Renal Tubule Tumor Induction by Tertiary-Butyl Alcohol

Douglas McGregor*,1 and Gordon C. Hard{dagger}

* Toxicity Evaluation Consultants (TEC), 102 rue Duguesclin, 69006 Lyon, France; and {dagger} American Health Foundation, 1 Dana Road, Valhalla, New York 10595

ABSTRACT

The article highlighted in this issue is "{alpha}2u-Globulin Nephropathy, Renal Cell Proliferation, and Dosimetry of Inhaled tert-Butyl Alcohol in Male and Female F-344 Rats" by Susan J. Borghoff, Judith S. Prescott, Derek B. Janszen, Brian A. Wong, and Jeffrey I. Everitt (pp. 176–186).

The identification of substances that are potential human carcinogens has been heavily reliant upon long-term, high exposure experiments with rodents. To this end, a relatively small number of rat and mouse strains are used, at least some of which develop neoplasms through mechanisms that appear to have little, if any, relevance for human hazard identification or risk assessment. The acceptance for a particular chemical of such species-specific mechanisms depends on the quality and quantity of evidence available on that chemical. The article of Borghoff et al. in this issue adds evidence for the criteria for a species-/strain-specific mechanism to the action of tertiary-butanol (TBA) in male rats. It does not address the mechanisms by which other tumors might arise in TBA-treated rats or mice.

The larger usage of TBA is in the synthesis of isobutylene and tertiary-methyl butyl ether (MTBE). There is the possibility of widespread human exposure to MTBE, due to its use as a petrol/gasoline additive to enhance the octane rating of unleaded fuels and reduce air pollution. It is as a result of the synthesis of MTBE that the general population receives potential exposure to TBA, since this is one of the primary metabolites of MTBE. Consequently, the appropriate evaluation of TBA has significant implications for the human health risk evaluation not only of TBA but also of MTBE itself.

A two-year drinking water exposure study conducted by the US National Toxicology Program (NTP, 1995Go) has led to the conclusion that TBA induces renal tubule cell adenomas in male rats and to the hypothesis that the mechanism is the induction of {alpha}2u-globulin nephropathy. {alpha}2u-Globulin is an 18.5 kDa protein synthesized by the liver of adult male rats (about 50 mg/day) that is freely filtered by the glomeruli. About 60% of the filtered portion undergoes tubular reabsorption and catabolism, and 40% is excreted in the urine (Neuhaus et al, 1986). The nephropathy evolves due to the modification of the protein in blood by a chemical ligand, so that the complex reabsorbed by the P2 segment of the proximal renal tubules is rendered more resistant to proteolysis and accumulates in the phagolysosomes of the tubule cells. This accumulation is recognized histologically as hyaline droplets, leading to lysosomal overload and the shedding of cells into the lumen. Sustained tubule cell regeneration throughout chronic exposure to the eliciting chemical ensues and tumors are presumed to arise from within this proliferating cell population. Fundamental to this hypothesis is the specificity of {alpha}2u-globulin to male rats of conventional strains. Thus, male NCI Black Reiter (NBR) rats, which do not synthesize {alpha}2u-globulin in the liver, do not develop {alpha}2u-globulin nephropathy and are not susceptible to renal tubule cell tumor promotion by chemicals that cause this tumor in male rats of other strains (Dietrich and Swenberg, 1991aGo, bGo). In addition, conventional laboratory mice, which do not synthesize {alpha}2u-globulin, are resistant to the renal toxicity of the chemicals that induce {alpha}2u-globulin nephropathy, but transgenic mice genetically modified to synthesize {alpha}2u-globulin can develop this syndrome (Lehman-McKeeman and Caudill, 1994Go).

Criteria have been proposed for identifying chemicals causing renal tubule cell tumors through an {alpha}2u-globulin-associated response in male rats. A set of criteria proposed by the International Agency for Research on Cancer is as follows (IARC, 1999Go):

Only 7 of 40 chemicals and mixtures that have been recorded as inducng hyaline droplets in male rat kidney meet these stringent criteria fully (Swenberg and Lehman-McKeeman, 1999). Many or all of the remaining chemicals may well act by this mechanism, but additional data are needed to fulfill the requirements of the criteria. The U.S. EPA has identified a similar, but less extensive set of criteria for designating a chemical as an inducer of {alpha}2u-globulin accumulation (Baetcke et al., 1991Go).

TBA has been tested directly for carcinogenicity in Fischer 344 rats and B6C3F1 mice in a two-year study in which exposure was via drinking water containing TBA at concentrations of up to 5 mg/ml for male rats, 10 mg/ml for female rats and 20 mg/ml for male and female mice (NTP, 1995Go). There were no renal tubule tumors in any groups of female rats or male or female mice. There were, however, renal tubule cell adenomas in all groups of male rats, but a statistically significant increase in male rat renal tubule cell tumors was achieved only when the results of step sectioning of the kidney were included. These findings were judged to provide some evidence of carcinogenic activity. In groups of 50 male rats exposed to TBA in drinking water at 0, 1.25, 2.5 and 5 mg/ml, the standard evaluation (a single sagittal and a single transverse section) found 1, 3, 4, and 3 adenomas or carcinomas, respectively. The additional step sectioning at 1 mm intervals combined with the standard sectioning provided aggregate tumor incidences of 8, 13, 19 (p < 0.01) and 13 renal tubule adenoma- and carcinoma-bearing rats per group of 50, respectively. Statistical significance was also achieved (p < 0.05) at the high dose when one tumor found at an interim sacrifice was included in the total. It is noted that step sectioning has transformed a hitherto rare or unusual tumor type in control rats into a common one. Renal tubule hyperplasia was present in the aggregated sections of 14, 20, 17 and 25 (p < 0.01) rats per group of 50, respectively. In contrast, in females, renal tubule hyperplasia developed in a single rat in the highest dose group, probably representing an incidental finding.. Foci of linear mineralization in the renal medulla was present in the highest TBA-exposed male groups, and this lesion has been specifically and consistently reported as a long-term consequence of hyaline droplet nephropathy (Hard et al., 1993Go). In a preliminary 13-week study there was an increased incidence of hyaline droplets and angular, crystalline structures associated with the hyaline droplets within renal tubule epithelium and tubule lumina in groups of rats exposed to TBA in drinking water at 2.5 mg/ml and higher. Thus, there were already indications that an {alpha}2u-globulin nephropathy might be induced by TBA, although the tumorigenic response was weak. Indirectly, a low potency of this chemical is consistent with results from experiments with the parent compound, MTBE. Renal tubule adenomas were increased in male, but not female, Fischer 344 rats exposed to MTBE by inhalation (Chun et al., 1992Go), whereas no renal cell tumors were reported in a lifetime gavage study of MTBE with Sprague-Dawley rats (Belpoggi et al., 1995Go). Thus, the result is not reproducible either because it is spurious or because it is only a weak and variable effect.

Four of the criteria for the {alpha}2u-globulin-associated mechanism listed above (1, 2, 3 and 5) have already been met for TBA (see NTP, 1995; Williams et al., 2000). Further evidence for a role of {alpha}2u-globulin nephropathy in TBA-associated male rat renal tumorigenesis is provided by the studies of Borghoff et al. reported in this issue. They exposed male and female Fischer 344 rats by inhalation in whole-body exposure chambers to 0, 250, 450 and 1750 ppm TBA 6 h per day for 10 days to assess {alpha}2u-globulin nephropathy and renal cell proliferation. The highest exposure in this study provided a dose similar to the penultimate dose of the NTP 90-day drinking water study, in which the highest dose produced, in comparison, a significant decrease in protein droplet accumulation. This effect could be due to toxicity and one might speculate that one possible mechanism could be an estrogen-like suppression of hepatic {alpha}2u-globulin synthesis, which would be consistent with known controlling factors (Roy et al., 1975Go).

In this study, protein (hyaline) droplets observed microscopically were stained with Mallory Heidenhain, a nonspecific protein stain. The protein droplet score (a product of severity and percentage of the tubules affected) increased with TBA dose and was significantly higher in the 1750 ppm group than in the air-exposed controls. In male rats, specific immunohistochemical staining for {alpha}2u-globulin was slightly greater in TBA treated rats than in controls, but a dose-related increase in stain intensity was not evident. No response was observed with female rats of any group. The concentration of {alpha}2u-globulin in homogenized kidney cytosol, measured using an ELISA with mouse monoclonal antibody, was significantly increased by about 60% in male rats exposed to 1750 ppm TBA, whereas the concentrations in male rats of the lower exposure concentration groups were not different from the controls. The concentrations in female rats were very low in all groups. While this result correlates with the nonspecific protein staining, it does not correlate well with the {alpha}2u-globulin immunohistochemical staining result. This could be because the cytosolic measurement of {alpha}2u-globulin by ELISA is a more quantitative and reproducible technique than is immunohistochemical staining.

The best exposure-related phenomenon shown in this study is the increase in cell proliferation, which was assessed after immunohistochemical staining for 5-bromo-2-deoxyuridine (BrdU) delivered from an osmotic pump for 3.5 days before rats were killed. This shows a very clear, statistically significant dose-related increase in the labeling index in epithelial cells of the renal cortex of male rats at all doses, but absolutely no response in female rats. The results, however, also show an apparent dislocation of cell proliferation from {alpha}2u-globulin accumulation, since proliferation occurred at doses lower than those at which protein accumulation was recognized. Therefore, while there is no doubt that cell proliferation, hyperplasia and adenoma formation are all specific to the male sex among rats, and at least the latter two features do not occur in mice, the role of {alpha}2u-globulin accumulation at low TBA doses remains to be demonstrated. It may be that the cell proliferation observed at the low doses would not have been sustained, if exposures had continued beyond 10 days, in contrast to the proliferation observed at higher doses when protein accumulation occurred. The discrepancy could also reflect a difference in sensitivities of the methods employed and/or the time course of the different responses, the one (DNA synthesis) involving a multi-day infusion of label and the other ({alpha}2u-globulin detection) capturing a single early time-point in a chronic process. Nevertheless, a nongenotoxic, sex- and species-specific mechanism remains the most probable explanation for the TBA-induced renal tubule adenomas.

The results obtained with TBA are remarkably similar to those obtained with MTBE, delivered to male and female rats by inhalation for 10 days at concentrations of 0, 413, 1516 and 3013 ppm (Prescott-Mathews et al., 1997Go). With MTBE there was also a statistically significant increase in protein droplets only at the two higher concentrations, a lack of a clear increase in immunohistochemically stained {alpha}2u-globulin and a significant increase in cytosolic {alpha}2u-globulin only at the highest dose, but a very clear increase in cortical labeling index over the whole dose range. The similarities suggest very reasonably that the {alpha}2u-globulin nephropathy of MTBE is due to TBA, although the similarity could simply be what might be expected of two independent but weakly active compounds. Besides providing important new information, this well-conducted study reminds us there will always remain questions to be answered. In evaluation of hazards and risks, therefore, the art is in judging what constitutes a reasonable weight of evidence based on the availability of reliable data. In this respect the link between TBA (and MTBE) and the {alpha}2u-globulin mode of action is strengthening.

NOTES

1 To whom correspondence should be addressed. E-mail: mcgregor{at}iarc.fr. Back

REFERENCES

Baetcke, K. P., Hard, G. C., Rodgers, I. S., McGaughy, R. E. and Tahan, L. M. (1991). {alpha}2u-globulin: Association with chemically induced renal toxicity and neoplasia in the male rat. (EPA/625/3–91/019) Risk Assessment Forum, U.S. Environmental Protection agency (U.S. EPA), Washington, D.C.

Belpoggi, F., Soffritti, M., and Maltoni, C. (1995). Methyl tertiary-butyl ether (MTBE) – A gasoline additive – causes testicular and lympho-haematopoietic cancers in rats. Toxicol. Ind. Health 11, 119–149.[ISI][Medline]

Chun, J. S., Burleigh-Flayer, H. D., and Kintigh, W. J. (1992). Methyl Tertiary Butyl Ether: Vapor Inhalation Oncogenicity Study in Fischer 344 Rats. Report No. 91N0013B. Bushy Run Research Center, Export, Pennsylvania.

Dietrich, D. R., and Swenberg, J. A. (1991a). NCI-Black-Reiter (NBR) male rats fail to develop renal disease following exposure to agents that induce {alpha}2u-globulin ({alpha}2u) nephropathy. Fundam. Appl. Toxicol. 16, 749–762.[ISI][Medline]

Dietrich D. R. and Swenberg, J. A. (1991b). The presence of {alpha}2u-globulin is necessary for d-limonene promotion of male rat kidney tumors. Cancer Res. 51, 3512–3521.[Abstract]

Hard, G. C., Rodgers, I. S., Baetcke, K. P., Richards, W. L., McGaughy, R. E., and Valcovic, L. R. (1993). Hazard evaluation of chemicals that cause accumulation of {alpha}2u-globulin, hyaline droplet nephropathy, and tubule neoplasia in the kidneys of male rats. Environ. Health Perspect. 99, 313–349.[ISI][Medline]

IARC (1999). Consensus Report. In Species Differences in Thyroid, Kidney and Urinary Bladder Carcinogenesis (C. C. Capen, E. Dybing, J. M. Rice, and J. D. Wilbourn, Eds.), pp. 5–9. IARC Sci. Pub. No. 147. International Agency for Research on Cancer, Lyon, France.

Lehman-McKeeman, L. D., and Caudill, D. (1994) d-Limonene induced hyaline droplet nephropathy in {alpha}2u-globulin transgenic mice. Fundam. Appl. Toxicol. 23, 562–568.[ISI][Medline]

Neuhaus, O.W. (1986) Renal reabsorption of low molecular weight proteins in adult male rats: {alpha}2u-globulin. Proc. Soc. Exp. Biol. Med. 182, 531–539.[Abstract]

NTP (1995) NTP Technical Report on the Toxicology and Carcinogenesis Studies of t-Butyl Alcohol (CAS No. 75–65–0) in F344/N Rats and B6C3F1 Mice (Drinking Water Studies). NTP TR 436. NIH Publ. No. 95–3167. National Toxicology Program, National Institutes of Health.

Prescott-Mathews, J. S., Wolf, D. C., Wong, B. A., and Borghoff, S. J. (1997). Methyl tert-Butyl ether causes {alpha}2u-globulin nephropathy and enhanced renal cell proliferation in male Fischer-344 rats. Toxicol. Appl. Pharmacol. 143, 301–314.[ISI][Medline]

Roy, A. K., McMinn, D. M., and Biswas, N. M. (1975). Estrogenic inhibition of the hepatic synthesis of {alpha}2u-globulin in the rat. Endocrinology 97, 1501–1508.[Abstract]

Swenberg, J. A., and Lehman-McKeeman, L. D. (1999). {alpha}2-Urinary globulin-associated nephropathy as a mechanism of renal tubule cell carcinogenesis in male rats. In Species Differences in Thyroid, Kidney and Urinary Bladder Carcinogenesis (Capen, C.C., Dybing, E., Rice, J.M. and Wilbourn, J.D., Eds.). pp 95–118. IARC Sci. Pub. No. 147. International Agency for Research on Cancer, Lyon, France.

Williams, T. M., Howell, E. R., Mooney, E. C., and Borghoff, S. J. (2000). Characterization of tert-butyl alcohol binding to {alpha}2u-globulin. Toxicologist 54, 401 (abstract).





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