Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Metabolism and nephrotoxicity of Indan in male fischer 344 rats M. P. Servé , M. J. Ferry , K. O. Yu , C. T. Olson & D. W. Hobson To cite this article: M. P. Servé , M. J. Ferry , K. O. Yu , C. T. Olson & D. W. Hobson (1990) Metabolism and nephrotoxicity of Indan in male fischer 344 rats, Journal of Toxicology and Environmental Health, 29:4, 409-416, DOI: 10.1080/15287399009531401 To link to this article: http://dx.doi.org/10.1080/15287399009531401
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METABOLISM AND NEPHROTOXICITY OF INDAN IN MALE FISCHER 344 RATS M. P. Servé, M. J. Ferry, K. O. Yu Department of Chemistry, Wright State University, Dayton, Ohio C. T. Olson, D. W. Hobson
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Battelle Memorial Institute, Columbus, Ohio
Indan, a component of fuels, solvents, and varnishes, is metabolized in male Fischer 344 rats to 1-indanol, 2-indanol, 5-indanol, 1-indanone, 2-indanone, 2-hydroxy-1indanone, cis-1,2-indandiol, and trans-1,2-indandiol. The metabolites were identified using the techniques of gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The rats treated with indan demonstrated the classic lesions of hydrocarbon-induced nephropathy. The kidney damage produced was less than that found for tetralin and other branched-chain acyclic hydrocarbons.
INTRODUCTION The cyclic molecules eis- and frans-decalin (Olson et al., 1986) and tetralin (Servé et al., 1989) have been shown to produce a specific nephropathy consisting of hyaline droplet formation in the proximal tubules of male rats. Long-term exposure to these chemicals or hydrocarbon-based fuels resulted in additional damage consisting of tubular segments near the corticomedullary junction being blocked and dilated with casts of necrotic cell debris, resulting in cell death and in some cases tumor formation (Gaworski et al., 1984). It has recently been reported that the normally nonnephrotoxic monocyclic hydrocarbon cyclohexane can produce the similar renal protein pathology when a branched alkyl side chain such as tertiary butyl or isopropyl is attached to the cyclohexane ring (Henningsen et al., 1987,1988). Studies of the hyaline droplets found in the rat proximal tubule after hydrocarbon exposure have concluded that the protein a2u-globulin is the main component (Kanerva et al., 1987; Olson et al., 1987). A recent report showed that exposure to unleaded gasoline did not alter the a2uglobulin level in aged male rats, in whom the capacity to synthesize the The authors wish to thank the U.S. Air Force for their generous funding of this work under grant number AFOSR-87-0108. Requests for reprints should be sent to M. P. Servé, Department of Chemistry, Wright State University, Dayton, OH 45435.
409 Journal of Toxicology and Environmental Health, 29:409-416, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
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protein had diminished (Murty et al., 1988). The authors suggested that young male rats approximately 3.5 mo old were the most susceptible to the hyaline droplet nephropathy and that the susceptibility abated with age. A review of the work on a2u-globulin and the difficulty in extrapolation of the rat data to humans was recently published (Swenberg et al., 1989). Examination of the chemical structures of the alkyl-substituted cyclohexanes, tetralin, and the isomeric decalins revealed that all the molecules possess a nonplanar chair configuration. This chair structure may alter the metabolic biotransformation of the cyclic compounds by inhibiting the fit of the oxidative enzymes or the conjugation enzymes that are necessary for the urinary excretion of the hydrocarbons. The resulting prolonged time in which the hydrocarbon, or a metabolite of the hydrocarbon, remains in the animal could be a vital factor in the production of nephrotoxicity. Cyclic molecules, which do not possess the chair configuration, have not been examined for their ability to induce the aforementioned nephrotoxicity. In order to see whether a planar cyclic hydrocarbon would be less toxic to the kidneys, it was decided to look at the nephrotoxic potential of indan. Indan (QH^) is a common cyclic hydrocarbon found as a constituent of naphtha solvents (Hibino and Suzumura, 1955) and as a component of reformed gasolines (Bohlman et al., 1965). Indan is a planar molecule with a boiling point of 178°C, which is within 20°C of the boiling points of tetralin and the decalins. The volatility of the hydrocarbon is important since low-boiling molecules such as cyclohexane are easily exhaled by the animal and metabolism and excretion of the xenobiotic through the kidneys are reduced with a resulting diminution of nephrotoxicity. An examination of the metabolism and nephrotoxicity potential of indan should yield information concerning the effect of hydrocarbon structure on the extent of renal damage and the type of biotransformation reactions facilitated (Fig. 1). MATERIALS AND METHODS
1-lndanol, 2-indanol, 5-indanol, 2-indanone, indene, 4-chromanone, dihydrocoumarin, 5-methoxy-1-indanone, hexamethyldisilazane, and trimethylsilyl iodide were purchased from the Aldrich Chemical Company (Milwaukee, Wis.); 1-indanone and indan were obtained from the Lancaster Synthesis Ltd. (Windham, N.H.). The indan was distilled to >99% purity as indicated by GC. 2-Hydroxy-1-indanone formed by treating 1indanone with hexamethyldisilazane and trimethylsily! iodide (Miller and McKean, 1979). 3-Hydroxy-1-indanone was prepared by oxidation of 3bromo-1-indanone (Treibs and Schroth, 1961) according to the literature procedure (Undheim and Nilsen, 1975). 4-Hydroxy-1-indanone was synthesized by heating dihydrocoumarin and aluminum chloride (Louden and Razdan, 1954). 5-Hydroxy-1-indanone was isolated from the reaction
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INDAN
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FIGURE 1. Scheme 1.
of 5-methoxy-1-indanone and aluminum bromide. 6-Hydroxy-1-indanone was prepared by the cyclization of p-methoxy-3-phenylpropanoic acid (Louden and Razdan, 1954). The fusion of 4-chromanone and aluminum chloride yielded 7-hydroxy-1-indanone (Louden and Razdan, 1954). The eis- and frans-indan-1,2-diol were prepared according to the literature procedure (Balsamo et al., 1974). Animals, Housing, and Exposure Conditions
Eighteen Fischer 344 male rats (Charles River Breeding Laboratories, Wilmington, Mass.) weighing 270 ± 13 g were divided into 2 groups of 9 treated rats and 9 control rats. The rats were maintained in a controlled environment with temperatures of 74 ± 2°F, relative humidity of 55 ± 10%, and light cycle of 12 h on and 12 h off. When urine was not being collected using metabolism cages, rats were housed three to a polycarbonate cage with hardwood chips for bedding. Feed and water were available ad libitum. Rats were dosed by gavage with neat indan at 240 mg/kg body weight. A dosage of 480 mg indan/kg resulted in the death of all animals within 2 d. (All the rats that perished due to overdosing showed a red coloration in their urines that was determined to be a mixture of coproporphyrin and uroporphyrin, two precursors of heme. Blood tests on the rats showed that the animals were anemic. No autopsy was performed.) Dosing was continued on alternate days for 2 wk to facilitate metabolite identification and to rapidly induce any potential renal toxic effects. (More frequent dosing of the rats with indan can cause an alteration in their eating habits, which in turn can lead to severe weight loss and other complications.) Nine control rats were dosed with equivalent volumes of water over the same period. At 24 h following the final indan dose, the rats were sacrificed by halothane overdose and the kidneys excised. One kidney was used for histopathologic examination while the other kidney was homogenized in distilled water to look for any indan metabolites that might be embedded in the kidney itself. The kidney homogenate was processed using the same procedure as used for the urine (vide infra). Histopathologic
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examination was performed on paraffin-embedded kidney sections stained with hematoxylin and eosin. Tissues from exposed rats were compared to those from controls for the presence of characteristic lesions of hydrocarbon-induced nephropathy, including hyaline droplet formation, tubular cysts, and papillary calcification. Lesions were graded by pathologists for degree of severity.
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Isolation of Urinary Metabolites
For the first 48 h following initial dosing, rats were placed in metabolism cages where the urine was collected and then frozen until analyzed. The pH of urine samples was adjusted to 4.0, and 0.5 ml glucuronidase/sulfatase (specific activity of 5.5 and 1.5 units/ml, respectively; Calbiochem, Lajolla, Calif.) was added to 5.0-ml aliquots. This quantity of enzyme was in excess of that needed for complete hydrolysis of conjugates, as determined by varying the quantities added to different aliquots of samples. The solutions were shaken at 37°C for 16 h, cooled at room temperature, and filtered through diatomaceous earth columns (Clin Elut, Analytichem International, Harbor City, Calif.) using neat méthylène chloride as eluent. Unconjugated metabolites of indan were isolated by direct extraction of a second 5.0-ml aliquot of urine with méthylène chloride. The urine extract samples were then analyzed by gas chromatography and gas chromatography/mass spectrometry. Urinary Metabolite Identification
The méthylène chloride extracts of the urine metabolites were analyzed on a gas Chromatograph equipped with a flame ionization detector (model 5880A Hewlett-Packard Corp., Avondale, Pa.) and using a 25 m x 0.22 m ID carbowax 20M fused silica column (Hewlett-Packard Corp., Avondale, Pa.). A 60°C oven temperature was maintained for 1 min after sample injection and then programmed at 2°C/min to 180°C. Detector and injection port temperatures were 250 and 200°C, respectively. Helium was used as the carrier gas, with a linear velocity of 22 cm/s at 100°C and a split ratio of 5 : 1 . Metabolite identification was conducted with a Hewlett-Packard 5985 gas chromatograph/mass spectrometer system and a 4 ft x 2 mm ID 3% SP-1000 and 100/200 Supelco-port glass column (Supelco Corp., Bellefonte, Pa.) with a helium flow rate of 28 ml/min. The oven temperature was held at 100°C for 1 min and then programmed at 10°C/min to 200°C. The mass spectrometer was a quadrupole instrument operated in the electron impact mode at a voltage of 70 eV and with an ion source temperature of 200°C. Identification of both treated and control sets of urinary metabolites was confirmed by comparing mass spectra fragmentation patterns with the fragmentation patterns of purchased or synthesized compounds. Quantitation of the metabolites was accom-
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TABLE 1. Urinary and Kidney Metabolites of Fischer 344 Male Rats Dosed with Indan
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Relative amounts of metabolites recovered Metabolite
0-48 h
Final3
1-lndanol 2-lndanol 5-Indanol 1-lndanone 2-lndanone 2-Hydroxy-1-indanone 3-Hydroxyl-1-indanone c/'s-1,2-lndandiol trans-1,2-lndandiol
6
b
a
1.1 1.1 2.7 3.2 50 27.8 4.3 9.1
± ± ± ± ± ± ± ±
0.2 0.3 0.4 1 8 5 2 2
1.0 0.9 2.9 3.4 52 26 4.8 11.4
± 0.2 ± 0.4 ± 0.4 ± 0.8 ± 8 ± 5 ±1.8 ± 2
Urine collection following last dosing. Could not be separated from 4-methylphenol, present in urine of control rats.
fc
pushed by taking a weighed sample of the individual metabolites (100 mg), adding a weighed amount of dodecane (50 mg), and determining the relative gas Chromatographie peak areas. A weighed sample of dodecane was then added to the urine samples before analysis, and the ratio of the gas Chromatograph area was compared to yield the relative molar abundances. RESULTS Metabolites Fischer 344 male rats exposed by gavage to indan produced the urinary metabolites listed in Table 1. The values listed in Table 1 represent the sum of free and conjugated urinary metabolites, although GC analysis of ¡ndan-dosed rat urines not treated with glucuronidase/sulfatase showed the absence of indan or its metabolites. With the exception of 5indanol, all of the other metabolites showed that oxidative attack had occurred on the saturated five-membered ring of indan. Metabolic oxidation of the cyclopentane ring produced a variety of monoalcohols, ketones, hydroxyketones, and diols. There was no trace of any disubstituted metabolites in which oxidation had occurred on each ring. Analysis of the kidney homogenates showed the presence of 1-indanol and 1indanone in the ratio of 1 : 2.5. Histopathology When compared to controls, male Fischer 344 rats given indan showed a mild increase in cytoplasmic hyaline droplets in the epithelial
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cells of the proximal convoluted tubules of the nephrons. The morphology of the hyaline droplets varied from elongated, crystalline forms to homogeneous spheroids of assorted dimensions. Also present within the proximal convoluted tubules were foci of cellular degeneration, where epithelial cells exhibited increased cytoplasmic basophilia and vesicular nuclei. Not seen were intratubular casts, overt glomerular changes, and significant inflammation. The kidneys of the control rats showed no unusual changes.
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DISCUSSION The extensive metabolism of indan on the nonaromatic ring paralleled the metabolism of tetralin (Servé et al., 1989). The simple ketones 1and 2-indanone were isolated as metabolites from indan, but the analogous ketones 1- and 2-tetralone were not isolated from tetralin. 1,4Tetralindiol was observed to be present in tetralin-exposed rats, while indan produced no indan-1,3-diol. Both tetralin and indan yielded a 2hydroxy-1-ketone metabolite and a metabolite in which one benzylic position had been oxidized to a ketone while the other benzylic carbon had been converted to an alcohol. The identification of 1-indanone from the homogenized kidney extracts of the indan-exposed rats that showed renal damage corresponds with the finding of eis- and frans-2-decalones in the kidney extracts of male rats dosed with eis- and frans-decalin. No metabolites were found from the kidney homogenates of male rats dosed with tetralin. Histopathologic studies revealed that the damage done to the kidneys by indan was typical of that reported for other hydrocarbons. However, the severity of the damage, with respect to hyaline droplet formation, intratubular cast formation, and inflammation, was less than that found in male rats exposed to tetralin and the decalins. Since one of the major differences in structure between ¡ndan and the other cyclic hydrocarbons tetralin and the decalins is the planar structure of the indan molecule, it is possible that this stereochemical situation permits easier excretion of the indan metabolite from the animal. It has recently been suggested that in the case of 2,2,4-trimethylpentane, the protein droplet nephropathy may not depend on chemical binding to a2u-globulin, but on conformational changes in the protein structure produced by the xenobiotic chemical or one of its metabolites (Borghoff et al., 1989). A mechanism similar to the branched-chain acyclic hydrocarbons may be involved in the hydrocarbon-induced nephropathy of the various cyclic hydrocarbons; for example, a prolonged lifetime of the hydrocarbon metabolite in the kidney increases the opportunity for the metabolite to bind with the a2u-globulin in such a manner that the a2u-globulin conformation is altered and cannot be broken down by lysozymal enzymes. A
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facilitation of the conjugation of the metabolite with glucuronic acid or sulfate could in turn speed the removal of the metabolite from the kidneys and lessen the opportunity for the metabolite to interact with the a2u-globulin and initiate the hydrocarbon nephrotoxicity. The approach and reaction of the conjugating enzyme systems may be made easier because of the planar structure of the indan molecule. Once conjugation has occurred, excretion would be expected to soon follow and the potential for hydrocarbon would be reduced.
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REFERENCES Balsamo, A., Berti, G., Crotti, P., Ferretti, M., Macchia, B., and Macchia, F. 1974. The anomalous steric course of ring opening reactions of indene oxide. A reexamination. J. Org. Chem. 39:2596-2598. Bohlman, D., Wehner, K., and Leibnitz, E. 1965. Investigation of the components of heavy reformed fractions, especially concerning the production of aromatic hydrocarbons. J. Prakt. Chem. 29:215-229. Borghoff, S. J., Upton, P. B., and Swenberg, J. A. 1989. Characteristic of 2,2,4-trimethyl-2-pentanol binding to α 2u -globulin and other compounds that cause protein droplet nephropathy. Toxicologist 9:79. Gaworski, C. L., MacEwen, J. D., Vernot, E. H., Bruner, R. H., and Cowan, M. J. 1984. Comparison of the subchronic inhalation toxicity of petroleum and oil shale JP5 jet fuels. In Advances in Modern Environmental Toxicology, vol. VII, Renal Effects of Petroleum Hydrocarbons, ed. M. A. Mehlman, pp. 33-48. Princeton, N.J.: Princeton Scientific. Henningsen, G. M., Yu, K. O., Salomon, R. A., Ferry, M. J., Lopez, I., Roberts, J. A., and Servé, M. P. 1987. The metabolism of t-butylcyclohexane in Fischer 344 male rats with hyaline droplet nephropathy. Toxicol. Lett. 39:313-318. Henningsen, G. M., Salomon, R. A., Yu, K. O., Lopez, I., Roberts, J. A., and Servé, M. P. 1988. Metabolism of nephrotoxic isopropylcyclohexane in male Fischer 344 rats. J. Toxicol. Environ. Health 24:19-25. Hibino, O., and Suzumura, H. 1955. Constituents of solvent naphtha. Coal Tar 7:301-304. Kanerva, R. L., Ridder, G. M., Stone, L. C., and Alden, C. L. 1987. Characterization of spontaneous and decalin-induced hyaline droplets in kidneys of adult male rats. Food Chem. Toxicol. 25:63-82. Louden, J. D., and Razdan, R. K. 1954. Rearrangements of chromanones and dihydrocoumarins by aluminum chloride. J. Chem. Soc. 4299-4303. Miller, R. D., and McKean, D. R. 1979. The facile silylation of aldehydes and ketones using trimethylsilyl iodide: An exceptionally simple procedure for the generation of thermodynamically equilibrated trimethylsilylenol ethers. Synthesis 730-732. Murty, C. V. R., Olson, M. J., Garg, B. D., and Roy, A. K. 1988. Hydrocarbon-induced hyaline droplet nephropathy in male rats during senescence. Toxicol. Appl. Pharmacol. 96:380-392. Olson, C. T., Yu, K. O., and Servé, M. P. 1986. The metabolism of cis- and trans-decalin in Fischer 344 rats. J. Toxicol. Environ. Health 18:285-292. Olson, M. J., Manicini, M. A., Garg, B. D., and Roy, A. K. 1987. Accumulation of a 2u -globulin in the renal proximal tubules of male rats exposed to unleaded gasoline. Toxicol. Appl. Pharmacol. 90:43-51. Servé, M. P., Llewelyn, B. M,, Yu, K. O., Olson, C. T., and Hobson, D. W. 1989. The metabolism and nephrotoxicity of tetralin in male Fischer 344 rats. J. Toxicol. Environ. Health 21:267-275. Swenberg, J. A., Short, B., Borghoff, S., Strasser, J., and Charbonneau, M. 1989. The comparative pathobiology of α 2u -globulin nephropathy. Toxicol. Appl. Pharmacol. 97:35-46.
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Treibs, W., and Schroth, W. 1961. 2-Indanone. Pseudoaromatic compounds from 2-indanone. Liebigs Ann. Chemie 639:204-213. Undheim, K., and Nilsen, B. P. 1975. 2,3-Epoxyindanone, synthesis and reactions. Acta Chem. Scand. B 29:503-506.
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Received May 26, 1989 Accepted November 8, 1989
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Metabolism and nephrotoxicity of Indan in male fischer 344 rats M. P. Servé , M. J. Ferry , K. O. Yu , C. T. Olson & D. W. Hobson To cite this article: M. P. Servé , M. J. Ferry , K. O. Yu , C. T. Olson & D. W. Hobson (1990) Metabolism and nephrotoxicity of Indan in male fischer 344 rats, Journal of Toxicology and Environmental Health, 29:4, 409-416, DOI: 10.1080/15287399009531401 To link to this article: http://dx.doi.org/10.1080/15287399009531401
Published online: 20 Oct 2009.
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Article views: 7
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [ECU Libraries]
Date: 03 October 2015, At: 17:04
METABOLISM AND NEPHROTOXICITY OF INDAN IN MALE FISCHER 344 RATS M. P. Servé, M. J. Ferry, K. O. Yu Department of Chemistry, Wright State University, Dayton, Ohio C. T. Olson, D. W. Hobson
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Battelle Memorial Institute, Columbus, Ohio
Indan, a component of fuels, solvents, and varnishes, is metabolized in male Fischer 344 rats to 1-indanol, 2-indanol, 5-indanol, 1-indanone, 2-indanone, 2-hydroxy-1indanone, cis-1,2-indandiol, and trans-1,2-indandiol. The metabolites were identified using the techniques of gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The rats treated with indan demonstrated the classic lesions of hydrocarbon-induced nephropathy. The kidney damage produced was less than that found for tetralin and other branched-chain acyclic hydrocarbons.
INTRODUCTION The cyclic molecules eis- and frans-decalin (Olson et al., 1986) and tetralin (Servé et al., 1989) have been shown to produce a specific nephropathy consisting of hyaline droplet formation in the proximal tubules of male rats. Long-term exposure to these chemicals or hydrocarbon-based fuels resulted in additional damage consisting of tubular segments near the corticomedullary junction being blocked and dilated with casts of necrotic cell debris, resulting in cell death and in some cases tumor formation (Gaworski et al., 1984). It has recently been reported that the normally nonnephrotoxic monocyclic hydrocarbon cyclohexane can produce the similar renal protein pathology when a branched alkyl side chain such as tertiary butyl or isopropyl is attached to the cyclohexane ring (Henningsen et al., 1987,1988). Studies of the hyaline droplets found in the rat proximal tubule after hydrocarbon exposure have concluded that the protein a2u-globulin is the main component (Kanerva et al., 1987; Olson et al., 1987). A recent report showed that exposure to unleaded gasoline did not alter the a2uglobulin level in aged male rats, in whom the capacity to synthesize the The authors wish to thank the U.S. Air Force for their generous funding of this work under grant number AFOSR-87-0108. Requests for reprints should be sent to M. P. Servé, Department of Chemistry, Wright State University, Dayton, OH 45435.
409 Journal of Toxicology and Environmental Health, 29:409-416, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
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protein had diminished (Murty et al., 1988). The authors suggested that young male rats approximately 3.5 mo old were the most susceptible to the hyaline droplet nephropathy and that the susceptibility abated with age. A review of the work on a2u-globulin and the difficulty in extrapolation of the rat data to humans was recently published (Swenberg et al., 1989). Examination of the chemical structures of the alkyl-substituted cyclohexanes, tetralin, and the isomeric decalins revealed that all the molecules possess a nonplanar chair configuration. This chair structure may alter the metabolic biotransformation of the cyclic compounds by inhibiting the fit of the oxidative enzymes or the conjugation enzymes that are necessary for the urinary excretion of the hydrocarbons. The resulting prolonged time in which the hydrocarbon, or a metabolite of the hydrocarbon, remains in the animal could be a vital factor in the production of nephrotoxicity. Cyclic molecules, which do not possess the chair configuration, have not been examined for their ability to induce the aforementioned nephrotoxicity. In order to see whether a planar cyclic hydrocarbon would be less toxic to the kidneys, it was decided to look at the nephrotoxic potential of indan. Indan (QH^) is a common cyclic hydrocarbon found as a constituent of naphtha solvents (Hibino and Suzumura, 1955) and as a component of reformed gasolines (Bohlman et al., 1965). Indan is a planar molecule with a boiling point of 178°C, which is within 20°C of the boiling points of tetralin and the decalins. The volatility of the hydrocarbon is important since low-boiling molecules such as cyclohexane are easily exhaled by the animal and metabolism and excretion of the xenobiotic through the kidneys are reduced with a resulting diminution of nephrotoxicity. An examination of the metabolism and nephrotoxicity potential of indan should yield information concerning the effect of hydrocarbon structure on the extent of renal damage and the type of biotransformation reactions facilitated (Fig. 1). MATERIALS AND METHODS
1-lndanol, 2-indanol, 5-indanol, 2-indanone, indene, 4-chromanone, dihydrocoumarin, 5-methoxy-1-indanone, hexamethyldisilazane, and trimethylsilyl iodide were purchased from the Aldrich Chemical Company (Milwaukee, Wis.); 1-indanone and indan were obtained from the Lancaster Synthesis Ltd. (Windham, N.H.). The indan was distilled to >99% purity as indicated by GC. 2-Hydroxy-1-indanone formed by treating 1indanone with hexamethyldisilazane and trimethylsily! iodide (Miller and McKean, 1979). 3-Hydroxy-1-indanone was prepared by oxidation of 3bromo-1-indanone (Treibs and Schroth, 1961) according to the literature procedure (Undheim and Nilsen, 1975). 4-Hydroxy-1-indanone was synthesized by heating dihydrocoumarin and aluminum chloride (Louden and Razdan, 1954). 5-Hydroxy-1-indanone was isolated from the reaction
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INDAN
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FIGURE 1. Scheme 1.
of 5-methoxy-1-indanone and aluminum bromide. 6-Hydroxy-1-indanone was prepared by the cyclization of p-methoxy-3-phenylpropanoic acid (Louden and Razdan, 1954). The fusion of 4-chromanone and aluminum chloride yielded 7-hydroxy-1-indanone (Louden and Razdan, 1954). The eis- and frans-indan-1,2-diol were prepared according to the literature procedure (Balsamo et al., 1974). Animals, Housing, and Exposure Conditions
Eighteen Fischer 344 male rats (Charles River Breeding Laboratories, Wilmington, Mass.) weighing 270 ± 13 g were divided into 2 groups of 9 treated rats and 9 control rats. The rats were maintained in a controlled environment with temperatures of 74 ± 2°F, relative humidity of 55 ± 10%, and light cycle of 12 h on and 12 h off. When urine was not being collected using metabolism cages, rats were housed three to a polycarbonate cage with hardwood chips for bedding. Feed and water were available ad libitum. Rats were dosed by gavage with neat indan at 240 mg/kg body weight. A dosage of 480 mg indan/kg resulted in the death of all animals within 2 d. (All the rats that perished due to overdosing showed a red coloration in their urines that was determined to be a mixture of coproporphyrin and uroporphyrin, two precursors of heme. Blood tests on the rats showed that the animals were anemic. No autopsy was performed.) Dosing was continued on alternate days for 2 wk to facilitate metabolite identification and to rapidly induce any potential renal toxic effects. (More frequent dosing of the rats with indan can cause an alteration in their eating habits, which in turn can lead to severe weight loss and other complications.) Nine control rats were dosed with equivalent volumes of water over the same period. At 24 h following the final indan dose, the rats were sacrificed by halothane overdose and the kidneys excised. One kidney was used for histopathologic examination while the other kidney was homogenized in distilled water to look for any indan metabolites that might be embedded in the kidney itself. The kidney homogenate was processed using the same procedure as used for the urine (vide infra). Histopathologic
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examination was performed on paraffin-embedded kidney sections stained with hematoxylin and eosin. Tissues from exposed rats were compared to those from controls for the presence of characteristic lesions of hydrocarbon-induced nephropathy, including hyaline droplet formation, tubular cysts, and papillary calcification. Lesions were graded by pathologists for degree of severity.
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Isolation of Urinary Metabolites
For the first 48 h following initial dosing, rats were placed in metabolism cages where the urine was collected and then frozen until analyzed. The pH of urine samples was adjusted to 4.0, and 0.5 ml glucuronidase/sulfatase (specific activity of 5.5 and 1.5 units/ml, respectively; Calbiochem, Lajolla, Calif.) was added to 5.0-ml aliquots. This quantity of enzyme was in excess of that needed for complete hydrolysis of conjugates, as determined by varying the quantities added to different aliquots of samples. The solutions were shaken at 37°C for 16 h, cooled at room temperature, and filtered through diatomaceous earth columns (Clin Elut, Analytichem International, Harbor City, Calif.) using neat méthylène chloride as eluent. Unconjugated metabolites of indan were isolated by direct extraction of a second 5.0-ml aliquot of urine with méthylène chloride. The urine extract samples were then analyzed by gas chromatography and gas chromatography/mass spectrometry. Urinary Metabolite Identification
The méthylène chloride extracts of the urine metabolites were analyzed on a gas Chromatograph equipped with a flame ionization detector (model 5880A Hewlett-Packard Corp., Avondale, Pa.) and using a 25 m x 0.22 m ID carbowax 20M fused silica column (Hewlett-Packard Corp., Avondale, Pa.). A 60°C oven temperature was maintained for 1 min after sample injection and then programmed at 2°C/min to 180°C. Detector and injection port temperatures were 250 and 200°C, respectively. Helium was used as the carrier gas, with a linear velocity of 22 cm/s at 100°C and a split ratio of 5 : 1 . Metabolite identification was conducted with a Hewlett-Packard 5985 gas chromatograph/mass spectrometer system and a 4 ft x 2 mm ID 3% SP-1000 and 100/200 Supelco-port glass column (Supelco Corp., Bellefonte, Pa.) with a helium flow rate of 28 ml/min. The oven temperature was held at 100°C for 1 min and then programmed at 10°C/min to 200°C. The mass spectrometer was a quadrupole instrument operated in the electron impact mode at a voltage of 70 eV and with an ion source temperature of 200°C. Identification of both treated and control sets of urinary metabolites was confirmed by comparing mass spectra fragmentation patterns with the fragmentation patterns of purchased or synthesized compounds. Quantitation of the metabolites was accom-
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TABLE 1. Urinary and Kidney Metabolites of Fischer 344 Male Rats Dosed with Indan
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Relative amounts of metabolites recovered Metabolite
0-48 h
Final3
1-lndanol 2-lndanol 5-Indanol 1-lndanone 2-lndanone 2-Hydroxy-1-indanone 3-Hydroxyl-1-indanone c/'s-1,2-lndandiol trans-1,2-lndandiol
6
b
a
1.1 1.1 2.7 3.2 50 27.8 4.3 9.1
± ± ± ± ± ± ± ±
0.2 0.3 0.4 1 8 5 2 2
1.0 0.9 2.9 3.4 52 26 4.8 11.4
± 0.2 ± 0.4 ± 0.4 ± 0.8 ± 8 ± 5 ±1.8 ± 2
Urine collection following last dosing. Could not be separated from 4-methylphenol, present in urine of control rats.
fc
pushed by taking a weighed sample of the individual metabolites (100 mg), adding a weighed amount of dodecane (50 mg), and determining the relative gas Chromatographie peak areas. A weighed sample of dodecane was then added to the urine samples before analysis, and the ratio of the gas Chromatograph area was compared to yield the relative molar abundances. RESULTS Metabolites Fischer 344 male rats exposed by gavage to indan produced the urinary metabolites listed in Table 1. The values listed in Table 1 represent the sum of free and conjugated urinary metabolites, although GC analysis of ¡ndan-dosed rat urines not treated with glucuronidase/sulfatase showed the absence of indan or its metabolites. With the exception of 5indanol, all of the other metabolites showed that oxidative attack had occurred on the saturated five-membered ring of indan. Metabolic oxidation of the cyclopentane ring produced a variety of monoalcohols, ketones, hydroxyketones, and diols. There was no trace of any disubstituted metabolites in which oxidation had occurred on each ring. Analysis of the kidney homogenates showed the presence of 1-indanol and 1indanone in the ratio of 1 : 2.5. Histopathology When compared to controls, male Fischer 344 rats given indan showed a mild increase in cytoplasmic hyaline droplets in the epithelial
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cells of the proximal convoluted tubules of the nephrons. The morphology of the hyaline droplets varied from elongated, crystalline forms to homogeneous spheroids of assorted dimensions. Also present within the proximal convoluted tubules were foci of cellular degeneration, where epithelial cells exhibited increased cytoplasmic basophilia and vesicular nuclei. Not seen were intratubular casts, overt glomerular changes, and significant inflammation. The kidneys of the control rats showed no unusual changes.
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DISCUSSION The extensive metabolism of indan on the nonaromatic ring paralleled the metabolism of tetralin (Servé et al., 1989). The simple ketones 1and 2-indanone were isolated as metabolites from indan, but the analogous ketones 1- and 2-tetralone were not isolated from tetralin. 1,4Tetralindiol was observed to be present in tetralin-exposed rats, while indan produced no indan-1,3-diol. Both tetralin and indan yielded a 2hydroxy-1-ketone metabolite and a metabolite in which one benzylic position had been oxidized to a ketone while the other benzylic carbon had been converted to an alcohol. The identification of 1-indanone from the homogenized kidney extracts of the indan-exposed rats that showed renal damage corresponds with the finding of eis- and frans-2-decalones in the kidney extracts of male rats dosed with eis- and frans-decalin. No metabolites were found from the kidney homogenates of male rats dosed with tetralin. Histopathologic studies revealed that the damage done to the kidneys by indan was typical of that reported for other hydrocarbons. However, the severity of the damage, with respect to hyaline droplet formation, intratubular cast formation, and inflammation, was less than that found in male rats exposed to tetralin and the decalins. Since one of the major differences in structure between ¡ndan and the other cyclic hydrocarbons tetralin and the decalins is the planar structure of the indan molecule, it is possible that this stereochemical situation permits easier excretion of the indan metabolite from the animal. It has recently been suggested that in the case of 2,2,4-trimethylpentane, the protein droplet nephropathy may not depend on chemical binding to a2u-globulin, but on conformational changes in the protein structure produced by the xenobiotic chemical or one of its metabolites (Borghoff et al., 1989). A mechanism similar to the branched-chain acyclic hydrocarbons may be involved in the hydrocarbon-induced nephropathy of the various cyclic hydrocarbons; for example, a prolonged lifetime of the hydrocarbon metabolite in the kidney increases the opportunity for the metabolite to bind with the a2u-globulin in such a manner that the a2u-globulin conformation is altered and cannot be broken down by lysozymal enzymes. A
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facilitation of the conjugation of the metabolite with glucuronic acid or sulfate could in turn speed the removal of the metabolite from the kidneys and lessen the opportunity for the metabolite to interact with the a2u-globulin and initiate the hydrocarbon nephrotoxicity. The approach and reaction of the conjugating enzyme systems may be made easier because of the planar structure of the indan molecule. Once conjugation has occurred, excretion would be expected to soon follow and the potential for hydrocarbon would be reduced.
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REFERENCES Balsamo, A., Berti, G., Crotti, P., Ferretti, M., Macchia, B., and Macchia, F. 1974. The anomalous steric course of ring opening reactions of indene oxide. A reexamination. J. Org. Chem. 39:2596-2598. Bohlman, D., Wehner, K., and Leibnitz, E. 1965. Investigation of the components of heavy reformed fractions, especially concerning the production of aromatic hydrocarbons. J. Prakt. Chem. 29:215-229. Borghoff, S. J., Upton, P. B., and Swenberg, J. A. 1989. Characteristic of 2,2,4-trimethyl-2-pentanol binding to α 2u -globulin and other compounds that cause protein droplet nephropathy. Toxicologist 9:79. Gaworski, C. L., MacEwen, J. D., Vernot, E. H., Bruner, R. H., and Cowan, M. J. 1984. Comparison of the subchronic inhalation toxicity of petroleum and oil shale JP5 jet fuels. In Advances in Modern Environmental Toxicology, vol. VII, Renal Effects of Petroleum Hydrocarbons, ed. M. A. Mehlman, pp. 33-48. Princeton, N.J.: Princeton Scientific. Henningsen, G. M., Yu, K. O., Salomon, R. A., Ferry, M. J., Lopez, I., Roberts, J. A., and Servé, M. P. 1987. The metabolism of t-butylcyclohexane in Fischer 344 male rats with hyaline droplet nephropathy. Toxicol. Lett. 39:313-318. Henningsen, G. M., Salomon, R. A., Yu, K. O., Lopez, I., Roberts, J. A., and Servé, M. P. 1988. Metabolism of nephrotoxic isopropylcyclohexane in male Fischer 344 rats. J. Toxicol. Environ. Health 24:19-25. Hibino, O., and Suzumura, H. 1955. Constituents of solvent naphtha. Coal Tar 7:301-304. Kanerva, R. L., Ridder, G. M., Stone, L. C., and Alden, C. L. 1987. Characterization of spontaneous and decalin-induced hyaline droplets in kidneys of adult male rats. Food Chem. Toxicol. 25:63-82. Louden, J. D., and Razdan, R. K. 1954. Rearrangements of chromanones and dihydrocoumarins by aluminum chloride. J. Chem. Soc. 4299-4303. Miller, R. D., and McKean, D. R. 1979. The facile silylation of aldehydes and ketones using trimethylsilyl iodide: An exceptionally simple procedure for the generation of thermodynamically equilibrated trimethylsilylenol ethers. Synthesis 730-732. Murty, C. V. R., Olson, M. J., Garg, B. D., and Roy, A. K. 1988. Hydrocarbon-induced hyaline droplet nephropathy in male rats during senescence. Toxicol. Appl. Pharmacol. 96:380-392. Olson, C. T., Yu, K. O., and Servé, M. P. 1986. The metabolism of cis- and trans-decalin in Fischer 344 rats. J. Toxicol. Environ. Health 18:285-292. Olson, M. J., Manicini, M. A., Garg, B. D., and Roy, A. K. 1987. Accumulation of a 2u -globulin in the renal proximal tubules of male rats exposed to unleaded gasoline. Toxicol. Appl. Pharmacol. 90:43-51. Servé, M. P., Llewelyn, B. M,, Yu, K. O., Olson, C. T., and Hobson, D. W. 1989. The metabolism and nephrotoxicity of tetralin in male Fischer 344 rats. J. Toxicol. Environ. Health 21:267-275. Swenberg, J. A., Short, B., Borghoff, S., Strasser, J., and Charbonneau, M. 1989. The comparative pathobiology of α 2u -globulin nephropathy. Toxicol. Appl. Pharmacol. 97:35-46.
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Treibs, W., and Schroth, W. 1961. 2-Indanone. Pseudoaromatic compounds from 2-indanone. Liebigs Ann. Chemie 639:204-213. Undheim, K., and Nilsen, B. P. 1975. 2,3-Epoxyindanone, synthesis and reactions. Acta Chem. Scand. B 29:503-506.
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Received May 26, 1989 Accepted November 8, 1989
