Chemical Industry Institute of Toxicology, Research Triangle Park, NC, 27709, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations: BB, sodium barbital; BrdU, bromodeoxyuridine.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Every time DNA replicates it does so at less than 100% fidelity, which provides an opportunity for a mistake to be made and a somatic mutation to occur (3,4). Some non-genotoxic compounds can increase cancer incidence by increasing proliferation in transformed or intermediate cells. There is evidence that induction of increased cell proliferation can increase the risk of developing cancer (3,5,6). Increasing the proliferation rate of intermediate cells can increase the risk of cancer with time (7). Affecting the kinetics of intermediate cells is efficient in affecting tumor incidence because small changes in the rates of cell replication over cell death rates can lead to large changes in tumor incidence (7).
A xenobiotic can promote the development of tumors by several mechanisms. Tumor promoters may target initiated cells rather than non-initiated parenchymal cells for mitogenesis (4,8). The parenchymal cell may undergo hyperplasia as a reparative response to cytotoxicity (4). The initiated cells may have different growth control mechanisms than non-initiated cells and their response to mitogens or toxins may be different (4). Or, quite possibly, a particular promoter may work through more than one mechanism.
A rat model of hereditary renal cell cancer was first described by Eker (9). These hereditary renal tumors are due to a germline mutation in the tumor suppressor tuberous sclerosis 2 gene (Tsc2) (10,11). Tsc2 has been shown to be involved in the pathogenesis of renal cell carcinoma in humans and rats (12). The mutation in the Eker rat is an insertion of DNA into the 3'-portion of the Tsc2 gene. This insertional mutation results in premature stopping of transcription and elimination of the distal portion of the Tsc2 gene (13,14). Since this is a germline mutation, all the cells in the kidney are considered initiated in that they already have the first mutation in developing a renal cell tumor.
Sodium barbital (BB) has been used to promote intestinal, hepatic, thyroid, urinary bladder and renal tumor growth in rats alone or after treatment with various initiators (1522). Male F344 rats promoted with 4000 p.p.m. BB in the diet developed renal carcinomas. The promoting effect was evident as a greater incidence of large renal tubular tumors after 52 weeks treatment. The incidences of dysplastic tubules and preneoplastic lesions were not different between treated and control animals, suggesting that the targets for the promoting activity of BB are the dysplastic lesions that arise from proximal tubule epithelium (23,24). Rats given BB had a positive correlation between increased cell proliferation in proximal tubules and the degree of nephropathy, with a 2-fold increase in proliferation over controls after 52 and 72 weeks treatment (23).
Prolonged estrogen treatment in Tsc2 mutant (Eker) rats enhanced the development of hereditary renal cell tumors and increased the severity of nephropathy, with a 2-fold greater number of preneoplastic and neoplastic renal lesions compared with untreated Eker rats (25). Ovariectomizing Eker rats resulted in 33% fewer renal lesions and was protective for nephropathic changes compared with the unmanipulated control group (25). These data illustrated an association between increased renal toxicity and hereditary renal tumor development. Treatment with a mutagenic chemical also increased the multiplicity of hereditary renal tumors in Eker rats (26). The present study was designed to identify a concentration of the nephrotoxicant and tumor promoter BB that increased proximal tubule cell proliferation with minimal nephrotoxicity and then to use that concentration to determine if enhanced cell proliferation in the absence of severe cytotoxicity is sufficient to increase tumor development in a genetically susceptible animal.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Osmotic minipumps (Alzet, Palo Alto, CA) containing a 20 mg/ml solution of bromodeoxyuridine (BrdU) (CAS no. 59-14-3; Sigma) in saline were implanted in the morning 3 days before euthanasia and necropsy to assess alterations in cell proliferation. After 3 or 8 weeks BB treatment, six rats from each dose group were euthanized by pentobarbital anesthesia, exanguinated and then necropsied. The kidneys were examined, weighed and fixed in 10% neutral buffered formalin. Kidneys were cut mid-sagittaly and placed in cassettes, each with a section of duodenum for confirmation that BrdU was present in the tissues. The kidneys were processed to paraffin by routine methods and sectioned at 5 µm. The sections were either stained with hematoxylin and eosin or reacted with anti-BrdU antibody and counterstained with hematoxylin (27).
The hematoxylin and eosin stained sections were examined by light microscopy for evidence of toxicity. Severity of toxicity was determined by counting the number of foci of regenerating tubules per kidney section. Kidney sections were also reacted with anti-BrdU antibody for quantitation of labeling indices.
Quantitation of BrdU immunostaining was performed using analytical Cytology/Histology Recognition Information System (CHRIS) software provided by Sverdrup Technologies (Ft Walton Beach, FL). Initial images were captured on an optical diskette using Image-1 (Universal Imaging Corp.,Westchester, PA). Resolution of images was 1024x768 displaying 256 gray levels on a color monitor driven by a Groundhog Graphics VGA card. A total of 14 fields from the renal cortex were analyzed per animal, seven fields from each kidney. Every attempt was made to limit observations to proximal tubules, avoiding glomeruli and other non-proximal tubule cell populations. All fields were obtained using a 20x objective. One field per group was calibrated by identifying labeled nuclei, unlabeled nuclei and cytoplasm. The calibrated field was then reviewed for labeled and unlabeled cells by CHRIS. The animal used for calibration was then identified as a Set Default for the group and all animals in the group were analyzed according to the parameters set for that animal. After calibration for each group, the study was processed by CHRIS. Fields were then reviewed and edits made as necessary. The final labeling indices per field, per animal and per group were then reported.
Statistical analysis was performed on labeling index data using the William's test, a trend test that determines which points in a series are significantly different from the control value. For determining the statistical significance of basophilic foci measurements, a two-way analysis of variance (ANOVA), comparing duration of exposure and concentration, was performed. Results were considered significant at P < 0.05.
Tumor promotion study
Unbalanced treatment groups were present in the experiment because the mutant status of individual rats was unknown at the outset of the experiment and a population-based design was initially used (26). At the completion of the in-life phase of the study, a published description of the molecular changes responsible for the Eker mutation allowed a PCR-based analysis to identify the Tsc2 status of individual animals (10,11). This allowed only confirmed Tsc2 mutant animals to be included in the final evaluation. The details of the PCR technique are presented elsewhere (25).
From the preliminary subchronic study it was determined that the dosage of BB causing the greatest amount of cell proliferation with least toxicity was 500 p.p.m. in the feed. Therefore, that concentration was used as the high dose and 100 p.p.m. as the low dose. A total of 194 male Long-Evans rats from a mutant Tsc2 carrier colony were randomly assigned to treatment groups by weight, 56 in the control and in the 100 p.p.m. treatment groups and 82 in the 500 p.p.m. group. The additional 26 animals in the 500 p.p.m. group were used as a recovery group. The recovery group received 500 p.p.m. BB until 6 months of age and were then placed on the control diet until 12 months of age. The same exposure conditions and feed analysis methods were used for the chronic study as described above.
At 6 and 12 months of age the rats were euthanized by pentobarbital anesthesia, exanguinated and necropsied. Kidneys were removed, examined macroscopically, weighed and fixed in 10% buffered formalin. The numbers and sizes of macroscopically visible renal masses were recorded. The kidneys were sectioned mid-sagittaly and placed in separate cassettes. The kidneys were processed by routine methods to paraffin, sectioned at 5 µm and stained with hematoxylin and eosin. The kidneys were examined for preneoplastic and neoplastic microscopic renal lesions and severity of nephropathy as previously described (25). Macroscopic masses and microscopic lesions were counted and reported independently of each other.
Analyses for two types of responses were performed: whether or not there was any occurrence of each lesion and the number of lesions (multiplicity) that occurred. Fisher's exact test was used for the analysis of presence of a lesion. If the overall test was significant, Fisher's exact test was also used to test for significance of relevant treatment subsets. Lesion multiplicity data was first square root transformed and then analyzed by one-way ANOVA. If the overall F test for equal means was significant, the TukeyKramer test for all pairwise comparisons was applied. The significance level for all analyses was 0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tumor promotion study
Untreated Eker rats had time-dependent significant increases in number and incidence of atypical hyperplasias and tumors and a significantly increased incidence in macroscopically visible masses (Tables IIV). These data confirm that spontaneous lesions develop early, grow over time and form tumors as previously reported. (27)
|
|
|
|
Rats treated for 10.5 months with 100 p.p.m. BB had significantly increased numbers of macroscopic masses and total tumors compared with those treated for 4.5 months (Tables II and IV). Rats treated with 500 p.p.m. BB for 10.5 months also had significantly increased numbers of macroscopic masses and significantly more carcinomas, but significantly fewer atypical tubules, compared with those treated for 4.5 months (Tables II and V
).
A biologically significant finding in this study was that there was no statistically significant difference between the numbers of rats with lesions or the multiplicity of lesions in any category between any of the groups of animals necropsied at 12 months of age (Tables IV and V). There was no statistically significant difference in any parameter measured between animals treated for 4.5 months with 500 p.p.m. BB and necropsied at 6 months of age and any of the groups necropsied at 12 months of age (Tables IIV
). In addition, the recovery group, treated with 500 p.p.m. BB for 4.5 months and necropsied at 12 months of age, was not different from any of the other groups necropsied at 12 months of age (Tables IV and V
).
The severity of nephropathy was slightly increased in the rats treated with 500 p.p.m. BB for 10.5 months compared with the other groups necropsied at 12 months of age (Table VI).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tumor development can be enhanced by increasing the number of cells at risk, such as with a mutagenic event, increasing the mitotic activity of a tissue resulting in a decreased opportunity for DNA repair or by a combination of these events (29,30). Cell turnover times are fairly low for many organs, such as the kidney, and spontaneous rates of somatic gene mutation are extremely small compared with the average cell turnover time (31). DNA replication does not have 100% fidelity, resulting in mistakes in critical genes that can lead to tumor formation (3,32). When cells proliferate there is a chance that a critical molecular event can occur, resulting in an increased risk of developing cancer (3,7,32). The total number of DNA replications probably has a greater influence on the tumor process than proliferation rate (32). The number of cell replications is a function of the proliferation rate and the number of cells in the population at risk (3,32). In the case of the Eker rat model, all renal epithelial cells are considered to be intermediate cells.
Compounds such as BB, which are not directly mutagenic, may increase cancer incidence by increasing proliferation in the transformed or intermediate cell population. Merely increasing the number of proliferating cells may be a mechanism by which an agent increases the risk of developing cancer (3,4,6,30,33). Even a doubling of the percentage of replicating cells may significantly increase the probability that cancer will arise in the target cell population (33). Subchronic exposure to BB at 500 p.p.m. in the feed resulted in a doubling of the percentage of cells in S phase in the target population of the kidney that develops renal tumors. While this exposure regimen did increase the number of renal tumors present after 4.5 months treatment, merely increasing the numbers of proliferating cells was not sufficient to increase the number of renal tumors after longer treatment. These data indicate that enhancing proliferation rates without additional cell stressors is not sufficient by itself to increase tumor numbers, even in a susceptible population.
Cancer may develop from chronic exposure to a xenobiotic that is not directly mutagenic by increasing the proliferation of normal or intermediate cells (4,6,7). This increased proliferation could increase transition rates from normal to intermediate or intermediate to malignant genotypes and phenotypes (7). Xenobiotics that increase the number of proliferating intermediate cells without affecting transition rates may increase cancer risk with time, and even small changes in the rate of cell proliferation over cell death rate can lead to large changes in tumor incidence (7). The Eker rat model of renal cancer has a mutation that allows increased susceptibility and sensitivity to renal carcinogenesis in all the cells, which means that all the cells in the kidney are intermediate cells because the rats are born with a proposed initiating mutation in the renal cancer process. The concentrations of BB used in the present study promoted spontaneous tumors, so that they reached a size described as adenoma earlier.
Treatment with BB caused a concentration-related significant increase in the labeling index in the renal cortex and outer medulla of male F344 rats (34). In all the studies using BB as a tumor promoter, there has not been a concentration-dependent response for tumor promotion in male rats treated with concentrations ranging from 500 to 4000 p.p.m. (34). All concentrations used appeared to have comparable promoting activity. Previous studies have shown concentration- and time-related BB-induced nephrotoxicity (35). BB given at 4000 p.p.m. in the feed resulted in a 6270% incidence of dysplastic tubules and a 45% incidence in rats treated with 500 p.p.m. (35). BB-induced tumor incidence appeared to be time rather than concentration dependent (35). The present subchronic study supports the previous work showing a concentration-dependent increase in nephrotoxicity in rats treated with BB and also indicates that 500 p.p.m. BB given in the feed is sufficient to act as a renal tumor promoter. In the Eker rat, the inherited mutation in the Tsc2 gene dramatically decreased the time to tumor. This model is useful in detecting renal carcinogens and renal tumor promoters. However, it is necessary to examine multiple time points to determine whether a particular xenobiotic is a carcinogen or tumor promoter.
While elevated cell proliferation does increase the likelihood of tumor formation, increased cell proliferation associated with mild toxicity was probably not sufficient to increase tumor formation in the present study. In some studies increased renal cell proliferation associated with mild nephrotoxicity was unrelated to tumor development (36,37). Others have shown that increased severity of nephropathy was associated with increased frequency of atypical hyperplasias and adenomas in the renal cortex (38). The presence of proliferative lesions was closely correlated with severity of nephropathy (38). Continual administration of cytotoxicants can generate a hyperproliferative state characterized by persistent cell death and subsequent regeneration. This cytotoxicproliferative response is an obligatory step in the cancer process such that concentrations that produced no cytotoxicity would not be associated with increased risk of cancer (39). In a previous study where female Eker rats were chronically treated with 17ß-estradiol the treatment resulted in severe nephrotoxicity and increased numbers of renal tumors (25). In the same study there was a dramatic decrease in spontaneous nephropathy and an associated decrease in preneoplastic and neoplastic renal lesions when female Eker rats were ovariectomized (25). The concentrations of BB given in the present study did not dramatically enhance nephrotoxicity and did not increase the multiplicity of preneoplastic and neoplastic renal lesions. Taken together, these studies indicate that for compounds not directly mutagenic there has to be a minimum level of ongoing cytotoxicity associated with regenerative proliferation to enhance the cancer process beyond promoting spontaneous lesions.
The results of the present study suggest that particular attention needs to be paid to the duration of treatment with respect to tumor latency when designing experiments using genetically susceptible or transgenic rodent models. If only the 6 month data were available in the present study, then the interpretation of those data would be that BB is a complete renal carcinogen that increases the multiplicity of both preneoplastic and neoplastic renal lesions. If only the 12 month data were available, then the interpretation would be that BB has no effect on renal carcinogenesis. This points out the necessity of designing studies appropriately when examining poorly characterized xenobiotics in genetically enhanced rodent models. The final interpretation of the potential hazard of a xenobiotic may be as much a function of the design of the study as of the properties of the compound.
In conclusion, the present study has shown that Eker rat hereditary renal tumors are sensitive to the promotional effects of BB. Without significant nephrotoxicity, there was no increase in the multiplicity of renal tumors. The interpretation of a study using genetically susceptible animals can be time dependent and this fact is very important when designing studies using transgenic or knockout animals. The present study and previous work have shown that the Eker rat model of hereditary renal cancer responds to mutagenic and non-mutagenic renal carcinogens and renal tumor promoters.
![]() |
Notes |
---|
2 To whom correspondence should be addressed at present address: US EPA, MD-68, 86 TW Alexander, Research Triangle Park, NC 27711, USA Email: wolf.doug{at}epa.gov
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|