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Interactions of aflatoxin B1 and blood components of various species in vitro: Interconversion of aflatoxin B1 and aflatoxicol in the blood
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
67, 292-30
1 ( 1983)
Interactions of Aflatoxin B, and Blood Components of Various Species in Vitro: Interconversion of Aflatoxin B, and Aflatoxicol in the Blood SUSUMUKUMAGAI,~
NAOKONAKANO,ANDKAGEAKI
AIBARA
Department of Biomedical Research on Food, National Institute of Health, Kamiosaki, Shinagawa-ku, Tokyo, Japan Received June 24, 1982; accepted October 21, 1982 Interactions of Aflatoxin B, and Blood Components of Various Species in Vitro: Interconversion of Aflatoxin B, and Aflatoxicol in the Blood. KUMAGAI, S., NAKANO, N., AND AIBARA, K. ( 1983). Toxicol. Appl. Pharrnacol. 67,292-301. The fate of athttoxin B1 (AFB,) in the blood of various species of animals was studied in vitro. Examination of the distribution of radioactivity in blood incubated with [“C]AFT$ at 37°C showed that high levels of radioactivity were associated with blood cells. The radioactivity was readily removed from the blood cells by washing with fresh plasma, indicating loose binding of AFB, to blood cells. Most of the radioactivity in plasma was bound to protein. These results suggest that a large part of the AFB, in blood in vivo may be carried not only by the plasma proteins but also by the blood cells. When chloroform extracts of plasma of [“C]AFB,-treated mouse, rat, duckling, and hamster blood were developed by thin-layer chromatography, hi levels of radioactivity were found in both the AFB, region and the aflatoxicol (AFL) region. Incubation of blood with nonradioactive AFB, and AFL showed marked interconversion of AFB, and AFL in the blood of rats, hamsters, mice, and Mongolian gerbils, but not in the blood of guinea pigs, rhesus monkeys, squirrel monkeys, or humans. Interconversion occurred in red blood cell suspensions but not in plasma, indicating that the red blood cells are responsible for AFB,-AFL interconversion in the blood.
The aflatoxins are secondary metabolites of Aspergillus flaws, aflatoxin Br (APB,) being the most potent hepatotoxic and hepatocarcinogenic compound among these metabolites. There is a large species variation in suscep tibility to APB1 poisoning. The rabbit and duckling are the most sensitive to the acute effect of APB1 (Patterson, 1973). The rat, duckling, and rainbow trout are highly susceptible to the carcinogenic effect of APB, (Newberne and Butler, 1969; Hsieh et al., 1977). In the human, hepatitis and liver cancer because of afIatoxin intake have been suggested by epidemiologic studies (Krishnamachari et al., 1975; Wogan, 1973) though comparative susceptibility remains uncertain. To elucidate the mechanisms of toxic and carcinogenic actions of atlatoxins, many stud’
To
whom all correspondence should be addressed.
004 1408X/83/020292-
10.$03.00/O
Copyright 0 1983 by Academic Press, Inc. All fights of r~pmduction in any form reserved.
292
ies have focused on phenomena occurring inside the liver, which is the primary target organ of the toxins, but only a few on phenomena outside the liver especially on the fate of aflatoxin in the blood. Although binding of APB, to plasma proteins, which may affect the distribution and elimination of the toxin, has been studied in some species (Bassir and Bababunmi, 1973; Ueno et al., 1980), little is known about the binding of the toxin to blood cells or the biotransformation of the toxin by the blood. In the present study, the fate of APB, in the blood was studied in vitro with blood from various animal species. METHODS Animals. Wistar rats (8 to 11 weeks and 1 to 2 years old), Fischer rats (8 to 16 weeks old), ICR mice (7 weeks old), BALB/c mice (7 to 9 weeks old), C57BL mice (7 to 9 weeks old), golden hamsters (3 and 6 months old), Mongolian gerbils (4 months old), Khaki-Campbell ducklings
AFB,-AFL
INTERCONVERSION
(500 g), Hartley guinea pigs (2 months old), squirrel monkeys (adult), rhesus monkeys (adult), and humans (adult) were used. Fischer rats were castrated under ether anesthesia at 15 weeks and used at 16 weeks. Chemicals. Aflatoxins B,, MI (AIM,), Bz. (AFBz,), and aflatoxicol (AFL) were purchased from Makor Chemical Ltd. (Jerusalem, Israel) and dissolved in benzene:acetonitrile (98:2). [‘4C]AFB, (specific activity, 40 mCi/mmol, labeled in all carbons except the extranuclear-OCH,) dissolved in methanol was purchased from Moravek Biochemicals, Inc. (California). Blood sampling. Venous blood was taken from the vein at the elbow of humans and from the femoral vein of monkeys, both without anesthesia, and from the vena cava of other animals under light ether anesthesia. Arterial blood was taken from the aorta of rats under ether anesthesia. Immediately after sampling, all blood samples were mixed with heparin (20 units/ml). Preparation of red blood cells. After the plasma was separated by centrifugation at 3000 rpm for 10 min, white blood cells and platelets were discarded, and the red blood cells were washed three times at 0 to 4°C with 5 vol of saline buffered to pH 7.2 with 15 mM 2-amino-2-(hydroxymethyl)- 1,3-propanediol (Tris) containing 1 mM glucose (White and Rothstein, 1973). Preparation ofheated blood. Rat blood was diluted lofold with buffered saline and a portion of the diluted blood was heated in boiled water for 15 min. Unheated diluted blood was used as a control. Incubation. After evaporation to dryness under Nz gas, 200 ng of [14C]AFB,, 200 ng of [“‘C]AFB, mixed with 1500 ng of nonradioactive AFB, , or 400 or 2000 ng of nonradioactive AFB, or AFL was incubated aerobically with whole blood, plasma, red blood cells suspended in buffered saline (50% hematocrit), or heated or unheated diluted blood in a shaking water bath at 37°C. Unless otherwise stated, 0.6 ml of each sample was used. The dried aflatoxins and the blood samples were mixed rapidly before incubation. Preliminary experiments showed that dried AFB, was almost completely redissolved in the blood. Dried [i4C]AFBI (1700 ng) and blood in test tubes were mixed rapidly; the blood was removed, and the test tubes were washed with chloroform and perchloric acid. Only 1.6 -t 0.3% (n = 3) of the total radioactivity was contained in the washings. Immediately after incubation, plasma or buffered saline was separated from the blood cells by centrifugation at 3000 rpm for 5 min. Preparation of protein-free fraction of the plasma. Ultrafiltration employing a Micropartition System (Amicon Co., Massachusetts) was used to separate protein-bound and -free fractions of plasma. In accordance with the op erating instructions, the ultrafiltrate was obtained by centrifugation of the cell containing plasma at 3000 rpm for 15 min. No protein bands of the ultratiltrate were found on electrophoresis plates (cellulose acetate plates, Helena Lab., USA) stained by Ponceau 3R, while the same amount of plasma showed several protein bands. Extraction
and
thin-layer
chromatography
(TLC).
293
IN BLOOD
Plasma, buffered saline, or distilled water was extracted three times with 30 vol of chloroform. After being evap orated at 40°C under N2 gas, the extracts were quantitatively transferred with chloroform onto TLC plates. Known amounts of authentic AFBi, AIM,, AFB,, and AFL were cochromatographed. TLC plates of silica gel 60 (Merck, Darmstadt, West Germany) and TLC plastic sheets of silica gel 60 (Merck), both without fluorescent indicator, were used for separating nonradioactive metabolites and 14C metabolites, respectively. The developing solvent was chloroform-acetone-water (88: 12: 1.5) (Stubblefield et al., 1969) or ethyl ether (Patterson and Roberts, 1971). Fluorescent spots on the plates were visualized under ultraviolet light (365 nm). Determination of radioactivity. Radioactivity was determined in a scintillation counter (Type LS-2OOB, Beckman). Plasma, buffered saline, and the ultrafdtrate of plasma were counted after being mixed with 10 ml of ACS II (Amex&am Corp., Arlington Heights, Ill.). The blood cells and the aqueous layer of plasma were mixed with perchloric acid and hydrogen peroxide, warmed in the oven at 70°C for 30 to 60 min (Mahin and Lotberg, 1966), mixed with 10 to 15 ml of ACS II, and counted. TLC plastic sheets containing radioactive metabolites were cut into six portions, corresponding to the region containing both AFM, and AFB&, the AFL region, the AFB, region, the origin, the region between AFB, and the solvent front, and the region between AFBz, and AFL. After each portion was transferred to a counting vial containing 1 to 2 ml of water, silica was removed from the plastic sheet by an ultrasonic cleaner (Branson Instrument Co., Connecticut) and a mixer (Thennomix Co., Ltd., Tokyo, Japan). The silica-water suspension was mixed with 10 to 15 ml of ACS II and counted. Analysis of nonradioactive AFBl and AFL. AFB, and AFL concentrations on TLC were measured in a spectrophotofluorometer equipped with a TLC scanner (Hitachi Co., Japan). Standard curves were obtained with cochromatographed authentic aflatoxins. Since the maximum activation and emission wavelengths for AFB, were about 363 and 427 nm, respectively, and those for AFL were about 328 and 422 nm, respectively, the wavelengths were set at the corresponding values. The range of measurement was 10 to 150 ng of aflatoxin. Absorption of ultraviolet light by AFB, and AFL bands scraped from TLC plates was determined in a standard spectrophotometer (Type 181, Hitachi Co.) and a double-wave length double-beam spectrophotometer (Type 556, Hitachi Co.). Statistics. The statistical significance of differences between means was determined by Student’s t test.
RESULTS
Distribution [‘4C]AFB,
and
Biotransformation
of
Mouse, rat, hamster, guinea pig, duckling, and human blood were incubated with
294
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[14C]AFB,. Radioactivity contained in the chloroform extracts of plasma, in the residual aqueous layer, and in blood cells was determined. Radioactivity in the chloroform extracts of plasma was calculated from that contained in the six portions of the TLC plates. Distribution of radioactivity in the blood differed among species (Fig. 1). In the blood of rats of both sexes and in mice, higher radioactivity was found in the chloroform extracts of plasma than in blood cells. A similar relationship was observed in human blood, though the difference in radioactivity between these two fractions was much smaller than that seen in rat blood. In hamster and guinea pig blood, the reverse relationship was observed. In duckling blood, radioactivity in the chloroform extract and in blood cells was similar when low concentrations of AFB, were used, but blood cells showed higher radioactivity than extracts when high concentrations of AFBl were used. Radioactivity in the aqueous layer was lower than that in the other two fractions for all animal species, but it was relatively high in the human blood. After being incubated with [‘4C]AFB, (200 ng/0.6 ml) for 10 min, blood of Wistar rats (1 to 2 years of age, male) and hamsters (3 to 6 months of age, female) was separated into blood cells and plasma, and the blood cells were washed repeatedly with an equal volume of fresh plasma of the corresponding animal species. Radioactivity in each plasma and washed blood cell specimen was counted. Most of the radioactivity associated with blood cells was transferred to plasma by washing three times. The proportion of radioactivity remaining in blood cells was 7.0% (n = 2) for hamster blood and 4.9% (n = 2) for rat blood. Profiles of radioactivity on TLC plates of chloroform extracts of plasma (Fig. 2) show that the proportion of radioactivity in the AFB, region of the total chloroform extractable radioactivity decreased with incubation time. The pattern of transformation of [‘4C]AFB, differed among animal species. In mouse, rat, and hamster blood, conversion of AFB, to the metabolite in the AFL region took place. The percentage of the metabolite
AND AIBARA
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FIG. 2. Biotransformation of [?]AFB, in blood and plasma incubated with [‘4C]AFB,. Blood samples were those shown in Fig. 1. *M and F indicate male and female, respectively. **a, origin; b, AFBz, and AFMI; C, the region between AFBI, and AFL: d, AFL; e, Al%,; f, the region between AFB, and the solvent front. The value of AFL at 10 min was significantly lower (p < 0.05) in mouse blood than in the rat and hamster blood.
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FIG. 3. Proportion of radioactivity of the protein-free plasma to total radioactivity in the plasma. Blood samples were those shown in Fig. 1 for the rat, hamster, guinea pig, duckling, and human. For the mouse, blood was taken from eight animals. Blood samples were incubated with 200 ng of [‘%]AFB, or 200 ng of [‘%]AFBi plus 1500 ng of AFB, for 10, 60, or 120 min. Each value was obtained from radioactivity in the plasma (40 ~1) and the plasma ultrafiltrate (40 ~1). *M and F indicate male and female, respectively.
formed after a IO-min incubation was lower in mouse blood than in rat or hamster blood. In duckling blood, the same pattern was observed when low concentrations of AFB, were used, but not when high concentrations were used. A single blue spot was observed under ultraviolet light in the AFL region when high radioactivity was found in this region. With increase in the incubation time, radioactivity in the region less polar than AFB, increased gradually in guinea pig blood as did that at the origin in human blood. For comparison, mouse, rat, duckling, hamster, or human plasma or distilled water was incubated with [14C]AFB, for 120 min. About 90% of the chloroform extractable radioactivity was found in the AFBl region (Fig. 2).
In another series of experiments, radioactivity of protein-free plasma was determined by the ultrafiltration technique, after blood was incubated with [‘4C]AFB,. Only a small proportion of the radioactive compounds in the plasma was free of protein. The proportion of radioactivity in the ultrafiltrate to the
total radioactivity in the plasma was lower for the mouse, rat, and hamster than for the human, guinea pig, and duckling (Fig. 3). 1oc Male
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FIG 4. AFB,-AFL interconversion in arterial and venous blood. Four female and three male lo-week-old Fischer rats were used. Arterial and venous blood was taken from the same animals and each was incubated with 400 ng of AFB, or AFL for 1 hr. Chloroform extracts of plasma of the incubated blood were developed on TLC (chloroform-acetone-water) and assayed for both AFB, and AFL. X ? SE (SE is shown by vertical bars) of the proportion of AFB, when AFL was used, or that of AFL when AFB, was used, to the total amount of AFB, and AFL is shown. *V and A indicate venous and arterial blood, respectively. No significant difference (P -z 0.05) was found between the male and female blood or between the arterial and venous blood.
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FIG. 5. AFEt,-AFL interconversion in the blood of different animal species. Hamster blood was incubated for 15, 30, 60, or 120 min, and that of the other species for 10,60, or 120 min, with 2000 ng of AFB, or AFL. AFR, and AFL in chloroform extracts of plasma were separated by TLC (chloroform-acetone-water). Each abscissa shows time (0 to 120 min), and vertical bars show the incubation time. Each point shows the mean value expressed as the proportion of AFR, when AFL was used, or AFL when AFB, was used, to the total amount of AFR, and AFL. Number of animals (n) and number of blood samples for each point (N): Wistar rat (8, 11 weeks), n = 3-4, N = 2-4, pooled, Wistar rat (1 to 2 years), n = 1, N = 2-4; Fischer rat (10, 16 weeks), n = 3-4, N = 2-4, pooled; castrated Fischer rat, n = 3, N = 2, pooled; Mongolian gerbil, n = 5, N = 2, pooled, hamster, n = 4, N = 2, pooled, ICR mice, n = 18 (female) or 20 (male), N = 3-4; squirrel monkey, n = 3, N = 3, pooled; rhesus monkey, n = 1 (male) or 2 (female), N = 2, pooled (female); human, n = 1, N = 2; duckling, n = 1, N = 2. AFL formed from AFB, was not found or was below the detectable level in blood of the male squirrel monkey and of the rhesus monkeys and humans of both sexes after incubation for 10, 60, and 120 min, and in the blood of ducklings of both sexes after incubation for 10 min. AFl3, formed from AFL was not found or was below the detectable level in the blood of the male squirrel monkey and the humans and ducklings of both sexes after incubation for 10 min. The interconversion in the blood of female 16-week-old Fischer rat and the conversion of AFR, to AFL in the blood of the male duckling, both at 120 min incubation, were not determined. *M, F, m, and f indicate male, female, and castrated male and female, respectively. **Age or body weight: w, m, and yr indicate weeks, months, and years of age, respectively; g indicates body weight. Significant difference (p < 0.05) in values at 10 min between mice and rats (8 to 16 weeks old): AFL formed from AFR,, male and female ICR mice vs male and female lO- and 16-week-old Fischer rats, and male and female I-weekold Wistar rats; AFR, formed from AFL, male and female ICR mice vs male and female lo- and 16-weekold Fischer rats, and male &week-old and female 1l-week-old Wistar rats. Significant difference between sexes of rats was not found except in values at 120 min of 8-week-old Wistar rats. 297
298
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FIG. 6. Distribution of radioactivity in red blood cell suspensions obtained from pooled blood of four adult (8 to 10 weeks) or two aged ( 1 to 2 years) Wistar rats. After duplicate samples were incubated with 200 ng of [ ‘%JAFB , (left panel of each pair) or 200 ng of [‘%]AFB, plus 1500 ng of AFB, (right panel of each pair) for 10, 60, or 120 mitt, the blood cells and the buffered saline were separated and the blood cells were washed with fresh buffered saline. The mean values of the percentage of radioactivity in the chloroform extracts of the buffered saline (B), residual aqueous layer (0), buffered saline after washing the blood cells (O), and the washed blood cells (0) are shown. Radioactivity in the chloroform extracts was calculated from that on TLC plates.
Interconversion of Nonradioactive AFBI and AFL The chloroform extracts of plasma which were separated after incubation of rat venous blood with AFB, or AFL gave two fluorescent spots on TLC plates developed in chloroform-acetone-water or ethyl ether. Their Rf values were identical with cochromatographed authentic AFB, or AFL in both solvent systems. Ultraviolet absorption peaks for these metabolites, which were obtained by incubating 4 to 8 ml of blood with 40 to 50 pg of AFBi or AFL and then separating the chloroform extracts of the plasma on TLC, were identical with authentic AFB, and AFL (AFB,, peak at 223, 265, and 362 nm in ethanol; AFL, peak at 254,261, and 332 nm in ethanol). No difference in the rate of conversion was observed between the arterial and venous blood of rats (Fig. 4). Neither formation of AFL from AFBi nor of AFB, from AFL was observed after the incubation of heated, diluted blood, whereas both were observed after the incubation of unheated, diluted blood.
AND AIBARA
AFB,-AFL interconversion also was examined in venous blood from nine animal species. Blood from Wistar rats, Fischer rats, hamsters, Mongolian gerbils, and ICR mice was highly active; blood of the other species was inactive or possessed only slight activity (Fig. 5). AFB,-AFL interconversion also occurred in blood of BALB/c and C57BL mice after a 1-hr incubation. AFL formed from AI+ was 22.6, 21.9, 21.6, or 13.0% of the total AFB , and AFL in male or female BALB/ c mice, or male or female C57BL mice, respectively. AFB, formed from AFL was 52.0, 49.4, 48.8, and 47.1% of the total AFB, and AFL in male or female BALB/c mice, or male or female C57BL mice, respectively. The interconversion did not occur in blood of guinea pigs of either sex. Neither sex nor castration altered the interconversion in rat blood (Figs. 4, 5). The interconversion favored AFB, formation, however, in the blood of aged rats (Fig. 5). The percentage of metabolites formed after a lo-min incubation was higher in rat blood than in mouse blood (ICR) (Fig. 5), indicating that the interconversion occurred more rapidly in the blood of rats than in mice. When plasma was used instead of whole blood, no spots other than the substrate were found on TLC plates, or only an undetectable amount of AFBl was formed from AFL, presumably because of contamination by blood cells.
Fate of [‘4C]AFB,, Nonradioactive AFB, , and AFL in Red Blood Cell Suspension Red blood cell suspensions were incubated with [14C]AFB1 and after being removed from the buffered saline, the red blood cells were washed with an equal volume of fresh buffered saline. Examination of the distribution of radioactivity showed that 60 to 75% of the radioactivity was retained in the blood cells even after they were washed with fresh buffered saline (Fig. 6). High radioactivity was found in the AFB, and AFL regions on TLC after incubation for 10 or 60 min, but it was found only in the AFB, region after 120 min incubation (Fig. 7). Radioactivity in the AFB, region after 120 min incubation was higher
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than after 60 min incubation, indicating that AFB,-AFL interconversion took place and that it favored AFB, formation at 120 min. Incubation of nonradioactive aflatoxins with red blood cell suspensions also resulted in AFBi-AFL interconversion (Fig. 8). AFB, formation from AFL increased and the reverse conversion decreased with time. For comparison, blood that was kept at 0 to 4°C for 1 hr preceding incubation was examined for interconversion because preparing the red blood cell suspension required the same condition. Interconversion was observed after 2 hr of incubation (AFB1 formed from AFL and AFL formed from AFB, were 74.7 and 22.0% of the total amount of AFB, and AFL, respectively), but only AFBi was found after incubation for 3 hr whether AFB, or AFL was the substrate.
plasma. Since most radioactivity in the plasma was in the plasma protein fraction, the distribution of AFB, between plasma and cells may be determined by the propensity of the toxin for binding to plasma protein and blood cells. AFB, given po has been found in the systemic blood within 1 hr (Butler and Clifford, 1965; Dalezios and Hsieh, 1973; Wong and Hsieh, 1978). The concentration of AFBl in the blood in the present study was within th, range of blood concentration of radioactivity 100
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DISCUSSION When whole blood was incubated with [‘4C]AFB,, high levels of radioactivity were found in the blood cell fraction in all animal species examined, although there were species differences in the ratio of radioactivity in blood cells to that in plasma. When blood cells suspended in buffered saline were used, radioactivity in the blood cell fraction was much higher than in the buffered saline traction. The blood cells retained high levels of radioactivity even after being washed with fresh buffered saline. These results suggest that AFB, may bind some component of the blood cell. Radioactivity associated with the blood cells could be liberated by washing with fresh
10
30
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(min)
FIG. 8. AFB,-AFL interconversion by red blood cell suspensions. After duplicate samples of red blood cell suspensions of male Wistar rats were incubated with 2000 ng of AFB, or AFL for 10, 30, or 60 min, the buffered saline was separated from the blood cells, and the chloroform extracts of the buffered saline were assayed for both AFB, and AFL. The developing solvent for TLC was chloroform-acetone-water. Mean values of the proportion of AFL when AFB, was used, or AFB, when AFL was used, to the total amount of AFB, and AFL are shown.
300
KUMAGAI,
NAKANO,
equivalent to those of AFB, found in vivo after administration of radioactive AFB, (Dalezios and Hsieh, 1973; Wong and Hsieh, 1978). After being absorbed from the gastrointestinal tract, AFBi is transported by portal blood to the liver. The toxin which is transported via the lymphatic route, if any, or the toxin which failed to enter the liver would also reach the liver or other target organs after circulating in the systemic blood. The distribution of radioactivity in blood found in the present study suggests that AFB, is carried by both blood cells and plasma protein, and that the contribution of the blood cells as a carrier may be larger than that of plasma protein in some species, such as the hamster and guinea pig. Incubation of blood with [14C]AFB, or nonradioactive AFB, or AFL showed marked interconversion of AFBl and AFL in rat, mouse, hamster, and Mongolian gerbil blood. In duckling blood, marked conversion of AFT+ to AFL was noted when low concentrations of [14C]AFB, were used, but no interconversion or limited interconversion was observed in the presence of high concentrations of the toxin. Red blood cell suspensions, but not plasma, from rats were active in Al%,-AFL interconversion, indicating that the red blood cells are responsible for the reaction. Enzyme systems in the red blood cells may be involved because heated blood was inactive. The AFBIAFL interconversion has also been observed in vitro in the soluble fraction of livers from several species of animals (Patterson and Roberts, 1972). In that study, AFL formation from AFB, increased at first but then decreased (Patterson and Roberts, 1972). Similar changes were noted in the incubated whole blood or red blood cell suspension in the present study; AFB, formation from AFL and AFL formation from AFB, both increased at first, but thereafter an increase in AFBi formation and a decrease in AFL formation took place, regardless of which toxin was used as substrate. AFL has been reported to be carcinogenic to the rat and rainbow trout (Schoenhard et al., 1981; Nixon et al., 1981), mutagenic
AND AIBARA
(Wong and Hsieh, 1976) and acutely toxic in the rabbit (Detroy and He&tine, 1970), though less potent than AFB, . AFB, and AFL have both been found in systemic blood of rats given one of these substances iv (Wong et al., 1979; Wong and Hsieh, 1980), but AFL has not been detected in the blood of mice and monkeys given AFB, iv (Wong and Hsieh, 1980). The presence of both AFBl and AFL in the blood observed in vivo may reflect mainly the AFBi-AFL interconversion in the blood rather than that in the liver, because the presence of AFL in the blood after AFB, administration correlates with the AI%,-AFL interconversion in the blood but not with that in the liver. The interconversion took place less rapidly in mouse blood than in rat blood, and the blood of monkey was inactive, whereas no marked difference in the conversion activity in the liver among these species has been reported (Patterson and Roberts, 197 1; Hsieh et al., 1977; Roebuck and Wogan, 1977; Salhab and Edwards, 1977). No sex difference in rats was observed either in distribution of [14C]AFB, in the blood or in AFBi-AFL interconversion in the blood, suggesting that the fate of AFB, in the blood is not involved in the mechanism of sex difference in the action of AFB, in the rat (Wogan, 1973; Gurtoo and Motycka, 1976). Also the lack of a definitive relation between species susceptibility to the carcinogenic action of AFB, and species variation in the fate of AFBl in the blood indicates that the fate of AFBl in the blood may not be a key factor in susceptibility to the carcinogenic action of AFB,. In contrast, species variation in the fate of AFBl in the blood in part correlates with species difference in the acute toxicity of AFBi . The proportion of radioactivity in the protein-free plasma to total plasma radioactivity was smaller in the mouse, rat, and hamster, all of which are relatively resistant, than in the guinea pig and duckling, both of which are acutely susceptible for the toxin. The large proportion of free AFBi in the plasma may account for the efficient transfer of the toxin to liver cells and consequently for the high
AFB,-AFL
INTERCONVERSION
susceptibility to the acute effects of AFBl . The activity of AFB,-AFL interconversion in the blood was high in the resistant species, but its meaning remains uncertain, If AFL formation is an “aflatoxin reservoir” as suggested by others (Patterson, 1973; Campbell and Hayes, 1976), the AFBI-AFL interconversion in the blood may modify the toxic action of AFB, _ ACKNOWLEDGMENTS The authors thank Dr. T. Suzuki of Institute of Research and Development, Yamanouchi Pharmaceutical Company, for providing squirrel and rhesus monkey blood, Mrs. Y. Yamamoto for carrying out electrophoresis, Dr. T. Asano for the other experimental animals, and Miss K. Shiromizu and Mr. Y. Ito for their technical assistance.
REFERENCES BASSIR, O., AND BABABLJNMI, E. A. (1973). The binding of allatoxin Br with serum albumin, B&hem. Pharmacol.
22, 132- 134.
BUTLER, W. H., AND CLIFFORD, J. I. (1965). Extraction of allatoxin from rat liver. Nature (London) 5, 10451046. CAMPBELL, T. C., AND HAYES, J. R. (1976). The role of aflatoxin metabolism in its toxic lesion. Toxicol. Appl. Pharmacol.
35, 199-222.
DALEZIOS, J. I., AND HSIEH, D. P. H. (1973). Excretion and metabolism of orally administered aflatoxin B, by rhesus monkeys. Food Cosmet. Toxicol. 11, 605-6 16. DETROY, R. W., AND HESSELTINE, C. W. (1970). Aflatoxicol: Structure of a new transformation product of atlatoxin B, . Canad. J. Biochem. 48, 830-832. GURTOO, H. L., AND MOTYCKA, L. (1976). Effect of sex difference on the in vitro and in vivo metabolism of aflatoxin B, by the rat. Cancer Res. 36, 4663-467 I. HSIEH, D. P. H., WONG, Z. A., WONG, J. J., MICHAS, C., AND RUEBNER, B. H. (1977). Comparative metabolism of aflatoxin. In Mycotoxins in Human and Animal Health (J. V. Rodricks, C. W. He&tine, and M. A. Mehlman, eds.), pp. 37-50. Pathotox, Park Forest South, Ill. KRISHNAMACHARI, K. A. V. R., BHAT, R. V., NAGARAJAN, V., AND TILAK, T. B. G. (1975). Hepatitis due to aflatoxicosis. An outbreak in western India. Lancer 10, 1061-1063.
MAHIN, D. T., AND LOFBERG, R. T. (1966). A simplified method of sample preparation for determination of tritium, carbon-14, or sulfur-35 in blood or tissue by liquid scintillation counting. Anal. Biochem. 16,500-509. NEWBERNE, P. M., AND BUTLER, W. H. (1969). Acute and chronic effectsof aflatoxin on the liver of domestic and laboratory animals: A review. Cancer Res. 29,236250.
IN BLOOD
301
NIXON, J. E., HENDRICKS, J. D., PAWLOWSKI, N. E., LOVELAND, P. M., AND SINNHUBER,R. 0. (1981). Carcinogenicity of aflatoxicol in Fischer 344 rats. J. Nat. Cancer Inst. 66, 1159-l 163. PATTERSON, D. S. P. (1973). Metabolism as a factor in determining the toxic action of the aflatoxins in different animal species. Food Cosmet. Toxicol. 11; 287294.
PATTERSON, D. S. P., AND ROBERTS, B. A. ( 197 1). The in vilro reduction of aflatoxins Br and Bz by soluble avian liver enzymes. Food Cosmet. Toxicol. 9, 829837.
PATTERSON, D. S. P., AND ROBERTS, B.A. (1972). Aflatoxin metabolism in duck-liver homogenates: The relative importance of reversible cyclopentenone reduction and hemiacetal formation. Food Cosmet. Toxicol. 10, 501-512.
ROEBUCK, B. D., AND WOGAN, G. N. (1977). Species comparison of in vitro metabolism of atlatoxin Bi. Cancer Res. 37, 1649-1656. SALHAB, A. S., AND EDWARDS,G. S. (1977). Comparative in vitro metabolism of aflatoxicol by liver preparations from animals and humans. Cancer Res. 37,1016-1021. SCHOENHARD, G. L., HENDRICKS, J. D., NIXON, J. E., LEE, D. J., WALES, J. H., SINNHUBER, R. O., AND PAWLOWSKI, N. E. (1981). Aflatoxicol-induced hepatocellular carcinoma in rainbow trout (Salmo gairdneri) and the synergistic effectsof cyclopropenoid fatty acids. Cancer Res. 41, 101 I-1014. STUBBLEFIELD, R. D., SHANNON, G. M., AND SHCTWELL, 0. L. (1969). Aflatoxins: Improved resolution by thin layer chromatography. J. Assoc. Ofl Anal. Chem. 52,669-672. I., FRIEDMAN, L., AND STONE, C. L. (1980). Speties difference in the binding of aflatoxin B, to hepatic macromolecules. Toxicol. Appl. Pharmacol. 52, 177-
UENO,
180.
WHITE, J. F., AND ROTHSTEIN, A. (1973). The interaction of methyl mercury with erythrocytes. Toxicol. Appl. Pharmacol.
26, 370-384.
WOGAN, G. N. (1973). Aflatoxin carcinogenesis. In Methods in Cancer Research (H. Busch, ed.), Vd. VII pp. 309-344. Academic Press, New York. WONG, Z. A., DECAD, G. M., BYARD, J. L., AND HSIEH, D. P. H. (I 979). Conversion of aflatoxicol to aflatoxin B, in rats in vivo and primary hepatocyte culture. Food Cosmet.
Toxicol.
17, 48 l-486.
WONG, J. J., AND HSIEH, D. P. H. (1976). Mutagenicity of aflatoxins related to their metabolism and carcinogenic potential. Proc. Nat. Acad. Sci. USA 73, 22412244.
WONG, Z. A., AND HSIEH, D. P. H. (1978). Aflatoxicol: Major aflatoxin B, metabolite in rat plasma. Science 200, 325-327.
WONG, Z. A., AND HSIEH, D. P. H. (1980). The comparative metabolism and toxicokinetics of aflatoxin B, in the monkey, rat and mouse. Toxicol. Appl. Pharmacol. 55, 115-125.
AND
APPLIED
PHARMACOLOGY
67, 292-30
1 ( 1983)
Interactions of Aflatoxin B, and Blood Components of Various Species in Vitro: Interconversion of Aflatoxin B, and Aflatoxicol in the Blood SUSUMUKUMAGAI,~
NAOKONAKANO,ANDKAGEAKI
AIBARA
Department of Biomedical Research on Food, National Institute of Health, Kamiosaki, Shinagawa-ku, Tokyo, Japan Received June 24, 1982; accepted October 21, 1982 Interactions of Aflatoxin B, and Blood Components of Various Species in Vitro: Interconversion of Aflatoxin B, and Aflatoxicol in the Blood. KUMAGAI, S., NAKANO, N., AND AIBARA, K. ( 1983). Toxicol. Appl. Pharrnacol. 67,292-301. The fate of athttoxin B1 (AFB,) in the blood of various species of animals was studied in vitro. Examination of the distribution of radioactivity in blood incubated with [“C]AFT$ at 37°C showed that high levels of radioactivity were associated with blood cells. The radioactivity was readily removed from the blood cells by washing with fresh plasma, indicating loose binding of AFB, to blood cells. Most of the radioactivity in plasma was bound to protein. These results suggest that a large part of the AFB, in blood in vivo may be carried not only by the plasma proteins but also by the blood cells. When chloroform extracts of plasma of [“C]AFB,-treated mouse, rat, duckling, and hamster blood were developed by thin-layer chromatography, hi levels of radioactivity were found in both the AFB, region and the aflatoxicol (AFL) region. Incubation of blood with nonradioactive AFB, and AFL showed marked interconversion of AFB, and AFL in the blood of rats, hamsters, mice, and Mongolian gerbils, but not in the blood of guinea pigs, rhesus monkeys, squirrel monkeys, or humans. Interconversion occurred in red blood cell suspensions but not in plasma, indicating that the red blood cells are responsible for AFB,-AFL interconversion in the blood.
The aflatoxins are secondary metabolites of Aspergillus flaws, aflatoxin Br (APB,) being the most potent hepatotoxic and hepatocarcinogenic compound among these metabolites. There is a large species variation in suscep tibility to APB1 poisoning. The rabbit and duckling are the most sensitive to the acute effect of APB1 (Patterson, 1973). The rat, duckling, and rainbow trout are highly susceptible to the carcinogenic effect of APB, (Newberne and Butler, 1969; Hsieh et al., 1977). In the human, hepatitis and liver cancer because of afIatoxin intake have been suggested by epidemiologic studies (Krishnamachari et al., 1975; Wogan, 1973) though comparative susceptibility remains uncertain. To elucidate the mechanisms of toxic and carcinogenic actions of atlatoxins, many stud’
To
whom all correspondence should be addressed.
004 1408X/83/020292-
10.$03.00/O
Copyright 0 1983 by Academic Press, Inc. All fights of r~pmduction in any form reserved.
292
ies have focused on phenomena occurring inside the liver, which is the primary target organ of the toxins, but only a few on phenomena outside the liver especially on the fate of aflatoxin in the blood. Although binding of APB, to plasma proteins, which may affect the distribution and elimination of the toxin, has been studied in some species (Bassir and Bababunmi, 1973; Ueno et al., 1980), little is known about the binding of the toxin to blood cells or the biotransformation of the toxin by the blood. In the present study, the fate of APB, in the blood was studied in vitro with blood from various animal species. METHODS Animals. Wistar rats (8 to 11 weeks and 1 to 2 years old), Fischer rats (8 to 16 weeks old), ICR mice (7 weeks old), BALB/c mice (7 to 9 weeks old), C57BL mice (7 to 9 weeks old), golden hamsters (3 and 6 months old), Mongolian gerbils (4 months old), Khaki-Campbell ducklings
AFB,-AFL
INTERCONVERSION
(500 g), Hartley guinea pigs (2 months old), squirrel monkeys (adult), rhesus monkeys (adult), and humans (adult) were used. Fischer rats were castrated under ether anesthesia at 15 weeks and used at 16 weeks. Chemicals. Aflatoxins B,, MI (AIM,), Bz. (AFBz,), and aflatoxicol (AFL) were purchased from Makor Chemical Ltd. (Jerusalem, Israel) and dissolved in benzene:acetonitrile (98:2). [‘4C]AFB, (specific activity, 40 mCi/mmol, labeled in all carbons except the extranuclear-OCH,) dissolved in methanol was purchased from Moravek Biochemicals, Inc. (California). Blood sampling. Venous blood was taken from the vein at the elbow of humans and from the femoral vein of monkeys, both without anesthesia, and from the vena cava of other animals under light ether anesthesia. Arterial blood was taken from the aorta of rats under ether anesthesia. Immediately after sampling, all blood samples were mixed with heparin (20 units/ml). Preparation of red blood cells. After the plasma was separated by centrifugation at 3000 rpm for 10 min, white blood cells and platelets were discarded, and the red blood cells were washed three times at 0 to 4°C with 5 vol of saline buffered to pH 7.2 with 15 mM 2-amino-2-(hydroxymethyl)- 1,3-propanediol (Tris) containing 1 mM glucose (White and Rothstein, 1973). Preparation ofheated blood. Rat blood was diluted lofold with buffered saline and a portion of the diluted blood was heated in boiled water for 15 min. Unheated diluted blood was used as a control. Incubation. After evaporation to dryness under Nz gas, 200 ng of [14C]AFB,, 200 ng of [“‘C]AFB, mixed with 1500 ng of nonradioactive AFB, , or 400 or 2000 ng of nonradioactive AFB, or AFL was incubated aerobically with whole blood, plasma, red blood cells suspended in buffered saline (50% hematocrit), or heated or unheated diluted blood in a shaking water bath at 37°C. Unless otherwise stated, 0.6 ml of each sample was used. The dried aflatoxins and the blood samples were mixed rapidly before incubation. Preliminary experiments showed that dried AFB, was almost completely redissolved in the blood. Dried [i4C]AFBI (1700 ng) and blood in test tubes were mixed rapidly; the blood was removed, and the test tubes were washed with chloroform and perchloric acid. Only 1.6 -t 0.3% (n = 3) of the total radioactivity was contained in the washings. Immediately after incubation, plasma or buffered saline was separated from the blood cells by centrifugation at 3000 rpm for 5 min. Preparation of protein-free fraction of the plasma. Ultrafiltration employing a Micropartition System (Amicon Co., Massachusetts) was used to separate protein-bound and -free fractions of plasma. In accordance with the op erating instructions, the ultrafiltrate was obtained by centrifugation of the cell containing plasma at 3000 rpm for 15 min. No protein bands of the ultratiltrate were found on electrophoresis plates (cellulose acetate plates, Helena Lab., USA) stained by Ponceau 3R, while the same amount of plasma showed several protein bands. Extraction
and
thin-layer
chromatography
(TLC).
293
IN BLOOD
Plasma, buffered saline, or distilled water was extracted three times with 30 vol of chloroform. After being evap orated at 40°C under N2 gas, the extracts were quantitatively transferred with chloroform onto TLC plates. Known amounts of authentic AFBi, AIM,, AFB,, and AFL were cochromatographed. TLC plates of silica gel 60 (Merck, Darmstadt, West Germany) and TLC plastic sheets of silica gel 60 (Merck), both without fluorescent indicator, were used for separating nonradioactive metabolites and 14C metabolites, respectively. The developing solvent was chloroform-acetone-water (88: 12: 1.5) (Stubblefield et al., 1969) or ethyl ether (Patterson and Roberts, 1971). Fluorescent spots on the plates were visualized under ultraviolet light (365 nm). Determination of radioactivity. Radioactivity was determined in a scintillation counter (Type LS-2OOB, Beckman). Plasma, buffered saline, and the ultrafdtrate of plasma were counted after being mixed with 10 ml of ACS II (Amex&am Corp., Arlington Heights, Ill.). The blood cells and the aqueous layer of plasma were mixed with perchloric acid and hydrogen peroxide, warmed in the oven at 70°C for 30 to 60 min (Mahin and Lotberg, 1966), mixed with 10 to 15 ml of ACS II, and counted. TLC plastic sheets containing radioactive metabolites were cut into six portions, corresponding to the region containing both AFM, and AFB&, the AFL region, the AFB, region, the origin, the region between AFB, and the solvent front, and the region between AFBz, and AFL. After each portion was transferred to a counting vial containing 1 to 2 ml of water, silica was removed from the plastic sheet by an ultrasonic cleaner (Branson Instrument Co., Connecticut) and a mixer (Thennomix Co., Ltd., Tokyo, Japan). The silica-water suspension was mixed with 10 to 15 ml of ACS II and counted. Analysis of nonradioactive AFBl and AFL. AFB, and AFL concentrations on TLC were measured in a spectrophotofluorometer equipped with a TLC scanner (Hitachi Co., Japan). Standard curves were obtained with cochromatographed authentic aflatoxins. Since the maximum activation and emission wavelengths for AFB, were about 363 and 427 nm, respectively, and those for AFL were about 328 and 422 nm, respectively, the wavelengths were set at the corresponding values. The range of measurement was 10 to 150 ng of aflatoxin. Absorption of ultraviolet light by AFB, and AFL bands scraped from TLC plates was determined in a standard spectrophotometer (Type 181, Hitachi Co.) and a double-wave length double-beam spectrophotometer (Type 556, Hitachi Co.). Statistics. The statistical significance of differences between means was determined by Student’s t test.
RESULTS
Distribution [‘4C]AFB,
and
Biotransformation
of
Mouse, rat, hamster, guinea pig, duckling, and human blood were incubated with
294
KUMAGAI,
NAKANO,
[14C]AFB,. Radioactivity contained in the chloroform extracts of plasma, in the residual aqueous layer, and in blood cells was determined. Radioactivity in the chloroform extracts of plasma was calculated from that contained in the six portions of the TLC plates. Distribution of radioactivity in the blood differed among species (Fig. 1). In the blood of rats of both sexes and in mice, higher radioactivity was found in the chloroform extracts of plasma than in blood cells. A similar relationship was observed in human blood, though the difference in radioactivity between these two fractions was much smaller than that seen in rat blood. In hamster and guinea pig blood, the reverse relationship was observed. In duckling blood, radioactivity in the chloroform extract and in blood cells was similar when low concentrations of AFB, were used, but blood cells showed higher radioactivity than extracts when high concentrations of AFBl were used. Radioactivity in the aqueous layer was lower than that in the other two fractions for all animal species, but it was relatively high in the human blood. After being incubated with [‘4C]AFB, (200 ng/0.6 ml) for 10 min, blood of Wistar rats (1 to 2 years of age, male) and hamsters (3 to 6 months of age, female) was separated into blood cells and plasma, and the blood cells were washed repeatedly with an equal volume of fresh plasma of the corresponding animal species. Radioactivity in each plasma and washed blood cell specimen was counted. Most of the radioactivity associated with blood cells was transferred to plasma by washing three times. The proportion of radioactivity remaining in blood cells was 7.0% (n = 2) for hamster blood and 4.9% (n = 2) for rat blood. Profiles of radioactivity on TLC plates of chloroform extracts of plasma (Fig. 2) show that the proportion of radioactivity in the AFB, region of the total chloroform extractable radioactivity decreased with incubation time. The pattern of transformation of [‘4C]AFB, differed among animal species. In mouse, rat, and hamster blood, conversion of AFB, to the metabolite in the AFL region took place. The percentage of the metabolite
AND AIBARA
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FIG. 3. Proportion of radioactivity of the protein-free plasma to total radioactivity in the plasma. Blood samples were those shown in Fig. 1 for the rat, hamster, guinea pig, duckling, and human. For the mouse, blood was taken from eight animals. Blood samples were incubated with 200 ng of [‘%]AFB, or 200 ng of [‘%]AFBi plus 1500 ng of AFB, for 10, 60, or 120 min. Each value was obtained from radioactivity in the plasma (40 ~1) and the plasma ultrafiltrate (40 ~1). *M and F indicate male and female, respectively.
formed after a IO-min incubation was lower in mouse blood than in rat or hamster blood. In duckling blood, the same pattern was observed when low concentrations of AFB, were used, but not when high concentrations were used. A single blue spot was observed under ultraviolet light in the AFL region when high radioactivity was found in this region. With increase in the incubation time, radioactivity in the region less polar than AFB, increased gradually in guinea pig blood as did that at the origin in human blood. For comparison, mouse, rat, duckling, hamster, or human plasma or distilled water was incubated with [14C]AFB, for 120 min. About 90% of the chloroform extractable radioactivity was found in the AFBl region (Fig. 2).
In another series of experiments, radioactivity of protein-free plasma was determined by the ultrafiltration technique, after blood was incubated with [‘4C]AFB,. Only a small proportion of the radioactive compounds in the plasma was free of protein. The proportion of radioactivity in the ultrafiltrate to the
total radioactivity in the plasma was lower for the mouse, rat, and hamster than for the human, guinea pig, and duckling (Fig. 3). 1oc Male
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FIG 4. AFB,-AFL interconversion in arterial and venous blood. Four female and three male lo-week-old Fischer rats were used. Arterial and venous blood was taken from the same animals and each was incubated with 400 ng of AFB, or AFL for 1 hr. Chloroform extracts of plasma of the incubated blood were developed on TLC (chloroform-acetone-water) and assayed for both AFB, and AFL. X ? SE (SE is shown by vertical bars) of the proportion of AFB, when AFL was used, or that of AFL when AFB, was used, to the total amount of AFB, and AFL is shown. *V and A indicate venous and arterial blood, respectively. No significant difference (P -z 0.05) was found between the male and female blood or between the arterial and venous blood.
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FIG. 5. AFEt,-AFL interconversion in the blood of different animal species. Hamster blood was incubated for 15, 30, 60, or 120 min, and that of the other species for 10,60, or 120 min, with 2000 ng of AFB, or AFL. AFR, and AFL in chloroform extracts of plasma were separated by TLC (chloroform-acetone-water). Each abscissa shows time (0 to 120 min), and vertical bars show the incubation time. Each point shows the mean value expressed as the proportion of AFR, when AFL was used, or AFL when AFB, was used, to the total amount of AFR, and AFL. Number of animals (n) and number of blood samples for each point (N): Wistar rat (8, 11 weeks), n = 3-4, N = 2-4, pooled, Wistar rat (1 to 2 years), n = 1, N = 2-4; Fischer rat (10, 16 weeks), n = 3-4, N = 2-4, pooled; castrated Fischer rat, n = 3, N = 2, pooled; Mongolian gerbil, n = 5, N = 2, pooled, hamster, n = 4, N = 2, pooled, ICR mice, n = 18 (female) or 20 (male), N = 3-4; squirrel monkey, n = 3, N = 3, pooled; rhesus monkey, n = 1 (male) or 2 (female), N = 2, pooled (female); human, n = 1, N = 2; duckling, n = 1, N = 2. AFL formed from AFB, was not found or was below the detectable level in blood of the male squirrel monkey and of the rhesus monkeys and humans of both sexes after incubation for 10, 60, and 120 min, and in the blood of ducklings of both sexes after incubation for 10 min. AFl3, formed from AFL was not found or was below the detectable level in the blood of the male squirrel monkey and the humans and ducklings of both sexes after incubation for 10 min. The interconversion in the blood of female 16-week-old Fischer rat and the conversion of AFR, to AFL in the blood of the male duckling, both at 120 min incubation, were not determined. *M, F, m, and f indicate male, female, and castrated male and female, respectively. **Age or body weight: w, m, and yr indicate weeks, months, and years of age, respectively; g indicates body weight. Significant difference (p < 0.05) in values at 10 min between mice and rats (8 to 16 weeks old): AFL formed from AFR,, male and female ICR mice vs male and female lO- and 16-week-old Fischer rats, and male and female I-weekold Wistar rats; AFR, formed from AFL, male and female ICR mice vs male and female lo- and 16-weekold Fischer rats, and male &week-old and female 1l-week-old Wistar rats. Significant difference between sexes of rats was not found except in values at 120 min of 8-week-old Wistar rats. 297
298
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FIG. 6. Distribution of radioactivity in red blood cell suspensions obtained from pooled blood of four adult (8 to 10 weeks) or two aged ( 1 to 2 years) Wistar rats. After duplicate samples were incubated with 200 ng of [ ‘%JAFB , (left panel of each pair) or 200 ng of [‘%]AFB, plus 1500 ng of AFB, (right panel of each pair) for 10, 60, or 120 mitt, the blood cells and the buffered saline were separated and the blood cells were washed with fresh buffered saline. The mean values of the percentage of radioactivity in the chloroform extracts of the buffered saline (B), residual aqueous layer (0), buffered saline after washing the blood cells (O), and the washed blood cells (0) are shown. Radioactivity in the chloroform extracts was calculated from that on TLC plates.
Interconversion of Nonradioactive AFBI and AFL The chloroform extracts of plasma which were separated after incubation of rat venous blood with AFB, or AFL gave two fluorescent spots on TLC plates developed in chloroform-acetone-water or ethyl ether. Their Rf values were identical with cochromatographed authentic AFB, or AFL in both solvent systems. Ultraviolet absorption peaks for these metabolites, which were obtained by incubating 4 to 8 ml of blood with 40 to 50 pg of AFBi or AFL and then separating the chloroform extracts of the plasma on TLC, were identical with authentic AFB, and AFL (AFB,, peak at 223, 265, and 362 nm in ethanol; AFL, peak at 254,261, and 332 nm in ethanol). No difference in the rate of conversion was observed between the arterial and venous blood of rats (Fig. 4). Neither formation of AFL from AFBi nor of AFB, from AFL was observed after the incubation of heated, diluted blood, whereas both were observed after the incubation of unheated, diluted blood.
AND AIBARA
AFB,-AFL interconversion also was examined in venous blood from nine animal species. Blood from Wistar rats, Fischer rats, hamsters, Mongolian gerbils, and ICR mice was highly active; blood of the other species was inactive or possessed only slight activity (Fig. 5). AFB,-AFL interconversion also occurred in blood of BALB/c and C57BL mice after a 1-hr incubation. AFL formed from AI+ was 22.6, 21.9, 21.6, or 13.0% of the total AFB , and AFL in male or female BALB/ c mice, or male or female C57BL mice, respectively. AFB, formed from AFL was 52.0, 49.4, 48.8, and 47.1% of the total AFB, and AFL in male or female BALB/c mice, or male or female C57BL mice, respectively. The interconversion did not occur in blood of guinea pigs of either sex. Neither sex nor castration altered the interconversion in rat blood (Figs. 4, 5). The interconversion favored AFB, formation, however, in the blood of aged rats (Fig. 5). The percentage of metabolites formed after a lo-min incubation was higher in rat blood than in mouse blood (ICR) (Fig. 5), indicating that the interconversion occurred more rapidly in the blood of rats than in mice. When plasma was used instead of whole blood, no spots other than the substrate were found on TLC plates, or only an undetectable amount of AFBl was formed from AFL, presumably because of contamination by blood cells.
Fate of [‘4C]AFB,, Nonradioactive AFB, , and AFL in Red Blood Cell Suspension Red blood cell suspensions were incubated with [14C]AFB1 and after being removed from the buffered saline, the red blood cells were washed with an equal volume of fresh buffered saline. Examination of the distribution of radioactivity showed that 60 to 75% of the radioactivity was retained in the blood cells even after they were washed with fresh buffered saline (Fig. 6). High radioactivity was found in the AFB, and AFL regions on TLC after incubation for 10 or 60 min, but it was found only in the AFB, region after 120 min incubation (Fig. 7). Radioactivity in the AFB, region after 120 min incubation was higher
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than after 60 min incubation, indicating that AFB,-AFL interconversion took place and that it favored AFB, formation at 120 min. Incubation of nonradioactive aflatoxins with red blood cell suspensions also resulted in AFBi-AFL interconversion (Fig. 8). AFB, formation from AFL increased and the reverse conversion decreased with time. For comparison, blood that was kept at 0 to 4°C for 1 hr preceding incubation was examined for interconversion because preparing the red blood cell suspension required the same condition. Interconversion was observed after 2 hr of incubation (AFB1 formed from AFL and AFL formed from AFB, were 74.7 and 22.0% of the total amount of AFB, and AFL, respectively), but only AFBi was found after incubation for 3 hr whether AFB, or AFL was the substrate.
plasma. Since most radioactivity in the plasma was in the plasma protein fraction, the distribution of AFB, between plasma and cells may be determined by the propensity of the toxin for binding to plasma protein and blood cells. AFB, given po has been found in the systemic blood within 1 hr (Butler and Clifford, 1965; Dalezios and Hsieh, 1973; Wong and Hsieh, 1978). The concentration of AFBl in the blood in the present study was within th, range of blood concentration of radioactivity 100
r
DISCUSSION When whole blood was incubated with [‘4C]AFB,, high levels of radioactivity were found in the blood cell fraction in all animal species examined, although there were species differences in the ratio of radioactivity in blood cells to that in plasma. When blood cells suspended in buffered saline were used, radioactivity in the blood cell fraction was much higher than in the buffered saline traction. The blood cells retained high levels of radioactivity even after being washed with fresh buffered saline. These results suggest that AFB, may bind some component of the blood cell. Radioactivity associated with the blood cells could be liberated by washing with fresh
10
30
60
(min)
FIG. 8. AFB,-AFL interconversion by red blood cell suspensions. After duplicate samples of red blood cell suspensions of male Wistar rats were incubated with 2000 ng of AFB, or AFL for 10, 30, or 60 min, the buffered saline was separated from the blood cells, and the chloroform extracts of the buffered saline were assayed for both AFB, and AFL. The developing solvent for TLC was chloroform-acetone-water. Mean values of the proportion of AFL when AFB, was used, or AFB, when AFL was used, to the total amount of AFB, and AFL are shown.
300
KUMAGAI,
NAKANO,
equivalent to those of AFB, found in vivo after administration of radioactive AFB, (Dalezios and Hsieh, 1973; Wong and Hsieh, 1978). After being absorbed from the gastrointestinal tract, AFBi is transported by portal blood to the liver. The toxin which is transported via the lymphatic route, if any, or the toxin which failed to enter the liver would also reach the liver or other target organs after circulating in the systemic blood. The distribution of radioactivity in blood found in the present study suggests that AFB, is carried by both blood cells and plasma protein, and that the contribution of the blood cells as a carrier may be larger than that of plasma protein in some species, such as the hamster and guinea pig. Incubation of blood with [14C]AFB, or nonradioactive AFB, or AFL showed marked interconversion of AFBl and AFL in rat, mouse, hamster, and Mongolian gerbil blood. In duckling blood, marked conversion of AFT+ to AFL was noted when low concentrations of [14C]AFB, were used, but no interconversion or limited interconversion was observed in the presence of high concentrations of the toxin. Red blood cell suspensions, but not plasma, from rats were active in Al%,-AFL interconversion, indicating that the red blood cells are responsible for the reaction. Enzyme systems in the red blood cells may be involved because heated blood was inactive. The AFBIAFL interconversion has also been observed in vitro in the soluble fraction of livers from several species of animals (Patterson and Roberts, 1972). In that study, AFL formation from AFB, increased at first but then decreased (Patterson and Roberts, 1972). Similar changes were noted in the incubated whole blood or red blood cell suspension in the present study; AFB, formation from AFL and AFL formation from AFB, both increased at first, but thereafter an increase in AFBi formation and a decrease in AFL formation took place, regardless of which toxin was used as substrate. AFL has been reported to be carcinogenic to the rat and rainbow trout (Schoenhard et al., 1981; Nixon et al., 1981), mutagenic
AND AIBARA
(Wong and Hsieh, 1976) and acutely toxic in the rabbit (Detroy and He&tine, 1970), though less potent than AFB, . AFB, and AFL have both been found in systemic blood of rats given one of these substances iv (Wong et al., 1979; Wong and Hsieh, 1980), but AFL has not been detected in the blood of mice and monkeys given AFB, iv (Wong and Hsieh, 1980). The presence of both AFBl and AFL in the blood observed in vivo may reflect mainly the AFBi-AFL interconversion in the blood rather than that in the liver, because the presence of AFL in the blood after AFB, administration correlates with the AI%,-AFL interconversion in the blood but not with that in the liver. The interconversion took place less rapidly in mouse blood than in rat blood, and the blood of monkey was inactive, whereas no marked difference in the conversion activity in the liver among these species has been reported (Patterson and Roberts, 197 1; Hsieh et al., 1977; Roebuck and Wogan, 1977; Salhab and Edwards, 1977). No sex difference in rats was observed either in distribution of [14C]AFB, in the blood or in AFBi-AFL interconversion in the blood, suggesting that the fate of AFB, in the blood is not involved in the mechanism of sex difference in the action of AFB, in the rat (Wogan, 1973; Gurtoo and Motycka, 1976). Also the lack of a definitive relation between species susceptibility to the carcinogenic action of AFB, and species variation in the fate of AFBl in the blood indicates that the fate of AFBl in the blood may not be a key factor in susceptibility to the carcinogenic action of AFB,. In contrast, species variation in the fate of AFBl in the blood in part correlates with species difference in the acute toxicity of AFBi . The proportion of radioactivity in the protein-free plasma to total plasma radioactivity was smaller in the mouse, rat, and hamster, all of which are relatively resistant, than in the guinea pig and duckling, both of which are acutely susceptible for the toxin. The large proportion of free AFBi in the plasma may account for the efficient transfer of the toxin to liver cells and consequently for the high
AFB,-AFL
INTERCONVERSION
susceptibility to the acute effects of AFBl . The activity of AFB,-AFL interconversion in the blood was high in the resistant species, but its meaning remains uncertain, If AFL formation is an “aflatoxin reservoir” as suggested by others (Patterson, 1973; Campbell and Hayes, 1976), the AFBI-AFL interconversion in the blood may modify the toxic action of AFB, _ ACKNOWLEDGMENTS The authors thank Dr. T. Suzuki of Institute of Research and Development, Yamanouchi Pharmaceutical Company, for providing squirrel and rhesus monkey blood, Mrs. Y. Yamamoto for carrying out electrophoresis, Dr. T. Asano for the other experimental animals, and Miss K. Shiromizu and Mr. Y. Ito for their technical assistance.
REFERENCES BASSIR, O., AND BABABLJNMI, E. A. (1973). The binding of allatoxin Br with serum albumin, B&hem. Pharmacol.
22, 132- 134.
BUTLER, W. H., AND CLIFFORD, J. I. (1965). Extraction of allatoxin from rat liver. Nature (London) 5, 10451046. CAMPBELL, T. C., AND HAYES, J. R. (1976). The role of aflatoxin metabolism in its toxic lesion. Toxicol. Appl. Pharmacol.
35, 199-222.
DALEZIOS, J. I., AND HSIEH, D. P. H. (1973). Excretion and metabolism of orally administered aflatoxin B, by rhesus monkeys. Food Cosmet. Toxicol. 11, 605-6 16. DETROY, R. W., AND HESSELTINE, C. W. (1970). Aflatoxicol: Structure of a new transformation product of atlatoxin B, . Canad. J. Biochem. 48, 830-832. GURTOO, H. L., AND MOTYCKA, L. (1976). Effect of sex difference on the in vitro and in vivo metabolism of aflatoxin B, by the rat. Cancer Res. 36, 4663-467 I. HSIEH, D. P. H., WONG, Z. A., WONG, J. J., MICHAS, C., AND RUEBNER, B. H. (1977). Comparative metabolism of aflatoxin. In Mycotoxins in Human and Animal Health (J. V. Rodricks, C. W. He&tine, and M. A. Mehlman, eds.), pp. 37-50. Pathotox, Park Forest South, Ill. KRISHNAMACHARI, K. A. V. R., BHAT, R. V., NAGARAJAN, V., AND TILAK, T. B. G. (1975). Hepatitis due to aflatoxicosis. An outbreak in western India. Lancer 10, 1061-1063.
MAHIN, D. T., AND LOFBERG, R. T. (1966). A simplified method of sample preparation for determination of tritium, carbon-14, or sulfur-35 in blood or tissue by liquid scintillation counting. Anal. Biochem. 16,500-509. NEWBERNE, P. M., AND BUTLER, W. H. (1969). Acute and chronic effectsof aflatoxin on the liver of domestic and laboratory animals: A review. Cancer Res. 29,236250.
IN BLOOD
301
NIXON, J. E., HENDRICKS, J. D., PAWLOWSKI, N. E., LOVELAND, P. M., AND SINNHUBER,R. 0. (1981). Carcinogenicity of aflatoxicol in Fischer 344 rats. J. Nat. Cancer Inst. 66, 1159-l 163. PATTERSON, D. S. P. (1973). Metabolism as a factor in determining the toxic action of the aflatoxins in different animal species. Food Cosmet. Toxicol. 11; 287294.
PATTERSON, D. S. P., AND ROBERTS, B. A. ( 197 1). The in vilro reduction of aflatoxins Br and Bz by soluble avian liver enzymes. Food Cosmet. Toxicol. 9, 829837.
PATTERSON, D. S. P., AND ROBERTS, B.A. (1972). Aflatoxin metabolism in duck-liver homogenates: The relative importance of reversible cyclopentenone reduction and hemiacetal formation. Food Cosmet. Toxicol. 10, 501-512.
ROEBUCK, B. D., AND WOGAN, G. N. (1977). Species comparison of in vitro metabolism of atlatoxin Bi. Cancer Res. 37, 1649-1656. SALHAB, A. S., AND EDWARDS,G. S. (1977). Comparative in vitro metabolism of aflatoxicol by liver preparations from animals and humans. Cancer Res. 37,1016-1021. SCHOENHARD, G. L., HENDRICKS, J. D., NIXON, J. E., LEE, D. J., WALES, J. H., SINNHUBER, R. O., AND PAWLOWSKI, N. E. (1981). Aflatoxicol-induced hepatocellular carcinoma in rainbow trout (Salmo gairdneri) and the synergistic effectsof cyclopropenoid fatty acids. Cancer Res. 41, 101 I-1014. STUBBLEFIELD, R. D., SHANNON, G. M., AND SHCTWELL, 0. L. (1969). Aflatoxins: Improved resolution by thin layer chromatography. J. Assoc. Ofl Anal. Chem. 52,669-672. I., FRIEDMAN, L., AND STONE, C. L. (1980). Speties difference in the binding of aflatoxin B, to hepatic macromolecules. Toxicol. Appl. Pharmacol. 52, 177-
UENO,
180.
WHITE, J. F., AND ROTHSTEIN, A. (1973). The interaction of methyl mercury with erythrocytes. Toxicol. Appl. Pharmacol.
26, 370-384.
WOGAN, G. N. (1973). Aflatoxin carcinogenesis. In Methods in Cancer Research (H. Busch, ed.), Vd. VII pp. 309-344. Academic Press, New York. WONG, Z. A., DECAD, G. M., BYARD, J. L., AND HSIEH, D. P. H. (I 979). Conversion of aflatoxicol to aflatoxin B, in rats in vivo and primary hepatocyte culture. Food Cosmet.
Toxicol.
17, 48 l-486.
WONG, J. J., AND HSIEH, D. P. H. (1976). Mutagenicity of aflatoxins related to their metabolism and carcinogenic potential. Proc. Nat. Acad. Sci. USA 73, 22412244.
WONG, Z. A., AND HSIEH, D. P. H. (1978). Aflatoxicol: Major aflatoxin B, metabolite in rat plasma. Science 200, 325-327.
WONG, Z. A., AND HSIEH, D. P. H. (1980). The comparative metabolism and toxicokinetics of aflatoxin B, in the monkey, rat and mouse. Toxicol. Appl. Pharmacol. 55, 115-125.