Toxicology and carcinogenesis studies of N,N-dimethyl-p-toluidine (CAS No. 99-97-8) in F344/N rats and B6C3F1/N mice (gavage studies).

N,N-dimethyl-p-toluidine was nominated for toxicology and carcinogenesis studies by the National Cancer Institute based on the potential for human exposure through its use in dental materials and bone cements and the lack of toxicity and carcinogenicity data. Male and female F344/N rats and B6C3F1/N mice were administered N,N-dimethyl-p-toluidine (greater than 99% pure) in corn oil by gavage for 3 months or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and Escherichia coli, mouse peripheral blood, and mouse and rat liver. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were administered 0, 62.5, 125, 250, 500, or 1,000 mg N,N-dimethyl-p-toluidine/kg body weight in corn oil by gavage, 5 days per week for 14 weeks. Additional groups of 10 male and 10 female rats (clinical pathology study) were administered the same doses, 5 days per week for 25 days. On day 88, blood was collected from core study rats for hemoglobin and methemoglobin analyses only. All 1,000 mg/kg male and female rats and one 500 mg/kg male rat died by study day 3. Mean body weights of all surviving dosed groups of males and females were significantly less than those of the vehicle controls. Clinical findings associated with exposure to N,N-dimethyl-p-toluidine included cyanosis, abnormal breathing, and lethargy in groups administered 250 mg/kg or greater. Methemoglobinemia appeared to be the primary hematologic toxic response, and many other lesions could be explained as secondary to methemoglobin formation including Heinz body formation; a macrocytic, hypochromic, responsive anemia; and increased hematopoietic cell proliferation in the spleen and bone marrow. In general, hematologic changes were dose-related and occurred at both evaluated timepoints in all dosed groups. Anemia was evidenced by decreases in hematocrit values, hemoglobin concentrations, and erythrocyte counts; erythrocyte macrocytosis was characterized by increases in mean cell volume and mean cell hemoglobin values; erythrocyte hypochromia was evidenced by decreases in mean cell hemoglobin concentration values; and an erythropoietic response to the anemia was characterized by substantially increased reticulocyte and nucleated erythrocyte counts. Liver weights of all surviving dosed groups of males and females were significantly greater than those of the vehicle controls. Kidney weights of all surviving dosed groups of females were significantly greater than those of the vehicle controls. There were significant decreases in left cauda epididymis and left epididymis weights in 250 mg/kg males. There was a dose-related decrease in the number of cycling females, with only four females in the 250 mg/kg group having regular cycles and females in the 125 and 250 mg/kg groups spending a significantly higher proportion of time in extended diestrus compared to the vehicle control group. In the surviving groups of rats, there were significantly increased incidences of pigmentation in the liver of all dosed groups, hepatocyte hypertrophy in groups administered 125 mg/kg or greater, and hepatocyte necrosis in 62.5, 250, and 500 mg/kg females. In the olfactory epithelium of the nose, there were dose-related increases in the incidences and severities of degeneration in all dosed groups and significantly increased incidences of metaplasia in the 250 and 500 mg/kg groups. In the respiratory epithelium of the nose, there were significantly increased incidences of hyperplasia and squamous metaplasia in all of the groups administered 125 mg/kg or greater. The incidences of glandular hyperplasia of the nose were significantly increased in males and females administered 125, 250, or 500 mg/kg. In the spleen, there were significantly increased incidences of capsule fibrosis, congestion, mesothelial hypertrophy, and lymphoid follicle atrophy primarily in groups administered 125 mg/kg or greater. Hematopoietic cell proliferation and pigmentation were increased in severity in treated groups. In the kidney, there were significantly increased incidences of nephropathy (females), pigmentation (males and females), papillary necrosis (males and females), and mineralization (males). Other treatment-related lesions included inflammation of the forestomach in males, mesenteric lymph node atrophy in females, and bone marrow hyperplasia in males and females. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were administered 0, 15, 30, 60, 125, or 250 mg N,N-dimethyl-p-toluidine/kg body weight in corn oil by gavage, 5 days per week for 14 weeks. All 250 mg/kg male and female mice (except for one male mouse) died before day 10, and three males and two females administered 125 mg/kg died before the end of the study. The final mean body weight of 125 mg/kg males and the mean body weight gains of 125 mg/kg males and females were significantly less than those of the vehicle controls. Clinical findings associated with administration of N,N-dimethyl-p-toluidine included abnormal breathing, thinness, lethargy, cyanosis, and ruffled fur in 125 and 250 mg/kg males and females. Methemoglobinemia appeared to be the primary hematologic toxic response; however there were less severe erythron changes compared to the 3-month study in rats. In females, no erythron changes were detected up to 125 mg/kg. In males, inconsistent and minor decreases in hematocrit values, hemoglobin concentrations, and erythrocyte counts, and increased reticulocyte counts occurred in groups administered 60 mg/kg or greater. Methemoglobin values were minimally increased in males and females administered 30 mg/kg or greater. Heinz bodies were slightly increased in 60 mg/kg females, 125 mg/kg males and females, and the one surviving 250 mg/kg male; Heinz body formation was considered secondary to methemoglobin formation. Liver weights of all dosed groups of mice were significantly greater than those of the vehicle controls. In the surviving groups of mice, there were significantly increased incidences of bronchiolar epithelium degeneration, bronchiolar epithelium regeneration, and peribronchiolar chronic active inflammation in the lung of 125 mg/kg groups, and histiocytic infiltrates of the alveoli in 125 mg/kg females. In the nose, there were significantly increased incidences of glandular hyperplasia and olfactory epithelium metaplasia in the 125 mg/kg groups and olfactory epithelium degeneration in 60 mg/kg females and 125 mg/kg males and females. In the thymus, the incidences of thymocyte necrosis in the 125 mg/kg groups were significantly increased. In the liver, the severities of cytoplasmic vacuolization of the hepatocytes were increased in dosed groups of males and females. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered 0, 6, 20, or 60 mg N,N-dimethyl-p-toluidine/kg body weight in corn oil by gavage, 5 days per week for 104 or 105 weeks. Additional groups of 10 male and 10 female rats (clinical pathology study) were administered the same doses for 86 days. Survival of 60 mg/kg males was significantly less than that of the vehicle controls. Mean body weights of 60 mg/kg males and females were more than 10% less than those of the vehicle controls after week 61 and week 33, respectively. Clinical findings included signs of pallor in 60 mg/kg females and hyperactivity and boxing behavior in 20 mg/kg females and 60 mg/kg males and females. The hematology findings at the 3-month timepoint were consistent with those in the 3-month study in rats which indicated that methemoglobinemia was the primary hematologic toxic response. In the 20 and 60 mg/kg groups, there were dose-related decreases in hematocrit values, hemoglobin concentrations, and erythrocyte counts. There were similar trends toward erythrocyte macrocytosis and hypochromia and increased erythropoiesis as seen in the 3-month study. While the magnitudes of the erythron decreases were not sufficient to classify the responses as anemias, the patterns of the erythron changes were identical to those in the 3-month study. In the liver of 60 mg/kg males and females, there were significantly increased incidences of hepatocellular carcinoma and hepatocellular adenoma or hepatocellular carcinoma (combined). Numerous nonneoplastic liver lesions occurred in dosed males and females primarily in the 20 and 60 mg/kg groups. In the nose, there were significantly increased incidences of transitional epithelium adenoma and transitional epithelium adenoma or carcinoma (combined) in 60 mg/kg males; transitional epithelium adenoma also occurred in female rats administered 6 or 60 mg/kg. In the nose, there were significantly increased incidences of nonneoplastic lesions in the olfactory, respiratory, and transitional epithelia of dosed rats. These lesions occurred with the greatest incidence and severity in the 60 mg/kg groups. The incidences of inflammation and nerve atrophy were significantly increased in males and females administered 60 mg/kg. There were increased incidences of follicular cell adenoma or carcinoma (combined) of the thyroid gland in all dosed groups of males, and an increased incidence of follicular cell adenoma in 20 mg/kg females. In the spleen, there were significantly increased incidences of hematopoietic cell proliferation in all dosed groups of males and females. The incidences of congestion and mesothelial hypertrophy of the capsule were significantly increased in 60 mg/kg males and all dosed groups of females. There were also significantly increased incidences of capsular fibrosis and atrophy of the lymphoid follicle in the 60 mg/kg groups. The incidences of pigmentation were significantly increased in all dosed groups of males and in 60 mg/kg females. In all dosed groups of female rats, there were significantly increased incidences of nephropathy. (ABSTRACT TRUNCATED)

Molecular study of signaling-pathway genes in experimental rat thyroid carcinoma.

INTRODUCTION: A study was conducted on histological patterns and biomolecular changes in Goitrogen-induced experimental rat thyroid tumors. The link between the histological types observed and N-ras, B-raf, and PI3KCA gene mutations widely reported in human thyroid cancers was explored. MATERIAL AND METHODS: An analysis was done on paraffin-embedded tumor tissue sections from Wistar rats receiving 1% potassium perchlorate (KClO(4)) added to the ad libitum drinking-water supply over an 18-month period. Three experimental subgroups were formed, each comprising 10 thyroids: subgroup I (control) consisted of thyroids from untreated controls; subgroups II and III (experimental) consisted of thyroids from KClO(4)-treated rats, displaying capsular, vascular, or both invasion but no metastasis (II), or distant metastasis (III). DNA was extracted from paraffin-embedded tissues. To test for the genetic mutations most widely reported in human thyroid cancers, exon 1 of the N-ras gene, exons 9 and 20 of the PI3KCA gene, and exon 15 of the B-raf gene were amplified and sequenced. RESULTS: All tumors were of the follicular type. None of the 20 experimental rat thyroids displayed the expected gene mutations reported in humans. However, 90% of them contained four new B-raf gene mutations and all were silent and did not cause an amino acid substitution in the protein chain. CONCLUSIONS: Biomolecular analysis suggested that N-ras, PI3KCA, and B-raf gene mutations may not be involved in thyroid tumor formation using the experimental procedure applied in this study. But the four mutations in B-raf, though without functional repercussions, may be a specific marker for this tumor type.

Involvement of glycogen synthase kinase-3beta signaling and aberrant nucleocytoplasmic localization of retinoblastoma protein in tumor promotion in a rat two-stage thyroid carcinogenesis model.

To investigate the cell cycle kinetics during the tumor promotion process induced by hypothyroidism in a rat model of thyroid follicular cell carcinogenesis, immunohistochemical analysis of cell cycle molecules and related signaling molecules was performed in conjunction with analysis of cell proliferation activity in an initiation-promotion model. Male F344 rats were injected with N-bis(2-hydroxypropyl)nitrosamine, and one week later treated with 6-propyl-2-thiouracil (PTU) at 12ppm in the drinking water for 4, 10 or 15 weeks. At each time point, proliferative lesions increased the expression of cyclin A, cyclin D, cyclin E and cyclin-dependent kinase (Cdk)-2, in association with the development of lesion stages from the early focal hyperplasia to the late carcinoma, while a subpopulation of proliferative lesions showed decreased numbers of both cell division cycle-2- and Ki-67-positive cells at week 15 compared with that at week 10, suggesting a reduced promoting effect of serum thyroid-stimulating hormone in the sensitive cellular population after long-term exposure to PTU. On the other hand, increased immunolocalization of phosphorylated and inactive glycogen synthase kinase (GSK)-3beta was observed in a subpopulation of proliferative lesions, in parallel with the cyclins and Cdk2. Nuclear immunoreactivity of phosphorylated and inactive retinoblastoma (Rb) protein was also increased in association with lesion development, with carcinomas showing increased cytoplasmic localization. The results suggest that proliferative lesions activate the cell cycle machinery following tumor promotion via a regulatory mechanism involving inactivation of GSK3beta and Rb protein, the latter signaling mechanism involving its aberrant nucleocytoplasmic transport for the acquisition of a malignant phenotype.

Inhibitory effects of aminoguanidine on thyroid follicular carcinoma development in inflamed capsular regions of rats treated with sulfadimethoxine after N-bis(2-hydroxypropyl)nitrosamine-initiation.

We have reported that thyroid capsular thickening with inflammation induced by an antithyroidal agent, sulfadimethoxine (SDM), might play a role in the development of invasive follicular carcinomas in rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). Inducible nitric oxide synthase (iNOS) expressed in the inflamed capsular regions further appeared to be implicated in the tumor progression. In the present study, the effects of an iNOS inhibitor, aminoguanidine (AG), on thyroid carcinogenesis were examined. F344 male rats were treated with SDM in drinking water (0.1%) with or without concomitant dietary administration of AG (0.2%) for 4 and 10 weeks after subcutaneous injection of DHPN at 2800 mg/kg bodyweight. At week 4, thyroid capsular thickening with inflammation was observed and iNOS-positive foci were found in the inflamed regions. In addition, single-strand DNA-positive inflammatory cells were scattered among neighboring follicular cells, indicating some cellular damage, at least partly in association with iNOS induction. Concurrent dietary administration of AG with SDM treatment slightly decreased the number of single-strand DNA-positive cells but did not alter the incidence and multiplicity of iNOS-positive foci in the inflamed capsular regions at week 4. At week 10, however, invasive follicular carcinomas predominantly arose in the thickened capsule in the DHPN-SDM-treated rats, and AG administration decreased (P < 0.05) their multiplicity. The carcinoma cells were partly positive for iNOS. These results thus suggested that iNOS induction in both inflammatory and tumor cells might play pivotal roles in tumor progression in this DHPN-SDM rat model.

Mechanism of trifluralin-induced thyroid tumors in rats.

Trifluralin, an herbicide, has been reported to cause a significant increase in thyroid follicular cell tumors in male Fischer 344 rats. This study was designed to determine the mechanism of thyroid hyperactivity after trifluralin exposure. A group of 15 male Fischer 344 rats were exposed to trifluralin-fortified (6500 ppm) diet for 2 weeks. The time weighted average daily intake of trifluralin was 441+/-77 mg/kg/day. Ten rats of the group were sacrificed and the sera analyzed for T3, T4, and TSH levels. The livers were also analyzed for selected T4-specific UGT gene expression and total UGT enzyme activity. In the trifluralin treated rats, the serum T3 and T4 levels decreased by 17% and 90%, respectively and TSH increased by 37% more than the control rats. Trifluralin-induced total hepatic UGT enzymes (2.4-fold) and mRNA expression of selected hepatic UGT isozymes (UGT1A1, 1.4-fold; UGT1A6, 6.4-fold; UGT2B1, 3.7-fold). For the remaining 5 rats in the group, bile was collected for 2 h and analyzed for free and conjugated T3 and T4. The total amount of T4 in bile more than doubled in trifluralin treated rats. Trifluralin treatment increased bile flow, caused a 3.2-fold increase in biliary elimination of conjugated T4 and 63% increase in conjugated T3. Based on these data, the decrease in total serum T3 and T4 levels in the trifluralin treated rats was due to enhanced peripheral metabolism and an increase in bile flow that results in a compensatory increase in TSH synthesis and secretion. The increased levels of TSH with chronic exposure to trifluralin would exert a continuous stimulation of the thyroid gland leading to cellular hypertrophy and proliferation predisposing to the development of follicular cell tumors in rats.

Depression of T cell-mediated immunity reduces sulfadimethoxine-induced capsular inflammation and inhibits associated development of invasive thyroid follicular cell carcinomas in rats.

We previously demonstrated that thyroid capsular inflammation induced by continuous treatment with the antithyroidal agent sulfadimethoxine is associated with development of invasive follicular cell carcinomas in rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). The inflammatory changes are characterized by large numbers of macrophages and lymphocytes as well as fibroblasts and we hypothesized that it might be enhanced by interplay between macrophages and T cells. To clarify this hypothesis, a comparative study was conducted between athymic nude (rnu/rnu) rats and euthymic (rnu/+) littermates initiated with DHPN (2800 mg/kg, s.c.) followed by sulfadimethoxine treatment in drinking water (0.1%) for 10 weeks. In rnu/+rats, marked capsular thickening with inflammation was induced along with invasive follicular cell carcinomas (2.8 +/- 1.3/rat). In rnu/rnu rats, limited fibrous capsular thickening was noted with or without minimal inflammatory change, and the multiplicity of invasive carcinomas was significantly lower (1.1 +/- 1.0/rat, P < 0.01). Inducible nitric oxide synthase expression in the inflamed lesions was detected in three of 10 rnu/+rats but in none of the rnu/rnu animals. The results thus suggest that development of invasive carcinomas is enhanced by capsular inflammation mediated by T cells, and inducible nitric oxide synthase induction may play a role in tumor progression.

DNA fragmentation and DNA repair synthesis induced in rat and human thyroid cells by chemicals carcinogenic to the rat thyroid.

Five chemicals that are known to induce in rats thyroid follicular-cell adenomas and carcinomas were assayed for their ability to induce DNA damage and DNA repair synthesis in primary cultures of human thyroid cells. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the same Comet assay, were obtained after a 20-h exposure to the following subtoxic concentrations of the five test compounds: methimazole from 2.5 to 10mM; nitrobenzene, potassium bromate, N,N'-diethylthiourea and ethylenethiourea from 1.25 to 5mM. Under the same experimental conditions, DNA repair synthesis, as evaluated by quantitative autoradiography, was present in potassium bromate-exposed thyroid cells from all the three donors and in those from two of three donors with either nitrobenzene or ethylenethiourea, but did not match the criteria for a positive response in thyroid cells from any of the donors with methimazole and N,N'-diethylthiourea. Consistently with their ability to induce thyroid tumors, all the five test compounds, administered p.o. in rats in a single dose corresponding to 1/2 LD50, induced a statistically significant degree of DNA fragmentation in the thyroid. These findings suggest that the five test compounds might be carcinogenic to thyroid in humans.

Acrylamide, an in vivo thyroid carcinogenic agent, induces DNA damage in rat thyroid cell lines and primary cultures.

Chronic treatment of rats with acrylamide induces various tumors among which thyroid tumors are the most frequent. The aim of the present study was to develop an in vitro model of acrylamide action on thyroid cells to allow the investigation of the mechanism of this tumorigenic action. The first part of the study considered as targets, characteristics of thyroid metabolism, which could explain the thyroid specificity of acrylamide action: the cAMP mitogenic effect and the important H2O2 generation by thyroid cells. However, acrylamide did not modulate H2O2 or cAMP generation in the thyroid cell models studied. No effect on thyroid cell proliferation was observed in the rat thyroid cell line FRTL5. On the other hand, as shown by the comet assay, acrylamide induced DNA damage, as the positive control H2O2 in the PC Cl3 and FRTL5 rat thyroid cell lines, as well as in thyroid cell primary cultures. The absence of effect of acrylamide on H2AX histone phosphorylation suggests that this effect does not reflect the induction of DNA double strand breaks. DNA damage leads to the generation of mutations. It is proposed that such mutations could play a role in the carcinogenic effect of acrylamide. The mechanism of this effect can now be studied in this in vitro model.

Development of invasive follicular cell carcinomas in a rat thyroid carcinogenesis model: biological impact of capsular inflammation and reduced cyclooxygenase-2 expression.

We have previously reported that thyroid capsular inflammation induced by sulfadimethoxine (SDM), a goitrogen, might play a role in development of invasive follicular cell adenocarcinomas in rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). The present study was designed to examine the role of cyclooxygenase (COX)-2, widely known to be up-regulated in inflammatory states, during chemically induced rat thyroid carcinogenesis. Male F344 rats received a subcutaneous DHPN (2800 mg/kg) injection, and 1 week later were allowed free access to drinking water containing antithyroidal propylthiouracil (PTU, 0.003%) or SDM (0.1%) for 4 or 10 weeks. Control groups receiving goitrogen alone and no treatment were also included. At week 4, diffuse follicular cell hyperplasia was induced in all PTU- and SDM-treated groups, along with fibrous capsular thickening and capsular thickening with inflammation, respectively. Additionally, multiple focal follicular cell hyperplasias and adenomas were observed in the DHPN + PTU and DHPN + SDM cases. At week 10, adenocarcinomas invasive to the capsule and restricted to the capsular adjacent region, were frequent in the DHPN + SDM group, but not observed in the animals given DHPN + PTU. Western blots and immunohistochemistry revealed constitutive COX-2 expression in non-neoplastic follicular cells of the control and all of the PTU- and SDM-treated rats. However, COX-2 reactivity was significantly reduced or negative in the preneoplastic/neoplastic lesions in the DHPN-treated groups. In fibrous or inflamed thickened capsules, only a few component cells with inflammatory elements were positive for COX-2, and there was no significant difference in this regard between the PTU and SDM treatments. The present results suggest that capsular inflammation could play a role in development of invasive carcinomas, but COX-2 expression does not make a major contribution.

Increase in thyroid follicular cell tumors in nelfinavir-treated rats observed in a 2-year carcinogenicity study is consistent with a rat-specific mechanism of thyroid neoplasia.

The carcinogenic potential of nelfinavir mesylate (nelfinavir) was evaluated in a 2-year oral (gavage) study on Sprague-Dawley rats at dose levels of 0 (control), 0 (vehicle control), 100, 300 and 1000 mg/kg per day. At the end of the treatment, increased incidences of thyroid follicular cell hyperplasia and neoplasms were observed at 300 (males) and 1000 mg/kg per day (both sexes). There were no other treatment-related effects and no tumors at other sites. Results from previous studies indicated a number of effects in the liver and thyroid, as well as metabolic profiles that suggested nelfinavir might cause thyroid hyperplasia/neoplasia secondary to hormone imbalance by altering thyroid hormone disposition. To investigate this hypothesis, the effects of nelfinavir on gene expression in rat hepatocytes and liver slices (in vitro), thyroxine plasma clearance, and thyroid gland function were evaluated. Compared to controls, gene expression analyses demonstrated an increased expression of glucuronyltransferase (UDPGT) and CYP450 3A1 in nelfinavir-treated rat hepatocytes and liver slices. In rats treated with nelfinavir (1000 mg/kg per day) for 4 weeks, liver weights and centrilobular hepatocellular hypertrophy were increased and minimal to mild diffuse thyroid follicular cell hypertrophy and follicular cell hyperplasia were evident in the thyroid gland. Thyroid-stimulating hormone (TSH) levels were significantly increased (three-fold), while tri-iodothyronine (T3)/tetra-iodothyronine (T4) and reverse T3(rT3) levels were unchanged, indicating that a compensated state to maintain homeostasis of T3/T4 had been achieved. Plasma 125I-thyroxine clearance was increased and the plasma thyroxine AUC0-48 was decreased (24%) compared to control. In conclusion, these data indicate that thyroid neoplasms observed in the nelfinavir-treated rats were secondary to thyroid hormone imbalance. Increased thyroxine clearance contributes to the effects of nelfinavir on thyroid gland function and is probably a result of UDPGT induction that leads to elevated TSH levels in the rat and eventual thyroid neoplasia. These results are consistent with a well-recognized rat-specific mechanism for thyroid neoplasms.

Sequential analysis of development of invasive thyroid follicular cell carcinomas in inflamed capsular regions of rats treated with sulfadimethoxine after N-bis(2-hydroxypropyl)nitrosamine-initiation.

A 2-stage thyroid follicular carcinogenesis model in rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN) is widely used to detect modifying effects of chemicals on thyroid carcinogenesis. A number of goitrogens are known to strongly promote carcinogenesis, and the carcinomas often originate adjacent to the thyroid capsule and show invasive growth into the capsule or adjacent tissues. To clarify mechanisms of progression to invasive carcinomas, we sequentially evaluated histopathological and immunohistochemical characteristics of thyroids in male F344 rats treated with sulfadimethoxine (SDM, 0.1% in drinking water) for 0-10 weeks beginning 1 week after DHPN initiation (2800 mg/kg body weight, single s.c. injection). In DHPN-SDM-treated rats, multiple focal hyperplasias and adenomas developed in thyroid follicular parenchyma at weeks 4 to 6. Apart from the proliferative lesions, capsular thickening with inflammatory cell infiltration, mainly consisting of macrophages, and migration of follicular epithelium into the capsule were also observed. Focal hyperplasias/adenomas adjacent to the capsule progressively developed to invasive carcinomas at weeks 6 to 10. In thyroid parenchyma, malignant lesions were seldom observed. With SDM-treatment alone, although no neoplastic lesions were observed, capsular thickening with inflammation and epithelial migration resulted in intracapsular residual follicles. Intracapsular residual follicular cells as well as invasive and intrathyroidal carcinoma cells generally showed increased cell proliferative activity, coincidental with cytoplasmic/nuclear positivity for beta-catenin. These results suggested that beta-catenin activation related to capsular inflammation may play a role in development of invasive carcinomas but is insufficient for tumor formation by itself. Whether this is associated with mutations in the beta-catenin gene remains to be clarified.

Ki-ras mutational analysis in rat follicular-cell proliferative lesions of the thyroid gland induced by radioactive iodine and potassium perchlorate.

Although in both human and experimental pathology ras mutations have been related to the origin and progression of follicular-cell tumours, reports differ considerably with respect to the frequency of such mutations. The present paper reports, using direct sequencing, the incidence of Ki-ras mutations (codons 12 and 13) in follicular-cell carcinomas of the thyroid gland in Wistar rats induced by administration of radioactive iodine and potassium perchlorate. Direct sequencing revealed no mutations in the amplified gene segment of any of the 72 carcinoma samples studied. This absence of mutations agrees with some and is in sharp contrast with other previous reports in the literature, both for experimental animals and in studies of human thyroid follicular-cell carcinoma. Our results suggest that Ki-ras activation via mutations at codons 12 and 13 is neither a constant event nor an early event in the development of rat thyroid follicular-cell carcinoma.

Synergistic interaction between excess caffeine and deficient iodine on the promotion of thyroid carcinogenesis in rats pretreated with N-bis(2-hydroxypropyl)nitrosamine.

The combined effects of caffeine (1,3,7-trimethylxanthine) with iodine deficiency (ID) were examined in a rat two-stage thyroid carcinogenesis model using N-bis(2-hydroxypropyl)nitrosamine (DHPN). Male F344 rats were divided into 6 groups each consisting of 10 animals, and received a single s.c. injection of 2800 mg/kg DHPN. From 1 week after the DHPN initiation, the rats were respectively fed a basal diet in which the protein was exchanged for 20% gluten, containing 1500 ppm caffeine + ID, 300 ppm caffeine + ID, 60 ppm caffeine + ID, 1500 ppm caffeine or ID or a basal diet alone for 12 weeks. Relative thyroid weights were significantly (P < 0.05) increased due to the development of proliferative lesions induced by the ID diet as compared to the DHPN-alone group value, which was enhanced by caffeine, albeit without statistical significance. Relative pituitary weights were significantly (P < 0.05) increased with 300 or 1500 ppm caffeine + ID as compared to the DHPN-alone group value. Serum thyroid stimulating hormone (TSH) levels were slightly increased by ID, an effect which was further enhanced by 300 or 1500 ppm caffeine. Serum thyroxine (T(4)) levels were slightly increased by caffeine or ID alone, but decreased by caffeine with ID. Histopathologically, thyroid follicular carcinomas were found only in the 1500 ppm caffeine + ID group, although thyroid follicular adenomas were detected in all the ID-treated groups. The multiplicity of focal thyroid follicular hyperplasias was significantly (P < 0.05) increased by 1500 ppm caffeine. These results indicate that caffeine may synergistically promote thyroid carcinogenesis with ID partially through a pituitary-dependent pathway in rats, implying the possible implication of routine caffeine intake in the promotion of thyroid carcinogenesis.

Promoting effects of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone on rat glandular stomach carcinogenesis initiated with N-methyl-N'-nitro-N-nitrosoguanidine.

The modifying effects of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a mutagenic by-product in chlorinated water, on the development of glandular stomach cancers were investigated in Wistar rats. A total of 120 males, 6 weeks of age, were divided into six groups. After initiation with 100 ppm N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) solution and 5% NaCl diet for 8 weeks, 30 rats each in groups 1-3 were given MX in the drinking water at concentrations of 30, 10, or 0 ppm for the following 57 weeks. Ten animals each in groups 4-6 were administered the MX without prior carcinogen exposure. There were no statistical significant differences in final body weights between the groups. The incidences and multiplicities of adenocarcinomas in the glandular stomachs were significantly higher (P < 0.05) in the initiated 30 ppm MX group than those in the MNNG/NaCl group. The incidences of atypical hyperplasias in the glandular stomachs were also significantly increased (P < 0.05 or 0.01) by the MX treatments. With their multiplicity, the effects were clearly dose dependent. Interestingly, the 30 ppm MX alone itself induced atypical hyperplasias in the pylorus, although the incidences and severity were low. Moreover, MX showed a tendency to enhance the development of intrahepatic cholangiocellular tumors and thyroid follicular cell tumors in the MNNG-treated animals. The results of the present study thus indicate that MX exerts promoting effects when given during the postinitiation phase of two-stage glandular stomach carcinogenesis in rats.

Mode of carcinogenic action of pesticides inducing thyroid follicular cell tumors in rodents.

Of 240 pesticides screened for carcinogenicity by the U.S. Environmental Protection Agency Office of Pesticide Programs, at least 24 (10%) produce thyroid follicular cell tumors in rodents. Thirteen of the thyroid carcinogens also induce liver tumors, mainly in mice, and 9 chemicals produce tumors at other sites. Some mutagenic data are available on all 24 pesticides producing thyroid tumors. Mutagenicity does not seem to be a major determinant in thyroid carcinogenicity, except for possibly acetochlor; evidence is less convincing for ethylene thiourea and etridiazole. Studies on thyroid-pituitary functioning, including indications of thyroid cell growth and/or changes in thyroxine, triiodothyronine, or thyroid-stimulating hormone levels, are available on 19 pesticides. No such antithyroid information is available for etridiazole, N-octyl bicycloheptene dicarboximide, terbutryn, triadimefon, and trifluralin. Of the studied chemicals, only bromacil lacks antithyroid activity under study conditions. Intrathyroidal and extrathyroidal sites of action are found: amitrole, ethylene thiourea, and mancozeb are thyroid peroxidase inhibitors; and acetochlor, clofentezine, fenbuconazole, fipronil, pendimethalin, pentachloronitrobenzene, prodiamine, pyrimethanil, and thiazopyr seem to enhance the hepatic metabolism and excretion of thyroid hormone. Thus, with 12 pesticides that mode of action judgments can be made, 11 disrupt thyroid-pituitary homeostasis only; no chemical is mutagenic only; and acetochlor may have both antithyroid and some mutagenic activity. More information is needed to identify other potential antithyroid modes of thyroid carcinogenic action.

Risk assessment of thyroid follicular cell tumors.

Thyroid follicular cell tumors arise in rodents from mutations, perturbations of thyroid and pituitary hormone status with increased stimulation of thyroid cell growth by thyroid-stimulating hormone (TSH), or a combination of the two. The only known human thyroid carcinogen is ionizing radiation. It is not known for certain whether chemicals that affect thyroid cell growth lead to human thyroid cancer. The U.S. Environmental Protection Agency applies the following science policy positions: 1) chemically induced rodent thyroid tumors are presumed to be relevant to humans; 2) when interspecies information is lacking, the default is to assume comparable carcinogenic sensitivity in rodents and humans; 3) adverse rodent noncancer thyroid effects due to chemically induced thyroid-pituitary disruption are presumed to be relevant to humans; 4) linear dose-response considerations are applied to thyroid cancer induced by chemical substances that either do not disrupt thyroid functioning or lack mode of action information; 5) nonlinear thyroid cancer dose-response considerations are applied to chemicals that reduce thyroid hormone levels, increase TSH and thyroid cell division, and are judged to lack mutagenic activity; and 6) nonlinear considerations may be applied in thyroid cancer dose-response assessments on a case-by-case basis for chemicals that disrupt thyroid-pituitary functioning and demonstrate some mutagenic activity. Required data for risk assessment purposes is mode of action information on mutagenicity, increases in follicular cell growth (cell size and number) and thyroid gland weight, thyroid-pituitary hormones, site of action, correlations between doses producing thyroid effects and cancer, and reversibility of effects when dosing ceases.

Recent developments in the investigation of thyroid regulation and thyroid carcinogenesis.

This review covers new mechanistic information spanning the past 10 years relevant to normal and abnormal thyroid growth and function that may assist in the risk assessment of chemicals inducing thyroid follicular cell neoplasia. Recent studies have shown that thyroid regulation occurs via a complex interactive network mediated through several different messenger systems. Increased thyroid-stimulating hormone (TSH) levels activate the signal transduction pathways to stimulate growth and differentiation of the follicular cell. The important role of TSH in growth as well as in function helps to explain how disruptions in the thyroid-pituitary axis may influence thyroid neoplasia in rodents. New investigations that couple mechanistic studies with information from animal cancer bioassays (e. g., sulfamethazine studies) confirm the linkage between prolonged disruption of the thyroid-pituitary axis and thyroid neoplasia. New initiation/promotion studies in rodents also support the concept that chronic stimulation of the thyroid induced by goitrogens can result in thyroid tumors. Some of these studies confirm previous suggestions regarding the importance of chemically induced thyroid peroxidase inhibition and the inhibition of 3,3',5, 5'-tetraiodothyronine (T4, thyroxine) deiodinases on disruption of the thyroid-pituitary axis leading to thyroid neoplasia. Some comparative physiologic and mechanistic data highlight certain differences between rodents and humans that could be expected to confer an increased vulnerability of rodents to chronic hypersecretion of TSH. New data from epidemiologic and molecular genetic studies in humans contribute further to an understanding of thyroid neoplasia. Acute exposure to ionizing radiation, especially in childhood, remains the only verified cause of thyroid carcinogenesis in humans. Iodine deficiency studies as a whole remain inconclusive, even though several new studies in humans examine the role of dietary iodine deficiency in thyroid cancer. Specific alterations in gene expression have been identified in human thyroid neoplasia, linked to tumor phenotype, and thus oncogene activation and tumor-suppressor gene inactivation may also be factors in the development and progression of thyroid cancer in humans. An analysis by the U.S. EPA Risk Assessment Forum, prepared as a draft report in 1988 and completed in 1997, focused on the use of a threshold for risk assessment of thyroid follicular tumors. New studies, involving several chemicals, provide further support that there will be no antithyroid activity until critical intracellular concentrations are reached. Thus, for chemically induced thyroid neoplasia linked to disruptions in the thyroid-pituitary axis, a practical threshold for thyroid cancer would be expected. More information on thyroid autoregulation, the role of oncogene mutations and growth factors, and studies directly linking persistently high TSH levels with the sequential cellular development of thyroid follicular cell neoplasia would provide further confirmation.

Follicular cell carcinoma of the thyroid in a child after bone marrow transplantation for acute lymphoblastic leukemia.

Follicular cell carcinoma (FCC) of the thyroid is rarely found during childhood. We report a 12 1/2-year-old girl with acute lymphoblastic leukemia, who developed rapidly growing FCC of the thyroid, 3 years after bone marrow transplantation. The role of chemotherapy in the induction of such secondary tumors after transplantation is discussed, and a proposal for the approach to these patients is suggested.