Douglas Caudill

d-Limonene-induced male rat specific nephrotoxicity: evaluation of the association between d-limonene and α2u-globulin

d-Limonene is a naturally occurring monoterpene, which when dosed orally, causes a male rat-specific nephrotoxicity manifested acutely as the exacerbation of protein droplets in proximal tubule cells. Experiments were conducted to examine the retention of [14C]d-limonene in male and female rat kidney, to determine whether d-limonene or one or more of its metabolites associates with the male rat-specific protein, alpha 2u-globulin, and if so, to identify the bound material. The results indicated that, 24 hr after oral administration of 3 mmol d-limonene/kg, the renal concentration of d-limonene equivalents was approximately 2.5 times higher in male rats than in female rats. Equilibrium dialysis in the presence or absence of sodium dodecyl sulfate indicated that approximately 40% of the d-limonene equivalents in male rat kidney associated with proteins in a reversible manner, whereas no significant association was observed between d-limonene equivalents and female rat kidney proteins. Association between d-limonene and male rat kidney proteins was characterized by high-performance gel filtration and reverse-phase chromatography. Gel filtration HPLC indicated that d-limonene in male rat kidney is associated with a protein fraction having a molecular weight of approximately 20,000. Separation of alpha 2u-globulin from other kidney proteins by reverse-phase HPLC indicated that d-limonene associated with a protein present only in male rat kidney which was definitively identified as alpha 2u-globulin by amino acid sequencing. The major metabolite associated with alpha 2u-globulin was d-limonene-1,2-oxide. Parent d-limonene was also identified as a minor component in the alpha 2u-globulin fraction. Thus, d-limonene, and more specifically d-limonene-1,2-oxide, associates with alpha 2u-globulin in a reversible manner in male rat kidney. This interaction may be responsible for excessive accumulation of alpha 2u-globulin in kidneys of male rats exposed to d-limonene.

Lysosomal degradation of α2u-globulin and α2u-globulin-xenobiotic conjugates☆

Abstract A diverse group of chemicals cause a male rat-specific nephrotoxicity in which α2u-globulin accumulates in renal lysosomes. It has been suggested that these chemicals bind to the protein and decrease its degradation by lysosomal proteinases. To test this hypothesis, the lysosomal degradation of native α2u-globulin and that to which d -limonene, d -limonene-1,2-oxide, isophorone, 1,4-dichlorobenzene, and 2,5-dichlorophenol were bound was studied. α2u-Globulin was purified from male rat urine, and male rat renal cortical lysosomes, isolated by differential centrifugation, served as the proteolytic enzyme source. Pepstatin, an inhibitor of aspartic acid proteinases, and leupeptin, an inhibitor of cysteine proteinases, reduced α2u-globulin degradation to 28 ± 8 and 17 ± 5% of control, respectively, whereas addition of both inhibitors decreased α2u-globulin degradation to 8 ± 1% of control values. These results indicate that both classes of endopeptidases are important in the degradation of α2u-globulin. Under the incubation conditions used, 30% of native α2u-globulin was degraded in a 4-hr period. Conjugates of the protein were made for in vitro binding experiments. Binding of d -limonene and 1,4-dichlorobenzene to α2u-globulin did not alter the degradation of the protein, whereas binding of d -limonene-1,2-oxide, 2,5-dichlorophenol, and isophorone decreased α2u-globulin degradation by 33%. These results indicate that not all chemicals which have been shown to bind in vivo to α2u-globulin alter the in vitro lysosomal degradation of the protein. However, in all cases, one metabolite of each hyaline droplet inducer did alter degradation of α2u-globulin, suggesting that a decrease in lysosomal degradation is involved in the accumulation of this protein in male rat kidney lysosomes.

α2u-globulin is the only member of the lipocalin protein superfamily that binds to hyaline droplet inducing agents

The rate-limiting step in chemically induced, male rat-specific hyaline droplet nephropathy is the reversible binding of a xenobiotic to alpha 2u-globulin. In this study, equilibrium saturation binding experiments were conducted to evaluate the in vitro binding of d-limonene-1,2-oxide (dLO) and 2,4,4-trimethyl-2-pentanol (TMP-OH) to alpha 2u-globulin and members of the alpha 2u-globulin protein superfamily. Both dLO and TMP-OH bound to alpha 2u-globulin, with Scatchard analysis yielding dissociation constants of 5.6 and 6.4 x 10(-7) M, respectively. The Bmax for binding (nmol bound/mg protein) was 50.7 and 61.1 for dLO and TMP-OH, respectively, yielding a molar ratio of approximately 1 for both ligands. The ability of dLO and TMP-OH to bind to human-derived alpha 1-acid glycoprotein, rat-derived retinol-binding protein, human protein-1, and bovine beta-lactoglobulin was also studied. These superfamily proteins are generally abundant in plasma, are freely filtered across the glomerulus, and can bind a wide range of ligands. However, neither dLO nor TMP-OH bound to any of the superfamily proteins. In contrast, under identical experimental conditions, alpha 1-acid glycoprotein did bind progesterone (Kd = 10(-6) M), whereas both beta-lactoglobulin and retinol-binding protein bound retinol (Kd = 10(-8) M for both proteins). These results indicate that, under conditions where alpha 2u-globulin superfamily proteins bind to established ligands, the proteins do not interact with hyaline droplet inducing agents. Thus, the interaction between male rat-specific nephrotoxicants and alpha 2u-globulin is unique to this protein. More importantly, these results provide direct evidence that the presence of the alpha 2u-globulin superfamily proteins does not predispose humans to develop hyaline droplet nephropathy and renal cancer from this class of chemicals.

Biochemical basis for mouse resistance to hyaline droplet nephropathy: Lack of relevance of the α2u-globulin protein superfamily in this male rat-specific syndrome

It is well-established that binding of a chemical to alpha 2u-globulin is the rate-limiting step in the development of male rat-specific hyaline droplet nephropathy. Mice synthesize mouse urinary protein (MUP), a protein which is very similar to alpha 2u-globulin, but this protein does not render the mouse sensitive to a similar renal toxicity. Therefore, the purpose of the present study was to determine the biochemical basis for mouse resistance to hyaline droplet nephropathy. Male Fischer 344 rats and B6C3F1 mice excreted 12.24 +/- 0.60 and 14.88 +/- 0.99 mg of alpha 2u-globulin and MUP daily, indicating that quantitative differences in protein excretion were not involved in the species specificity of the nephropathy. With d-limonene as a model hyaline droplet inducing agent, both rat and mouse liver microsomes oxidized the terpene to its 1,2-epoxide (the metabolite that binds reversibly to alpha 2u-globulin in vivo), demonstrating that metabolic differences do not determine the mouse resistance to this lesion. In spite of the formation of the epoxide intermediate, no binding of [14C]d-limonene equivalents to mouse kidney proteins was observed. In contrast, about 40% of the d-limonene equivalents in male rat kidney was reversibly bound to renal proteins. The renal reabsorption of alpha 2u-globulin and MUP was markedly different, as rats reabsorbed about 60% of the total filtered load of alpha 2u-globulin, but MUP was not reabsorbed by the mouse kidney. Given the absence of MUP in mouse kidney, in vitro equilibrium saturation binding studies were also conducted to determine whether MUP could bind the epoxide metabolite. alpha 2u-Globulin bound [14C]d-limonene-1,2-oxide with an apparent Kd of 4 x 10(-7) M. However, under identical experimental conditions, MUP failed to bind the epoxide. These data indicate that two major biochemical differences between alpha 2u-globulin and MUP contribute to mouse resistance to hyaline droplet nephropathy. Under both in vivo and in vitro conditions, MUP does not bind d-limonene-1,2-oxide, the rate-limiting step in the development of the nephropathy. However, even if MUP did bind the epoxide, the fact that it is not reabsorbed into the mouse kidney precludes its involvement in a syndrome involving renal protein overload. Finally, the absence of an interaction between d-limonene, a model hyaline droplet inducer, and the protein most similar to alpha 2u-globulin suggests that no other protein in the alpha 2u-globulin superfamily is likely to cause hyaline droplet nephropathy in other species.

Effects of musk xylene and musk ketone on rat hepatic cytochrome P450 enzymes

The purpose of the present work was to characterize the effect of musk xylene (MX) and musk ketone (MK) treatment on rat hepatic cytochrome P450 enzymes. Male F344 rats were dosed orally with MX (10, 50 or 200 mg/kg) or MK (20, 100 or 200 mg/kg) for 7 days, after which CYP1A, 2B and 3A enzyme activities and protein levels were determined. MX treatment resulted in a two- to four-fold increase in the activity of CYP1A, 2B and 3A enzymes. For CYP1A and 3A, these changes were consistent with small increases in immunoreactive proteins. However, for CYP2B, despite only a three-fold increase in enzyme activity, protein levels were increased nearly 50-fold relative to control. This induction occurred by transcriptional activation of the CYP2B1 gene as evidenced by increased steady state CYP2B1 mRNA levels. In contrast to MX, MK treatment increased CYP2B activity, protein and mRNA levels. However MK treatment also increased CYP1A enzyme activity nearly 30-fold higher than control rats, a profile that was markedly different from MX, and very different from its effects in mice (Stuard, S.B., Caudill, D., Lehman-Mc-Keeman, L.D., 1997. Characterization of the effects of musk ketone on mouse cytochrome P450 enzymes. Fund. Appl. Toxicol. 40, 264-271). These results indicate that in rats, MX is an inducer of CYP2B enzymes, but these enzymes are not functionally active. In contrast, MK also induces CYP2B enzymes, with no concurrent inactivation. MK also exhibits a unique pattern of cytochrome P450 induction by increasing both CYP1A and CYP2B in rats.

Hyaline droplet nephropathy resulting from exposure to 3,5,5-trimethylhexanoyloxybenzene sulfonate

Acute oral dosing of 3,5,5-trimethylhexanoyloxybenzene sulfonate (THBS) to adult male and female rats causes a male rat-specific nephrotoxicity manifested as exacerbation of hyaline droplet formation. This chemical is structurally distinct from the volatile hydrocarbons known to cause male rat-specific kidney lesions. Therefore, to classify THBS as a hyaline droplet-inducing agent, experiments were conducted to determine whether [14C]THBS equivalents bound to alpha 2 mu-globulin and caused the protein to accumulate in male rat kidney cortex. Two-dimensional gel electrophoretic separation of male rat kidney proteins indicated that alpha 2u-globulin levels in kidney increased 24 hr after a single oral dose of THBS (500 mg/kg). Furthermore, a sex-dependent retention THBS was noted as there was approximately 10 times more THBS equivalents in male rat kidney than in female rat kidney. Equilibrium dialysis experiments indicated that 40% of THBS equivalents bound reversibly to male rat kidney proteins, whereas no interaction between THBS and female rat kidney proteins was detected. Specific binding of THBS to alpha 2mu-globulin was determined by anion-exchange HPLC after which metabolites in the alpha 2u-globulin fraction were identified by gas chromatography with parallel radioactivity-mass spectrometry and mass spectrometry-matrix isolation Fourier-transform infrared analysis. Four metabolites of THBS were found in this protein fraction, and the major component (approximately 70%) was identified as the cis gamma-lactone of 3,5,5-trimethylhexanoic acid. Experiments were also conducted in mice to determine whether THBS bound to any mouse kidney proteins, particularly mouse urinary protein. The results indicated that there was no interaction between THBS and mouse urinary protein, a protein which shares significant homology with alpha 2u-globulin. These results indicate that THBS treatment exacerbates hyaline droplet formation in male rat kidneys by binding to alpha 2mu-globulin, thereby causing the protein to accumulate in the renal cortex. The interaction between THBS and alpha 2mu-globulin appears to be unique to this male rat-specific protein as THBS does not interact with a very similar protein found in mice.

Quantitation of urinary α2u-globulin and albumin by reverse-phase high performance liquid chromatography

A rapid, reproducible, and sensitive high-performance liquid chromatography (HPLC) method for the quantitation of alpha 2u-globulin, the major urinary protein excreted by adult male rats, and albumin has been developed. Total urinary proteins, isolated by a simple Sephadex G-25 gel filtration step, are separated and quantitated by reverse-phase HPLC on a C4 Macrosphere 300 column. The proteins are separated and eluted with a two-step gradient of acetonitrile in aqueous trifluoroacetic acid. Detection limits of 9 and 25 micrograms/mL of urine were established for albumin and alpha 2u-globulin, respectively. Quantitation of urinary excretion of the two proteins in young adult male and female rats and aging male rats showed that values obtained with this method compared favorably with values from previously developed immunological techniques. To quantitate total urinary protein excretion, we modified the Bradford protein assay to use rat urinary protein as standard. Given the established importance of alpha 2u-globulin in the development of male rat-specific nephrotoxicity and nephrocarcinogenicity, these methods should be useful for studying the renal handling of this protein under normal and nephrotoxic conditions.

The metabolism and disposition of 3,4,4'-trichlorocarbanilide in the intact and bile duct-cannulated adult and in the newborn rhesus monkey (M. mulatta).

Abstract The metabolism and disposition of intravenously infused radioactive 3,4,4′-trichloro[ 14 C]carbanilide (TCC) in the adult and newborn rhesus monkey have been evaluated. In adult animals the major metabolic reactions were N -glucuronide formation or ring hydroxylation followed by conjugation to glucuronic acid or sulfuric acid. Removal of 14 C from the plasma was biphasic; TCC and the N -glucuronides accounted for the fast phase, and the O -sulfate conjugates accounted for the slow phase. The major urinary metabolites were the N -glucuronides of TCC. The tissue residue of 14 C was low in the monkeys and was limited primarily to tissues that are active in drug metabolism (liver, kidneys, and lungs). The bile was the major route of elimination with glucuronide conjugates as the major radioactive component. Enterohepatic circulation was extensive but did not affect the plasma concentrations or the elimination kinetics of TCC-derived material from the plasma. The newborn monkey also metabolized TCC by ring hydroxylation or N -glucuronidation. The plasma kinetics were similar to those observed in adults as was the tissue distribution. Unlike the adult, there were only very low amounts of O -glucuronides and, instead, high amounts of O -sulfate conjugates. It is concluded that the infant monkey can readily metabolize and eliminate TCC.

Induction of mouse cytochrome P450 2B enzymes by amine metabolites of musk xylene: contribution of microsomal enzyme induction to the hepatocarcinogenicity of musk xylene.

Musk xylene (MX) is a synthetic nitromusk perfume ingredient that, although uniformly negative in genotoxicity testing, causes liver tumors in B6C3F1 mice. MX is also capable of inducing cytochrome P450 enzymes in a manner similar to that of phenobarbital (PB), which suggests that epigenetic mechanisms may be involved in the carcinogenic response. At the same time, MX is metabolized in vivo by nitroreduction, a reaction catalyzed by intestinal flora that yields aromatic amine metabolites. These amine metabolites are also capable of inactivating CYP2B10, the major cytochrome P450 enzyme induced by MX treatment. In the study reported here, the monoamine metabolites of MX, o‐ and p‐NH2‐MX, were evaluated for their potential to induce CYP2B10 and CYP1A2 mRNAs. Northern blot analyses indicated that both amines markedly induced CYP2B10 mRNA, whereas CYP1A2 mRNA, the enzyme implicated in the bioactivation of aromatic amines and frequently induced by aromatic amines, was induced only slightly, a response that was not different from that seen with PB. Induction of CYP2B10 mRNA suggested that the amine metabolites may contribute to the enzyme induction profile seen with MX treatment. To test this hypothesis, mice were treated with broad‐spectrum antibiotics (neomycin, tetracycline, and bacitracin) to eliminate the intestinal flora and prevent formation of o‐ and p‐NH2‐MX. In antibiotic‐treated mice treated with MX (200 mg/kg) for 4 d, no evidence of microsomal enzyme induction was observed, including no increases in liver weight, total cytochrome P450 content, or CYP2B protein levels. These results indicate that the amine metabolites of MX are responsible for the enzyme induction seen after MX administration. Thus, the biochemical and molecular effects of amine metabolites of MX are markedly different from those of other aromatic amines but very similar to those of PB. Therefore, it appears that MX is a non‐genotoxic chemical that may cause mouse liver tumors in a manner analogous to that of PB. Mol. Carcinog. 20:308–316, 1997.

Design of a gas chromatograph with parallel radioactivity and mass spectrometric detection: Application to the identification of the major metabolite of d-limonene associated with α2u-globulin

A Perkin Elmer 3920 gas chromatograph, equipped with a versatile inlet system (i.e. an injector/trap), was interfaced to a radioactivity detector and a mass-selective detector (H/P 5970B) to identify 14C-labeled compounds. The use of a pre-trap as a demountable, programmable-temperature injector, in conjunction with the injector/trap, allowed the introduction of 0.5-ml samples of rat kidney cytosol extracts to 0.32 mm I.D. capillary columns. The instrumentation greatly facilitated the identification of the major radiolabeled metabolite of d-limonene associated with the male rat-specific protein alpha 2u-globulin as 1,2-cis-d-limonene oxide.