Rat-specific Protein Research Articles

Assessing modes of action, measures of tissue dose and human relevance of rodent toxicity endpoints with octamethylcyclotetrasiloxane (D4)

Octamethylcyclotetrasiloxane (D4), a highly lipophilic, volatile compound with low water solubility, is metabolized to lower molecular weight, linear silanols. Toxicity has been documented in several tissues in animals following mixed vapor/aerosol exposures by inhalation at near saturating vapor concentrations or with gavage dosing in vegetable oil vehicles. These results, together with more mechanism-based studies and detailed pharmacokinetic information, were used to assess likely modes of action (MOAs) and the tissue dose measures of D4 and metabolites that would serve as key events leading to these biological responses. This MOA analysis indicates that pulmonary effects arise from direct epithelial contact with mixed vapor/aerosol atmospheres of D4; liver hypertrophy and hepatocyte proliferation arise from adaptive, rodent-specific actions of D4 with nuclear receptor signaling pathways; and, nephropathy results from a combination of chronic progresive nephropathy and silanol metabolites binding with alpha-2u globulin (a male rat specific protein). At this time, the MOAs of other liver effects – pigment accumulation and bile duct hyperplasia (BDH) preferentially observed in Sprague-Dawley (SD) rats- are not known. Hypothalamic actions of D4 delaying the rat mid-cycle gonadotrophin releasing hormone (GnRH) surge that result in reproductive effects and subsequent vaginal/uterine/ovarian tissue responses, including small increases in incidence of benign endometrial adenomas, are associated with prolongation of endogenous estrogen exposures due to delays in ovulation. Human reproduction is not controlled by a mid-cycle GnRH surge. Since the rodent-specific reproductive and the vaginal/uterine/ovarian tissue responses are not relevant for risk assessments in human populations, D4 should neither be classified as a CMR (i.e., carcinogenic, mutagenic, or toxic for reproduction) substance nor be regarded as an endocrine disruptor. Bile duct hyperplasia (BDH) and pigment accumulation in liver seen in SD rats are endpoints that could serve to define a Benchmark Dose (BMD) or No-Observed-Effect-Level (NOEL) for D4 although their human relevance remains uncertain.

Two types of deposits, hyaline droplets and eosinophilic bodies, associated with α2u-globulin accumulation in the rat kidney.

Alpha2u-globulin is an adult male rat-specific protein that accumulates spontaneously or inductively in the renal proximal tubular epithelium and forms microscopically observable deposits, which are generally referred to as “hyaline droplets,” whereas a specific type of deposits is referred to as “eosinophilic bodies” by Japanese toxicologic pathologists. We compared hyaline droplets and eosinophilic bodies using special stains including immunostaining for α2u-globulin and lysosome-associated membrane protein in spontaneously occurring and d-limonene-induced cases. Eosinophilic bodies appeared simultaneously and increased in parallel with the hyaline droplets in the induced case. In both of the spontaneous and induced cases, hyaline droplets and eosinophilic bodies were associated with α2u-globulin and lysosomes, although there were differences in the forms and staining properties that probably reflected the purity or density of α2u-globulin. According to the results, it is not necessary for eosinophilic bodies to be strictly distinguished from hyaline droplets, and it is reasonable to identify eosinophilic bodies as hyaline droplets in α2u-globulin nephropathy in routine toxicity studies, as they have been recognized to be a sequence of changes associated with accumulation of α2u-globulin.

Response to ‘Dr Raymond David, BASF Corporation’

We thank Dr Raymond David from BASF for his letter and are pleased to address his concerns. Regarding Dr David's first point about the irrelevance of the kidney toxicity to humans due to the accumulation of the rat-specific protein alpha-2-u-microglobulin, please note that the EU SCENIHR report that was cited in our article, as well as the 2008 report, refers to alpha-2-macroglobulin (page 74 in the 2007 report; page 44 in the Table 12 footnote to which Dr David refers, and page 81 in the 2008 report), and unlike alpha-2-u-microglobulin, alpha-2-macroglobulin is a human protease inhibitor.1, 2 It is possible that this was a mistake by EU SCENIHR. However, it should be noted that the available literature regarding the health effects of DINCH was limited to summaries by BASF and the review in the EU SCENIHR report of unreferenced and unpublished animal studies submitted by BASF.1, 2 The publication of toxicological studies would enable independent evaluation of study results and prevent misinterpretation.

Association of Botulinum Neurotoxin Serotype A Light Chain with Plasma Membrane-bound SNAP-25

The Clostridium botulinum neurotoxins (BoNTs) cleave SNARE proteins, which inhibit binding and thus fusion of neurotransmitter vesicles to the plasma membrane of peripheral neurons. BoNTs comprise an N-terminal light chain (LC) and C-terminal heavy chain, which are linked by a disulfide bond. There are seven serotypes (A-G) of BoNTs based upon immunological neutralization. Although the binding and entry of BoNT/A into neurons has been subjected to considerable investigation, the intracellular events that allow BoNT/A to efficiently cleave SNAP-25 within neurons is less well understood. Earlier studies showed that intracellular LC/A bound to the plasma membrane of neurons. In this study, intracellular LC/A is shown to directly bind SNAP-25 on the plasma membrane. Solid phase binding showed that the N-terminal residues of LC/A bound residues 80-110 of SNAP-25, which was also observed in cultured neurons. Association of the N-terminal 8 amino acids of LC/A and residues 80-110 of SNAP-25 also enhanced substrate cleavage. These findings explain how LC/A associates with SNAP-25 on the plasma membrane and provide a basis for LC/A cleavage of SNAP-25 within the SNARE complex.

Physiologically based pharmacokinetic rat model for methyl tertiary-butyl ether; comparison of selected dose metrics following various MTBE exposure scenarios used for toxicity and carcinogenicity evaluation

There are a number of cancer and toxicity studies that have been carried out to assess hazard from methyl tertiary-butyl ether (MTBE) exposure via inhalation and oral administration. MTBE has been detected in surface as well as ground water supplies which emphasized the need to assess the risk from exposure via drinking water contamination. This model can now be used to evaluate route-to-route extrapolation issues concerning MTBE exposures but also as a means of comparing potential dose metrics that may provide insight to differences in biological responses observed in rats following different routes of MTBE exposure. Recently an updated rat physiologically based pharmacokinetic (PBPK) model was published that relied on a description of MTBE and its metabolite tertiary-butyl alcohol (TBA) binding to α2u-globulin, a male rat-specific protein. This model was used to predict concentrations of MTBE and TBA in the kidney, a target tissue in the male rat. The objective of this study was to use this model to evaluate the dosimetry of MTBE and TBA in rats following different exposure scenarios, used to evaluate the toxicity and carcinogenicity of MTBE, and compare various dose metrics under these different conditions. Model simulations suggested that although inhalation and drinking water exposures show a similar pattern of MTBE and TBA exposure in the blood and kidney (i.e. concentration–time profiles), the total blood and kidney levels following exposure of MTBE to 7.5 mg/ml MTBE in the drinking water for 90 days is in the same range as administration of an oral dose of 1000 mg/kg MTBE. Evaluation of the dose metrics also supports that a high oral bolus dose (i.e. 1000 mg/kg MTBE) results in a greater percentage of the dose exhaled as MTBE with a lower percent metabolized to TBA as compared to dose of MTBE that is delivered over a longer period of time as in the case of drinking water.

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.

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.

Cloning of a rat-specific long PCP4/PEP19 isoform

We report the identification of a cDNA that encodes a putative protein of 94 amino acids and expected molecular weight of 10.7 kDa, the C-terminal half of which is identical to that of PEP19, a small, brain-specific protein involved in Ca++/calmodulin signaling. The novel rat-specific protein, tentatively named long PEP19 isoform (LPI), is the product of alternative splicing of the rat PCP4 gene encoding PEP19. We found that antibodies raised against the first 13 N-terminal amino acids of LPI, not present in PEP19, recognize a protein enriched in the developing rat brain.

Changes in serum α2u‐globulin levels in castrated male rats treated with testosterone propionate in a Hershberger assay

The Hershberger assay has been proposed as a candidate screening test method for the detection of androgenic and anti-androgenic chemicals and is being validated presently under the test guideline programme of the Organization for Economic Cooperation and Development (OECD). Rat alpha2u-globulin is male rat-specific protein appearing in their serum and urine, and the protein is known to be induced by androgens. We investigated the usefulness of measuring serum alpha2u-globulin levels as a parameter for the androgenic activity of chemicals tested in the Hershberger assay. The serum alpha2u-globulin level was measured after the administration of testosterone propionate at dosages of 0, 20, 100 or 500 microg kg(-1) day(-1) for ten consecutive days in the castrated male rats. The ventral prostate, balbocavernosus/levator ani muscles, glans penis and Cowper's gland were collected and weighed. Although all the androgen-responsive organ weights were increased significantly at dosages of 100 and 500 microg kg(-1) day(-1), the serum alpha2u-globulin level was increased significantly only at a dosage of 500 microg kg(-1) day(-1). These results show that the serum alpha2u-globulin level may be a useful biomarker for detecting androgenic activity caused by test chemicals, but it is less sensitive than the organ weights of androgen-responsive tissues in the Hershberger assay.

Binding of Perfluorooctanoic Acid to Rat Liver‐form and Kidney‐form α2u‐Globulins

Perfluorooctanoic acid (PFOA) is an organic fluorochemical and is reported to have a long half‐life in human blood. Its urinary elimination in rats is markedly sex‐dependent, and characterized by significantly longer plasma half‐life of PFOA in male rats than in females. It has been postulated that male‐specific PFOA binding protein(s) is responsible for the long half‐life of PFOA in male rats. In this paper, two male rat specific proteins, liver‐ and kidney‐form α2u‐globulins (A2UL and A2UK), were purified from male rat urine and kidney, respectively. The binding of these two proteins to PFOA was investigated using ligand blotting, electrospray ionization mass spectrometry and fluorescence competitive binding assay. The results revealed that both A2UL and A2UK were able to bind PFOA in vitro under physiological conditions, and that PFOA and a fluorescent‐labeled fatty acid shared the same binding site on both A2UL and A2UK. The binding affinities, however, are relatively weak. The estimated dissociation constants are in the 10− 3 M range, indicating that bindings of PFOA to either A2UL or A2UK cannot adequately explain the sex‐dependent elimination of PFOA in rats, and it is unlikely that PFOA‐A2Uk binding would induce A2U nephropathy as seen with, for example, 1,4‐dichlorobenzene.

The role of α2u-globulin in ochratoxin A induced renal toxicity and tumors in F344 rats

The mycotoxin ochratoxin A (OTA) was shown to be a potent kidney carcinogen in rats demonstrating a marked sex difference in the response. Compared to female rats, male rats had a 10-fold higher incidence of kidney carcinomas. The objective of this study was to investigate whether this sex difference in tumor response is due to an exacerbation of effect resulting from the interaction of the male rat specific urinary protein α2u-globulin ( α2u) with OTA. Male and female rats were treated by oral gavage with OTA (1 mg/kg per day), d-limonene (dL; 1650 mg/kg per day) as a positive control or corn oil for 7 consecutive days. OTA induced severe renal lesions predominantly in the P 3 region of the proximal tubules. The lesions consisted of necrotic cells and cell exfoliations. No hyaline droplets were found in the P 2 segment following OTA treatment, whereas dL induced the expected accumulation of droplets. The results suggest that OTA induced kidney lesions are in all characteristic points different from the known α2u-nephropathy induced by dL. Based on these experiments the male rat specific protein α2u does not seem to be involved in the mechanism(s) leading to the high tumor incidence observed in OTA exposed male rats.

Subchronic Toxicity (90-Day) Study withpara-Nonylphenol in Rats

As a component to the risk assessment process forpara-nonylphenol (NP; CASRN 84852-15-3), a 90-day study was conducted in rats following U.S. EPA TSCA guidelines and Good Laboratory Practice regulations. NP was administered to four groups of rats at dietary concentrations of 0, 200, 650, or 2000 ppm which corresponded to approximate dietary intakes of 0, 15, 50, or 150 mg/kg/day, respectively. There were 25 rats/sex/group in the control and high-dose groups and 15 rats/sex/group in the low- and middose groups. Ten of the 25 rats/sex in the control and high-dose groups were designated as recovery animals and were maintained on control diets for 4 weeks after completion of the 90-day exposure period to assess the reversibility of any effects which might be observed. To evaluate for the possible weak estrogen-like activity that has been reported for NP in a number of screening assays, estrous cyclicity was monitored using vaginal cytology during Week 8 of the study, and sperm count, motility, and morphology were evaluated at termination. In-life effects from NP exposure were limited to small decreases in body weight and food consumption in the 2000-ppm dose group. Postmortem measurements at Week 14 indicated a dose-related kidney weight increase in males and a decrease in renal hyaline globules/droplets in males from the high-dose group. The kidney weights showed complete recovery following the 4-week postdosing recovery period. Due to the small magnitude of the changes (i.e., all weights were within or near laboratory historical control values) and the lack of correlating clinical or histopathological changes, the kidney weight alterations were not considered toxicologically significant. The biological significance of reduced hyaline in the kidneys of male rats from the high-dose group is uncertain. Renal tubular hyaline is associated with the rat-specific protein, α-2u-globulin, and, therefore, this finding was not considered toxicologically relevant to humans. No other effects attributable to NP were observed. No changes were observed for estrous cycling, sperm evaluations, or effects on endocrine organs. NP, therefore, did not manifest any estrogen-like activity as measured in these parameters at dietary concentrations as high as 2000 ppm, the maximum dose administered in this study. Based on the minor findings for the 2000-ppm dose group, the NOAEL (no-observed-adverse-effect level) for NP in this study is considered to be 650 ppm in the diet, corresponding to an approximate intake of 50 mg/kg/day.

In VitroUptake of Methyltert-Butyl Ether in Male Rat Kidney: Use of a Two-Compartment Model to Describe Protein Interactions

Methyltert-butyl ether (MTBE) is a gasoline additive that causes renal tumors in male rats. In the process of measuring chemical specific parameters necessary to develop a quantitative dosimetry model of MTBE in rats, the uptake of MTBE was found to be 5.5 times greater in male than in female F-344 rat kidney homogenate. The objectives of this study were to characterize the factor(s) that influences the high uptake of MTBE into male rat kidneyin vitroand to develop a system to evaluate the interaction of MTBE with the male rat-specific protein, α2u-globulin (α2u). The uptake of MTBE in male, but not female, rat kidney homogenate was found to be dependent on protein and chemical concentrations. When [14C]MTBE was incubated with male rat kidney homogenate, radioactivity coeluted with the total protein fraction on a gel filtration column. An interaction between [14C]MTBE and male rat kidney proteins was not found under conditions of dialysis or anion exchange chromatography. A two-compartment vial equilibration model was used to assess the interaction between MTBE and α2u. Using this system, the dissociation constant for MTBE and α2u was estimated to be 2.15 × 10−4m, which is in the range of other chemicals known to bind to α2u and cause α2u-mediated nephropathy.d-Limonene oxide was used to validate this two-compartment vial equilibration system. These findings illustrate a technique useful in estimating the dissociation constant for a volatile chemical and a protein, as well as explain the process that contributes to the uptake of MTBE into male rat kidney homogenatein vitro.A description of the weak interaction between MTBE and α2u will be used to refine a physiologically based pharmacokinetic model to describe the target tissue (kidney) concentrations of MTBE.

Review of the carcinogenic potential of gasoline.

This review examines the animal, human, and mechanistic studies that precede the new studies reported in this volume. Wholly vaporized unleaded gasoline was found to produce a dose-dependent increase in renal carcinoma in male rats and an excess above background incidence of hepatocellular tumors in female mice in the high-dose group. Mechanistic studies suggest that gasoline is not mutagenic and that the probable mechanism for the male rat renal tumors involves a rat-specific protein, alpha 2u-globulin, whose binding with highly branched aliphatic compounds results in renal tubule cell death and, in turn, a proliferative sequence that increases renal tubule tumors. Human evidence generated predominantly from studies of refinery workers does not support a kidney or liver cancer risk in humans. The current epidemiologic database is inadequate to access leukemia risk from low-level benzene exposure from gasoline. Studies of gasoline-exposed workers that incorporate quantitative exposure information are needed.

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 α2u-globulin and caused the protein to accumulate in male rat kidney cortex. Two-dimensional gel electrophoretic separation of male rat kidney proteins indicated that α2u-globulin levels in kidney increased 24 hr after a single oral dose of THBS (500 mg/kg). Furthermore, a sex-dependent retention of THBS was noted as there was approximately 10 times more THBS equivalent 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 α2u-globulin was determined by anion-exchange HPLC after which metabolites in the α2u-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 ggg-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 siggificant homology with α2u-globulin. These results indicate that THBS treatment exacerbates hyaline droplet formation in male rat kidneys by binding to α2u-globulin, thereby causing the protein to accumulate in the renal cortex. The interaction between THBS and α2u-globulin appears to be unique to this male rat-specific protein as THBS does not interact with a very similar protein found in mice.

Design of a gas chromatograph with parallel radioactivity and mass spectrometric detection

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.

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 ratspecific 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, α 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 α 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 α 2u-globulin by amino acid sequencing. The major metabolite associated with α 2u-globulin was d-limonene-1,2-oxide. Parent d-limonene was also identified as a minor component in the α 2u-globulin fraction. Thus, d-limonene, and more specifically d-limonene-1,2-oxide, associates with α 2u-globulin in a reversible manner in male rat kidney. This interaction may be responsible for excessive accumulation of α 2u-globulin in kidneys of male rats exposed to d-limonene.

Rapid postexposure decay of α2u‐globulin and hyaline droplets in the kidneys of gasoline‐treated male rats

Unleaded gasoline induces nephropathy, characterized by rapid accumulation of hyaline (protein resorption) droplets in epithelial cells of the renal proximal convoluted tubules, only in male rats. The hepatic synthesis of the male rat-specific protein alpha 2u-globulin, a constituent of renal hyaline droplets, is unaltered by gasoline treatment (Olson et al., 1987). Renal alpha 2u-globulin content increased to 210% of control within 18 h of a single oral dose of gasoline (2.0 ml/kg); maximal levels (320% of control) were attained following gasoline administration for 3 d. Increases in renal alpha 2u-globulin caused by gasoline were accompanied by concurrent proliferation of hyaline droplets. However, within 3 d of terminating gasoline administration renal alpha 2u-globulin content decreased to the same level as that in unexposed rats, although renal hyaline droplet number returned to pretreatment levels somewhat more slowly. The conjoint effect of postexposure recovery and estradiol (an inhibitor of hepatic alpha 2u-globulin synthesis) administration was also determined in male rats. On postexposure d 3, 6, and 9, estradiol treatment (1 mg/kg, sc, 4 d, starting on d 9 of gasoline treatment) decreased renal alpha 2u-globulin content to 75%, 59%, and 48%, respectively, of that in rats allowed to recover from gasoline with no hormone treatment. Hepatic alpha 2u-globulin content in estradiol-treated rats was decreased by 74%, 97%, and 96% at the same intervals. Estradiol treatment during recovery from gasoline also appeared to increase the removal of accumulated hyaline droplets from the renal cortex. Thus, accumulation of alpha 2u-globulin-containing hyaline droplets after subacute exposure of male rats to gasoline is rapidly reversible, dependent on continuous exposure to gasoline and maintenance of the normal rate of hepatic alpha 2u-globulin synthesis. These results emphasize the dynamic state of renal cortical hyaline droplets and suggest strongly that gasoline hydrocarbons cause hyaline droplet accumulation by prolonging the half-time of degradation of alpha 2u-globulin.

Identification of urinary metabolites in rats exposed to the nephrotoxic agent 2,3,4-trimethylpentane.

Branched-chain hydrocarbon-induced nephropathy in male rats was examined using 2,3,4-trimethylpentane. Lesions are elected only in male rats, not in females or in controls. Mechanisms of nephropathy may be the interaction of metabolites with the male rat-specific protein alpha 2u globulin. The identified urinary metabolites of 2,3,4-trimethylpentane in male rats given the hydrocarbon by gavage are 2,3,4-trimethyl-1-pentanol, 2,3,4-trimethyl-2-pentanol and 2,3,4-trimethyl-1-pentanoic acid. Of the C8-isomers, 2,3,4-trimethylpentane dosing leads to the highest incidence of kidney damage in male rats.

Carbohydrate composition of specific rat epididymal protein.

Previous results demonstrated that androgen-dependent rat specific epididymal proteins (SEP) were bound to spermatozoa during their maturation in the epididymis. This paper describes the purification of glycoprotein DE, which constitutes 40% of SEP, and the identification and semiquantitative determination of the sugars forming its oligosaccharide chain. Affinity chromatography on Sepharose-Concanavalin A produced a sample of D-E 95% pure in which 10.5 g of sugar were present per 100 g protein. The percentual composition of the oligosaccharide was D-mannose 19%; D-galactose 3%; N-acetyl-D-glucosamine 33%; N-acetyl-neuraminic acid 31% and D-glucose 13%.