Accumulation Of Protein Droplets Research Articles

Ethyl tertiary-Butyl Ether: A Toxicological Review

A number of oxygenated compounds (oxygenates) are available for use in gasoline to reduce vehicle exhaust emissions, reduce the aromatic compound content, and avoid the use of organo-lead compounds, while maintaining high octane numbers. Ethyl tertiary-butyl ether (ETBE) is one such compound. The current use of ETBE in gasoline or petrol is modest but increasing, with consequently similar trends in the potential for human exposure. Inhalation is the most likely mode of exposure, with about 30% of inhaled ETBE being retained by the lungs and distributed around the body. Following cessation of exposure, the blood concentration of ETBE falls rapidly, largely as a result of its metabolism to tertiary-butyl alcohol (TBA) and acetaldehyde. TBA may be further metabolized, first to 2-methyl-1,2-propanediol and then to 2-hydroxyisobutyrate, the two dominant metabolites found in urine of volunteers and rats. The rapid oxidation of acetaldehyde suggests that its blood concentration is unlikely to rise above normal as a result of human exposure to sources of ETBE. Single-dose toxicity tests show that ETBE has low toxicity and is essentially nonirritant to eyes and skin; it did not cause sensitization in a maximization test in guinea pigs. Neurological effects have been observed only at very high exposure concentrations. There is evidence for an effect of ETBE on the kidney of rats. Increases in kidney weight were seen in both sexes, but protein droplet accumulation (with α2u-globulin involvement) and sustained increases in cell proliferation occurred only in males. In liver, centrilobular necrosis was induced in mice, but not rats, after exposure by inhalation, although this lesion was reported in some rats exposed to very high oral doses of ETBE. The proportion of liver cells engaged in S-phase DNA synthesis was increased in mice of both sexes exposed by inhalation. ETBE has no specific effects on reproduction, development, or genetic material. Carcinogenicity studies have been conducted with ETBE, TBA, and ethanol (included in this review as an endogenous precursor of acetaldehyde in the absence of TBA). A single experiment with ETBE in rats and several experiments with ethanol in rats and mice were not considered adequate for an evaluation of ETBE carcinogenicity. In male rats only, TBA induced α2u-globulin nephropathy-related renal tubule adenomas. These are generally considered to have no human relevance. In addition, increases in thyroid follicular cell adenoma incidence were associated with TBA treatment in female mice. This result lacks independent confirmation and is not supported by experiments in which similar or higher internal doses of TBA were delivered.

Sex differences in the metabolism of (+)- and (-)-limonene enantiomers to carveol and perillyl alcohol derivatives by cytochrome p450 enzymes in rat liver microsomes.

(+)-Limonene is reported to cause nephropathy in male rats, but not in female rats and other species of animals including mice, rabbits, guinea pigs, and dogs. Male rats contain high levels of alpha2u-globulin in kidneys, and it has been shown that limonene and/or its metabolites are able to bind noncovalently to alpha2u-globulin, resulting in an accumulation of protein droplets in the renal tubules. In this study, we investigated whether (+)- and (-)-limonene enantiomers are differentially metabolized by liver microsomes of male and female rats. (+)- and (-)-limonene enantiomers were found to be oxidized to their respective trans-carveol (6-hydroxylation) and perillyl alcohol (7-hydroxylation) derivatives in greater amounts by liver microsomes of male rats than those of female rats. The limonene hydroxylation activities were not detected in liver microsomes of rat fetuses and were increased developmentally after birth, only in male rats. Treatment of male rats with phenobarbital significantly increased liver microsomal 6-hydroxylation activities with both enantiomers whereas beta-naphthoflavone, isosafrole, and pregnenolone 16alpha-carbonitrile did not cause such effects. Anti-P450 2C9 which cross-reacts with rat P450 2C11 inhibited limonene hydroxylations catalyzed by liver microsomes of untreated male rats, and it was also found that anti-P450 2B1 suppressed the activities catalyzed by liver microsomes of phenobarbital-treated rats. Possible roles of P450 2C11 and P450 2B1 in the limonene hydroxylation activities were supported by the experiments with purified rat liver P450s in reconstitution systems and with recombinant rat P450s in Trichoplusia ni. Our present results showing that there are sex-related differences in the oxidative metabolism of limonene enantiomers by liver microsomes may provide useful information on the basis of limonene-induced toxicities in different animal species.

Alpha 2u-Globulin nephropathy, renal cell proliferation, and dosimetry of inhaled tert-butyl alcohol in male and female F-344 rats.

tert-Butyl alcohol (TBA) has been shown to cause kidney tumors in male rats following chronic administration in drinking water. The objective of the present study was to determine whether TBA induces alpha 2u-globulin (alpha 2u) nephropathy (alpha 2u-N) and enhanced renal cell proliferation in male, but not female, F-344 rats, and whether the dosimetry of TBA to the kidney is gender specific. Male and female F-344 rats were exposed to 0, 250, 450, or 1750 ppm TBA vapors 6 h/day for 10 consecutive days to assess alpha 2u-nephropathy and renal cell proliferation and for 1 and 8 days to evaluate the dosimetry of TBA following a single and repeated exposure scenario. Protein droplet accumulation was observed in kidneys of male rats exposed to 1750 ppm TBA, with alpha 2u-globulin immunoreactivity present in these protein droplets. A statistically significant increase in alpha 2u concentration in the kidney, as measured by an enzyme-linked immunosorbent assay, was observed in male rats exposed to 1750 ppm TBA with a exposure-related increase in renal cell proliferation. Renal alpha 2u concentration was positively correlated with cell proliferation in male rat kidney. No histological lesions or increased renal cell proliferation was observed in female rats exposed to TBA compared to controls. The TBA kidney:blood ratio was higher at all concentrations and time points in male rats compared with female rats, which suggests that TBA is retained longer in male rat kidney compared with female rat kidney. Together these data suggest that TBA causes alpha 2u-N in male rats, which is responsible for the male rat-specific increase in renal cell proliferation.

Methyltert-Butyl Ether Causes α2u-Globulin Nephropathy and Enhanced Renal Cell Proliferation in Male Fischer-344 Rats

Methyltert-butyl ether (MTBE), a fuel additive blended into unleaded gasoline to decrease carbon monoxide emissions, induces renal tumors in male, but not female, rats exposed by inhalation to ≥3000 ppm MTBE. A number of chemicals that induce male rat-specific renal tumors also cause a syndrome unique to male rats referred to as α2u-globulin nephropathy (α2u-N). The objective of the present study was to determine if MTBE induces an α2u-N and renal cell proliferation in male F-344 rats. Male and female F-344 rats were exposed to MTBE vapors of 0, 413, 1516, or 3013 ppm for 6 hr/day for 10 consecutive days. Significant proximal tubule necrosis and protein droplet accumulation were observed in kidneys from male rats exposed to 1516 and 3013 ppm MTBE. Significantly greater labeling indices were observed in all groups of MTBE-exposed male rats. α2u-Globulin immunoreactivity was present in and confined to protein droplets in male rat kidney. A mild dose-related increase in α2u concentration in the kidney, as measured by an enzyme-linked immunosorbent assay, was observed in male rats exposed to MTBE, with a statistically significant increase in α2u concentration in male rats exposed to 3013 ppm MTBE. There was a strong positive correlation (r= 0.994) with exposure concentration between cell proliferation and α2u concentration in male rat kidney. No significant differences were observed in female rats for any of these responses. Further analysis of kidney cytosol failed to demonstrate the accumulation of any protein besides α2u in MTBE-exposed male rat kidney. These findings demonstrate that MTBE causes a mild induction of α2u-N and enhanced renal cell proliferation in male, but not female, F-344 rats, suggesting a role for α2u-N in renal tumorigenesis.

Induction of protein droplet ( α2 μ-globulin) nephropathy in male rats after short-term dosage with 1,8-cineole and l-limonene

As part of a series of short-term studies on peppermint oil constituents for their possible induction of the encephalopathy found with peppermint oil, 1,8-cineole and l-limonene were studied. Groups of 10 male Wistar rats were given 0, 500, or 1000 mg 1,8-cineole/kg body wt./day or 0, 800, or 1600 mg l-limonene/kg body wt./day for 28 days. 1,8-Cineole and l-limonene both induced accumulation of protein droplets containing α 2 μ -globulin in proximal tubular epithelial cells in male rats. These results suggest that both 1,8-cineole and l-limonene possibly belong to the group of chemicals characterized by their induction of excessive α 2 μ -globulin accumulation. Neither of the 2 substances induced encephalopathy.

Subchronic Toxicity Study of Dicyclopentadiene Vapor in Rats

Fischer 344 rats were exposed by inhalation to 0, 1, 5 or 50 ppm dicyclopentadiene (DCPD) vapor 6 hr/day, 5 days/week for 13 weeks, followed by a 13-week recovery period. Animals were euthanized following completion of exposure at 2, 6, or 13 weeks and at postexposure weeks 4 or 13. No mortality, overt signs, body weight changes, hematologic or clinical chemistry values were related to DCPD exposure. In the high-exposure male rats, relative liver weights were significantly increased but with no accompanying histopathologic changes. Exposure to DCPD produced adverse kidney effects in male, but not female, rats as evidenced by the excretion of epithelial cells in the urine. Histologic changes were localized to the proximal tubules of the kidney and included increased accumulation of protein droplets, regenerative epithelium, and the presence of intraluminal proteinaceous material. In addition, several alterations in renal function were observed. Urinary Na+ excretion rates were decreased and urinary K+ excretion rates were increased throughout the exposure period; however, glucose was not present in the urine, and creatinine clearance was normal. The ability of the kidney to concentrate urine was also impaired. After the recovery period, many of the treatment-related kidney effects were not observed, including the presence of hyaline droplets in the proximal tubules and epithelial cells in the urine. These findings indicate an overall low degree of systemic toxicity following subchronic inhalation exposure of dicyclopentadiene at exposure levels up to 50 ppm. The only effect that was observed was a male rat-specific nephropathy that is characteristic of the hyaline droplet nephropathy produced by a diverse group of compounds.

An alternative hypothesis on the role of chemically induced protein droplet (α2u-globulin) nephropathy in renal carcinogenesis

Based on associations between the accumulation of protein droplets containing α2u-globulin in proximal tubular epithelial cells and increased incidences of renal tubular neoplasms in male rats, it has been suggested that the carcinogenicity of chemicals that cause α2u-globulin nephropathy is unique to animals that synthesize this protein. Chemicals that caused α2u-globulin nephropathy and renal carcinogenicity in male rats have not been shown to produce renal tumors in animals that lack the capability for hepatic α2u-globulin systhesis, including female rats, male NBR rats, or mice of either sex. Because humans do not synthesize α2u-globulin it has been suggested that chemicals which cause renal toxicity associated with α2u-globulin accumulation do not pose an increased cancer risk to humans. In this review on the association between α2u-globulin nephropathy and renal carcinogenesis, it is apparent that (a) there are data inconsistent with the hypothesis linking these occurrences, (b) alternative mechanisms of renal toxicity and carcinogenicity are plausible, (c) data on quantitative dose-response correspondences between the various stages of α2u-globulin nephropathy and renal carcinogenicity are limited, and (d) a greater understanding of the molecular changes occurring during renal carcinogenesis is needed before assuming that the current hypothesis is correct. Future research aimed at resolving issues raised in this paper should help determine whether or not the association between α2u-globulin nephropathy and renal carcinogenesis represents a cause-and-effect relationship.

Male rat specific nephrotoxicity resulting from subchronic administration of hexachlorobenzene

Male rats are more sensitive to the nephrocarcinogenic effect of hexachlorobenzene (HCB) than are female rats. The purpose of this study was to shed light on this phenomenon by investigating mechanisms of subchronic nephrotoxicity of HCB. Groups of rats were administered HCB in corn oil (po) at 100 mg/kg, 5 days per week for 15 days or at 50 mg/kg, 5 days per week for 50 days. Urine was collected on Days 1, 8, and 15 for the 15-day treatment and on Day 50 for the 50-day treatment. Glucosuria, proteinuria, and enzymuria (gamma-glutamyl transpeptidase) were measured to assess renal function. Twenty-four hours after the last HCB treatment, the animals were killed and kidneys were removed for histopathological evaluation. Urine analyses showed no indication of renal dysfunction in treated animals compared to controls during the 15-day treatment. However, histology of male rat kidneys revealed degenerative and regenerative cellular foci accompanied by an increased accumulation of protein droplets in epithelial cells of the proximal tubules. The same histological observations were also made in male rats after a 50-day HCB treatment but this time they were accompanied by renal function alterations. In female rats, no such renal functional or histological alterations were observed. The histopathological observations in male rats correspond well with the protein droplet nephropathy; the latter is characteristic of the accumulation in kidney cells of alpha 2u-globulin probably caused by the reversible binding of a chemical to alpha 2u that renders the protein indigestible to kidney proteases. alpha 2u-Globulin was measured in the cytosol of male rats and was found to be increased 11-fold compared to controls. Also, HCB was found to be bound reversibly to alpha 2u. These results suggest that HCB induces a male rat specific nephropathy that could explain the higher incidence of kidney tumors in male rats compared to female rats.

Perchloroethylene-induced rat kidney tumors: An investigation of the mechanisms involved and their relevance to humans

Lifetime exposure to perchloroethylene by inhalation has been shown to cause a low incidence of renal tumors in male rats. The mechanisms responsible for the induction of these tumors have been investigated following exposure of rats to perchloroethylene by oral gavage (1500 mg/kg for up to 42 days) or by inhalation (400 ppm for 28 days). Comparisons have been made between rats and mice in vivo and between rats, mice, and humans in vitro. High doses of perchloroethylene given by gavage have been shown to be toxic to the rat kidney, causing increases in urinary markers of kidney damage. A marked accumulation of protein droplets (α-2u-globulin) was seen in the P 2 segment of the kidney proximal tubules. This response were not seen after inhalation exposure to 400 ppm perchloroethylene for 28 days and hence may not be associated with the tumors seen at this dose level. Protein droplet formation was seen after exposure to 1000 ppm perchloroethylene, suggesting that 400 ppm is below the threshold dose required to induce this response. Perchloroethylene has been shown to be metabolized by glutathione conjugation in the liver, resulting in the formation of a mutagenic cysteine conjugate which is activated by the kidney enzyme β-lyase. Levels of the mercapturic acid of perchloroethylene have been compared in rat and mouse urine. The enzyme kinetics of hepatic glutathione conjugation and renal β-lyase activation have been compared in rat, mouse, and human tissues in vitro. Results of these studies are consistent with the rat being the species susceptible to kidney tumors. Although human kidney was shown to contain β-lyase, glutathione conjugation of perchloroethylene could not be detected in human liver. Perchloroethylene-induced male rat kidney tumors may be a result of chronic toxicity, protein droplet nephropathy, and genotoxicity from the β-lyase pathway. These mechanisms appear to have little relevance to humans.

Potential role of α-2μ-globulin, protein droplet accumulation, and cell replication in the renal carcinogenicity of rats exposed to trichloroethylene, perchloroethylene, and pentachloroethane

Trichloroethylene (TCE), perchloroethylene (PER), and pentachloroethane (PENT) are used extensively as industrial solvents. These agents cause an increased incidence of renal tumors in male, but not female, rats. Male and female F-344 rats were gavaged for 10 days with TCE (1000 mg/kg), PER (1000 mg/kg), and PENT (150 mg/kg) to determine if chlorinated hydrocarbon-induced changes in levels of renal α-2μ-globulin (α2μ), protein droplet accumulation (PDA), and cell replication were male rat specific. The animal strain, dose, and route of administration were the same as previous chronic bioassays in order to better understand the relationship between α2μ, PDA, and cell replication to the sex-specific renal carcinogenicity. In male rats, increases in protein droplet and crystalloid accumulation in the cytoplasm of the P 2 segment of the proximal tubule were evident after PER and more notably PENT administration. Cell replication rates in male rats increased specifically in the histologically damaged P 2 segments after PER or PENT exposure. Protein droplets and cell replication did not differ from controls in TCE-treated male rats or in female rats treated with TCE, PER, or PENT. Immunohistochemical staining for α2μ revealed a marked correlation between the presence of α2μ and the protein droplets. Renal α2μ concentrations in male rats increased after PER or PENT but not TCE administration. The protein droplet nephropathy induced in male rats after PER and PENT treatment appears identical to that observed with other male-rat-specific renal carcinogens such as unleaded gasoline. The differences observed in male and female rats after chlorinated hydrocarbon exposure suggest that increases in cell replication may be directly linked to the male-rat-specific protein α2μ. Since compensatory cell division is postulated to affect all stages of the carcinogenic process, the increased incidence of renal tumors in male rats after PER or PENT treatment may be related to nephrotoxicity and resulting enhanced cell replication. Mechanisms involved in TCE-induced renal carcinogenicity appear to be different from PER- and PENT-induced renal carcinogenicity.

Assessment of the covalent binding potential of 2,2,4-trimethylpentane to rat α2u-globulin

Subchronic exposure of male rats to the nephrotoxin 2,2,4-trimethylpentane (TMP) causes an accumulation of protein droplets in the epithelial cells of the renal cortex. Experimental evidence suggests that these droplets contain α 2u-globulin, a low-molecular-weight protein found specifically in the urine of male rats. It has been proposed that aldehyde metabolites of TMP form Schiff base adducts with the lysine groups of α 2u-globulin and thereby inhibit renal lysosomal processing of the protein. Accordingly, the ability of TMP and its metabolites to covalently bind to α 2u-globulin was examined. As a model, a [ 14C]formaldehyde -α 2u - globulin Schiff base was formed. This protein adduct was stabilized by reduction with cyanoborohydride and could be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein analysis by SDS-PAGE demonstrated that hepatocytes isolated from male Fischer-344 rats produced significant quantities of α 2u-globulin in culture, whereas hepatocytes from female rats did not. A 15-hr exposure of metabolically competent, primary cultures of male rat hepatocytes to [ 14C]TMP (0.1 and 0.5%, v/v), followed by reduction with cyanoborohydride, dialysis, and analysis with SDS-PAGE, revealed no evidence of radiolabeled α 2u-globulin. When [ 14C]TMP was administered to an adult male Fischer-344 rat (300 mg/kg, ig) 22, 16, and 10 hr before sacrifice, 16% of the administered radioactivity was eliminated in the urine as TMP metabolites. Analysis as above showed no TMP-derived radioactivity in fractions containing α 2u-globulin from liver, blood, kidney cortex, or urine. The absence of a detectable covalent interaction between TMP and α 2u-globulin following in vitro or in vivo exposure suggests that a TMP- α 2u-globulin adduct is not responsible for the excessive formation of protein droplets in the renal cortex of exposed male rats.