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14
Disposition of [ C]furan in the male F344 rat b
a
Leo T. Burka , Kelly D. Washburn & Richard D. Irwin
a
a
National Institute of Environmental Health Sciences , Research Triangle Park, North Carolina b
National Institute of Environmental Health Sciences , P.O. Box 12233, Research Triangle Park, NC, 27709 Published online: 19 Oct 2009.
To cite this article: Leo T. Burka , Kelly D. Washburn & Richard D. Irwin (1991) Disposition of 14
[ C]furan in the male F344 rat, Journal of Toxicology and Environmental Health, 34:2, 245-257, DOI: 10.1080/15287399109531564 To link to this article: http://dx.doi.org/10.1080/15287399109531564
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DISPOSITION OF [14C]FURAN IN THE MALE F344 RAT Leo T. Burka, Kelly D. Washburn, Richard D. Irwin
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National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina
In a recently completed 2-yr bioassay, furan was found to induce cholangiocarcinomas at high incidence in rats. The disposition of single and multiple gavage doses of [2,514 C]furan has been determined in male F344 rats to aid in interpretation of that study. In the 24 h after dosing about 80% of the furan-derived radioactivity was eliminated, primarily via urine and expired air. [14C]Carbon dioxide was a major metabolite, indicating that furan ring opening followed by complete oxidation of at least one of the labeled carbons was a major part of the overall metabolism of furan. Liver contained more furan-derived radioactivity by far than other tissues after 24 h. Approximately 80% of the radioactivity in liver was not extracted by organic solvents and was associated with protein. There was either no binding to DNA or the furan-DNA adduct was not stable to the isolation procedure. Repeated daily administration of [14C]furan resulted in a more or less linear increase in covalent binding through four doses; at this point the amount of nonextractable radioactivity plateaus. Urine contained at least 10 metabolites, again indicating extensive metabolism of the furan ring. From the data obtained in this study it is clear that furan is metabolized to reactive species, apparently primarily in liver, and these intermediates react with protein. The hepatotoxicity resulting from furan exposure may be due to the reaction of furan metabolites with liver macromolecules; the presence of some of these reactive metabolites following chronic exposure to furan may result in cholangiocarcinomas.
INTRODUCTION Furan is the parent for a class of industrially important chemicals that include furfural, furfuryl alcohol, and their derivatives. Furan, furfural, and furfuryl alcohol are widely used in the production of corrosion-resistant polymers, high-temperature laminates, in the formation of foundry molds, as solvents, and for a variety of other applications (Kirk-Othmer, 1978; Quaker Oats, 1974). Because of large production volume and extensive industrial use, the National Toxicology Program (NTP) has been conducting studies to evaluate the toxicity and carcinogenic potential of furan, furfural, and furfuryl alcohol, as well as several other compounds that contain a furan ring as part of their structure. We thank Steven Vo for his excellent technical assistance. Requests for reprints should be sent to Leo T. Burka, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709.
245 Journal of Toxicology and Environmental Health, 34:245-257,1991 Copyright © 1991 by Hemisphere Publishing Corporation
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In NTP prechronic studies with F344 rats, furan was significantly more hepatotoxic than either furfural or furfuryl alcohol, causing extensive distortion of gross liver structure and substantial microscopic pathology at doses one-tenth those required for furfural- or furfuryl alcohol-induced hepatotoxicity (NTP, 1989,1991). In 2-yr carcinogenicity studies in F344 rats, furfural administered at doses of 30 or 60 mg/kg induced cholangiocarcinomas in 2 high-dose males and dysplasia with fibrosis, a percursor lesion to cholangiocarcinoma, in 2 additional highdose males (NTP, 1989). Furan, administered by gavage in corn oil at doses of 2, 4, or 8 mg/kg, induced cholangiocarcinomas at high incidences in all dose levels in male rats, and therefore was a much more potent carcinogen than furfural (NTP, 1991). Studies with F344 rats have demonstrated that at dose levels comparable to those used in the 2-yr study, [14C]furfural is rapidly converted to furoic acid, furanacrylic acid, and furoylglycine, most likely by nonmixed-function oxidase-dependent pathways, and eliminated in urine (Irwin et al., 1985) as has been reported for humans (Sedivec and Flek, 1978; Flek and Sedivec, 1978). However, the disposition and metabolism of furan have not been well resolved. Therefore in the present study we have examined the disposition of [14C]furan in male F344 rats. For these studies furan was administered by gavage in corn oil at 8 mg/kg, the highest dose level used in the 2-yr carcinogenicity study. MATERIALS AND METHODS Chemicals Furan (99 + % pure) was purchased from Aldrich Chemical Co. (Milwaukee, Wis.). [2,5-14C]Furan (specific activity 56 mCi/mmol) was obtained from Amersham (Arlington Heights, III.) and was 99% radiochemically pure. Animals Male Fischer 344 rats were obtained from Charles River Breeding Laboratories (Wilmington, Mass.) and housed in facilities with an ambient temperature of 21-22°C, relative humidity 50 ± 10%, and 12-h light/dark cycle. Animals were given pelleted diet and water ad libitum. Treatment The desired amount of furan was dissolved in corn oil (5 ml corn oil/kg body weight). Radiolabeled furan was diluted as needed with nonlabeled furan to administer approximately 10 /zCi/kg body weight. Due to the volatility of furan, the dosing solution was prepared immediately prior to administration. Animals were dosed once per day for up to 8 consecutive days. Animals that were to be observed for 24 h were
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housed immediately in individual glass metabolism cages (MetabowlMark III, Jencons Ltd., Hertfordshire, England), with free access to water and food and an air flow maintained at 0.3-0.4 l/min. This housing allows for separate collection of urine and feces 5s well as trapping of expired CO2 and volatiles. Animals that were to receive multiple doses or that were to be observed for more than 24 h were housed in plastic metabolism cages that allowed for separate collection of urine and feces.
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Tissue Distribution Treated animals were killed at time points ranging from 1 to 8 d after dosing. At least three animals were used for each time point. At necropsy, animals were immediately dissected and each of the major tissues was removed, weighed, and stored at — 20°C until assayed. The radioactivity in each tissue was analyzed by combustion of triplicate 100-mg samples to 14CO2 in a Packard Tri-Carb sample oxidizer (Packard Instrument Co., Downers Grove, III.). The 14CO2 was counted in a Beckman LS-9800 liquid scintillation counter (Beckman Co., Fullerton, Calif.). Samples of urine were counted directly without combustion. Body composition estimates for blood and muscle were 8 and 50%, respectively (Matthews and Anderson, 1975), and for adipose and skin, 11 and 16%, respectively (Birnbaum et al., 1980). Fecal samples were air-dried, weighed, and ground to a fine powder with a mortar and pestle. Triplicate 100-mg samples were oxidized and the radioactivity was determined as described above. Carbon Dioxide Collection and Volatiles Collection Total air flow through the glass metabolism cages was passed through three traps. The first was an activated carbon trap (Environmental Compliance Corp., Venetia, Pa.) for collection of volatile compounds. The second and third traps each contained 400 ml of a mixture of ethylene glycol monomethyl ether and ethanolamine (7 : 3 v/v) for CO2 collection. The traps were changed at predetermined times. Triplicate aliquots from the CO2 traps were counted after mixing with Aquasol (NEN Research Products, Boston). The carbon traps were extracted with toluene and an aliquot of the extract was counted. Analysis of Expired Volatiles Expired volatiles, adsorbed by the activated carbon traps and eluted with toluene, were analyzed by high-performance liquid chromatography (HPLC). A 250 x 4.6 mm Rainin Microsorb C-18 column (Rainin Instrument Co., Woburn, Mass.) was used with a flow rate of 1.5 ml/min and a linear gradient of 99.9 : 0.1 water/acetic acid to 59.9 : 0.1 : 40 water/acetic acid/acetonitrile in 28 min. Under these conditions furan
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had a retention time of 12.5 min. Detection was by a radiochemical flow detector (Flo-one Beta, Radiomatic Instrument Co., Tampa, Fla.).
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Covalent Binding of [14C]Furan to Liver Macromolecules Covalent binding to DNA was assayed by the method of Marmur (1961) as modified by Anderson et al. (1981). Protein covalent binding was measured by homogenizing liver with 3 volumes of methanol, centrifuging, and washing the pellet with additional methanol until the radioactivity in the wash was twice background or less and then washing with an additional 3 volumes of ethyl acetate. The pellet was then dissolved in 0.5 N NaOH; triplicate aliquots were acidified with acetic acid and counted in a scintillation counter. The protein content was determined in triplicate aliquots by the method of Lowry et al. (1951). Analysis of Urinary Metabolites Urine from treated rats was acidified with 10 /il of acetic acid/ml and centrifuged at low g. HPLC analysis was performed using a Rainin Microsorb C-18 column at a flow rate of 1.5 ml/min and a linear gradient from 99 :1 water/acetic acid to 74 :1 : 25 water/acetic acid/acetonitrile in 28 min. Statistical Analysis Statistical analysis was performed using a pairwise comparison of variance (one-sided t test). Values were considered statistically significant at p < .05. RESULTS During the first 24 h after dosing, the expired air, urine, and feces were all significant routes of elimination of furan-derived radioactivity (Fig. 1). Approximately 14% of the administered dose was expired as unchanged furan, most (11% of the dose) within the first hour after dosing. Twenty-six percent of the administered dose was expired as 14 CO2, virtually all of which was eliminated in the first 12 h. Radioactivity in urine and feces accounted for about 20 and 22% of the administered dose, respectively. After 24 h, 19% of the administered radioactivity remained in the tissues. Distribution of furan-derived radioactivity in several tissues is shown in Table 1. After 24 h, the highest concentration of radioactivity by far was found in the liver (307 nmol eq/g tissue or 13% of the dose). Lesser concentrations were found in kidney and the gastrointestinal (Gl) tract. Lung, a target tissue for several furan-containing compounds, contained a relatively low concentration of radioactivity, lower than blood. A total of 15 tissues or organs were assayed for radioactivity. The
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30-
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UJ
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o TIME (hr) 14
FIGURE 1. Elimination of [ C]furan-derived radioactivity as carbon dioxide (open squares), in feces (open circles), in urine (closed squares), and as expired volatiles (closed circles) following a single 8-mg/kg dose. Points are means ± SD from three animals.
concentrations in the unreported tissues ranged from 1.0 to 4.3 nmol eq/g of tissue. Elimination of radioactivity after a single dose of [14C]furan was followed for 8 d. In this time 20 and 25% of the dose was eliminated in urine (Fig. 2) and feces (Fig. 3), respectively. Concentrations of radioactivity in liver, kidney and blood were also followed for 8 d (Fig. 4). Elimination of radioactivity from the liver appeared to follow first-order kinetics with a half-life of 1.8 d. The kinetics for elimination of radioactivity from kidney and blood appeared more complex than first TABLE 1. Tissue Radioactivity 24 Hours After an 8-mg/kg Dose of [14C]Furan Tissue
nmol eq/g Tissue
Liver Kidney Large intestine Small intestine Glandular stomach Forestomach Blood Lung
307 60 25 13 6.3 6.1 5.5 3.5
a
± 93 ± 12 ± 5 ± 3 ± 0.7 ±1.8 ± 0.7 ± 1.6
Mean ± standard deviation of three animals.
Percent dose 13 0.45 0.13 0.15 0.07 0.02 0.42 0.02
± ± ± ± ± ± ± ±
3a 0.8 0.03 0.04 0.07 0.01 0.0 0.01
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UJ CO
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1 2
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TIME (days) 14
FIGURE 2. Elimination of [ C]furan-derived radioactivity in urine following a single 8-mg/kg dose (squares) and daily 8-mg/kg doses for 8 d (circles). Points are means ± SD from 3-15 animals.
30-
TJ UJ U)
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FIGURE 3. Elimination of [14C]furan-derived radioactivity in feces following a single 8-mg/kg dose (squares) and daily 8-mg/kg doses for 8 d (circles). Points are means ± SD from 3-15 animals.
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If If
O o O E
10-
TIME (days) 14
FIGURE 4. Concentration of [ C]furan-derived radioactivity in (A) liver, (B) kidney, and (C) blood following a single 8-mg/kg dose (squares) and 8 daily 8-mg/kg doses (circles). Points are means ± SD from three animals.
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order. The blood concentration of radioactivity remained more or less constant during the 8 d following a single 8-mg/kg dose. The effect of multiple dosing on the distribution and elimination of 14 C was also investigated. Animals received daily doses of 8 mg/kg [14C]furan for up to 8 d. Groups of 3 were sacrificed 24 h after the last dose of 2,4, or 8 doses. The concentration of furan-derived radioactivity increased in liver, kidney, and blood with multiple doses (Fig. 4). The concentration appeared to increase linearly in kidney and blood during the first 4 d, but was nonlinear by d 8. The increase in concentration of radioactivity in the liver appeared to be nonlinear by d 4. The concentration of furan-derived radioactivity increased about six-fold in blood and kidney after eight doses compared to a single dose; the concentration increased fourfold in liver. The percent of the total administered radioactivity eliminated in feces was the same for daily doses for 8 d as for one dose followed for 8 d. In Fig. 3, the plots represent the percent of the total amount of radioactivity administered that was eliminated in the feces up to the reported time. If multiple dosing does not alter an elimination pathway, then the two plots should coincide in this format. For example, after 8 d in animals receiving 8 daily doses of [14C]furan, the actual amount of radioactivity administered was 8 times the amount given the singledose group, but the cumulative percent of the total radioactivity eliminated in feces was the same for both groups. The percent of total administered radioactivity eliminated in urine showed an upward trend after 4 d in the multiple-dose experiment. After 8 d, 33% of the total accumulated dose had been eliminated in urine compared to 20% in the single-dose 8-d experiment. The differences in urinary elimination between single and multiple doses on d 7 and 8 are statistically significant. The nature of the radioactive species present in liver and plasma was also investigated. At 24 h, HPLC analysis of plasma or an extract of liver showed no furan to be present. Only 20% of the radioactivity was extractable from liver by organic solvents; the remaining radioactivity was assumed to be "covalently bound" to tissue macromolecules. Extraction of liver homogenate with phenol-chloroform-amyl alcohol resulted in most of the radioactivity being in the protein interlayer. There was some radioactivity in the DNA-containing aqueous layer, but after precipitating the DNA, treating the resolubilized DNA with RNase and protease, and reprecipitating the DNA, the radioactivity was essentially at background. Radioactivity in the protein layer was not removed upon further extraction with organic solvents. Covalent binding of radioactivity to protein was assayed by exhaustive organic solvent extraction of liver homogenate (Fig. 5). The radioactivity remaining 24 h after a single 8-mg/kg dose of [14C]furan corresponded to 2.2 nmol eq of furan per gram of liver. The amount of unextractable radioactivity in protein after
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DISPOSITION OF FURAN IN MALE RATS
TIME (days) FIGURE 5. Covalent binding of [14C]furan-derived radioactivity to liver protein following a single 8-mg/kg dose (squares) and 8 daily 8-mg/kg doses (circles). Points are means ± SD from three animals.
a single dose decreased during 8 d in the same manner as the total tissue radioactivity (Fig. 5). Between the fourth and eighth dose the unextractable radioactivity in the multiple-dose experiment reached a plateau. HPLC analysis of urine collected during the 24 h following an 8mg/kg dose of [14C]furan revealed extensive metabolism. The radiochromatogram contained at least 10 peaks and several of these appeared to be poorly resolved mixtures (Fig. 6). The pattern of metabolites did not change appreciably in the multiple dose experiments. At this point none of the metabolites have been identified. The peaks eluting from the HPLC at 10-12,20, and 28 min (Fig. 6) were isolated, but unequivocal identification of metabolites could not be made. The nuclear magnetic resonance (NMR) spectrum (500 MHz) of each of the isolates contained resonances expected from a mercapturic acid, but the isolates were clearly mixtures. Further purification left insufficient material for NMR spectrometry. Attempts at obtaining FAB mass spectra were unsuccessful. DISCUSSION In the present study the disposition of [14C]furan in male F344 rats following daily 8 mg/kg doses for up to 8 d has been determined. After
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o UJ rUJ Q
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TIME (min) FIGURE 6. HPLC radiochromatogram of urine collected in the first 24 h following a single 8-mg/kg dose of [14C]furan. One hundred percent detector response is 200 cpm.
a single dose, most of the [MC]furan-derived radioactivity is eliminated in the 24 h following administration with about 40% of the radioactivity being eliminated in the expired air, 25% in the urine, and 20% in feces. Since furan is a volatile compound (bp 32°C), it was not unexpected that a substantial portion would be exhaled unchanged. In fact, about 14% of the furan was eliminated in this manner. However, more (26%) of the administered radioactivity was eliminated as 14CO2. Thus, a considerable portion of furan metabolism involves ring opening and oxidation to carbon dioxide. Assuming that the ring-opening process is the same as described for 2- and 3-methylfuran (Ravindranath et al., 1984), one would expect the initial ring-opened product to be c/s-2-butene-1,4-dial (maleic aldehyde). Oxidation of this dialdehyde would produce maleic acid, which has been shown to be rapidly metabolized to CO2 in rats, presumably involving enzymatic hydration to form malic acid (Taggart et al., 1962; Dreyer, 1985). Alternatively, it has been shown that maleic aldehyde is easily isomerized to fumaric aldehyde (Hufford et al., 1952). This dialdehyde could be further oxidized to fumaric acid, a citric acid cycle intermediate that would be oxidized to CO2. Most of the radioactivity remaining in the animal 24 h following a single 8-mg/kg dose was found in liver. Even tissues with large volumes, such as skin, muscle, and blood, contained two orders of magnitude less radioactivity on a percentage basis than liver. The presence of a large percentage of furan-derived radioactivity in liver is consistent with the observed hepatotoxicity of furan. High concentrations (nmol eq/g of tissue) of radioactivity compared to tissues and organs other than liver were found in kidney and the digestive tract. Lung, which retained relatively high concentrations of radioactivity following administration of
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[14C]-2-methylfuran (about 14% of the concentration found in liver) Ravindranath et al., 1986), retained approximately 1% of the concentration of radioactivity found in liver following treatment with furan. Thus, simple substitution of a methyl group on the furan ring can have a considerable effect on the distribution of these compounds. Covalent binding of furan-derived radioactivity to liver protein was evident from the amount of radioactivity remaining after solvent extraction of the tissue. Protein-bound radioactivity decreased following a single dose and increased following repeated doses in a manner similar to that observed for the concentration of radioactivity in whole tissue. Extraction of liver homogenate with chloroform-phenol-amyl alcohol resulted in a radioactive DNA fraction initially, but subsequent treatment of the DNA fraction with RNase, protease, reprecipitation, etc. resulted in loss of radioactivity in the DNA fraction. Since there was so much radioactivity associated with protein, it is probable that the radioactivity originally associated with the DNA fraction was in fact bound to incompletely separated protein. It may also be that the furan-DNA adduct, if formed, is not stable to further treatment by proteases, reprecipitation, etc. In an attempt to obtain higher levels of DNA-bound radioactivity, two rats were dosed 8 mg/kg, ip, with undiluted [14C]furan (56 mCi/mmol). Again, the radioactivity associated with the DNA layer was lost upon further processing. Incorporation of radioactivity from [14C]furan into the protein fraction probably results from formation of reactive species during furan metabolism. However, since citric acid cycle intermediates could result from furan metabolism, it is possible that amino acids synthesized from these precursors may incorporate radioactivity into protein. It would seem that there should be a more general tissue distribution of radioactivity if this were the explanation for incorporation, since the amino acids could be incorporated into proteins in tissues remote from their synthesis. In this study about 90% of the radioactivity remaining in the animal after 24 h was in the liver and most of this radioactivity was not extractable. For this reason we believe that the majority of the nonextractable radioactivity represents reaction with activated intermediates and not biosynthetic incorporation. While it is debatable whether covalent binding to protein is responsible for the cytotoxicity and necrosis associated with furan, the extent of covalent binding is certainly indicative of reactive species being formed during furan metabolism. The apparent lack of DNA binding found in this study is interesting since furan is carcinogenic under the conditions of the 2-yr bioassay (NTP, 1991). Furan is not a mutagen in the Ames Salmonella assay (Mortelmans et al., 1986), but it is positive in the mouse lymphoma-cell forward mutation assay (McGregor et al., 1988) and has been found to activate ras oncogenes in mouse liver (Reynolds et al., 1987). Induction of carcinogenesis by furan may result from
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a constant reaction of DNA with reactive species from furan metabolism at a level lower than detectable in this study, or, as suggested earlier (Reynolds et al., 1987), an indirect secondary genotoxic pathway resulting from a cytotoxic event. Indeed, furan-induced cell proliferation in rat liver at the highest dose administered in the 2-yr bioassay has recently been reported (Wilson et al., 1990, and in press). The differentiation between these and other possibilities will require more information.
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REFERENCES Anderson, M. W., Buroujerdi, M., and Wilson, A. G. E. 1981. Inhibition in vivo of the formation of adducts between metabolites of benzo(a)pyrene and DNA by butylated hydroxyanisole. Cancer Res. 41:4309-4315. Birnbaum, L. S., Decad, G. M., and Matthews, H. B. 1980. Disposition and excretion of 2,3,7,8tetrachlorodibenzofuran in the rat. Toxicol. Appl. Pharmacol. 55:342-352. Dreyer, J.-L. 1985. Isolation and biochemical characterization of maleic-acid hydratase, an ironrequiring hydro-lyase. Eur. J. Biochem. 150:145-154. Flek, J., and Sedivec, V. 1978. The absorption, metabolism and excretion of furfural in man. Int. Arch. Environ. Health 42:159-168. Hufford, D. L., Tarbell, D. S., and Koszalka, T. R. 1952. Maleic and fumaric dialdehydes, Δ4tetrahydrophthaladehyde and related compounds. J. Am. Chem. Soc. 74:3014-3018. Irwin, R. D., Enke, S. B., and Prejean, J. D. 1985. Urinary metabolites of furfural and furfuryl alcohol in F344/N rats. Toxicologist 5:240 (abstr. 960). Kirk-Othmer Encyclopedia of Chemical Technology. 1978. vol. 11, pp. 449-510. New York: John Wiley and Sons. Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Marmur, J. A. 1961. Procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3:208-218. Matthews, H. B., and Anderson, M. W. 1975. The distribution and excretion of 2,4,2´5´pentachlorobiphenyl in the rat. Drug Metab. Dispos. 3:211-219. McGregor, D. B., Brown, A., Cattanach, P., Edwards, I., McBride, D., Riach, C., and Caspary, W. J. 1988. Responses of the L5178Y tk + /tk + mouse lymphoma cell forward mutation assay. III. 72 coded chemicals. Environ. Mol. Mutagen. 12:85-154. Mortelmans, K., Haworth, S., Lawlor, T., Speck, W., Tainer, B., and Zeiger, E. 1986. Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals. Environ. Mutagen. 8(Suppl. 7):1119. National Toxicology Program. 1989. NTP Technical Report 382. Toxicology and carcinogenesis studies of furfural in F344 rats and B6C3F1 mice. Research Triangle Park, N.C.: National Toxicology Program. National Toxicology Program. 1991. NTP Technical Report. Toxicology and carcinogenesis studies of furan in F344 rats and B6C3F1 mice. Research Triangle Park, N.C.: National Toxicology Program. Quaker Oats. 1974. Furfural; General Information, Properties, Handling, Applications. Oak Brook, Ill.: Chemicals Division, The Quaker Oats Company, Bulletin 203C. Ravindranath, V., Burka, L. T., and Boyd, M. R. 1984. Reactive metabolites from the bioactivation of toxic methylfurans. Science 224:884-886. Ravindranath, V., McMenamin, M. G., Dees, J. H., and Boyd, M. R. 1986. 2-Methylfuran toxicity in rats—Role of metabolic activation in vivo. Toxicol. Appl. Pharmacol. 85:78-91. Reynolds, S. H., Stowers, S. J., Patterson, R. M., Maronpot, R. R., Aaronson, S. A., and Anderson, M. W. 1987. Activated oncogenes in B6C3F1 mouse liver tumors: Implications for risk assessment. Science 237:1309-1316.
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Sedivec, V., and Flek, J. 1978. Biologic monitoring of persons exposed to furfural vapors. Int. Arch. Environ. Health 42:42-49. Taggert, J. V., Angielski, S., and Morell, H. 1962. Complete oxidation of maleic acid via D(+)malate in kidney. Biochim. Biophys. Acta 58:141-144. Wilson, D. M., Goldsworthy, T. L., Popp, J. A., and Butterworth, B. E. 1990. Evaluation of genotoxicity, cytotoxicity and cell proliferation in hepatocytes from rats and mice treated with furan. Proc. AACR 81st Annual Meeting, abstr. 613. Wilson, D. M., Goldsworthy, T. L., Popp, J. A., and Butterworth, B. E. Evaluation of genotoxicity, pathology, and cell proliferation in livers of rats and mice treated with furan. Environ. Mol. Mutagen., in press.
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Received January 30, 1991 Accepted April 30, 1991
Journal of Toxicology and Environmental Health Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19
14
Disposition of [ C]furan in the male F344 rat b
a
Leo T. Burka , Kelly D. Washburn & Richard D. Irwin
a
a
National Institute of Environmental Health Sciences , Research Triangle Park, North Carolina b
National Institute of Environmental Health Sciences , P.O. Box 12233, Research Triangle Park, NC, 27709 Published online: 19 Oct 2009.
To cite this article: Leo T. Burka , Kelly D. Washburn & Richard D. Irwin (1991) Disposition of 14
[ C]furan in the male F344 rat, Journal of Toxicology and Environmental Health, 34:2, 245-257, DOI: 10.1080/15287399109531564 To link to this article: http://dx.doi.org/10.1080/15287399109531564
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DISPOSITION OF [14C]FURAN IN THE MALE F344 RAT Leo T. Burka, Kelly D. Washburn, Richard D. Irwin
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National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina
In a recently completed 2-yr bioassay, furan was found to induce cholangiocarcinomas at high incidence in rats. The disposition of single and multiple gavage doses of [2,514 C]furan has been determined in male F344 rats to aid in interpretation of that study. In the 24 h after dosing about 80% of the furan-derived radioactivity was eliminated, primarily via urine and expired air. [14C]Carbon dioxide was a major metabolite, indicating that furan ring opening followed by complete oxidation of at least one of the labeled carbons was a major part of the overall metabolism of furan. Liver contained more furan-derived radioactivity by far than other tissues after 24 h. Approximately 80% of the radioactivity in liver was not extracted by organic solvents and was associated with protein. There was either no binding to DNA or the furan-DNA adduct was not stable to the isolation procedure. Repeated daily administration of [14C]furan resulted in a more or less linear increase in covalent binding through four doses; at this point the amount of nonextractable radioactivity plateaus. Urine contained at least 10 metabolites, again indicating extensive metabolism of the furan ring. From the data obtained in this study it is clear that furan is metabolized to reactive species, apparently primarily in liver, and these intermediates react with protein. The hepatotoxicity resulting from furan exposure may be due to the reaction of furan metabolites with liver macromolecules; the presence of some of these reactive metabolites following chronic exposure to furan may result in cholangiocarcinomas.
INTRODUCTION Furan is the parent for a class of industrially important chemicals that include furfural, furfuryl alcohol, and their derivatives. Furan, furfural, and furfuryl alcohol are widely used in the production of corrosion-resistant polymers, high-temperature laminates, in the formation of foundry molds, as solvents, and for a variety of other applications (Kirk-Othmer, 1978; Quaker Oats, 1974). Because of large production volume and extensive industrial use, the National Toxicology Program (NTP) has been conducting studies to evaluate the toxicity and carcinogenic potential of furan, furfural, and furfuryl alcohol, as well as several other compounds that contain a furan ring as part of their structure. We thank Steven Vo for his excellent technical assistance. Requests for reprints should be sent to Leo T. Burka, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709.
245 Journal of Toxicology and Environmental Health, 34:245-257,1991 Copyright © 1991 by Hemisphere Publishing Corporation
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In NTP prechronic studies with F344 rats, furan was significantly more hepatotoxic than either furfural or furfuryl alcohol, causing extensive distortion of gross liver structure and substantial microscopic pathology at doses one-tenth those required for furfural- or furfuryl alcohol-induced hepatotoxicity (NTP, 1989,1991). In 2-yr carcinogenicity studies in F344 rats, furfural administered at doses of 30 or 60 mg/kg induced cholangiocarcinomas in 2 high-dose males and dysplasia with fibrosis, a percursor lesion to cholangiocarcinoma, in 2 additional highdose males (NTP, 1989). Furan, administered by gavage in corn oil at doses of 2, 4, or 8 mg/kg, induced cholangiocarcinomas at high incidences in all dose levels in male rats, and therefore was a much more potent carcinogen than furfural (NTP, 1991). Studies with F344 rats have demonstrated that at dose levels comparable to those used in the 2-yr study, [14C]furfural is rapidly converted to furoic acid, furanacrylic acid, and furoylglycine, most likely by nonmixed-function oxidase-dependent pathways, and eliminated in urine (Irwin et al., 1985) as has been reported for humans (Sedivec and Flek, 1978; Flek and Sedivec, 1978). However, the disposition and metabolism of furan have not been well resolved. Therefore in the present study we have examined the disposition of [14C]furan in male F344 rats. For these studies furan was administered by gavage in corn oil at 8 mg/kg, the highest dose level used in the 2-yr carcinogenicity study. MATERIALS AND METHODS Chemicals Furan (99 + % pure) was purchased from Aldrich Chemical Co. (Milwaukee, Wis.). [2,5-14C]Furan (specific activity 56 mCi/mmol) was obtained from Amersham (Arlington Heights, III.) and was 99% radiochemically pure. Animals Male Fischer 344 rats were obtained from Charles River Breeding Laboratories (Wilmington, Mass.) and housed in facilities with an ambient temperature of 21-22°C, relative humidity 50 ± 10%, and 12-h light/dark cycle. Animals were given pelleted diet and water ad libitum. Treatment The desired amount of furan was dissolved in corn oil (5 ml corn oil/kg body weight). Radiolabeled furan was diluted as needed with nonlabeled furan to administer approximately 10 /zCi/kg body weight. Due to the volatility of furan, the dosing solution was prepared immediately prior to administration. Animals were dosed once per day for up to 8 consecutive days. Animals that were to be observed for 24 h were
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housed immediately in individual glass metabolism cages (MetabowlMark III, Jencons Ltd., Hertfordshire, England), with free access to water and food and an air flow maintained at 0.3-0.4 l/min. This housing allows for separate collection of urine and feces 5s well as trapping of expired CO2 and volatiles. Animals that were to receive multiple doses or that were to be observed for more than 24 h were housed in plastic metabolism cages that allowed for separate collection of urine and feces.
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Tissue Distribution Treated animals were killed at time points ranging from 1 to 8 d after dosing. At least three animals were used for each time point. At necropsy, animals were immediately dissected and each of the major tissues was removed, weighed, and stored at — 20°C until assayed. The radioactivity in each tissue was analyzed by combustion of triplicate 100-mg samples to 14CO2 in a Packard Tri-Carb sample oxidizer (Packard Instrument Co., Downers Grove, III.). The 14CO2 was counted in a Beckman LS-9800 liquid scintillation counter (Beckman Co., Fullerton, Calif.). Samples of urine were counted directly without combustion. Body composition estimates for blood and muscle were 8 and 50%, respectively (Matthews and Anderson, 1975), and for adipose and skin, 11 and 16%, respectively (Birnbaum et al., 1980). Fecal samples were air-dried, weighed, and ground to a fine powder with a mortar and pestle. Triplicate 100-mg samples were oxidized and the radioactivity was determined as described above. Carbon Dioxide Collection and Volatiles Collection Total air flow through the glass metabolism cages was passed through three traps. The first was an activated carbon trap (Environmental Compliance Corp., Venetia, Pa.) for collection of volatile compounds. The second and third traps each contained 400 ml of a mixture of ethylene glycol monomethyl ether and ethanolamine (7 : 3 v/v) for CO2 collection. The traps were changed at predetermined times. Triplicate aliquots from the CO2 traps were counted after mixing with Aquasol (NEN Research Products, Boston). The carbon traps were extracted with toluene and an aliquot of the extract was counted. Analysis of Expired Volatiles Expired volatiles, adsorbed by the activated carbon traps and eluted with toluene, were analyzed by high-performance liquid chromatography (HPLC). A 250 x 4.6 mm Rainin Microsorb C-18 column (Rainin Instrument Co., Woburn, Mass.) was used with a flow rate of 1.5 ml/min and a linear gradient of 99.9 : 0.1 water/acetic acid to 59.9 : 0.1 : 40 water/acetic acid/acetonitrile in 28 min. Under these conditions furan
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had a retention time of 12.5 min. Detection was by a radiochemical flow detector (Flo-one Beta, Radiomatic Instrument Co., Tampa, Fla.).
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Covalent Binding of [14C]Furan to Liver Macromolecules Covalent binding to DNA was assayed by the method of Marmur (1961) as modified by Anderson et al. (1981). Protein covalent binding was measured by homogenizing liver with 3 volumes of methanol, centrifuging, and washing the pellet with additional methanol until the radioactivity in the wash was twice background or less and then washing with an additional 3 volumes of ethyl acetate. The pellet was then dissolved in 0.5 N NaOH; triplicate aliquots were acidified with acetic acid and counted in a scintillation counter. The protein content was determined in triplicate aliquots by the method of Lowry et al. (1951). Analysis of Urinary Metabolites Urine from treated rats was acidified with 10 /il of acetic acid/ml and centrifuged at low g. HPLC analysis was performed using a Rainin Microsorb C-18 column at a flow rate of 1.5 ml/min and a linear gradient from 99 :1 water/acetic acid to 74 :1 : 25 water/acetic acid/acetonitrile in 28 min. Statistical Analysis Statistical analysis was performed using a pairwise comparison of variance (one-sided t test). Values were considered statistically significant at p < .05. RESULTS During the first 24 h after dosing, the expired air, urine, and feces were all significant routes of elimination of furan-derived radioactivity (Fig. 1). Approximately 14% of the administered dose was expired as unchanged furan, most (11% of the dose) within the first hour after dosing. Twenty-six percent of the administered dose was expired as 14 CO2, virtually all of which was eliminated in the first 12 h. Radioactivity in urine and feces accounted for about 20 and 22% of the administered dose, respectively. After 24 h, 19% of the administered radioactivity remained in the tissues. Distribution of furan-derived radioactivity in several tissues is shown in Table 1. After 24 h, the highest concentration of radioactivity by far was found in the liver (307 nmol eq/g tissue or 13% of the dose). Lesser concentrations were found in kidney and the gastrointestinal (Gl) tract. Lung, a target tissue for several furan-containing compounds, contained a relatively low concentration of radioactivity, lower than blood. A total of 15 tissues or organs were assayed for radioactivity. The
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30-
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FIGURE 1. Elimination of [ C]furan-derived radioactivity as carbon dioxide (open squares), in feces (open circles), in urine (closed squares), and as expired volatiles (closed circles) following a single 8-mg/kg dose. Points are means ± SD from three animals.
concentrations in the unreported tissues ranged from 1.0 to 4.3 nmol eq/g of tissue. Elimination of radioactivity after a single dose of [14C]furan was followed for 8 d. In this time 20 and 25% of the dose was eliminated in urine (Fig. 2) and feces (Fig. 3), respectively. Concentrations of radioactivity in liver, kidney and blood were also followed for 8 d (Fig. 4). Elimination of radioactivity from the liver appeared to follow first-order kinetics with a half-life of 1.8 d. The kinetics for elimination of radioactivity from kidney and blood appeared more complex than first TABLE 1. Tissue Radioactivity 24 Hours After an 8-mg/kg Dose of [14C]Furan Tissue
nmol eq/g Tissue
Liver Kidney Large intestine Small intestine Glandular stomach Forestomach Blood Lung
307 60 25 13 6.3 6.1 5.5 3.5
a
± 93 ± 12 ± 5 ± 3 ± 0.7 ±1.8 ± 0.7 ± 1.6
Mean ± standard deviation of three animals.
Percent dose 13 0.45 0.13 0.15 0.07 0.02 0.42 0.02
± ± ± ± ± ± ± ±
3a 0.8 0.03 0.04 0.07 0.01 0.0 0.01
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UJ CO
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1 2
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FIGURE 2. Elimination of [ C]furan-derived radioactivity in urine following a single 8-mg/kg dose (squares) and daily 8-mg/kg doses for 8 d (circles). Points are means ± SD from 3-15 animals.
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FIGURE 3. Elimination of [14C]furan-derived radioactivity in feces following a single 8-mg/kg dose (squares) and daily 8-mg/kg doses for 8 d (circles). Points are means ± SD from 3-15 animals.
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If If
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FIGURE 4. Concentration of [ C]furan-derived radioactivity in (A) liver, (B) kidney, and (C) blood following a single 8-mg/kg dose (squares) and 8 daily 8-mg/kg doses (circles). Points are means ± SD from three animals.
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order. The blood concentration of radioactivity remained more or less constant during the 8 d following a single 8-mg/kg dose. The effect of multiple dosing on the distribution and elimination of 14 C was also investigated. Animals received daily doses of 8 mg/kg [14C]furan for up to 8 d. Groups of 3 were sacrificed 24 h after the last dose of 2,4, or 8 doses. The concentration of furan-derived radioactivity increased in liver, kidney, and blood with multiple doses (Fig. 4). The concentration appeared to increase linearly in kidney and blood during the first 4 d, but was nonlinear by d 8. The increase in concentration of radioactivity in the liver appeared to be nonlinear by d 4. The concentration of furan-derived radioactivity increased about six-fold in blood and kidney after eight doses compared to a single dose; the concentration increased fourfold in liver. The percent of the total administered radioactivity eliminated in feces was the same for daily doses for 8 d as for one dose followed for 8 d. In Fig. 3, the plots represent the percent of the total amount of radioactivity administered that was eliminated in the feces up to the reported time. If multiple dosing does not alter an elimination pathway, then the two plots should coincide in this format. For example, after 8 d in animals receiving 8 daily doses of [14C]furan, the actual amount of radioactivity administered was 8 times the amount given the singledose group, but the cumulative percent of the total radioactivity eliminated in feces was the same for both groups. The percent of total administered radioactivity eliminated in urine showed an upward trend after 4 d in the multiple-dose experiment. After 8 d, 33% of the total accumulated dose had been eliminated in urine compared to 20% in the single-dose 8-d experiment. The differences in urinary elimination between single and multiple doses on d 7 and 8 are statistically significant. The nature of the radioactive species present in liver and plasma was also investigated. At 24 h, HPLC analysis of plasma or an extract of liver showed no furan to be present. Only 20% of the radioactivity was extractable from liver by organic solvents; the remaining radioactivity was assumed to be "covalently bound" to tissue macromolecules. Extraction of liver homogenate with phenol-chloroform-amyl alcohol resulted in most of the radioactivity being in the protein interlayer. There was some radioactivity in the DNA-containing aqueous layer, but after precipitating the DNA, treating the resolubilized DNA with RNase and protease, and reprecipitating the DNA, the radioactivity was essentially at background. Radioactivity in the protein layer was not removed upon further extraction with organic solvents. Covalent binding of radioactivity to protein was assayed by exhaustive organic solvent extraction of liver homogenate (Fig. 5). The radioactivity remaining 24 h after a single 8-mg/kg dose of [14C]furan corresponded to 2.2 nmol eq of furan per gram of liver. The amount of unextractable radioactivity in protein after
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TIME (days) FIGURE 5. Covalent binding of [14C]furan-derived radioactivity to liver protein following a single 8-mg/kg dose (squares) and 8 daily 8-mg/kg doses (circles). Points are means ± SD from three animals.
a single dose decreased during 8 d in the same manner as the total tissue radioactivity (Fig. 5). Between the fourth and eighth dose the unextractable radioactivity in the multiple-dose experiment reached a plateau. HPLC analysis of urine collected during the 24 h following an 8mg/kg dose of [14C]furan revealed extensive metabolism. The radiochromatogram contained at least 10 peaks and several of these appeared to be poorly resolved mixtures (Fig. 6). The pattern of metabolites did not change appreciably in the multiple dose experiments. At this point none of the metabolites have been identified. The peaks eluting from the HPLC at 10-12,20, and 28 min (Fig. 6) were isolated, but unequivocal identification of metabolites could not be made. The nuclear magnetic resonance (NMR) spectrum (500 MHz) of each of the isolates contained resonances expected from a mercapturic acid, but the isolates were clearly mixtures. Further purification left insufficient material for NMR spectrometry. Attempts at obtaining FAB mass spectra were unsuccessful. DISCUSSION In the present study the disposition of [14C]furan in male F344 rats following daily 8 mg/kg doses for up to 8 d has been determined. After
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TIME (min) FIGURE 6. HPLC radiochromatogram of urine collected in the first 24 h following a single 8-mg/kg dose of [14C]furan. One hundred percent detector response is 200 cpm.
a single dose, most of the [MC]furan-derived radioactivity is eliminated in the 24 h following administration with about 40% of the radioactivity being eliminated in the expired air, 25% in the urine, and 20% in feces. Since furan is a volatile compound (bp 32°C), it was not unexpected that a substantial portion would be exhaled unchanged. In fact, about 14% of the furan was eliminated in this manner. However, more (26%) of the administered radioactivity was eliminated as 14CO2. Thus, a considerable portion of furan metabolism involves ring opening and oxidation to carbon dioxide. Assuming that the ring-opening process is the same as described for 2- and 3-methylfuran (Ravindranath et al., 1984), one would expect the initial ring-opened product to be c/s-2-butene-1,4-dial (maleic aldehyde). Oxidation of this dialdehyde would produce maleic acid, which has been shown to be rapidly metabolized to CO2 in rats, presumably involving enzymatic hydration to form malic acid (Taggart et al., 1962; Dreyer, 1985). Alternatively, it has been shown that maleic aldehyde is easily isomerized to fumaric aldehyde (Hufford et al., 1952). This dialdehyde could be further oxidized to fumaric acid, a citric acid cycle intermediate that would be oxidized to CO2. Most of the radioactivity remaining in the animal 24 h following a single 8-mg/kg dose was found in liver. Even tissues with large volumes, such as skin, muscle, and blood, contained two orders of magnitude less radioactivity on a percentage basis than liver. The presence of a large percentage of furan-derived radioactivity in liver is consistent with the observed hepatotoxicity of furan. High concentrations (nmol eq/g of tissue) of radioactivity compared to tissues and organs other than liver were found in kidney and the digestive tract. Lung, which retained relatively high concentrations of radioactivity following administration of
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[14C]-2-methylfuran (about 14% of the concentration found in liver) Ravindranath et al., 1986), retained approximately 1% of the concentration of radioactivity found in liver following treatment with furan. Thus, simple substitution of a methyl group on the furan ring can have a considerable effect on the distribution of these compounds. Covalent binding of furan-derived radioactivity to liver protein was evident from the amount of radioactivity remaining after solvent extraction of the tissue. Protein-bound radioactivity decreased following a single dose and increased following repeated doses in a manner similar to that observed for the concentration of radioactivity in whole tissue. Extraction of liver homogenate with chloroform-phenol-amyl alcohol resulted in a radioactive DNA fraction initially, but subsequent treatment of the DNA fraction with RNase, protease, reprecipitation, etc. resulted in loss of radioactivity in the DNA fraction. Since there was so much radioactivity associated with protein, it is probable that the radioactivity originally associated with the DNA fraction was in fact bound to incompletely separated protein. It may also be that the furan-DNA adduct, if formed, is not stable to further treatment by proteases, reprecipitation, etc. In an attempt to obtain higher levels of DNA-bound radioactivity, two rats were dosed 8 mg/kg, ip, with undiluted [14C]furan (56 mCi/mmol). Again, the radioactivity associated with the DNA layer was lost upon further processing. Incorporation of radioactivity from [14C]furan into the protein fraction probably results from formation of reactive species during furan metabolism. However, since citric acid cycle intermediates could result from furan metabolism, it is possible that amino acids synthesized from these precursors may incorporate radioactivity into protein. It would seem that there should be a more general tissue distribution of radioactivity if this were the explanation for incorporation, since the amino acids could be incorporated into proteins in tissues remote from their synthesis. In this study about 90% of the radioactivity remaining in the animal after 24 h was in the liver and most of this radioactivity was not extractable. For this reason we believe that the majority of the nonextractable radioactivity represents reaction with activated intermediates and not biosynthetic incorporation. While it is debatable whether covalent binding to protein is responsible for the cytotoxicity and necrosis associated with furan, the extent of covalent binding is certainly indicative of reactive species being formed during furan metabolism. The apparent lack of DNA binding found in this study is interesting since furan is carcinogenic under the conditions of the 2-yr bioassay (NTP, 1991). Furan is not a mutagen in the Ames Salmonella assay (Mortelmans et al., 1986), but it is positive in the mouse lymphoma-cell forward mutation assay (McGregor et al., 1988) and has been found to activate ras oncogenes in mouse liver (Reynolds et al., 1987). Induction of carcinogenesis by furan may result from
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a constant reaction of DNA with reactive species from furan metabolism at a level lower than detectable in this study, or, as suggested earlier (Reynolds et al., 1987), an indirect secondary genotoxic pathway resulting from a cytotoxic event. Indeed, furan-induced cell proliferation in rat liver at the highest dose administered in the 2-yr bioassay has recently been reported (Wilson et al., 1990, and in press). The differentiation between these and other possibilities will require more information.
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REFERENCES Anderson, M. W., Buroujerdi, M., and Wilson, A. G. E. 1981. Inhibition in vivo of the formation of adducts between metabolites of benzo(a)pyrene and DNA by butylated hydroxyanisole. Cancer Res. 41:4309-4315. Birnbaum, L. S., Decad, G. M., and Matthews, H. B. 1980. Disposition and excretion of 2,3,7,8tetrachlorodibenzofuran in the rat. Toxicol. Appl. Pharmacol. 55:342-352. Dreyer, J.-L. 1985. Isolation and biochemical characterization of maleic-acid hydratase, an ironrequiring hydro-lyase. Eur. J. Biochem. 150:145-154. Flek, J., and Sedivec, V. 1978. The absorption, metabolism and excretion of furfural in man. Int. Arch. Environ. Health 42:159-168. Hufford, D. L., Tarbell, D. S., and Koszalka, T. R. 1952. Maleic and fumaric dialdehydes, Δ4tetrahydrophthaladehyde and related compounds. J. Am. Chem. Soc. 74:3014-3018. Irwin, R. D., Enke, S. B., and Prejean, J. D. 1985. Urinary metabolites of furfural and furfuryl alcohol in F344/N rats. Toxicologist 5:240 (abstr. 960). Kirk-Othmer Encyclopedia of Chemical Technology. 1978. vol. 11, pp. 449-510. New York: John Wiley and Sons. Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Marmur, J. A. 1961. Procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3:208-218. Matthews, H. B., and Anderson, M. W. 1975. The distribution and excretion of 2,4,2´5´pentachlorobiphenyl in the rat. Drug Metab. Dispos. 3:211-219. McGregor, D. B., Brown, A., Cattanach, P., Edwards, I., McBride, D., Riach, C., and Caspary, W. J. 1988. Responses of the L5178Y tk + /tk + mouse lymphoma cell forward mutation assay. III. 72 coded chemicals. Environ. Mol. Mutagen. 12:85-154. Mortelmans, K., Haworth, S., Lawlor, T., Speck, W., Tainer, B., and Zeiger, E. 1986. Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals. Environ. Mutagen. 8(Suppl. 7):1119. National Toxicology Program. 1989. NTP Technical Report 382. Toxicology and carcinogenesis studies of furfural in F344 rats and B6C3F1 mice. Research Triangle Park, N.C.: National Toxicology Program. National Toxicology Program. 1991. NTP Technical Report. Toxicology and carcinogenesis studies of furan in F344 rats and B6C3F1 mice. Research Triangle Park, N.C.: National Toxicology Program. Quaker Oats. 1974. Furfural; General Information, Properties, Handling, Applications. Oak Brook, Ill.: Chemicals Division, The Quaker Oats Company, Bulletin 203C. Ravindranath, V., Burka, L. T., and Boyd, M. R. 1984. Reactive metabolites from the bioactivation of toxic methylfurans. Science 224:884-886. Ravindranath, V., McMenamin, M. G., Dees, J. H., and Boyd, M. R. 1986. 2-Methylfuran toxicity in rats—Role of metabolic activation in vivo. Toxicol. Appl. Pharmacol. 85:78-91. Reynolds, S. H., Stowers, S. J., Patterson, R. M., Maronpot, R. R., Aaronson, S. A., and Anderson, M. W. 1987. Activated oncogenes in B6C3F1 mouse liver tumors: Implications for risk assessment. Science 237:1309-1316.
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Sedivec, V., and Flek, J. 1978. Biologic monitoring of persons exposed to furfural vapors. Int. Arch. Environ. Health 42:42-49. Taggert, J. V., Angielski, S., and Morell, H. 1962. Complete oxidation of maleic acid via D(+)malate in kidney. Biochim. Biophys. Acta 58:141-144. Wilson, D. M., Goldsworthy, T. L., Popp, J. A., and Butterworth, B. E. 1990. Evaluation of genotoxicity, cytotoxicity and cell proliferation in hepatocytes from rats and mice treated with furan. Proc. AACR 81st Annual Meeting, abstr. 613. Wilson, D. M., Goldsworthy, T. L., Popp, J. A., and Butterworth, B. E. Evaluation of genotoxicity, pathology, and cell proliferation in livers of rats and mice treated with furan. Environ. Mol. Mutagen., in press.
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Received January 30, 1991 Accepted April 30, 1991
