Susan J. Borghoff

The comparative pathobiology of α2u-globulin nephropathy☆

Abstract α2u-Globulin nephropathy is an important toxicologic syndrome that occurs in male rats following exposure to a number of important industrial and environmental chemicals. A low, but significant incidence of renal neoplasia develops in male rats as a chronic sequela to the disease. Studies on the pathogenesis of α2u-globulin nephropathy have demonstrated that this protein is produced in large amounts in the male rat, that reversible binding occurs between chemicals and/or their metabolites and α2u-globulin, and that this complex is resistant to proteolytic hydrolysis, leading to accumulation in renal lysosomes and subsequent cytotoxicity and cell death. This results in marked exposure-related increases in cell proliferation that persist for at least one year, providing exposure continues. This sustained increase in renal cell proliferation can promote initiated cells to form preneoplastic foci and renal neoplasia in male rats. Since this syndrome is highly species and sex specific, it is important to determine the relevance of these data for human risk assessment. The scientific considerations involved in high to low dose and species to species extrapolation are discussed.

Characteristics of chemical binding to α2u-globulin, in vitro-Evaluating structure-activity relationships

alpha 2u-Globulin (alpha 2u) has been shown to accumulate in the kidneys of male rats treated with 2,2,4-trimethylpentane (TMP). 2,4,4-Trimethyl-2-pentanol (TMP-2-OH), a metabolite of TMP, is found reversibly bound to alpha 2u isolated from the kidneys of these treated rats. The objectives of the following study were to characterize the ability of [3H]TMP-2-OH to bind to alpha 2u in vitro and to determine whether other compounds that cause this protein to accumulate have the same binding characteristics. Although compounds that have been shown to cause the accumulation of alpha 2u in male rat kidneys compete in vitro with [3H]TMP-2-OH for binding to alpha 2u, they do so to varying degrees. The binding affinity (Kd) of the [3H]TMP-2-OH-alpha 2u complex was calculated to be on the order of 10(-7) M. The inhibition constant values (Ki) determined for d-limonene, 1,4-dichlorobenzene, and 2,5-dichlorophenol were all in the range 10(-4) M, whereas the Ki values for isophorone, 2,4,4- or 2,2,4-trimethyl-1-pentanol, and d-limonene oxide were determined to be in the range 10(-6) and 10(-7) M, respectively. TMP and 2,4,4- and 2,2,4-trimethylpentanoic acid did not compete for binding. This suggests that other factors, besides binding, are involved in the accumulation of alpha 2u. In this study the ability of a chemical to bind to alpha 2u was used as a measure of biological activity to assess structure-activity relationships among the chemicals tested and known to cause the accumulation of alpha 2u. The results so far suggest that binding is dependent on both hydrophobic interactions and hydrogen bonding.

Kinetics of selected di-n-butyl phthalate metabolites and fetal testosterone following repeated and single administration in pregnant rats

Human exposure to phthalic acid diesters occurs through a variety of pathways as a result of their widespread use in consumer products and plastics. Repeated doses of di-n-butyl phthalate (DBP) from gestation day (GD) 12 to 19 disrupt testosterone synthesis and male sexual development in the fetal rat. Currently little is known about the disposition of DBP metabolites, such as monobutyl phthalate (MBP) and its glucuronide conjugate (MBP-G), during gestation after repeated exposure to DBP. In order to gain a better understanding of the effect of repeated dosing on maternal and fetal metabolism and distribution, pregnant Sprague-Dawley rats were given a single dose of 500 mg/kg DBP on GD 19 or daily doses of 50, 100, and 500 mg/(kg day) from GD 12 to 19 via corn oil gavage. Dose-response evaluation revealed a non-linear increase in maternal and fetal plasma concentrations of MBP. Maternal and fetal MBP levels were slightly lower in animals after 8 days of dosing at 500 mg/(kg day). Fetal plasma MBP levels closely followed maternal plasma, while the appearance and elimination of MBP-G in fetal plasma were significantly delayed. MBP-G accumulated over time in the amniotic fluid. Inhibition of testosterone was rapid in fetal testes when exposed to DBP (500 mg/(kg day)) on GD 19. Within 24h, the level of inhibition in the fetus was similar between animals exposed to a single or multiple daily doses of 500 mg/(kg day). Examination of testosterone time-course data indicates a rapid recovery to normal levels within 24h post-dosing at DBP doses of 50 and 100 mg/(kg day), with a rebound to higher than normal concentrations at later time-points. MBP kinetics in fetal testes allows direct comparison of active metabolite concentrations and testosterone response in the fetal testes.

The role of regenerative cell proliferation in chloroform- induced cancer

Chloroform produces cancer by a nongenotoxic-cytotoxic mode of action, with no increased cancer risk expected at noncytotoxic doses. The default risk assessment for inhaled chloroform relies on liver tumor incidence from a gavage study with female B6C3F1 mice and estimates a virtually safe dose (VSD) at an airborne concentration of 0.000008 ppm of chloroform. In contrast, a 1000-fold safety factor applied to the NOAEL for liver cytotoxicity from inhalation studies yields a VSD of 0.01 ppm. This estimate relies on inhalation data and is more consistent with the mode of action of chloroform.

A comparison of European High Test gasoline and PS-6 unleaded gasoline in their abilities to induce α2u-globulin nephropathy and renal cell proliferation

Male Fischer-344 rats were administered European High Test gasoline (EHT) (50-500 mg/kg), PS-6 unleaded gasoline (UG) (16-500 mg/kg) or 2,2,4-trimethylpentane (TMP) (0.95-30 mg/kg) by gavage for ten consecutive days. To measure cell replication, rats were exposed to [3H]thymidine continuously over the last 7 days of the exposure period. Twenty-four hours after the final dose, protein droplet (PD) accumulation, alpha 2u-globulin (alpha 2u) concentration and the nuclear labeling index (LI), as a measure of cell replication, were measured in the kidneys of control and treated rats. Dose-related increases in PD, alpha 2u and cell replication were detected in the kidneys of rats treated with either gasoline mixture or TMP. The accumulation of PD and the increase in alpha 2u was greater in the kidneys of UG- and TMP-treated rats than in the kidneys of rats treated with EHT. These differences were attributed to the higher composition of branched hydrocarbons in UG, which have been shown to be the biologically active components for these endpoints. The extent of renal cell proliferation was similar in both EHT-, UG- and TMP-treated rats. This suggests that other components besides the branched hydrocarbons are responsible for the increased renal cell replication in EHT-treated rats.

Pharmacokinetics of tertiary butyl alcohol in male and female Fischer 344 rats

Tertiary butyl alcohol (TBA) is a small aliphatic alcohol with multiple industrial and scientific uses. A comprehensive pharmacokinetic profile for TBA has not been determined in rats. The purpose of this study was to fully characterize the pharmacokinetics of TBA in male and female F-344 rats following intravenous administration of 37.5, 75, 150 and 300 mg/kg TBA. TBA was observed to undergo a rapid distribution phase followed by a slower elimination phase. The steady-state volume of distribution for TBA was roughly 4.5 times greater than total body water, and the clearance was lower than the estimated glomerular filtration rate. The elimination of TBA appears to saturate at higher doses, as evidenced by a disproportional increase in area under the concentration-time curve and decreased rate of clearance.

Physiologically Based Pharmacokinetic Model of Methyl tertiary Butyl Ether and tertiary Butyl Alcohol Dosimetry in Male Rats Based on Binding to α2u-Globulin

Current physiologically based pharmacokinetic (PBPK) models for the fuel additive methyl tertiary butyl ether (MTBE) and its metabolite tertiary butyl alcohol (TBA) have not included a mechanism for chemical binding to the male rat-specific protein alpha2u-globulin, which has been postulated to be responsible for renal effects in male rats observed in toxicity and carcinogenicity studies with MTBE. The objective of this work was to expand the previously published models for MTBE to include binding to alpha2u-globulin in the kidney of male rats. In the model, metabolism of MTBE was assumed to occur only in the liver via two saturable pathways. TBA metabolism was assumed to occur only in the liver via one saturable, low-affinity pathway and to be inducible following repeated exposures. The binding of MTBE and TBA to alpha2u-globulin was modeled as saturable and competitive and was assumed to only affect the rate of hydrolysis of alpha2u-globulin in the kidney. The developed model characterized the differences in kidney concentrations of MTBE and TBA in male versus female rats from inhalation exposures to MTBE, as well as the observed changes in blood and tissue concentrations from repeated exposure to TBA. The model-predicted binding affinity of MTBE to alpha2u-globulin was greater than TBA, and the hydrolysis rate of chemically bound alpha2u-globulin was approximately 30% of the unbound protein. This PBPK model supports the role of MTBE and TBA binding to the male rat-specific protein alpha2u-globulin as essential for predicting concentrations of these chemicals in the kidney following exposure.

Methyl isobutyl ketone (MIBK) induction of α2u-globulin nephropathy in male, but not female rats

Male F-344 rats were administered corn oil (vehicle control), d-limonene (positive control, 300mg/kg), or MIBK (1000mg/kg) and female F-344 rats corn oil (vehicle control) or MIBK for 10 consecutive days by oral gavage. Approximately 24h after the final dose the kidneys were excised and the left kidney prepared and evaluated for histological changes including protein (hyaline) droplet accumulation, immunohistochemical staining for alpha2u-globulin (alpha2u), and proliferating cell nuclear antigen (PCNA) to quantitate renal cell proliferation. The right kidney was prepared for quantitation of total protein and alpha2u using an ELISA. MIBK elicited an increase in protein droplets, accumulation of alpha2u, and renal cell proliferation in male, but not female rats, responses characteristic of alpha2u-mediated nephropathy. MIBK produced identical histopathological changes in the male rat kidney when compared to d-limonene, an acknowledged inducer of alpha2u-nephropathy except that the grade of severity tended to be slightly lower with MIBK. MIBK did not induce any effects in female rats. Therefore, renal histopathology, along with the other measures of alpha2u accumulation, provides additional weight of evidence to support the inclusion of MIBK in the category of chemicals exerting renal effects through a alpha2u-nephropathy-mediated mode-of-action.

Development of a mechanism-based dosimetry model for 2,4,4-trimethyl-2-pentanol-induced α2u-globulin nephropathy in male Fischer 344 rats

A mechanism-based dosimetry model was developed to describe 2,4,4-trimethyl-2-pentanol (TMP-2-OH) dosimetry and renal alpha 2u-globulin (alpha 2u) nephropathy in the male Fischer 344 rat. Experimental data were collected to estimate the chemical-specific parameters (metabolic constants, tissue solubility, and oral absorption rate) necessary to describe TMP-2-OH dosimetry in male rats. The concentrations of alpha 2u and TMP-2-OH were measured in male rats up to 64 hr after a single oral dose of TMP-2-OH (6, 60, or 600 mg/kg). The model predicted the time course behavior of TMP-2-OH and alpha 2u in the kidney, but overestimated their renal concentrations by two or threefold. Simulations of renal alpha 2u concentration were sensitive to changes in TMP-2-OH-alpha 2u-binding affinity and degradation rate of the TMP-2-OH-protein complex. In contrast, simulation of the concentration of TMP-2-OH in the kidney was most sensitive to the amount of protein present. Oral absorption of TMP-2-OH was dose dependent. The model predicted that alpha 2u and TMP-2-OH concentration in the kidney is sensitive to changes in the rate of TMP-2-OH absorbed after oral administration. This model permitted a more rigorous evaluation than has previously been possible of the combination of protein characteristics and chemical dosimetry required for the accumulation of alpha 2u in the kidney of male rats. The behavior of the model is consistent with the qualitative aspects of the alpha 2u hypothesis. However, further characterization of alpha 2u distribution and renal hydrolysis will be required in order to fully characterize the hypothesis at the quantitative level.

14C-labeled Pulegone and Metabolites Binding to α2u-globulin in Kidneys of Male F-344 Rats∗

Pulegone is a major constituent of pennyroyal oil and a minor component of peppermint oil. Pulegone is biotransformed to menthofuran and menthones (diastereomeric menthone and isomenthone) in pennyroyal and peppermint as well as in rodents. Pulegone and menthofuran are hepatotoxic to rodents, and menthones are less toxic. The metabolism and disposition of pulegone and menthofuran were previously studied in rodents, and higher concentrations of pulegone- and menthofuran-derived radioactivity were observed in male than female rat kidney. One explanation is the association of pulegone and metabolites with a male rat-specific protein, α2u-globulin. To test this hypothesis, male and female rats were dosed orally with 14C-labeled pulegone (80 mg/kg, 120 μCi/kg) or menthofuran (60 mg/kg, 120 μCi/kg) or menthones (80 mg/kg, 120 μCi/kg) in corn oil, and the kidney cytosol was prepared 24 h after dosing. An equilibrium dialysis experiment showed that in all three studies the radioactivity was associated with kidney cytosol proteins of male but not female rats. The chemicals present in the male rat kidney cytosol after dialysis were extracted with dichloromethane and characterized by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC-MS). All parent compounds were detected, and the metabolites characterized included piperitone from pulegone or menthones treatment, menthones and possibly 8-hydroxymenthones from pulegone treatment, and mintlactones (diastereomeric mintlactone and isomintlactone) and 7a-hydroxymintlactone from menthofuran treatment. Analysis of the male rat kidney cytosol by a gel filtration column demonstrated that the retention was due to reversible binding of these chemicals with the male rat-specific protein α2u-globulin. However, binding of pulegone and/or metabolites to α2u-globulin did not produce accumulation of this protein in the kidney.