrew excreted a 40-fold greater amount of AFM1 per mg of creatinine than the rat over the 24 h postdosing period. These results show clear differences in the patterns and extent of oxidation of AFB1 by each species. Liquid chromatography/electrospray mass spectroscopy of AFM1-N7-guanine isolated from urine of AFB1-treated tree shrews is depicted in Figure 3. This study is the first identification and quantitation of the AFM1-N7-guanine adduct in biofluids obtained after in vivo exposure to AFB1. The top panel represents the LC separation of the urinary metabolites following isolation by immunoaffinity antibody columns as previously described (31). Panels A-C show AFM1-N7-guanine, AFB1-N7-guanine, and AFM1 eluting at 3.6, 5.6, and 11.3 min, respectively. The AFB2, used as an internal standard throughout the procedure, had a retention time of 15 min. Analysis of the guanine fragment m/z 152.1, resulting from the fragmentation of the protonated parent ions m/z 496.1 (AFM1-N7-guanine) and m/z 480.1 (AFB1-N7-guanine) was used to determine the amount of DNA adducts in urine. The fragment ion m/z 301.1, reflecting the loss of the carbonyl group (m/z 28) from the parent ion molecule, was used to integrate the amount of AFM1 in the urine extract. Standard curves of mass abundance created from authentic standards were used to quantify all unknowns. Panels D-F show the MS/MS spectra for the product ions. Figure 4 shows the comparison of urinary levels of excreted aflatoxin-DNA adducts 24 h after dosing. A greater amount of AFB1-N7-guanine was excreted into rat urine than AFM1-N7-guanine, 1.00 ( 0.21pmol/mg creatinine vs 0.29 ( 0.10 pmol/mg creatinine. Equal amounts of creatinine were excreted by both species over the 24 h
Figure 4. Urinary excretion of aflatoxin-DNA adducts (left) and the hepatic levels of aflatoxin DNA adducts (right) remaining at 24 h after dosing rats and tree shrews with 400 µg/kg AFB1 (right). Values are means ( SE; n ) 3 for rats and n ) 4 for tree shrews.
collection period. In tree shrews, the amounts of guanine adducts excreted in the urine are roughly equal, 0.60 ( 0.12 pmol/mg creatinine for the AFB1-N7-adduct and 0.69 ( 0.16 pmol/mg creatinine AFM1-N7-guanine. The level of aflatoxin-DNA adducts remaining in the liver 24 h after treatment is also shown in Figure 4. AFB1-N7guanine was the major form of DNA adduct found in rat liver at this time (35). Using LC/MS/MS, we were able to confirm the formation of an additional adduct, AFM1N7-guanine. The level of the AFB1-N7-guanine found in rat liver 24 h after dosing was approximately 15 times higher than AFM1-N7-guanine, 6.56 ( 2.41 pmol/mg DNA vs 0.42 ( 0.13 pmol/mg DNA. By contrast, the amount of AFB1-N7-guanine detected in tree shrew liver, 0.37 ( 0.07 pmol/mg DNA, was about half the level of the AFM1N7-guanine, 0.74 ( 0.14 pmol/mg DNA. Overall, the sum of the aflatoxin-guanine adducts (AFB1-N7-guanine + AFM1-N7-guanine) was 7-fold higher in rats than the corresponding adduct burden detected in tree shrews.
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Table 1. Incidence of Hepatic Foci, Adenomas, and HCCs estimated lifetime age at dose of autopsy aflatoxin (µg) (months) 375
24
3600
24
250 a
11-23
sex and no. per group
no. of animals with hepatic neoplasms (%) adenofoci adenoma carcinoma
tree shrews males, 8 0 (0) females, 8 4 (50) males, 7 7 (100) females, 8 8 (100)
0 (0) 0 (0) 2 (29) 1 (13)
0 (0) 0 (0) 1 (14) 0 (0)
ratsa males, 45 6 (13)
4 (9)
5 (11)
Ref 13.
The incidence of tumors in tree shrews following administration of AFB1 in two separate 24 month bioassays is summarized in Table 1. One of 16 animals treated with a cumulative dose of 3.6 mg of AFB1 over 4 months developed an adenocarcinoma, although all animals developed preneoplastic lesions in this time frame. Our earlier work (13) indicated that rats treated 5 days per week for 2 weeks (total dose of 250 µg/rat) yielded comparable incident rates of preneoplastic and neoplastic lesions within 2 years. However, tree shrews treated with a total dose of 375 µg/tree shrew did not develop adenocarcinomas and only a 50% incidence of preneoplastic lesions. Thus, these studies confirm that there are substantial quantitative differences in the susceptibility of these two species to the hepatocarcinogenic actions of AFB1. The tree shrew is, however, intrinsically responsive to AFB1.
Discussion Attempts to probe the mechanisms of hepatocarcinogenesis resulting from the synergistic relationship between chronic HBV infection and dietary AFB1 exposure observed in humans have been hampered by the lack of a representative animal model system. Although rats have been extensively used to examine AFB1 metabolism, mechanisms of hepatocarcinogenesis, and the effectiveness of chemopreventive agents to modulate tumor development, the inability to infect rodents with human HBV has impeded the study of hepatitis infection on tumorigenesis. Nonhuman viral-infected duck (36) and woodchuck models (37) as well as HBV transgenic mice (38) provide demonstrations of viral interactions with AFB1. T. belangeri chinensis, a Southeast Asian tree shrew, may be a more desirable model due to its single and combinatorial responses to AFB1 exposure and infection with human HBV (12, 25). As part of an overall attempt to evaluate this model as a surrogate for human hepatocarcinogenesis, we have characterized the metabolism of AFB1 in this species and compared it to the F344 rat. The major oxidized metabolite of AFB1 excreted in the urine of tree shrews is AFM1. The preferential formation of AFM1 in vitro by tree shrew microsomes and the 40fold greater urinary excretion of AFM1 as compared to the rat indicate significant quantitative differences in the oxidation of AFB1 between these two species. Like the tree shrew, in humans, AFM1 is a major metabolite excreted following AFB1 ingestion and may account for several percent of the ingested dose (39). CYP1A2 appears to play a predominant role in the metabolism of
AFB1 to AFM1 in humans and contributes to the activation of AFB1 to its 8,9-epoxide. Human CYP3A4 forms AFQ1 from AFB1 as well as the 8,9-epoxide (21). AFQ1 was detected in the rat liver microsomal incubations but not those of the tree shrew, indicating the absence of this isoform from the latter species. Moreover, the chemopreventive drug oltipraz, which is an inhibitor of human CYP1A2 (40) and reduces AFM1 excretion in exposed rats (41) and humans (42), also inhibits AFB1-N7-guanine formation and urinary excretion in tree shrews (25). Thus, it is tempting to speculate that a CYP1A2-like enzyme is primarily involved in aflatoxin oxidation in the tree shrew. Interestingly, the robust rate of formation of AFM1 in the tree shrew resulted in the excretion of a heretofore, unreported urinary metabolite, AFM1-N7-guanine. This DNA adduct was tentatively identified, but without rigorous characterization, in rat liver DNA shortly after administration of a high dose of [3H]AFB1 (0.6 mg/kg) (43). Subsequent studies have characterized the formation of AFM1-8,9-epoxide from human liver microsomes (44) and the mutagenicity of synthetic AFM1-8,9-epoxide in bacteria (45). In our study, direct confirmation of the adduct was possible by using specific LC/MS/MS techniques previously used to measure other AFB1 metabolites (31). As shown in Figure 3, the AFM1 adduct fragments were similar to the AFB1-N7-guanine adduct resulting in an AFM1 moiety and a guanine fragment. Quantitative measurement of fragment ions resulting from MS/MS ionization allows a greater degree of specificity and sensitivity as compared to traditional MS techniques as well as HPLC spectrophotometric methods. AFB1-8,9-epoxide has been demonstrated to exist as two stereoisomeric forms, endo-epoxide and exo-epoxide, with the latter being the DNA reactive species (16). Bujols et al. (45) determined that AFM1-8,9-epoxide prepared from dimethyldioxirane was largely (90%) in the exo configuration. Presumably, the AFM1-DNA adducts detected in vivo in the present study are also derived from exoepoxides, but the impact of stereochemistry of AFM1-8,9epoxide on DNA binding has not been examined. AFM1 is 10-fold less carcinogenic than AFB1 in animal bioassays but is equipotent as a hepatotoxicant (26, 27). In accord with the diminished carcinogenicity of AFM1, Bujols et al. (45) observed that AFM1-8,9-epoxide was mutagenic in Salmonella typhimurium strain TA-100 in the absence of S9 but that this activity was 3-fold lower than that observed using equimolar concentrations of AFB1-8,9-epoxide. Marien et al. (46) have suggested that the sequence specificity of AFB1-mediated DNA damage differs from that of AFM1. Thus, the amount and sites of the formed adducts, as well as their rates of removal, may influence their carcinogenic potencies. The level of liver DNA adduction per unit AFB1 dosage has been reported to correlate with species susceptibility (26, 47, 48). For example, rainbow trout (found to be highly sensitive to AFB1) have a 25-fold greater hepatic burden of DNA adducts than resistant coho salmon (9). Comparison of the total hepatic burden of AFB1-N7-guanine and AFM1N7-guanine adducts in rats and tree shrews suggests that the rat would be about seven times more susceptible to AFB1 carcinogenicity. Although there is limited doseresponse characterization of the carcinogenicity of AFB1 in the tree shrew, our results (Table 1) indicate that a cumulative dose of AFB1 that is an order of magnitude higher than that administered to rats provided similar
Detection of Aflatoxin M1-N7-Guanine in Vivo
final incidences of preneoplastic lesions and adenocarcinomas. Tumor multiplicity, however, was considerably lower in the tree shrew as compared to the rat. There is a discrepancy between the relative levels of residual DNA adducts detected in liver 24 h after dosing and the relative aggregate amounts of AFB1-N7-guanine and AFM1-N7-guanine excreted in the urines of these animals over this time period. In addition to the sizable amount of AFM1-N7-guanine found in tree shrew urine, upon comparing the concentration of N7-guanine products excreted in the urine to the level of total guanine adducts detected in the liver 24 h after treatment, disproportionately more adducts were found in the urine of tree shrews than rats when compared to hepatic adduct burdens. These differences may relate to differences in the halflives of the two adducts or on their effects on cell turnover. Perhaps the high yield of AFM1 in the tree shrew, while resulting in lower burdens of AFB1-N7guanine per unit dose, enhances the cytotoxic actions of aflatoxin on the hepatocytes of these animals, resulting in an overall higher initial rate of adduct elimination. In this regard, we have observed that administration of 400 µg/day, 5 days per week, to 14 tree shrews resulted in death between 3 and 8 weeks of dosing. At autopsy, the livers appeared small, hard, and greenish in color. Histopathology clearly confirmed massive hepatic parenchymal cell necrosis. Thus, the tree shrew is nearly comparable to the rat in terms of AFB1 cytotoxicity. Measurements of AFM1 have been used as markers of the genotoxic potential of aflatoxin exposures in humans. Correlations have been observed between levels of AFM1 excretion and levels of AFB1 in corn and peanut oil samples collected from residents of Guangxi Autonomous Region, P. R. C. (39). Several studies have also described associations between urinary excretion of AFM1 and risk of developing HCC (5, 7). Examination of urine samples collected from endemic regions of high aflatoxin exposure will be required to determine whether the AFM1-N7guanine adduct is formed and excreted to any extent in humans and what role it might play in the molecular dosimetry of aflatoxins.
Acknowledgment. This work was supported by NIH Grants CA39416 and ES06052 and NIEHS Center Grant ES03819.
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