Toxicological Sciences 53, 24-32 (2000)
Copyright © 2000 by the Society of Toxicology
Biotransformation and Male Rat-Specific Renal Toxicity of Diethyl Ethyl- and Dimethyl Methylphosphonate
Department of Toxicology, University of Würzburg, Versbacherstrasse 9, 97078 Würzburg, Germany; and * Clariant GmbH, Brüningstrasse 50, 65926 Frankfurt am Main, Germany
Received January 6, 1999; accepted August 30, 1999
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODRESULTSDISCUSSIONREFERENCES |
|---|
Dimethyl methylphosphonate (DMMP) is a widely used chemical. Diethyl ethylphosphonate (DEEP) has been proposed as a replacement for DMMP in several applications. A long-term carcinogenesis study with DMMP in rats and mice showed a significant increase in the incidence of kidney tumors after 2 years of exposure in male but not in female rats and both sexes of mice. DMMP is not genotoxic. Due to these findings, a role of
2u-globulin accumulation in organ-specific tumorigenicity may be possible. 2u-Globulin is a low-molecular-weight protein synthesized in male rats under androgen control. Several male rat specific renal carcinogens have been shown to bind to 2u-globulin and to impair the renal degradation of this protein. This impairment results in 2u-globulin accumulation in the kidney, lysosomal overload, cell death, cell proliferation, and finally, renal tumor induction. To further characterize the toxicology of DMMP and DEEP, we investigated the biotransformation of these compounds and their ability to induce 2u-globulin accumulation in kidney. Biotransformation of both DMMP and DEEP were studied in male and female rats after single oral doses of 50 and 100 mg/kg. 31P-NMR and GC/MS showed that unchanged DMMP was excreted with urine; methyl phosphonate was identified as the only metabolite in urine. Unchanged DEEP was also excreted with urine; in addition, ethyl ethylphosphonate and ethylphosphonate were urinary metabolites. The majority of the applied dose of both compounds was recovered in urine within 24 h indicating rapid absorption and excretion. No sex-differences in rates of formation or excretion of metabolites were seen. To investigate 2u-globulin accumulation in the kidney after DMMP and DEEP, male and female Fischer-344 rats were administered DMMP or DEEP daily for five consecutive days by gavage. DMMP doses were 500- and 1000-mg/kg body weight (bw); due to marked toxicity, daily DEEP dose of 50 and 100 mg/kg had to be used. Control rats received corn oil only and positive controls received five doses of 500-mg/kg bw trimethylpentane (TMP). Relative kidney weights were increased in male rats dosed with DMMP, DEEP, and TMP. 2u-Globulin in kidney cytosol was separated and quantified by capillary electrophoresis and by SDS-PAGE and Western blotting. In DMMP-, DEEP-, and TMP-treated rats, dose-dependent increases in the 2u-globulin content were observed by both methods in male, but not female rats. The increase of 2u-globulin accumulation was accompanied by the formation of protein droplets in the proximal tubules of male rats. These data demonstrate that the sex specific increase in kidney tumors by DMMP in male rats may be due to 2u-globulin accumulation and that similar toxic effects are to be expected from DEEP.
Key Words: dimethyl methylphosphonate (DMMP); diethyl ethylphosphonate (DEEP); trimethylpentane (TMP); 2|gu-globulin nephropathy; protein droplets.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODRESULTSDISCUSSIONREFERENCES |
|---|
Dimethyl methylphosphonate (DMMP) is used as a flame retardant, gasoline additive and as a nerve gas simulant (EPA, 1983
; NSWC, 1982). The compound has a low potential for toxicity after single administration (Dunnick et al., 1988). When given by gavage for 2 years in high doses to rats and mice, renal toxicity and induction of renal tumors was observed in male, but not in female rats (Dunnick et al., 1988). Renal toxicity was not seen in either sex in mice. DMMP is not mutagenic in bacteria (NTP, 1997). However, it caused kidney lesions in male rats similar to those seen after long-term administration of a variety of other chemicals including unleaded gasoline, hydrocarbon solvents, and dichlorobenzene (Charbonneau et al., 1989; Dunnick et al., 1988; Short et al., 1987; Swenberg, 1993; Swenberg et al., 1989). The likely mechanism by which these chemicals cause renal toxicity has been identified as 2u-globulin nephropathy.
Several lipophilic chemicals such as d-limonene and 2,2,4-trimethylpentane (TMP) (Alden, 1985, 1986; Alden et al., 1984; Charbonneau et al., 1987; Lehman-McKeeman et al., 1989; Lock et al., 1987) are known to reversibly bind to 2u-globulin. Binding of these chemicals or some of their metabolites (d-limonone-1,2-oxide and 2,2,4-trimethylpentan-2-ol) to 2u-globulin result in a protein-chemical complex, which is more resistant to lysosomal degradation (Charbonneau et al., 1987; Lehman-McKeeman et al., 1990b; Lock et al., 1987). The consequence of impaired digestion of 2u-globulin is accumulation of the protein in the lysosomes of the P2 segment of the nephron (Lehman-McKeeman et al., 1990a). Accumulation of the protein results in cell death, sloughing off of cells, and formation of granular casts consisting of dead cells. Single cell necrosis occurs in the P2-segment of the nephron at the site of protein accumulation and increased cell proliferation. Chronic cytolethality and cell replication is driving renal tumor formation (Hard et al., 1993; Short et al., 1989; Swenberg, 1993). This sex-specific mechanism of renal tumor formation is linked to protein (2u-globulin) droplet formation in the kidneys of male rats.
Diethyl ethylphosphonate (DEEP) has been suggested as a replacement for DMMP because of similarities in structure and physico-chemical properties. Since the mechanism of renal toxicity of DMMP and potential renal effects of DEEP and the biotransformation of these compounds are unknown, we investigated the metabolism and the induction of 2u-globulin accumulation in the kidney of male rats by both DMMP and DEEP. The results obtained show that both compounds are well absorbed in rats and both induce 2u-globulin nephropathy in male rats.
|
MATERIALS AND METHOD |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODRESULTSDISCUSSIONREFERENCES |
|---|
Chemicals.
DEEP (98%) was supplied by Bayer AG (Leverkusen, Germany); DMMP (99%) was purchased from Sigma-Aldrich (Deisenhofen, Germany). Methyl- and ethylphosphonic acid, methyl methylphosphonate, and ethyl ethylphosphonate were provided in 98% purity by Clariant GmbH (Frankfurt/Main, Germany). Bis-trimethylsilylacetamide was from Merck (Darmstadt, Germany). The monoclonal mouse anti-
2u-globulin antibody was a gift from Dr. H. Hildebrand, Bayer AG (Hildebrand et al., 1997). Horseradish peroxidase conjugated goat anti-rabbit IgG and streptavidin-horseradish peroxidase were from Amersham Life Sciences (Braunschweig, Germany). Enhanced chemiluminescence reagents were purchased from Pierce Chemical Company (Rockford, IL). Reagents for capillary electrophoresis were from BioRad (München, Germany). All other chemicals were obtained from commercial supplies in the highest purity available. GC-columns were purchased from J&W Scientific (Folsom, CA).
Animals and treatment.
Adult Fischer F344 rats aged 125–136 days (282–317 g) were obtained from Harlan Winkelmann (Borchen, Germany). Animals were kept at constant humidity and temperature (21°C) in the animal facility of the department, with a 12-hour light/dark cycle. Before the metabolism studies, the animals were allowed to become accustomed to the metabolic cages for 3 days and control urine was collected for 12 h before the exposures.
To study alkylphosphonate metabolism, male and female rats (4 per dose per sex) were orally administered the alkylphosphonates dissolved in corn oil (1 ml/kg bw). Dose levels for the metabolism studies were lower than those for the 2u-globulin-accumulation studies for DMMP, in order to be able to compare DMMP and DEEP biotransformation without reaching doses saturating biotransformation pathways. After dosing, animals were immediately transferred into metabolic cages (Macrolon®) and urine and feces were collected at 12-h intervals for 72 h. During collection, samples were kept at 4°C. Samples were stored at –20°C until analysis. To study renal 2u-globulin accumulation, rats (n = 5 of each sex per dose) were orally administered TMP (500 mg/kg/day for 5 days) or DMMP (500 and 1000 mg/kg/day for 5 days) or DEEP (50 and 100 mg/kg/day for 5 days) in corn oil (1 ml/kg bw) or corn oil only (controls). The DMMP doses were selected to compare with those used in the bioassay (NTP, 1997), lower doses of DEEP had to be used, due to its toxicity. Animals were sacrificed by cervical dislocation 4 h after the last dosing, and kidney cytosol was prepared. All animal experimentation was performed under permit from the local authorities and according to current legislative requirements.
Preparation of rat kidney cytosol.
Kidney cytosolic subcellular fractions of treated rats were prepared according to the method of Siekevitz (1962).
Protein determination.
Protein concentrations in liquid samples were determined by the method described by Bradford (1976) based on the formation of a protein-dye complex with Coomassie blue G-250, using the BioRad Protein Assay Kit (BioRad, München, Germany) and bovine gamma-globulin as a standard. Purification of 2u-globulin was performed as described (Pähler et al., 1999).
SDS–PAGE and immunoblotting.
Kidney cytosolic proteins were diluted with SDS sample buffer (0.125 M TRIS-HCl, pH 6.8, containing 10% [w/v], SDS, 20% [v/v] glycerol, 0.002 % [w/v] bromphenol blue and 10% [v/v] 2-mercaptoethanol) to a final concentration of 1 mg/ml. Samples were heated to 95°C for 10 min prior to resolving aliquots by SDS–PAGE on a Laemmli system with 12% resolving and 4.5% stacking gel at room temperature (Laemmli, 1970). Proteins were separated for 90 min at 30 mA from 8.4 x 7.0 mm minigels of 0.75 mm thickness, using BioRad MiniProtean II gel electrophoresis equipment. Separated proteins were transferred to nitrocellulose sheets (BioRad, TransBlot pure nitrocellulose 0.45 µm) by tank blotting, using 25 mM TRIS buffer containing 192 mM glycine and 10% (v/v) methanol for 200 V*h. After protein transfer, the sheets were blocked for 1 h at RT with phosphate-buffered saline (PBS; containing 10 mM Na2HPO4, 3 mM KH2PO4 and 137 mM NaCl, pH 7.4), containing 1% [w/v] casein and 0.02 % [w/v] thimerosal. After washing the sheets with 3 quick changes of PBS-T, then once for 15 min and twice for 5 min with PBS-T (PBS containing 0.05 % Tween 20), sheets were incubated with mouse monoclonal anti-#2u-globulin antibody (1:1 000 in PBS-T) overnight at 4°C. After additional washing steps as described above, a goat anti-mouse horeseradish peroxidase-conjugated second antibody was incubated (1:5000 in PBS-T) for 1 h at room temperature. After washing, visualization of recognized bands was performed by enhanced chemiluminescence detection on Hybond ECL film, using Pierce luminescence reagents. Integration of the specific bands was performed on a PC-compatible computer running GelPro3.1TM integration software, digitized on an Agfa Arcus II densitometer (Intas, Göttingen, Germany).
Capillary electrophoresis.
Kidney cytosol samples were prepared for capillary electrophoresis analysis in a final volume of 200 µl. To 100 µl of CE-SDS Protein Sample Buffer (BioRad) 70 µl of diluted kidney cytosol was added to yield a final protein concentration of 1 mg/ml. After addition of 20 µl of 150 mM DTT samples were heated to 95°C for 10 min prior to addition of 10 µl internal standard (benzoic acid, 1 mg/ml). SDS-protein complexes were separated in a hydrophilic sieving polymer solution, causing protein migration according to molecular size. The BioFocus 3000 automated capillary electrophoresis system (BioRad) and uncoated, fused silica capillaries (24 cm x 50 µm ID) were used for all separations. Samples were introduced into the polymer-filled capillary by electrophoretic injection (10 kV for 5 sec.). Separation was performed at 15 kV in negative mode at 15°C for 10 min. Prior to each run, the capillary was flushed for 90 s with 0.1 N HCl and 20 s with 0.1 N NaOH to remove any residual SDS and protein. Separation buffer was then introduced for 120 s in the high-pressure mode, and eluting proteins were monitored at 220 nm (Pähler et al., 1999). Calibration curves were established using purified 2u-globulin (Pähler et al., 1999).
Identification and quantitation of metabolites in urine.
Metabolite identification was performed by 31P-NMR. Urine samples (800 µl) were dissolved in 1 ml of D2O and analyzed by NMR without further workup. Metabolites were quantified by GC/MS. Urine samples (1.5 ml) were centrifuged at 500 x g. The internal standard (25 µl of 1-hydroxy-3-methyl-1H-phospholane oxide; Clariant, 100 mg/ml in bis-trimethylsilylacetamide) was added, and the supernatant was dried in GC-autosampler vials (2-ml) under reduced pressure. To the dry residues obtained, 1 ml of bis-trimethylsilylacetamide was added, and vials were capped and heated to 110°C for 90 min. From the solutions obtained, 20 µl were removed with a microliter syringe and diluted with 980-µl bis-trimethylsilylacetamide. These samples (2-µl) were then analyzed by GC/MS in the split mode (split ratio of 20:1). Samples were separated using helium as carrier gas (2 ml/min), and DB-1 coated, fused silica columns (30 m x 0.25 mm ID, film thickness 1 µm). A linear temperature gradient was applied, starting at an oven temperature of 55°C with a heating rate of 12°C/min for DMMP samples and 15°C/min for DEEP samples. Injector temperature was 200°C and transfer-line temperature was 280°C. During the separation, representative fragments (m/z 111, 167, and 191 for analyses of DEEP samples, m/z 93 for DMMP samples, and m/z 177 for the internal standard) were monitored with dwell times of 20 ms. Quantitation was performed relative to the internal standard and referenced to calibration curves obtained with known concentrations of the metabolites added to urine from control rats. The response of the method was linear in the range of sample concentrations encountered. When identical samples were repeatedly analyzed, deviations in the obtained results were lower than 10%. Excretions of DMMP and DEEP were also quantified by GC/MS. Urine (1 ml) was extracted with chloroform (1 ml), the chloroform phase was dried over Na2SO4, the internal standard was added as described above, and 2 µl of the chloroform phase was injected into the GC/MS. Samples were separated using helium (2 ml/min) as carrier gas on a DB-1-coated, fused silica column (30 m x 0.25 mm ID, film thickness 1 µm) using a linear temperature gradient and starting at an oven temperature of 55°C with a heating rate of 10°/min. During the separation, representative fragments (see above) were monitored with dwell times of 20 ms. Quantitation was performed relative to the internal standard and referenced to calibration curves obtained with known concentrations of the analytes added to urine samples from control rats.
Identification of chemicals bound to. 2u-globulin.
2u-Globulin from the kidneys of DEEP- and DMMP-treated male rats was isolated by anion-exchange HPLC (Pähler et al., 1997). The isolated protein gave only one peak when separated by capillary electrophoresis. For isolation, 7.5 ml of kidney cytosol from male rats treated with DMMP and DEEP, with a protein concentration of 5 mg/ml, was used. Isolated 2u-globulin was desalted, lyophilized, re-dissolved in water (1 ml), and 500 µl of a 10% solution of SDS in water was added. The obtained solution was extracted with 1 ml of chloroform after 30 min. The chloroform extracts were analyzed by GC/MS and DEEP and DMMP were identified as described above.
Instrumental analysis.
GC/MS analyses were performed on a Fisons MD 800 mass spectrometer coupled to a Carlo Erba 8000 series GC and equipped with an AS 800 autosampler (Fisons Instruments, Mainz, Germany). NMR-spectra were recorded with a Bruker AMX 400 at 161.98 Mhz. Chemical shifts were referenced to external H3PO4 (85% in D2O) assigned 
= 0 ppm.. Usually, 1024 scans were collected for Fourier-transformation.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODRESULTSDISCUSSIONREFERENCES |
|---|
Biotransformation of DEEP and DMMP
To study biotransformation, single doses of either DEEP or DMMP were orally administered to rats and urine samples were collected for 72 h. When urine samples were subjected to 31P-NMR to detect phosphorous-containing metabolites, urine samples from DMMP-treated rats showed two signals (Fig. 1A
) not present in control urine. The two signals had identical chemical shifts as authentic DMMP ( 38.5) and methyl methylphosphonate ( 28.3) when measured using control urine as matrix, suggesting that DMMP and methyl methylphosphonate are excreted in urine. The structure of the products was also confirmed by GC/MS of chloroform extracts (DMMP) or dried urine samples subjected to silylation (data not shown). Thus, DMMP and methyl methylphosphonate are identified as urinary excretion products in DMMP-treated rats. Analysis of urine samples from DEEP-treated rats by 31P-NMR showed three new NMR-signals identical in chemical shift to DEEP ( 30.8), ethyl ethylphosphonate ( 27.9), and ethylphosphonate ( 25.7) (Fig. 1B). The structures of these excretion products were also confirmed by GC/MS analysis of chloroform extracts (DEEP) or dried urine samples subjected to silylation (data not shown). With DMMP, methyl methylphosphonate was the major product recovered in urine of male rats. In female rats, unchanged DMMP was the major product excreted with urine. With DEEP, the monoester also was the major excretory product; however, in contrast to DMMP, further hydrolysis to ethylphosphonate occurred in low yields.
|
The major pathway of excretion for orally administered DMMP and DEEP is urine, in part both compounds are excreted as parent compound (due to having the same water solubility) and as metabolites (Tables 1 and 2
). Excretion is rapid with elimination half-lives between 3 and 6 h in rats. The concentrations of metabolites and parent compounds were below the limit of detection at 36 h after oral administration. After administration of both compounds, most of the administered doses were recovered in urine of female rats within 24 h after administration. In male rats, for both compounds, the recovery of metabolites in urine was significantly lower and accounted for only 58 to 74% of the administered doses within 24 h after administration (Tables 1 and 2).
|
|
Renal Effects of DMMP and DEEP
To study the potential role of
2u-globulin accumulation in DMMP toxicity and the effects of DEEP on the kidney, male and female rats were administered daily doses of DMMP or DEEP for 5 consecutive days. Due to massive toxicity of DEEP after a single dose of 500 mg/kg, the doses had to be reduced to 50 and 100 mg/kg. The signs of DEEP toxicity after the high doses were typical for intoxication with anti-cholinesterase agents and resulted in high mortality. After giving 100 mg/kg, no overt signs of toxicity were observed.
Examination of the relative kidney weights of DMMP- and DEEP-treated animals showed significant increases in male rats after both dose regimens of the phosphonates and after the positive control TMP (Table 3). Relative kidney weights of female rats treated with DMMP and TMP were not changed relative to controls. In addition, the signal representing 2u-globulin was not observed in the electropherograms from capillary electrophoresis of kidney cytosol from control, DMMP- or TMP-treated female rats (Table 3).
|
To quantify the content of
2u-globulin in the kidneys of treated male and female rats, cytosolic fractions from the kidney were analyzed for 2u-globulin content by SDS–PAGE and Western blotting, using an 2u-globulin-specific antibody, and by capillary electrophoresis (Pähler et al., 1999). In the Western blotting experiments, a significant increase relative to controls in the 2u-globulin content was observed in the renal cytosol of male rats treated with either DEEP or DMMP. The antibody did not detect 2u-globulin in female control rats nor in female rats treated with DMMP (Fig. 2).
|
The results obtained with the
2u-globulin-specific antibody were confirmed using CE-SDS as an independent procedure to detect and quantify 2u-globulin concentrations. As with the 2u-globulin specific antibody, CE-SDS did not detect 2u-globulin in control (Fig. 3A) or DMMP-treated female rats (Fig. 3B). In male rats, the peak representing 2u-globulin was significantly increased in DEEP- (data not shown) and DMMP-treated animals (Fig. 3D) as compared to controls (Fig. 3C). Purified 2u-globulin gave a single peak and eluted between 5.6 and 6.7 min, depending on the capillary used. The elution time of purified 2u-globulin was assessed at regular intervals. In male rats, the concentrations of 2u-globulin in renal cytosol were raised to up to 80% of total cytosolic protein, and differences between the 2 dose regimens of DMMP and DEEP were observed (Table 3
). After the higher doses, 2u-globulin accumulation reached the same levels with DEEP and DMMP as seen in rats treated with the positive control 2,2,4-trimethylpentane.
|
Histopathologic examinations of kidney sections of male rats administered DMMP, DEEP, or TMP all showed protein droplets in the P2-segment of the proximal tubules. Protein deposits were not observed in treated female rats (data not shown).
Identification of Chemicals Bound to 2u-Globulin
To identify the chemical bound to 2u-globulin, the 2u-globulin fraction was isolated by HPLC from renal cytosol of male rats treated with either DEEP or DMMP. Isolated cytosolic fractions were treated with SDS to liberate reversibly bound chemicals (Pähler et al., 1997), and bound chemicals were extracted into chloroform and identified by GC/MS using selected ion monitoring. In the chromatograms obtained (Figs. 4 A and 4B), peaks containing representative fragments for DMMP (Fig. 4A) and DEEP (Fig. 4B) in the expected relative intensities eluted at the retention time of the reference compounds under the chromatographic conditions applied. Moreover, the peaks in the purified 2u-globulin samples from male rat kidney also co-eluted with the authentic reference compounds when a different temperature gradient (8°/min) was used for gas chromatographic separation. These data demonstrate that the parent alkylphosphonates are likely to be the agents binding to 2u-globulin.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODRESULTSDISCUSSIONREFERENCES |
|---|
Data on the toxicity of alkyl phosphonates, which are widely used, are scarce, and studies on the biotransformation and excretion of these compounds are not available. This study was performed to obtain information on the biotransformation and excretion of DMMP and DEEP after oral administration, and to delineate a possible mechanism for the induction of renal tumors in male rats, the major toxic effect seen after chronic administration of DMMP.
The experimental data on the biotransformation and excretion show that both DMMP and DEEP are rapidly absorbed from the gastrointestinal tract and are rapidly eliminated with urine in rats, both as the parent compound and as metabolites. As observed with structurally related phosphoric acid esters, biotransformation of both DMMP and DEEP occurs by hydrolysis, to give alkyl phosphonate esters. Further hydrolysis to ethylphosphonate occurs only with DEEP. Likely, this reaction is catalyzed by esterases. With DMMP, only one of the phosphonate ester groups is cleaved to give methyl methylphosphonate, since this compound is highly water-soluble and may be rapidly excreted. With DMMP, further hydrolysis of ethyl ethylphosphonate occurs in part due to the higher lipophilicity. The sex differences in metabolite recovery from urine of treated rats, and the reduced recovery in male rats may be partly due to an increased retention of DMMP and DEEP in the kidney. This retention is due to binding to 2u-globulin (Charbonneau et al., 1987); however, sex differences in absorption or binding to other proteins may also be involved.
DMMP induced chronic renal toxicity and renal tumors in male, but not in female rats after long-term administration. The histological changes seen (protein droplet nephropathy) in the kidneys of treated male rats resembled those observed with several other chemicals where 2u-globulin nephropathy has been seen (Dunnick et al., 1988).
The quantitation of 2u-globulin in male rat kidney cytosol after treatment with a chemical has been shown to be a useful tool to demonstrate protein droplet formation in the nephron. Protein droplet formation is due to the accumulation of 2u-globulin because of reduced degradation after binding of a chemical.
Methods for the identification and quantitation of 2u-globulin in complex protein mixtures are often based on immunostaining in Western blot experiments, utilizing antibodies specific to 2u-globulin (Borghoff and Lagarde, 1993; Borghoff et al., 1990, 1991, 1992; Saito et al., 1996). These antibodies were applied to kidney cytosolic samples from rats treated both with DMMP or DEEP and demonstrated an increased 2u-globulin concentration, indicating protein droplets in male rats. In addition, accumulation of 2u-globulin in the kidneys of male, but not female, rats treated with DMMP or DEEP was demonstrated by CE-SDS. CE-SDS is a fast, sensitive, and accurate method, with high precision, for using in capillary UV-detection (Pähler et al., 1999). With this method, an increased area of the peak representing 2u-globulin was observed in the kidney cytosol of male rats treated with DMMP or DEEP. The observed 2u-globulin accumulation further supports the conclusion that DMMP and DEEP were present in the 2u-globulin fractions isolated by HPLC, under non-denaturing conditions, from the kidneys of treated male rats, and could be liberated by protein denaturation. The identification of DMMP and DEEP in 2u-globulin also suggests that the parent phosphonate esters are the agents binding to the lipophilic binding sites in 2u-globulin. In summary, all the methods applied suggest that DMMP and DEEP are capable of binding to 2u-globulin and that this binding is responsible for the formation of protein droplets in the kidneys of male rats.
A variety of chemicals has been demonstrated to bind to 2u-globulin, e.g., gasoline constituents, solvents, pesticides, food constituents, and drugs (Swenberg, 1993). They all cause male rat-specific nephrotoxicity and tumor induction. The formation of protein droplets within the proximal tubule cells and the binding to 2u -globulin are prerequisites to the development of the nephrotoxicity (Borghoff et al., 1990, 1992; Swenberg et al., 1989). The demonstration of 2u-globulin accumulation in the kidneys of male rats treated with DMMP suggests that 2u-globulin nephropathy is involved in the renal toxicity and tumorigenicity of DMMP in male rats. DEEP has not been investigated in long-term animal experiments for tumorigenicity. However, based on the observed 2u-globulin accumulation by this compound in male rats, it may also be expected that this compound induces renal tumor formation in male rats when given at high doses for a prolonged time.
When comparison is made between the concentrations of 2u-globulin in DEEP- and DMMP-treated animals with 2u-globulin concentrations in kidney induced by other agents, DMMP and DEEP, which, in even smaller doses, induced large increases in renal 2u-globulin concentration, seem to be relatively potent inducers of 2u-globulin accumulation.
Non-genotoxic chemicals that cause male rat-specific renal toxicty and tumors, and which have been shown to be mediated through accumulation of 2u-globulin within protein droplets in the kidney and have demonstrated binding of the chemical to 2u-globulin, are not considered a renal tumor risk in humans. 2u-Globulin is a male rat-specific protein that is not found in female rats, either sex of mice, or humans. Thus, the implicated mechanism of DMMP tumorigenicity to the rat kidney is very unlikely to occur in humans (Flamm and Lehman-McKeeman, 1991; Lehman-McKeeman, 1993; Lehman-McKeeman and Caudill, 1992; Swenberg, 1993).
|
|
NOTES |
|---|
1 To whom correspondence should be addressed. Fax: + 49(0)931/201–3865. E-mail: dekant{at}toxi.uni-wuerzburg.de.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODRESULTSDISCUSSIONREFERENCES |
|---|
Alden, C. L. (1985). Species, sex, and tissue specificity in toxicologic and proliferative responses. Toxicol. Pathol. 13, 135–140.[Medline]
Alden, C. L. (1986). A review of the unique male rat hydrocarbon nephropathy. Toxicol. Pathol. 14, 109–111.[Medline]
Alden, C. L., Kanerva, R. L., Ridder, G., and Stone, L. C. (1984). Renal effects of petroleum hydrocarbons. The pathogenesis of the nephrotoxicity of volatile hydrocarbons in the male rat. In Advances in Modern Environmental Toxicology (M. A. Mehlman, C. P.Hemstreet, J. J.Thorpe, and N. K.Weaver, Eds.), pp. 107–120. Princeton Scientific Publishing, New Jersey.
Borghoff, S. J., and Lagarde, W. H. (1993). Assessment of binding of 2,4,4-trimethyl-2-pentanol to low-molecular-weight proteins isolated from kidneys of male rats and humans. Toxicol. Appl. Pharmacol. 119, 228–235.[ISI][Medline]
Borghoff, S. J., Miller, A. B., Bowen, J. P., and Swenberg, J. A. (1991). Characteristics of chemical binding to 2u-globulin in vitro-evaluating structure-activity relationships. Toxicol. Appl. Pharmacol. 107, 228–238.[ISI][Medline]
Borghoff, S. J., Short, B. G., and Swenberg, J. A. (1990). Biochemical mechanisms and pathobiology of alpha 2u-globulin nephropathy. Annu. Rev. Pharmacol. Toxicol. 30, 349–367.[ISI][Medline]
Borghoff, S. J. H., Youtsey, N. L., and Swenberg, J. A. (1992). A comparison of European high-test gasoline and PS-6 unleaded gasoline in their abilities to induce alpha 2u-globulin nephropathy and renal cell proliferation. Toxicol. Lett. 63, 22–33.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein-dye binding. Anal. Biochem. 72, 248–254.[ISI][Medline]
Charbonneau, M., Lock, E. A., Strasser, J., Cox, M. G., Turner, M. J., and Bus, J. S. (1987). 2,2,4-Trimethylpentane-induced nephrotoxicity: I. Metabolic disposition of TMP in male and female Fischer 344 rats. Toxicol. Appl. Pharmacol. 91, 171–181.[Medline]
Charbonneau, M., Strasser, J., Jr., Lock, E. A., Turner, M. J., Jr., and Swenberg, J. A. (1989). Involvement of reversible binding to 2u-globulin in 1,4-dichlorobenzene-induced nephrotoxicity. Toxicol. Appl. Pharmacol. 99, 122–132.[ISI][Medline]
Dunnick, J. K., Eustis, S. L., and Haseman, J. K. (1988). Development of kidney tumors in the male F344/N rat after treatment with dimethyl methylphosphate. Fundam. Appl. Toxicol. 11, 91–99.[Medline]
EPA (U. S. Environmental Protection Agency) (1983). An Overview of Exposure Potential of Commercial Flame Retardants. EPA, Assessment Division, Washington, DC.
Flamm, W. G., and Lehman-McKeeman, L. D. (1991). The human relevance of the renal tumor-inducing potential of d-limonene in male rats: Implications for risk assessment. Regul. Toxicol. Pharmacol. 13, 70–86.[Medline]
Hard, G. C., Rodgers, I. S., Baetcke, K. P., Richards, W. L., McGaughy, R. E., and Valcovic, L. R. (1993). Hazard evaluation of chemicals that cause accumulation of alpha 2u-globulin, hyaline droplet nephropathy, and tubule neoplasia in the kidneys of male rats. Environ. Health Perspect. 99, 313–349.[ISI][Medline]
Hildebrand, H., Hartmann, E., Popp, A., and Bomhard, E. (1997). Quantitation of alpha(2)-microglobulin after administration of structurally divergent chemical compounds. Arch. Toxicol. 71, 351–359.[ISI][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–685.[Medline]
Lehman-McKeeman, L. D. (1993). Male rat-specific light hydrocarbon nephropathy. In Toxicology of the Kidney (J. B. Hook and R. S.Goldstein, Eds.), pp. 477–494. Raven Press, New York.
Lehman-McKeeman, L. D., and Caudill, D. (1992). Biochemical basis for mouse resistance to hyaline droplet nephropathy: Lack of relevance of the 2u-globulin protein superfamily in this male rat-specific syndrome. Toxicol. Appl. Pharmacol. 112, 214–221.[ISI][Medline]
Lehman-McKeeman, L. D., Caudill, D., Takigiku, R., Schneider, R. E., and Young, J. A. (1990a). Comparative disposition of d-limonene in rats and mice: relevance to male-rat-specific nephrotoxicity. Toxicol. Lett. 53, 193–195.[ISI][Medline]
Lehman-McKeeman, L. D., Rivera-Torres, M. I., and Caudill, D. (1990b). Lyosomal degradation of 2u-globulin and 2u-globulin-xenobiotic conjugates. Toxicol. Appl. Pharmacol. 103, 539–548.[Medline]
Lehman-McKeeman, L. D., Rodriguez, P. A., Takigiku, R., Caudill, D., and Fey, M. L. (1989). d-Limonene-induced male rat-specific nephrotoxicity: Evaluation of the association between d-limonene and 2u-globulin. Toxicol. Appl. Pharmacol. 99, 250–259.[ISI][Medline]
Lock, E. A., Charbonneau, M., Strasser, J., Swenberg, J. A., and Bus, J. S. (1987). 2,2,4-Trimethylpentane (TMP)-induced nephrotoxicity: II. The reversible binding of a TMP metabolite to a renal protein fraction containing 2u-globulin. Toxicol. Appl. Pharmacol. 91, 182–192.[ISI][Medline]
NSWC, U. S. (1982). Environmental assessment for testing NSWC advance-warning systems with DMMP (dimethyl methylphosphonate). Naval Surface Weapons Center, Washington, DC.
NTP (1997). Toxicology and carcinogenesis studies of dimethyl methylphosphonate (CAS No. 756–79–6) in F344/N rats and B6C3F1 mice (gavage studies). National Toxicology Program, TR-323.
Pähler, A., Birner, G., Ott, M. M., and Dekant, W. (1997). Binding of hexachlorobutadiene to 2u-globulin and its role in nephrotoxicity in rats. Toxicol. Appl. Pharmacol. 147, 372–380.[Medline]
Pähler, A., Blumbach, K., Herbst, J., and Dekant, W. (1999). Quantitation of 2u-globulin in rat kidney cytosol by capillary electrophoresis. Anal. Biochem. 267, 203–211.[Medline]
Saito, K., Uwagawa, S., Kaneko, H., Shiba, K., Tomigahara, Y., and Nakatsuka, I. (1996). 2u-globulins in the urine of male rats: A reliable indicator for alpha(2u)-globulin accumulation in the kidney. Toxicology 106, 149–157.[Medline]
Short, B. G., Burnett, V. L., Cox, M. G., Bus, I. S., and Swenberg, J. A. (1987). Site-specific renal cytotoxicity and cell proliferation in male rats exposed to petroleum hydrocarbons. Lab. Invest. 57, 564–577.[ISI][Medline]
Short, B. G., Burnett, V. L., and Swenberg, J. A. (1989). Elevated proliferation of proximal tubule cells and localization of accumulated 2u-globulin in F344 rats during chronic exposure to unleaded gasoline of 2,2,4-trimethylpentane. Toxicol. Appl. Pharmacol. 101, 414–431.[ISI][Medline]
Siekevitz, P. (1962). Preparation of microsomes and submicrosomal fractions: Mammalian. Methods Enzymol. 5, 61–68.
Swenberg, J. A. (1993). 2u-Globulin nephropathy: Review of the cellular and molecular mechanisms involved and their implications for human risk assessment. Environ. Health Perspect. 101, 39–44.
Swenberg, J. A., Short, B., Borghoff, S., Strasser, J., and Charbonneau, M. (1989). The comparative pathobiology of 2u-globulin nephropathy. Toxicol. Appl. Pharmacol. 97, 35–46.[ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||