* Battelle, Toxicology Northwest, Richland, Washington 99352; and
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Received September 25, 2002; accepted January 1, 2003
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ABSTRACT |
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Key Words: decalin (decahydronaphthalene); decalone; decalol; 2u-globulin; toxicokinetics.
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INTRODUCTION |
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Decalin is one of a series of organic compounds termed "chemicals that induce 2u-globulin accumulation" (CIGA; Borghoff and Lagarde, 1993
; Flamm and Lehman-McKeeman, 1991
; Hard et al., 1993
; Mao et al., 1998
; Ridder et al., 1990
; Swenberg et al., 1989
).
2u-Globulin is produced in the liver of young male rats (i.e., Fischer 344, Sprague-Dawley, Buffalo, and Norway Brown) at a rate of 90180 µg/g liver/h (Borghoff et al., 1990
). Female rat kidneys are practically devoid of this protein, having more than 100-fold less
2u-globulin than male rat kidneys. In male rats, the production of
2u-globulin peaks at puberty, representing
30% of total urinary protein compared to less than 10% for albumin (Neuhaus and Flory, 1978
). By 12 months of age, albumin in male rats represents
60% of the total urinary protein while
2u-globulin contributes only
7%, revealing that production and excretion of
2u-globulin substantially decreases with age. In addition to sex, species, and age effects, exposure to CIGA is known to cause excessive accumulation of
2u-globulin in renal tubular epithelial cells (Alden et al., 1984
; Gaworski et al., 1985
; Kanerva et al., 1987a
,b
; Mao et al., 1998
; Stone et al., 1987a
,b
). CIGA-induced nephrotoxicity is attributed to the accumulating
2u-globulin-ligand complex in the kidneys, which resists lysosomal degradation (Dietrich and Swenberg, 1991
; Lehman-McKeeman et al., 1990
).
Decalin was nominated by the National Institute of Environmental Health Sciences (NIEHS) for study by the National Toxicology Program because of its structural similarity to naphthalene and tetralin, high potential for consumer exposure, and lack of toxicity and carcinogenicity data. Prechronic (13-week) and chronic (2-year) studies were conducted in which male and female F344/N rats and B6C3F1 mice were exposed to concentrations up to 400 ppm decalin via whole-body inhalation. Findings from the rat studies (Dill et al., 2003) suggested that kidney lesions in male rats following repeated exposure to decalin for up to 13 weeks were linked to hyaline droplet nephropathy. The mean severity of chronic nephropathy clearly increased following chronic exposure to decalin in male rats, and increased nonneoplastic kidney lesions were considered responsible for increased carcinogenic effects on the renal cortical epithelium in chronically exposed male rats (Dill et al., 2003
).
A series of inhalation studies was conducted in conjunction with toxicity and carcinogenicity studies at exposure concentrations up to 400 ppm (single inhalation) or at doses up to 20 mg/kg (single iv administration). The objectives were to (1) characterize systemic elimination of decalin in rats and mice and evaluate disposition of decalin, its metabolites, and kidney 2u-globulin in young and old rats of both sexes following a single 6-h whole-body inhalation exposure at up to 400 ppm decalin and (2) assess disposition of each cis- or trans-decalin isomer, its metabolites, and kidney
2u-globulin in young male F344/N rats after iv administration at doses up to 20 mg/kg.
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MATERIALS AND METHODS |
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Generation and monitoring of decalin exposure.
Decalin was pumped through a preheater into the top of a heated glass column, where heated nitrogen entered from below to assist vaporization and transport of vapor out of the generator. Decalin vapor was transported to the chambers in heated lines and injected via a metering valve into the chamber inlet duct, where it was further diluted with conditioned air to achieve the desired vapor concentrations. Exposure concentrations were monitored every 24 min during the exposure by an on-line GC with flame ionization detection (FID; Hewlett-Packard [HP]-5890; Avondale, PA). The peaks for cis- and trans-decalin were integrated separately, and the calculated fraction of trans-decalin revealed no fractionation of the test chemical during exposure. The sum of the cis- and trans-decalin peak areas was used to calculate the concentration of decalin for each determination. Calibration of the on-line monitor was achieved by quantitative determination of decalin in exposure chamber samples collected with adsorbent gas sampling tubes (ORBO 101, Supelco, Bellfonte, PA).
Animals.
For the inhalation studies, young male and female F344/N rats and B6C3F1 mice (1213 weeks of age at study start) were obtained from Charles River Laboratories (Raleigh, NC). Old male and female F344/N rats (22 months of age at study start) were obtained from NIEHS (Research Triangle Park, NC). Animals were acclimated in environmentally controlled (humidity of 55 ± 15%; temperature of 75 ± 3°F) rooms for up to 2 weeks, with free access to NTP-2000 diet (irradiated pellets, Zeigler Bros., Gardners, PA) and fresh tap water. Prior to exposure, animals were placed in individual compartments in wire-mesh cage units and housed in environmentally controlled exposure chambers (Hazleton 2000, Lab Products, Inc., Aberdeen, MD). Animals had access to water, but not to food, during the exposure. A 12-h light/dark cycle (light starting at 0600 h) was maintained throughout the studies, except when light was turned on for bleeding during the dark cycle, at which time light was turned off immediately after blood collection. For the iv studies, male F344/N rats (
11 weeks old) were obtained from Taconic (Germantown, NY). Rats were housed in individual compartments of wire-mesh cage units on open racks prior to dosing, and in individual plastic metabolism cages (Lab Products, Inc., Seaford, DE) after dosing. Environmental conditions for the iv studies were identical to those employed for the inhalation studies.
Experimental Design
Study 1young rats and mice.
Young rats and mice of both sexes (13 weeks of age) were exposed once via whole-body inhalation to 25, 100, or 400 ppm decalin for 6 h. Blood samples were collected at < 5, 10, 20, 30, 60, 120, 240, 480, and 1440 min postexposure (3/sex/species/concentration/time point) from the retro-orbital sinus or vena cava in rats and from the supra-orbital sinus in mice, under 70% CO2 (30% air) anesthesia. Blood samples were stored at 70°C until analyzed for decalin. The blood decalin data from rats and mice were fit with a bi-exponential model and toxicokinetic parameters were estimated for decalin. Kidneys were collected from rats only at 2, 4, 8, and 24 h postexposure (3/sex/concentration/time point) and analyzed for (cis and trans)-decalin (also called decalin), (cis and trans)-2-decalone (also called decalone), and
2u-globulin.
Study 2 young and old rats.
Young (12 weeks of age) and old (
22 months of age) rats (5/sex/age/concentration/time point) were exposed once via whole-body inhalation to 25 or 400 ppm decalin for 6 h. Blood samples were collected immediately (
15 min [0 h]) and at 16 h postexposure from the retro-orbital sinus or vena cava, under 70% CO2 anesthesia. Rats assigned to the 16-h bleeding were placed in a metabolism cage (No. 2100-R, Lab Products, Inc., Seaford, DE) immediately following the exposure and urine was collected for 16 h before blood sampling. After blood collection, animals were sacrificed and kidneys were collected and analyzed for decalone and
2u-globulin. Urine volume and creatinine concentrations were measured and urine was subsequently stored at 70°C until analysis for 1- and 2-decalols (collectively called decalol).
A separate group of old male rats (22 months of age) was exposed once via whole-body inhalation to 25 or 400 ppm decalin for 6 h. Blood samples were collected at < 5, 30, 60, 120, 180, 240, 360, 540, and 720 min postexposure (3/concentration/time point) and analyzed for decalin. The 16-h blood concentration data from the old male rats, as described above, were included with these data before a bi-exponential model was fit to the data to estimate toxicokinetic parameters for decalin.
Study 3intravenous study in young rats using purified isomer formulations.
Young (11 weeks of age) male rats were dosed iv via the tail vein with 0, 2.5, 5, 10, and 20 mg/kg using formulations of purified cis- or trans-decalin in Emulphor:ethanol:water (1:1:8). Approximately 16 h after dosing, kidneys were collected and analyzed for decalin, decalone, and
2u-globulin.
Preparation of kidney homogenates.
Kidneys were weighed, frozen in liquid nitrogen, and stored at 70°C. Before analysis, kidneys were thawed on ice, a volume of 67 mM sodium/potassium phosphate buffer (pH 7.2) equivalent to twice the recorded fresh weight was added, and the sample was homogenized. Approximately 300 µl of kidney homogenate was analyzed for decalin and/or decalone. The remainder of the homogenate was centrifuged at 3000 x g for 15 min and the supernatant was analyzed for
2u-globulin.
Determination of decalin in blood and kidneys.
Blood samples were thawed, 100-mg aliquots were diluted with 43 mM NaHCO3 buffer (pH 11), (cis + trans)-decahydronaphthalene-d18 (decalin-d18; Aldrich Chemical Co.) was added as an internal standard, and the mixture was extracted with cyclohexane. Kidney homogenates were thawed on ice,
50-mg aliquots were spiked with (cis and trans)-decalin-d18 as an internal standard, and the mixture was extracted with cyclohexane.
The cyclohexane layer from each extract (blood or kidneys) was analyzed using an HP-5890 Series II gas chromatograph interfaced to an HP-5971A mass selective detector. Temperature-programmed separations (50300°C) were carried out on a fused-silica capillary column (DB-5; 30 m x 0.25 mm ID; film thickness, 0.25 µm; J&W Scientific; Folsom, CA). Selected ion-chromatograms were obtained by monitoring intense characteristic ions for (cis and trans)-decalin (m/z 138) and the internal standard, trans-decalin-d18 (m/z 156). Decalin concentrations are reported as the sum of total concentrations of (cis and trans)-isomers. However, trends in the relative amounts of cis- and trans-decalin in blood were evaluated as changes in the cis/trans ratio of the two isomers.
Determination of decalone in kidneys.
Kidney homogenates were thawed on ice and 100-mg aliquots were spiked with 2-decalone-1,1,3,3-d4 (2-decalone-d4; provided by John Cashman from the Seattle Biomedical Research Institute; Seattle, WA) as an internal standard. The mixture was extracted with cyclohexane and the organic layer was analyzed using an HP-5890 Series II gas chromatograph interfaced to an HP-5971A mass selective detector. Temperature-programmed separations (70270°C) were carried out on a fused silica capillary column (DB-1701; 30 m x 0.25 mm ID; film thickness, 0.25 µm; J&W Scientific). Selected ion-chromatograms were obtained by monitoring intense characteristic ions for (cis and trans)-2-decalone (m/z 152) and the internal standard, cis-2-decalone-d4 (m/z 156). Decalone concentrations are reported as the sum of the concentrations of (cis and trans)-2-decalone isomers. Where possible, trends in the relative amounts of cis- and trans-decalone in kidney were evaluated as changes in the cis/trans ratio of the two isomers.
Determination of 2u-globulin in kidneys.
2u-Globulin was purified from urine collected from untreated mature male rats and characterized as described in Mao et al.(1998)
. Calibration standards for the enzyme linked immunosorbent assay (ELISA) and liquid chromatography-electrospray ionization mass spectrometery (LC-ESI/MS) assay were prepared using purified
2u-globulin and aliquots of kidney homogenate prepared from untreated female rats in the buffers used for sample preparation. In the case of LC-ESI/MS, calibration standards were worked up along with the samples as described in Mao et al.(1998)
.
For the inhalation studies, the supernatants from kidney homogenates were analyzed for 2u-globulin using a competitive indirect ELISA (Borghoff et al., 1992
), using ascites fluid containing anti-
2u-globulin monoclonal antibodies (provided by Susan Borghoff, Chemical Industry Institute of Toxicology Centers for Health Research, Research Triangle Park, NC). Results are reported as the mass of
2u-globulin/g kidney.
For the iv studies, kidney homogenates were thawed and analyzed using LC-ESI/MS as described in Mao et al.(1998). Results are reported as nmol
2u-globulin/g kidney.
Determination of decalol in urine.
Urine samples were added to 0.2 M acetate buffer (pH 5.4) containing an internal standard (1,2,3,4-tetrahydro-1-naphthol) and incubated overnight at 37°C with ß-glucuronidase/arylsulfatase (Boehringer Mannheim, GmbH, Germany). Samples were extracted with cyclohexane and the decalol isomers were analyzed using GC/mass spectrometry (MS). Temperature programming (70250°C) was used to conduct separations on a fused silica capillary column (DB-WAX; 30-m x 0.25-mm ID; film thickness 0.25 µm; J&W Scientific). Selected ion-chromatograms were obtained by monitoring intense characteristic ions for the decalol (m/z 136) isomers and 1,2,3,4-tetrahydro-1-naphthol (m/z 130). An example of a selected ion-chromatogram of the eight possible decalol isomers is illustrated in Figure 2. Six urinary decalol isomers were identified as trans,cis-1-decalol, trans,trans-1-decalol, trans,trans-2-decalol, trans,cis-2-decalol, cis,cis-2-decalol (standards synthesized by John Cashman, Seattle Biomedical Research Institute), and cis,cis-1-decalol (Aldrich Chemical Co.). Standards for cis,trans-1-decalol and cis,trans-2-decalol were not available for identification. However, it was possible to identify these through the process of elimination and evidence in the literature (Elliott et al., 1966
; Olson et al., 1986
). Table 1
shows the identification of the eight isomers depicted in the chromatogram in Figure 2
.
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Statistics
Concentration comparison.
Concentrations of decalin in blood and kidneys, of decalone and 2u-globulin in kidneys, and of decalol in urine were analyzed by ANOVA for effects of sex, age, time point, and exposure concentration, using PROC GLM in SAS 6.12 (SAS Institute, Inc., Cary, NC). Differences were noted as significant at p
0.05.
Toxicokinetic modeling of systemic elimination of decalin.
Decalin toxicokinetic parameters were determined by fitting Equation 1 to the blood decalin concentration data using a nonlinear least-squares fitting program (SAS PROC NLIN; SAS Institute, Inc.).
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In Equation 1, C(t) is the blood decalin concentration at any postexposure time (t) and
and ß are the hybrid elimination rate constants (min-1) for the initial and terminal phases. A0 and B0 are the extrapolated intercepts on the ordinate (concentration) axis of each elimination phase, respectively. Estimates for these values, with their SEs were obtained directly from the model.
The elimination half-lives for the initial and terminal phases of the blood concentration versus time data (t1/2 and t1/2ß) were calculated as ln2/
or ln2/ß, respectively. The maximum blood decalin concentration (C0) was assumed to occur at t = 0 and was calculated as A0 + B0. The area under the blood concentration versus time curve was estimated to the last sampling time point (T; AUCT), using the trapezoidal rule. The AUC extrapolated to infinity (AUC
) was estimated using Equation 2:
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where CT is the blood decalin concentration measured at T.
The variability in blood concentrations increased with increasing exposure concentration. Statistical results from weights of 1/concentration and 1/concentration2 were reviewed as well as from unweighted analysis before selecting the most appropriate weighting functions. Weighting factors of [decalin blood concentration]-2 and [decalin blood concentration]-1 were used for rat and mouse blood data, respectively.
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RESULTS |
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Figure 4 reveals the behavior of the cis/trans ratio of decalin in blood collected from rats. The cis/trans ratio in males was near the initial ratio of the two isomers in the test chemical (0.54) and changed very little with exposure concentration and postexposure sampling time. However, the cis/trans ratio was slightly higher in females as compared to males.
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Time and/or sex differences were noted for kidney decalone and 2u-globulin concentrations (Table 3
). Both decalone and
2u-globulin concentrations in male rats were relatively constant over the 24-h post exposure interval. While kidney decalone concentrations in male rats increased marginally between 25 and 100 ppm and maintained similar levels at 400 ppm, there was no significant difference in the
2u-globulin concentrations across all exposure concentrations. In female rats, kidney decalone concentrations were near or below the experimental limit of quantitation (ELOQ) and the
2u-globulin concentration was more than 1000-fold lower than that in male rats.
The cis/trans ratio of kidney decalone was much higher than the cis/trans ratio of decalin in the test chemical in both males and females (Fig. 4). The cis/trans ratios of decalone in male kidneys increased with post-exposure time in the 25-ppm group, whereas they remained relatively constant with time in the 100 ppm group and decreased with time in 400 ppm group. Although the cis/trans ratios were sometimes greater in female than male rats, the decalone concentrations were also much lower in females compared to males (Table 3
).
Blood decalin concentrations in mice.
As with rats there were no significant differences between the blood elimination rates of cis- and trans-decalin between male and female mice (data not shown). Therefore, kinetics were calculated using the combined isomer concentrations. Compared to rats, decalin was more rapidly removed from blood in mice at the two lower exposure concentrations (Table 4; Fig. 5
), with a t1/2
of 612 min compared to 2335 min for rats. The nonlinearity in decalin elimination noted previously for rats was also observed in mice, but to a greater extent at 400 ppm. C0 and AUC
increased supra-proportionally with exposure concentration and estimates at 400 ppm were greater than those of rats in both sexes (e.g., [AUC
/exposure concentration] of 8.48.6 in mice versus 5.36.7 in rats). Additionally, in the 400-ppm groups t1/2
was protracted in both sexes of mice, whereas only female rats showed a similar trend in the 25- and 100-ppm groups. Protraction of t1/2
was apparent in the shapes of the initial elimination phase as the blood concentrations at 400 ppm declined more slowly and not in parallel to those of the lower exposure concentrations (Fig. 5
). Values for t1/2ß did not vary with exposure concentration in mice as all blood concentration curves declined in a parallel manner during terminal elimination phase. Unlike rats, there were no apparent sex differences in the kinetic parameters derived for mice.
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Study 2Young and Old Rats
Blood decalin concentrations.
Blood decalin concentrations immediately following the exposure (0 h) increased supra-proportionally between 25 and 400 ppm in all groups (Table 5). By 16 h postexposure, decalin concentrations were significantly lower in all groups, with values near the ELOQ (0.012 µg/g blood) at 25 ppm.
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The cis/trans ratios for decalin in blood were different between young and old rats. In young male and female rats, the cis/trans ratios for decalin in blood immediately after exposure and 16 h after exposure paralleled the behavior exemplified in Figure 4. In young rats the cis/trans ratios in both males and females were close to that in the test chemical, with those in the females being slightly higher than the males (e.g., 400 ppm at 16 h postexposure the cis/trans ratio in males was 0.55 ± 0.006 compared to 0.63 ± 0.010 in females). The cis/trans ratios for decalin in blood were very similar between old males and females at both exposure concentrations and time points (0 and 16 h postexposure), and varied only slightly from that in the test chemical. In old rats the cis/trans ratio was essentially identical between males and females (e.g., at 400 ppm at 16 h postexposure the cis/trans ratio in males was 0.620 ± 0.020 compared to 0.642 ± 0.010 in females).
Blood decalin elimination in old rats.
The blood decalin concentration-versus-time profile from old male rats was closely fit with a bi-exponential elimination model (Fig. 6; Table 6
). There were no remarkable differences in the elimination half-lives between the 25- and 400-ppm groups. The model-estimated values of C0 were comparable to those actually measured at 0 h postexposure (Table 5
), and increased supra-proportionally between 25 and 400 ppm (normalized C0 [C0/exposure concentration] increased
1.5-fold at 400 ppm). As seen with the young rats, it was the nonlinear increase in C0, not the elimination rate constants, which resulted in the supra-proportional increase in AUC
at 400 ppm [(AUC
/exposure concentration) increased
1.6-fold]. There was no consistent age difference in kinetic parameters for decalin elimination between old and young male rats in the 25- and 400-ppm groups.
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Kidney decalone concentrations for old male and all female rat groups were ELOQ (0.15 µg/g kidney) and only limited comparisons among these groups were possible. Unlike young male rats, decalone concentrations in old rats significantly decreased by 16 h postexposure at both exposure concentrations. Since kidney decalone concentrations were so low it was not possible to establish trends in the cis/trans ratios for old animals.
Kidney 2u-globulin concentrations.
Age and sex differences were clearly observed in kidney 2u-globulin concentrations (Table 5
), paralleling the trends that were observed for kidney decalone concentrations. Young male rats had greater
2u-globulin concentrations than old male and all (young and old) female groups at both exposure concentrations and sampling times. The
2u-globulin concentrations in young male rats at 25 ppm did not significantly change between 0 and 16 h, whereas those in the 400-ppm group increased by 16 h postexposure. Old male rats had significantly lower
2u-globulin concentrations than young male rats, but had slightly higher
2u-globulin concentrations than all (young and old) female rats. Kidney
2u-globulin concentrations in old male rats tended to decrease by 16 h postexposure at 400 ppm, paralleling a trend that was also observed with decalone concentration. The
2u-globulin concentrations for females were extremely low, with all group means less than
0.02 mg/g kidney.
Urinary decalol concentrations.
The 16-h urinary concentrations for total decalol isomers (normalized to creatinine) tended to increase proportionally with exposure concentration, but young male rats exhibited a less than proportional increase (Table 5). Unlike the aforementioned blood decalin, kidney decalone, and
2u-globulin concentrations, urinary decalol concentrations were consistently lower in young male rats compared to old male or all female rats especially at 400 ppm. This implied that in young male rats, decalin and its metabolite (decalone) were sequestered in kidneys and less decalin was converted and excreted as decalol in urine. Sex differences were significant at 400 ppm with different trends between the age groups; in young rats, females had higher decalol concentrations, whereas in old rats, males had higher concentrations. Therefore, among all rat groups, old male rats excreted the greatest, while young male rats excreted the least amount of decalol in urine.
The behavior of urinary decalol isomers was evaluated by examining the ratio of total cis isomers to total trans isomers. Note that in the following discussion cis and trans refer to the conformation of the decalin moiety rather than that of the alcohol moiety of the decalol metabolites. In young male and female rats the ratio of total cis/trans isomers was less than that seen in the test chemical, but males had a lower ratio than females (e.g., at 25 ppm the cis/trans ratio in males was 4.34 ± 0.040 versus a cis/trans ratio in females of 5.14 ± 0.018). However, in old rats this trend was reversed in that females had a lower decalol cis/trans ratio than males (e.g., at 25 ppm the cis/trans ration in males was 5.63 ± 0.026 versus a cis/trans ratio in females of 4.81 ± 0.031). Similar trends were observed at 400 ppm in both young and old rats.
Study 3Intravenous Study of cis- and trans-Decalin in Male Rats
Table 7 lists the decalin, decalone, and
2u-globulin concentrations in kidneys of young male rats administered increasing iv doses of either cis- or trans-decalin. These data indicate that young male rats accumulated 23 times as much
2u-globulin-bound decalone per unit dose when administered as cis-decalin compared to trans-decalin. Moreover at 10 mg/kg,
52% more
2u-globulin-bound decalin was found in the kidney when the dose was administered as trans-decalin as compared to cis-decalin. Accordingly, neither decalin nor decalone alone will account for the total bound quantity of
2u-globulin in the kidney. When both are considered, the molar ratio of
2u-globulin to total decalin plus decalone in the kidney is approximately 1.0.
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DISCUSSION |
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In young rats, females excreted significantly greater amounts of decalol in urine than males. This was likely due to decalols being excreted as such in female rats, without being further metabolized to decalone and retained in kidneys bound to 2u-globulin, as happens with young male rats (Fig. 1
). Either the rate or capacity of decalin metabolism to decalol isomers might also be different between the sexes, contributing to the higher urinary decalol excretion in females (Longacre, 1987
). An age difference in decalol excretion was obvious only with male rats, presumably due to the fact that little decalin or decalone were retained in kidneys of old rats due to the lack of
2u-globulin.
Decalin was detected in female kidneys, but at considerably lower concentrations than in male kidneys. These low concentrations in females might be a result of passive partitioning of decalin into the tissue and would reflect the blood decalin concentration, steadily decreasing with time. However, decalone concentrations in female kidneys were practically absent (near or below ELOQ), suggesting that metabolism of decalin to decalone might not occur in females to any significant extent (Olson et al., 1986). Additionally, there is no mechanism in the female rat kidneys for retaining decalone, whereas binding to
2u-globulin can lead to retention of decalone in males. Thus, decalin is predominantly metabolized to decalols in female rats, which conjugated forms are then excreted in urine.
In young male rats, elimination of decalin and especially of decalone from kidneys was noticeably delayed over the 24-h postexposure interval, even after a single exposure. When these kidney concentrations were compared on a molar basis, the relative ratios of decalin versus decalone concentrations varied with exposure concentration (data not shown), indicating that the dominant chemical responsible for binding 2u-globulin might change as the exposure concentration increases. This differential presence of decalin and decalone in male rat kidneys was more evident after repeated exposures (Dill et al., 2003
). Although the actual binding affinities to
2u-globulin were not measured, these findings indicate that the primary chemical moiety available for
2u-globulin binding in male rat kidneys might be decalone for exposure concentrations of up to 100 ppm with an increasing contribution of decalin at higher exposure concentrations. The basis for this was (1) metabolism of decalin to decalone could be saturated at high exposure concentrations, leaving more decalin available for
2u-globulin binding; (2) higher amount of decalin would leave less free
2u-globulin for decalone binding; and/or (3) formation of decalin-
2u-globulin complexes might further delay metabolism of decalin to decalone.
Kidney 2u-globulin concentrations in young male rats remained relatively constant over the 24-h postexposure period. Kidney
2u-globulin concentrations from male rats treated with either decalin isomer (1020 mg/g) were substantially higher than the background
2u-globulin concentration from untreated young male rats (
3 mg/g; Dill et al., 2003
). Considering proteolytic degradation of
2u-globulin (plasma t1/2 of 58 h; Lehman-McKeeman et al., 1990
), this high steady-state level of
2u-globulin was taken as evidence for the inhibition of catabolism, evidently due to binding to CIGA such as decalin and decalone. Inhibition of degradation would then be exacerbated following repeated exposures, as more CIGA are transported to kidneys and bind to
2u-globulin, as was observed with decalin exposure up to 13 weeks (Dill et al., 2003
).
The ability of a chemical to bind to 2u-globulin depends on several characteristics, including the presence of an electronegative atom on the ligand capable of hydrogen bonding, a lipophillic region, and/or a specific steric volume (Huwe et al., 1996
). The extent to which decalin, or its metabolite (decalone), participate in formation of
2u-globulin-ligand complexes is still unknown, and is likely a complex function of the rates of metabolism, elimination, distribution to kidney tissue, and relative binding affinities to
2u-globulin. Nonetheless, the decalin-
2u-globulin complex appears to be more labile than the decalone-
2u-globulin complex (based on the molar ratio over the exposure concentration; Dill et al., 2003
), suggesting a weaker binding constant for the former. This is evident in that decalin accumulation in kidneys only becomes predominant under conditions of high decalin concentration in the blood (saturated metabolism) and that decalin, but not
2u-globulin or decalone, appears to be readily eliminated from male rat kidneys. This behavior could be related to the absence of an electronegative element (oxygen) in decalin, resulting in lower
2u-globulin binding affinities for decalin compared to decalone.
Kidney decalone concentrations in old male rats were well above ELOQ immediately after exposure, but there was evidence for rapid elimination by 16 h postexposure. This again indicated that the presence of 2u-globulin is a prerequisite to its retention in kidneys. Not surprisingly, old male rats were more efficient in eliminating decalin as decalols than young male rats. Lack of significant sex differences in the disposition of decalin, decalone, or decalol in old rats was consistent with the lack of
2u-globulin in kidneys. Old male rats had slightly higher blood decalin concentrations at 0 and 16 h postexposure compared to old female rats, which overall led to higher kidney decalone as well as higher urinary decalol concentrations. However, considering the extreme sex-differences in young rats, the sex differences in old rats was insignificant by comparison.
Decalin is known to induce 2u-globulin accumulation in a species-, sex-, and age-specific manner (Hard et al., 1993
). In the single-iv administration study reported herein, administration of both cis and trans-decalin isomers led to
2u-globulin accumulation in male rat kidneys (Table 7
). Additionally, we also present evidence for both sex (Tables 3 and 5
) and age-related (Table 5
) differences in
2u-globulin accumulation in rat kidneys following a single-administration inhalation study. Dill et al.(2003)
reported that accumulation of
2u-globulin in kidney was directly responsible for nonneoplastic and neoplastic renal lesions in male rats following chronic exposure. These lesions are of potential concern because of the widespread human exposure to decalin. However, the risk for humans is markedly reduced because other species including humans do not produce this protein (Borghoff et al., 1990
; Hard et al., 1993
). Therefore, if nephropathy is only associated with chemically induced accumulation of
2u-globulin, then male rat-specific nephropathy should not be used in the hazard characterization for decalin.
The iv study using either cis- or trans-decalin is useful in the interpretation of the cis/trans ratios of decalin and its metabolites in vivo. Table 7 reveals that males accumulate more
2u-globulin bound decalone in the kidney when the dose is administered as the cis isomer rather than the trans isomer. These results were consistent with the increased cis/trans ratios of kidney decalone observed following inhalation exposure of young rats to mixed isomers of decalin (Dill et al., 2003
). This suggests either a greater affinity of
2u-globulin for cis-decalone, or that metabolism of cis-decalin occurs to a greater extent, or more rapidly, than trans-decalin. The behavior of the cis/trans ratio for decalols supports the conclusion that cis-decalone is produced in greater quantities than trans-decalone. Decalone is produced from decalol (Fig. 1
); therefore, if cis-decalone was produced in greater quantities than trans-decalone, a reduction in the cis/trans ratio for decalol isomers should be observed in males. A reduction of the cis/trans decalol isomer ratio was indeed observed in young males compared to females. Moreover, one would expect this behavior to be absent in older males and consistent with this expectation the cis/trans decalol isomer ratio was lower for males compared to females in young rats, but essentially identical in older rats.
Results reported in Table 7 are also useful in interpreting the behavior of cis- and trans-decalin bound by
2u-globulin in the kidney. With iv doses of 10 or 20 mg/kg, significantly more trans-decalin than cis-decalin was bound per unit dose. Accordingly, one would anticipate a reduction in the cis/trans ratio for decalin in the kidneys of males as compared to females and in the cis/trans ratio originally present in the test chemical, as was observed in younger animals administered a mixture of the two isomers.
The fact that the cis/trans ratio for decalin isomers in blood was lower for males than females is more difficult to explain. There was no statistical difference in the rate of disappearance of cis- versus trans-decalin in males or females. However, this simply may be a result of poor precision in the data. Although the kinetic data were not sufficiently precise to reveal differences in the rates of elimination of cis- and trans-decalin, when considering both cis- and trans-decalin together, elimination rates of decalin from blood tended to be slower in females than males, especially as exposure concentrations increased (Table 2).
Finally, as revealed by the molar ratios of kidney decalin, decalone, and 2u-globulin from the iv study (Table 7
), neither the quantity of decalin nor decalone was sufficient to account for the additional
2u-globulin above the background level seen in the kidney. However, when considered together, the amount of
2u-globulin was nearly equivalent to that for the combined amount of both decalin plus decalone in the rat kidney. Although this observation suggests that the stoichiometry of the
2u-globulin-decalin/decalone complex is 1:1, binding parameters between
2u-globulin and its ligands (decalin, decalone) were not measured.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Battelle, Toxicology Northwest, MS K4-16, P. O. Box 999, Richland, WA 99352. Fax: (509) 375-3649. E-mail: dillj{at}battelle.org.
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