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D Caudill

Publications and source records attributed to D Caudill.

At least 19 recordsLinked to original sources
PubMed2000

In vitro kinetics of coumarin 3,4-epoxidation: application to species differences in toxicity and carcinogenicity.

Coumarin, a natural product and fragrance ingredient, is a well recognized rat liver toxicant, and dietary administration at toxic dosages increased the incidence of rat cholangiocarcinomas and parenchymal liver-cell tumors in a chronic bioassay. Hepatotoxicity in rats is site- and species-specific, and is thought to result from the formation of coumarin 3,4-epoxide and its rearrangement product, o-hydroxyphenylacetaldehyde (o-HPA). The goals of the current study were to describe the in vitro kinetics of the metabolic activation of coumarin, and determine whether species differences in susceptibility to liver injury correlate with coumarin bioactivation determined in vitro. Coumarin 3,4-epoxidation was quantified via the formation of o-HPA in pooled hepatic microsomes from female B6C3F1 mice, male F344 rats, and individual humans (n = 12 subjects), and the apparent kinetic constants for o-HPA production were calculated using nonlinear regression and fitting to either a one-enzyme or two-enzyme model. Eadie-Hofstee analyses indicated that o-HPA formation was biphasic in both rat and mouse liver. Although the apparent high affinity K:(m) in rat and mouse liver microsomes was 38.9 and 47.2 microM, respectively, the overall rate of o-HPA formation was far greater in mouse than in rat liver microsomes. Furthermore, the total clearance (CL(int)) of coumarin via o-HPA formation in mouse liver microsomes was 4-fold greater than in rat liver microsomes. Since mice are relatively resistant to hepatotoxicity, the data indicated that rates of o-HPA formation in rat and mouse liver microsomes were not directly predictive of liver toxicity in vivo, and further suggested that o-HPA detoxification played a role in modulating coumarin-mediated toxicity. The current studies also indicated that coumarin 3,4-epoxidation in human hepatic microsomes was minimal. In human liver microsomes (n = 12), the kinetics of o-HPA formation were best described by a single enzyme model, with the K(m) for o-HPA formation ranging from 1320-7420 microM. In the most active human sample, the intrinsic clearance of coumarin via the 3,4-epoxidation pathway was 1/9 and 1/38 that of the rat and mouse, respectively. The in vitro kinetics of o-HPA formation, and in particular, the large quantities of coumarin required for o-HPA production in human liver microsomes, strongly suggest that humans are unlikely to produce toxicologically relevant concentrations of this metabolite following low level coumarin exposures.

Acetaldehyde↗
PubMed1999

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.

Animals↗
PubMed1999

Development of tolerance to Clara cell necrosis with repeat administration of coumarin.

Coumarin was identified as a mouse-lung carcinogen following oral gavage administration in a chronic bioassay, and was shown to cause the selective necrosis of terminal bronchiolar Clara (non-ciliated bronchiolar epithelial) cells in the mouse lung after acute administration. After oral gavage, a similar effect was not observed in the terminal bronchioles of rats, suggesting that coumarin-mediated Clara cell toxicity is a species-specific effect. Using coumarin dosages (50 and 200 mg/kg) and a dosing schedule modeled after the chronic bioassay, the current study examined the effects of repeated coumarin administration in mouse lung. A single dosage of coumarin (200 mg/kg) caused swelling of Clara cells and necrosis in mouse-lung terminal bronchioles. However, after 5 consecutive oral doses of coumarin (200 mg/kg), the mouse lung became tolerant to coumarin, and although areas of bronchiolar epithelial flattening and hyperplasia were noted, Clara cell necrosis was not observed. After 10 doses of coumarin, mouse lungs appeared nearly normal. Coumarin-mediated Clara cell injury is thought to result from the cytochrome P450-catalyzed formation of coumarin 3,4-epoxide and Western analysis of whole mouse lung microsomal P450 content indicated that, commensurate with Clara cell necrosis, many P450s were decreased. However, P450 levels appeared qualitatively normal in lung microsomes from tolerant mice. Similarly, coumarin epoxidation and 7-hydroxylation rates in whole lung microsomes from tolerant animals were similar to controls. To determine if animals tolerant to coumarin were tolerant to other Clara cell toxicants, a single toxic dose of naphthalene (200 mg/kg) was administered to coumarin-tolerant mice. Coumarin pretreatment reduced naphthalene-mediated Clara cell toxicity, supporting the hypothesis that tolerance may result from general biochemical and molecular changes and not exclusively from alterations in chemical metabolism.

Animals↗
PubMed1998

2-sec-butyl-4,5-dihydrothiazole is a ligand for mouse urinary protein and rat alpha 2u-globulin: physiological and toxicological relevance.

Mouse urinary protein (MUP) and alpha 2u-globulin are structurally homologous proteins that belong to a superfamily of ligand-binding proteins and represent the major urinary proteins excreted by adult male mice and rats, respectively. Although a variety of xenobiotics bind to alpha 2u-globulin and produce a male rat-specific hyaline droplet nephropathy, no endogenous ligand for this protein has been identified. Despite extensive sequence homology. MUP does not bind to hyaline droplet-inducing agents. While performing experiments with purified MUP, we observed that it presented with a strong, distinctive odor reminiscent of mouse urine. To determine whether this odor was the result of contamination or degradation or was attributed to an endogenous ligand bound to the protein, the protein was subjected to thermal desorption and any released volatile compounds were detected with a gas chromatograph equipped with an external sniff port and mass spectrometer. With this approach, two odorous compounds were detected at the sniff port by a human observer, but only one was present in sufficient mass to allow identification. This compound, which presented with the characteristic odor, was subsequently identified as 2-sec butyl-4,5-dihydrothiazole (DHT) by GC/MS/matrix isolation IR and NMR analyses. The identification of DHT was confirmed by comparing the chromatographic and spectral properties to those of the synthesized authentic compound. In direct contrast, purified urinary alpha 2u-globulin did not present with an obvious odor, and no volatile ligands were detected on this protein. Although DHT is a major endogenous ligand for MUP, it was also found to competitively inhibit the binding of [14C]d-limonene-1,2-epoxide to alpha 2u-globulin with relatively high affinity (Ki = 2.3 microM). When dosed orally to F344 rats, DHT (1 mmol/kg for 3 days) caused the characteristic exacerbation of hyaline droplets in male rat kidneys and increased renal levels of immunoreactive alpha 2u-globulin about threefold over control levels. These results indicate that despite structural homology, MUP and alpha 2u-globulin are distinguished by the presence of a volatile endogenous ligand only on the former, a distinction that may reflect differences in the physiological functions of the two proteins. Furthermore, although DHT can bind to both MUP and alpha 2u-globulin, renal toxicity was only observed in rats, thereby emphasizing the unique toxicological properties of alpha 2u-globulin in the development of hyaline droplet nephropathy.

Alpha-Globulins↗
PubMed1998

Selective Clara cell injury in mouse lung following acute administration of coumarin.

Coumarin is a known hepatotoxicant in laboratory animals, particularly rats. However, the mouse lung was identified as a major target organ in a chronic bioassay, with an oral gavage dosage of 200 mg/kg coumarin increasing the incidence of alveolar/bronchiolar adenomas and carcinomas. The purpose of the present work was to determine whether coumarin was acutely toxic in the mouse and rat lung. Male and female B6C3F1 mice were dosed orally by gavage with coumarin at 0, 10, 20, 50, 100, 150, and 200 mg/kg and lung toxicity was determined 24 h later by histological evaluation. The results indicated that coumarin dosages >/= 150 mg/kg caused selective injury to Clara cells in the distal bronchiolar epithelium. The time course of this injury was studied from 6 h to 7 days after a single dosage of coumarin (200 mg/kg). At 12 h after dosing, Clara cell swelling was apparent along with the onset of necrosis and bronchiolar epithelial disorganization. At 24-48 h, necrotic Clara cells were observed sloughed into the lumens of the terminal bronchioles, with concomitant thinning of the epithelium and flattening of the remaining ciliated cells. By 72-96 h, there was epithelial hypertrophy and hyperplasia, and by 7 days after dosing, the Clara cells had regenerated and the bronchiolar epithelial architecture appeared nearly normal. Unlike the mouse, oral administration of coumarin (200 mg/kg) caused severe hepatotoxicity in male F344 rats, seen histologically as centrilobular necrosis and associated with increases, up to 140-fold, in serum ALT, AST, and SDH levels. Clara cell toxicity was not observed in the distal bronchioles of treated rats. However, in the upper airways, coumarin treatment produced generalized epithelial necrosis involving both ciliated and nonciliated cells. 3,4-Dihydrocoumarin (DHC), which is not a mouse lung carcinogen, did not cause Clara cell injury when dosed to mice at 800 mg/kg. This finding suggests, because DHC lacks a 3,4-double bond, that bioactivation of coumarin to a 3,4-epoxide intermediate may contribute to mouse lung Clara cell toxicity. Collectively, the results indicate that coumarin is a Clara cell toxicant and establish the mouse lung as a target organ for coumarin toxicity. These new findings lay the foundation for studies to determine the mechanisms of coumarin-induced toxicity and carcincogenicity and to define the relevance of these effects to humans.

Administration, Oral↗
PubMed1997

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.

Amines↗
PubMed1997

Characterization of the effects of musk ketone on mouse hepatic cytochrome P450 enzymes.

Nitroaromatic musks, including musk ketone (MK; 2,6-dimethyl-3,5-dinitro-4-t-butylacetophenone), are chemicals used as perfume ingredients in household products, cosmetics, and toiletries. Musk xylene (MX; 1,3,5-trinitro-2-t-butylxylene), another nitromusk, is not genotoxic but has been reported to produce mouse liver tumors in a chronic bioassay. In addition, MX has been shown to both induce and inhibit mouse liver cytochrome P450 2B (CYP2B) isozymes. The ability of MX to inhibit CYP2B enzyme activity is attributable to inactivation of the enzyme by a specific amine metabolite. MK is structurally similar to MX, but lacks the nitro substitution that is reduced to the inactivating amine metabolite. Therefore, we hypothesized that MK would induce, but not inhibit, CYP2B isozymes. To test this hypothesis, and to evaluate the effects of MK on mouse liver cytochrome P450 enzymes, two sets of experiments were performed. To evaluate the ability of MK to induce cytochromes P450, mice were dosed daily by oral gavage at dosages ranging from 5 to 500 mg/ kg MK for 7 days. This treatment resulted in a pleiotropic response in mouse liver, including increased liver weight, increased total microsomal protein, and centrilobular hepatocellular hypertrophy. At the highest dose tested, MK caused a 28-fold increase in CYP2B enzyme activity and a small (approximately 2-fold) increase in both cytochromes P450 1A and 3A (CYP1A and CYP3A) enzyme activities over control levels. Protein and mRNA analyses confirmed the relative levels of induction for CYP2B, CYP1A, and CYP3A. In addition, the no-observable-effect level (NOEL) for CYP2B induction by MK was 20 mg/kg. To evaluate the ability of MK to inhibit phenobarbital-induced CYP2B activity, mice were given 500 ppm phenobarbital (PB) in the drinking water for 5 days to induce CYP2B isozymes, followed by a single equimolar (0.67 mmol/kg) oral gavage dose of either MK (198 mg/kg) or MX (200 mg/kg), and microsomes were prepared 18 h later. While MX inhibited more than 90% of the PB-induced CYP2B activity in the microsomes, MK caused only a small (about 20%) reduction in PB-induced CYP2B enzyme activity. These results indicate that, like MX. MK is a PB-type inducer of mouse liver CYP2B isozymes, but unlike MX, MK does not effectively inhibit PB-induced CYP2B enzyme activity.

Administration, Oral↗
PubMed1997

Induction and inhibition of mouse cytochrome P-450 2B enzymes by musk xylene.

Musk xylene (MX) (1,3,5-trinitro-2-t-butylxylene) is a nitromusk perfume ingredient that although uniformly negative in a battery of genotoxicity tests, produces a high incidence of liver tumors in mice. The purpose of this work was to characterize the profile and dose-response relationship of microsomal enzyme induction following exposure to MX. MX was dosed by gavage to male B6C3F1 mice for 7 days at 0, 1, 5, 10, 20, 50, 100, and 200 mg/kg after which microsomes were prepared. At 200 mg/kg, MX increased liver weight by about 65% and increased microsomal cytochrome P-450 content 2-fold over control. MX increased microsomal activity for O-dealkylation of 7-ethoxy and 7-methoxyresorufin 4- and 2-fold, respectively, and increased the N-demethylation of erythromycin approximately 2-fold. These results were generally consistent with increased CYP1A1, 1A2, and 3A protein levels determined by Western blotting. In contrast, whereas no increase in O-dealkylation of 7-pentoxyresorufin (PROD) was observed, MX treatment increased CYP2B protein levels about 25-fold over control at 200 mg/kg. Furthermore, a single dosage of MX (200 mg/kg) increased Cyp2b-10 mRNA to a maximal level and with a time course similar to phenobarbital (PB). To study inhibition of CYP2B enzymes in vivo, mice were treated with PB (0.05% in drinking water for 5 days), then given a single dosage of corn oil or MX (200 mg/kg) at 2 or 18 hr before necropsy. PB treatment increased PROD activity 25-fold, and at 2 hr after MX treatment (associated with peak plasma levels of MX), there was no change in the PB-induced PROD activity. However, at 18 hr, MX treatment decreased PROD activity by 90%. Despite the in vivo inhibition, in vitro studies indicated that MX did not cause mechanism-based inactivation of CYP2B enzymes. The potential for nitroreduction of MX (catalyzed by anaerobic intestinal bacteria) to contribute to the inhibition of CYP2B enzyme activity was evaluated in a separate group of PB-induced mice that were dosed orally with a regimen of broad spectrum antibiotics (neomycin, tetracyline, and bacitracin) to reduce gut flora prior to administration of MX. In these animals, MX (200 mg/kg) did not inhibit PB-induced PROD activity. In summary, MX treatment produced general hepatic changes consistent with induction of CYP2B enzymes in mice and caused a large increase in CYP2B protein and mRNA levels. These data indicate that MX is a PB-like inducer of cytochrome P-450 enzymes and may cause liver tumors in a manner analogous to PB. However, no increase in CYP2B enzyme activity was observed, suggesting that MX or metabolites of MX also inhibit this enzyme. When the intestinal flora was eliminated by antibiotic treatment, MX no longer inhibited the CYP2B enzyme, indicating that anaerobic bacteria are capable of metabolizing MX, and suggesting that amine metabolites formed by nitroreduction are involved in the inhibition of mouse CYP2B enzymes.

Administration, Oral↗
PubMed1997

Mechanism-based inactivation of mouse hepatic cytochrome P4502B enzymes by amine metabolites of musk xylene.

Musk xylene (2,4,6-trinitro-1-t-butylxylene; MX) is a synthetic nitromusk perfume ingredient that induces and inhibits mouse cytochrome P4502B (CYP2B) enzymes in vivo. The purpose of the present work was to determine whether amine metabolites of MX contributed to the enzyme inhibition and, if so, to define the nature and kinetics of this inhibition. When dosed orally to phenobarbital (PB)-treated mice, MX (200 mg/kg) inhibited > 90% of the PB-induced O-dealkylation of 7-pentoxyresorufin (PROD), and [14C]MX equivalents bound covalently to microsomal proteins. However, when this experiment was repeated in mice pretreated with antibiotics to eliminate the gastrointestinal flora, no decrease in PB-induced PROD activity and no covalent binding to microsomal proteins were observed. Thus, the ability of antibiotic treatment to eliminate the enzyme inhibition and covalent binding implicated amine metabolites of MX formed by nitroreduction in anaerobic intestinal flora as obligatory for these effects. Two monoamine metabolites of MX were synthesized to study enzyme inhibition directly. These metabolites were 2-amino-4,6-dinitro-1-t-butyl-xylene and 4-amino-2,6-dinitro-1-t-butylxylene, referred to as o-NH2-MX and p-NH2-MX, respectively, reflecting the position of the amine substitution relative to the t-butyl function. In the in vitro studies with PB-induced mouse liver microsomes, both amines inhibited PROD activity when preincubated in the absence of NADPH. However, only p-NH2-MX caused a time- and NADPH-dependent loss of PROD activity, and the inactivation rate was a pseudo-first-order process that displayed saturation kinetics. These results indicate that p-NH2-MX is a mechanism-based inactivator of mouse CYP2B enzymes. From kinetic analyses, the Ki was calculated to be 10.5 microM and the Kinact was 1.2 min-1. As final confirmation of the inhibitory effects of p-NH2-MX on mouse CYP2B enzymes, the amine (0.67 mmol/kg) was dosed orally to PB-induced mice. At 2 hr after dosing, p-NH2-MX inhibited essentially all of the PB-induced PROD activity, whereas an equimolar dosage of parent MX had no effect at this early time. Thus, although MX is an inducer of mouse CYP2B enzymes, an amine metabolite of MX is a mechanism-based inactivator of mouse CYP2B10. Furthermore, it is likely that the amine is responsible for the lack of functional CYP2B enzyme activity associated with induction of this enzyme by MX.

Animals↗
PubMed1995

Musk xylene induces and inhibits mouse hepatic cytochrome P-450 2B enzymes.

The purpose of this work was to characterize the effects of musk xylene on mouse hepatic microsomal enzyme activities. Male B6C3F1 mice were dosed for 7 days at 0 or 200 mg musk xylene/kg after which microsomes were prepared. Musk xylene treatment increased liver weight by 40%, caused hepatocellular hypertrophy and increased total cytochrome P-450 2-fold over control. Microsomes from musk xylene-treated mice showed increased activity for the dealkylation of ethoxy- and methoxyresorufin, results consistent with increased CYP1A1 and 1A2 protein levels determined by Western blotting. No increase in pentoxyresorufin-O-dealkylation activity was seen, but musk xylene treatment markedly increased CYP2B protein levels. Preliminary in vitro studies showed that musk xylene inhibited mouse CYP2B enzymes (IC50 approximately 1 microM), but did not affect the activities of CYP1A1 or 1A2. This inhibition was not NADPH-dependent. These results indicate that, in mice, musk xylene causes generalized hepatic changes similar to classical CYP2B inducers. However, musk xylene is also a potent inhibitor of the CYP2B enzymes.

Animals↗
PubMed1994

d-Limonene induced hyaline droplet nephropathy in alpha 2u-globulin transgenic mice.

d-Limonene is a hyaline droplet inducing agent and produces nephrotoxicity in male rats when the 1,2-epoxide metabolite binds to alpha 2u-globulin. Mice, which do not synthesize alpha 2u-globulin, are resistant to hyaline droplet nephropathy. In this study, the ability of d-limonene to cause hyaline droplet nephropathy in a transgenic mouse engineered to express alpha 2u-globulin was evaluated. The C57BL/6-derived mice excreted 0.4 +/- 0.1 mg alpha 2u-globulin/day, or approximately 16 mg alpha 2u-globulin/kg body wt. This represents about 30% of the amount excreted by adult male rats (11.9 +/- 1.1 mg/day or approximately 48 mg/kg). Transgenic mice excreted less mouse urinary protein (9.3 +/- 1.2 mg/day) than normal mice (15.1 +/- 1.6 mg/day). Unlike normal male rats, untreated transgenic mice did not show significant spontaneous hyaline droplet formation. Liver microsomes from naive transgenic mice oxidized d-limonene to the cis- and transisomers of the 1,2-epoxide, and following oral treatment with [14C]d-limonene reversible binding of d-limonene equivalents to renal cytosolic proteins was observed. Furthermore, with d-limonene treatment, hyaline droplets were observed in the transgenic mouse kidneys. These droplets, however, were much smaller in size than those seen in d-limonene-treated male rats. The accumulation of alpha 2u-globulin in the kidneys of transgenic mice and normal male rats before and after d-limonene treatment was analyzed by Western blotting. These results indicated that alpha 2u-globulin was present in the kidneys of the control transgenic mice, despite the lack of spontaneous hyaline droplet formation. After d-limonene treatment, approximately a three-fold increase in alpha 2u-globulin in the transgenic mouse kidney was observed, a response similar in magnitude to that seen in d-limonene-treated male rats. These results indicate that expression of alpha 2u-globulin in a species that does not normally develop hyaline droplet nephropathy is necessary and sufficient to render that species sensitive to this renal toxicity.

Alpha-Globulins↗
PubMed1992

Biochemical basis for mouse resistance to hyaline droplet nephropathy: lack of relevance of the alpha 2u-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.

Alpha-Globulins↗
PubMed1992

Alpha 2u-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.

Alpha-Globulins↗
PubMed1991

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.

Alpha-Globulins↗
PubMed1991

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 alpha 2u-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.

Administration, Oral↗
PubMed1991

Quantitation of urinary alpha 2u-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.

Albuminuria↗
PubMed1990

Lysosomal degradation of alpha 2u-globulin and alpha 2u-globulin-xenobiotic conjugates.

A diverse group of chemicals cause a male rat-specific nephrotoxicity in which alpha 2u-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 alpha 2u-globulin and that to which d-limonene, d-limonene-1,2-oxide, isophorone, 1,4-dichlorobenzene, and 2,5-dichlorophenol were bound was studied. alpha 2u-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 alpha 2u-globulin degradation to 28 +/- 8 and 17 +/- 5% of control, respectively, whereas addition of both inhibitors decreased alpha 2u-globulin degradation to 8 +/- 1% of control values. These results indicate that both classes of endopeptidases are important in the degradation of alpha 2u-globulin. Under the incubation conditions used, 30% of native alpha 2u-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 alpha 2u-globulin did not alter the degradation of the protein, whereas binding of d-limonene-1,2-oxide, 2.5-dichlorophenol, and isophorone decreased alpha 2u-globulin degradation by 33%. These results indicate that not all chemicals which have been shown to bind in vivo to alpha 2u-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 alpha 2u-globulin, suggesting that a decrease in lysosomal degradation is involved in the accumulation of this protein in male rat kidney lysosomes.

Alpha-Globulins↗