James A. Swenberg

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.

Elevated proliferation of proximal tubule cells and localization of accumulated α2u-globulin in F344 rats during chronic exposure to unleaded gasoline or 2,2,4-trimethylpentane

In order to better characterize the pathogenesis of alpha 2u-globulin (alpha 2uG) nephropathy, cell proliferation was quantitated within the three proximal tubule segments of the kidney (P1, P2, and P3) and proximal tubule segments affected by chronic progressive nephrosis (CPN) in male and female F344 rats exposed to 10, 70, or 300 ppm unleaded gasoline (UG) or 50 ppm 2,2,4-trimethylpentane (TMP) from 3 to 50 weeks. The P2 segment of male rats exposed to UG or TMP responded with dose-related increases in cell turnover (up to 11-fold) that persisted during chronic exposure. This proliferative response closely paralleled the extent and severity of immunohistochemically detectable alpha 2uG in the P2 segment. Neither alpha 2uG nor cytotoxicity was evident in cells of the P1 or P3 segment; however, cell proliferation was increased (up to 8-fold) for up to 22 weeks of exposure in the P3 segment. Increased numbers of proximal tubules affected by CPN were found in males exposed to UG or TMP for 22 or 48 weeks, compared to controls. These lesions contained epithelial cells that were highly proliferative. Control or treated female rats exhibited neither alpha 2uG nephropathy nor increases in P2 or P3 cell turnover, and the extent of CPN was greatly reduced as compared to male rats. The results of this and related studies suggest that chronic cell proliferation associated with alpha 2uG nephropathy and CPN in male rats exposed to UG or isoparaffinic components of UG, such as TMP, may be responsible for the sex- and species-specific nephrocarcinogenic effects of UG.

2,2,4-Trimethylpentane-induced nephrotoxicity: II. The reversible binding of a TMP metabolite to a renal protein fraction containing α2u-globulin

Trimethylpentane (TMP) produces nephrotoxicity in male but not in female rats. The toxicity is characterized by an increase in protein droplets in proximal convoluted tubular cells and an increase in the renal concentration of the male-rat-specific protein alpha 2u-globulin. Subcellular fractionation of the kidneys from male rats 24 hr after [3H]TMP administration showed that about 60% of the radiolabeled material was localized in the 116,000g supernatant. Column chromatography of this supernatant resolved the radioactivity into two components; one, which contained about 26% of the radiolabel, coeluted with alpha 2u-globulin and cross-reacted with an antibody specific for alpha 2u-globulin. The remaining component eluted in the low-molecular-weight range (less than 1000 Da) and was assumed to be TMP metabolites. Radiolabel from [3H]TMP in male rat urine also resolved into two components with about 0.1% of the radiolabel in urine coeluting with the alpha 2u-globulin-containing fraction. Radiolabel from TMP in male rat liver 116,000g supernatant and plasma and in female rat kidney 116,000g supernatant eluted as a single component in the low-molecular-weight range. Dialysis (1000-Da cutoff) of male kidney 116,000g supernatant led to a loss of the low-molecular-weight components, but nondialyzable radiolabel (about 20%) still coeluted with the alpha 2u-globulin after gel chromatography. Dialysis against 0.1% sodium dodecyl sulfate led to a loss of both the low- and high-molecular-weight radioactive material. These results suggested that the high-molecular-weight radioactive material was formed by the reversible binding of a radioactive component of TMP to a male-rat-specific protein. Gas chromatography-mass spectrometry of an ethyl acetate extract of the alpha 2u-globulin-containing fractions of TMP-treated male rat kidney 116,000g supernatant identified 2,4,4-trimethyl-2-pentanol as the only bound metabolite to alpha 2u-globulin. These studies provide the first evidence for a reversible binding between a metabolite of TMP and a male-rat-specific protein in the kidney and thus provide important insight delineating a potential mechanism of hydrocarbon-induced hyaline-droplet nephropathy.

Involvement of reversible binding to α2u-globulin in 1,4-dichlorobenzene-induced nephrotoxicity

Abstract Similarly to unleaded gasoline, 1,4-dichlorobenzene (1,4-DCB) administered for 2 years caused a dose-related increase in the incidence of renal tumors in male but not in female rats or in either sex of mice. Unleaded gasoline and 2,2,4-trimethylpentane (TMP), a component of unleaded gasoline, increased protein droplet formation and cell proliferation in male but not in female rat kidneys. These protein droplets contained, α 2u -globulin, a male rat-specific low-molecular-weight protein and 2,4,4-trimethyl-2-pentanol, a metabolite of TMP that was reversibly bound to this protein. Studies were undertaken to determine if 1,4-DCB produced similar effects; 1,2-DCB was used for comparison since it did not produce renal carcinogenesis in male rats. Gel filtration chromatography of a 116,000 g supernatant prepared from kidneys of 1,4-[ 14 C]DCB-treated rats showed that radiolabel coeluted with α 2u -globulin as one sharp peak as opposed to a multipeak pattern observed for 1,2-[ 14 C]DCB; the maximal quantity of radiolabel for 1,4-DCB was twice that for 1,2-DCB. Equilibrium dialysis of kidney cytosol in the presence or absence of sodium dodecyl sulfate demonstrated that the radiolabel was reversibly bound to α 2u -globulin; the amount for 1,4-[ 14 C]DCB-treated rats was almost twice as much as that for 1,2-[ 14 C]DCB-treated rats. 1,2-DCB was also shown to be covalently bound to renal α 2u -globulin, and covalently bound to liver and plasma high-molecular-weight proteins. 1,4-DCB and, to a minor extent, 2,5-dichlorophenol, the major metabolite of 1,4-DCB, were reversibly bound to renal α 2u -globulin from 1,4-DCB-treated rats. 1,4-DCB increased protein droplet formation in male but not in female rat kidneys, whereas equimolar doses of 1,2-DCB showed no effect in either sex. Renal cell proliferation, measured by [ 3 H]thymidine incorporation into renal DNA, was increased after 1,4-DCB but not after 1,2-DCB treatment. Nephrotoxicity and biochemical alterations induced by 1,4-DCB resemble those of unleaded gasoline and suggest that a similar mechanism is involved in the induction of α 2u -globulin nephropathy in male rats.

Histopathology and Cell Proliferation Induced by 2,2,4-Trimethylpentane in the Male Rat Kidney

Unleaded gasoline causes acute and chronic nephrotoxicity and renal tumors in male rats, but not female rats or mice of either sex. An active nephrotoxic component of unleaded gasoline has been identified as 2,2,4-trimethylpentane (TMP). The first objective of this study was to characterize light microscopic renal lesions induced in male F344 rats by a 21-day gavage regimen of 50 to 500 mg/kg TMP. The second objective was to localize and quantitate sites of renal cell proliferation induced by the same TMP dose regimens using histoautoradiographic analysis after [3H]thymidine incorporation. Light microscopic lesions in the proximal convoluted tubule consisted of protein droplet and crystalloid body accumulation, degeneration, and necrosis, and were similar to lesions noted in previous inhalation and gavage studies with other hydrocarbon compounds. The above renal lesions were not dose-related, although tubular dilation of thin limb segments with granular cell debris was dose-related. In cell proliferation studies TMP induced a non-dose-related five- to sixfold increase in the labelling index of the same proximal convoluted tubule portions (P2 segment) that contained severe crystalloid body accumulation, degeneration, and necrosis. Less pronounced, but statistically significant (p ≤ 0.05), increases in cell proliferation were also observed in other nephron segments, indicating a generalized regenerative response of the kidney to TMP. The cytotoxic and regenerative renal effects of TMP administered by gavage suggest that similar mechanisms may be involved in the induction of kidney tumors in male rats following chronic inhalation exposure to unleaded gasoline.

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.

Macromolecular adducts of ethylene oxide: a literature review and a time-course study on the formation of 7-(2-hydroxyethyl) guanine following exposures of rats by inhalation

The results of efforts to identify and quantify macromolecular adducts of ethylene oxide (ETO), to determine the source and significance of background levels of these adducts, and to generate molecular dosimetry data on these adducts are reviewed. A time-course study was conducted to investigate the formation and persistence of 7-(2-hydroxyethyl)guanine (7-HEG; Fig. 1) in various tissues of rats exposed to ETO by inhalation, providing information necessary for designing investigations on the molecular dosimetry of adducts of ETO. Male F344 rats were exposed 6 h/day for up to 4 weeks (5 days/wk) to 300 ppm ETO by inhalation. Another set of rats was exposed for 4 weeks to 300 ppm ETO, and then killed 1-10 days after cessation of exposures. DNA samples from control and treated rats were analyzed for 7-HEG using neutral thermal hydrolysis, HPLC separation, and fluorescence detection. The adduct was detectable in all tissues of treated rats following 1 day of ETO exposure and increased approximately linearly for 3-5 days before the rate of increase began to level off. Concentrations of 7-HEG were greatest in brain, but the extent of formation was similar in all tissues studied. The adduct disappeared slowly from DNA, with an apparent half-life of approx. 7 days. The shape of the formation curve and the in vivo half-life indicate that 7-HEG will approach steady-state concentrations in rat DNA by 28 days of ETO exposure. The similarity in 7-HEG formation in target and nontarget tissues indicates that the tissue specificity for tumor induction is due to factors in addition to DNA-adduct formation.

Association of inhaled [14C]methyl chloride with macromolecules from various rat tissues

Abstract Inhalation of 14 C-labeled methyl chloride (CH 3 Cl) by rats resulted in accumulation of radioactivity in lipid, RNA, DNA, and protein isolated from lung, liver, kidney, testes, brain, muscle, and intestine. At the end of a 6-hr exposure to 500 or 1500 ppm 14 CH 3 Cl, 8 to 20% of the radioactivity present in whole tissue homogenates was associated with acid-insoluble material. Most of this activity represented labeling of protein and lipid, although the concentration of 14 C was usually over 10-fold higher in nucleic acids, per mole of nucleotide, compared with protein, per amino acid residue. Sephasorb HP chromatography of DNA revealed that radioactivity derived from 14 CH 3 Cl was associated with normal purine bases and no methylated bases were detected. Similarly, 82% of the radioactivity present in the lipid fraction copurified with the major species of phospholipids upon thin-layer chromatography. Pretreatment of rats with cycloheximide reduced the amount of CH 3 Cl-derived radioactivity associated with tissue protein by 42 to 58%, whereas [ 3 H]leucine incorporation was inhibited by 75 to 85%, indicating that most, but not necessarily all, of the uptake of 14 CH 3 Cl into protein was dependent on normal protein synthesis. Pretreatment with methotrexate inhibited the uptake of 14 CH 3 Cl into lipid, acid-insoluble material, RNA, and DNA by 47, 64, 65, and 93%, respectively. Pretreatment with methanol inhibited 14 CH 3 Cl uptake into acid-insoluble material by 66%. These findings indicate that most of the 14 C appearing in macromolecules following inhalation of 14 CH 3 Cl arose through metabolic incorporation, stemming from metabolism of CH 3 Cl via the one-carbon pool. Methanol apparently competed with CH 3 Cl for entry into this pool, although the failure of ethanol, 4-methylpyrazole, or 3-amino-1,2,4-triazole to inhibit 14 CH 3 Cl incorporation indicated that CH 3 Cl was not metabolized to methanol per se. Methanol pretreatment also inhibited 14 CO 2 evolution from 14 CH 3 Cl, indicating that metabolism of CH 3 Cl via single-carbon pathways may be of major quantitative significance.

Histopathology of acute toxic response in rats and mice exposed to methyl chloride by inhalation

Abstract Histopathology of Acute Toxic Response in Rats and Mice Exposed to Methyl Chloride by Inhalation. Morgan, K.T., Swenberg, J.A., Hamm, T.E., Jr., Wolkowski-Tyl, R. and Phelps, M. (1982). Fundam. Appl. Toxicol. 2:293–299. Both sexes of one strain of rat (F344), two strains of mice (C3H and C57BL/6) and the cross (B6C3F1) of these 2 strains of mice were exposed by inhalation to methyl chloride for 6 hours per day for up to 12 days. Methyl chloride concentrations in air were 0, 500, 1000, or 2000 ppm for mice, and 0, 2000, 3500 or 5000 ppm for rats. All male B6C3F1 mice exposed to 2000 ppm were dead or moribund by day 2, and all male and female mice in the remaining 2000 ppm groups were moribund by day 5. Prior to death many of these mice exhibited ataxia, and hematuria with the latter occurring mainly in females. Treatment associated lesions in mice included hepatocellular degeneration and necrosis, degeneration and necrosis of proximal convoluted tubules and/or basophilic tubules in the renal cortex, and focal areas of necrosis of the internal granular layer of the cerebellum. Brain lesions were most severe in female C57BL/6 mice, while hepatocellular degeneration was most severe in male C57BL/6 and B6C3F1 strains. Approximately 50% of the male and female rats exposed to 5000 ppm were killed in extremis on day 5. The principal clinical signs, which were confined to the 5000 and 3500 ppm groups, included severe diarrhea, incoordination of the fore-limbs, and in a small number of animals, hind limb paralysis and convulsions. In rats, lesions were observed in the liver, kidney and brain which resembled those seen in mice but were generally less severe. Lesions observed in tissues examined only in rats included vacuolar degeneration of the zona fasciculata of the adrenal glands and degenerative changes in the seminiferous tubules and epididymis. Rats appeared to respond in a similar manner to mice but were more resistant to methyl chloride toxicity. These findings demonstrate species, strain and sex differences in susceptibility to methyl chloride.

Acute inhalation toxicity and sensory irritation of dimethylamine

The sensory irritation potential of dimethylamine (DMA) inhalation on male Fischer-344 rats and male Swiss-Webster mice was evaluated by measuring the reflex decrease in respiratory rate. In addition, the six hour LC50 for rats exposed to dimethylamine was established. Groups of 3 or 4 rats and mice were exposed for 10 minutes to concentrations of DMA ranging from 49 to 1576 ppm during which time the respiratory rate was monitored and recorded. Sensory irritation concentration-response curves were obtained and RD50 values (concentration which elicits a 50% decrease in respiratory rate) were determined to be 573 and 511 ppm for rats and mice, respectively. In another set of experiments seven groups of male rats were exposed to concentrations of DMA ranging from 600 to 6119 ppm for six hours. Mortality counts were made during and for 48 hours post exposure. The sic hour LC50 was determined to be 4540 ppm. Histopathologic examination of the respiratory tract revealed concentration related changes ranging from ulceration and necrosis to rhinitis, tracheitis, and emphysema. Overall, DMA was found to be less potent as a sensory irritant than other airborne irritants.