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.

Toxicologic evaluation of bumetanide, potent diuretic agent.

The studies reported represent the preclinical toxicologic evaluation of bumetanide. Toxicity testing in rats, rabbits, dogs, and baboons indicate that bumetanide is well tolerated in various species. The toxicity that was observed is, for the most part, the direct consequence of its potent diuretic activity and is similar to that produced by other "loop" diuretics. Bumetanide was fetotoxic at maternal toxic doses in the rabbit, but no evidence of teratogenic effects was observed in rabbits, rats, mice, or hamsters. The compound was devoid of mutagenic activity by the Ames test and was found to be noncarcinogenic in long-term rat studies.

Vitamin D3-induced proliferative lesions in the rat adrenal medulla.

Adrenal medullary hyperplasia and pheochromocytomas are induced in rats by a variety of non-genotoxic agents, and we have hypothesized that these agents induce lesions indirectly by stimulating chromaffin cell proliferation. Vitamin D3, which has not been previously associated with adrenal medullary proliferative lesions, is the most potent in vivo stimulus to chromaffin cell proliferation yet identified. The present investigation utilized the vitamin D3 model to prospectively test the relationship between mitogenicity and focal proliferative lesions in the adrenal medulla and to determine early events in the pathogenesis of these lesions. Charles River Crl:CD BR rats were treated with 0; 5000; 10,000; or 20,000 IU/kg/day of vitamin D3 in corn oil (5 ml/kg) by oral intubation. Rats were killed after 4, 8, 12, or 26 weeks of treatment, following a final week of labeling with bromodeoxyuridine (BrdU) using a mini-pump. Adrenal sections were double-stained for BrdU and phenylethanolamine-N-methyl transferase (PNMT) to discriminate epinephrine (E) from norepinephrine (NE) cells or for vesicular acetylcholine transporter (VAchT) to identify cholinergic nerve endings. Vitamin D3 caused a 4-5-fold increase in BrdU labeling at week 4, diminishing to a 2-fold increase by week 26. An initial preponderance of labeled E cells gave way to a preponderance of labeled NE cells. By week 26, 17/19 (89%) animals receiving the 2 highest doses of vitamin D3 had focal adrenal medullary proliferative lesions, in contrast to an absence of lesions in control rats. The lesions encompassed a spectrum including BrdU-labeled "hot spots" not readily visible on H and E sections, hyperplastic nodules, and pheochromocytomas. Lesions were usually multicentric, bilateral, and peripheral in location, and almost all were PNMT-negative. The lesions were not cholinergically innervated, suggesting autonomous proliferation. Hot spots, hyperplastic nodules, and pheochromocytomas appear to represent a continuum rather than separate entities. Their development might involve selective responses of chromaffin cell subsets to mitogenic signals, influenced by both innervation and corticomedullary interactions. A number of non-genotoxic compounds that induce pheochromocytomas in rats are known to affect calcium homeostasis. The results of this study provide further evidence to support the hypothesis that altered calcium homeostasis is indirectly involved in the pathogenesis of pheochromocytomas, via effects on chromaffin cell proliferation.

Thyroid gland neoplasia: non-genotoxic mechanisms.

There are two basic mechanisms whereby chemicals produce thyroid gland neoplasia in rodents. The first involves chemicals that exert a direct carcinogenic effect in the thyroid gland and the other involves chemicals which, through a variety of mechanisms, disrupt thyroid function and produce thyroid gland neoplasia secondary to hormone imbalance. There are important species differences in thyroid gland physiology between rodents and humans which may account for a marked species difference in the inherent susceptibility for neoplasia secondary to hormone imbalance. Thus, it is important to consider mechanism in the evaluation of potential cancer risks. There would be little if any risk for apparently nongenotoxic chemicals that act secondary to hormone imbalance at exposure levels that do not disrupt thyroid function. Further, the degree of thyroid dysfunction produced by a chemical would present a major toxicological problem before such exposure would increase the risk for neoplasia for humans.

Stereospecific assay and stereospecific disposition of racemic carprofen in rats.

A procedure was developed for the separation and selective quantitative determination of the (S)(+)- and (R)(-)-enantiomers of the racemic anti-inflammatory drug carprofen as their diastereomeric l-(-)-alpha-methylbenzylamides. These derivatives are obtained in equivalent yields by reacint purified 14C-carprofen from biological specimens with l-(-)-alpha-methylbenzylamine via the 1,1'-carbonyldiimidazole intermediate, followed by extraction and differential radiometric quantitation of the TLC-separated diastereomers. In the rat, the (R)(-)-carprofen enantiomer was eliminated from blood and secreted in the bile as the ester glucuronide at a rate approximately twice that of the (S)-(+)-enantiomer, resulting in the accumulation of the pharmacologically more active (S)(+)-enantiomer in the rat blood. Evidence for an additional process favoring the elimination of the (R)(-)-enantiomer in the rat was derived from pharmacokinetic data evaluation.

Mechanistic considerations for the relevance of animal data on thyroid neoplasia to human risk assessment.

There are two basic mechanisms whereby chemicals produce thyroid gland neoplasia in rodents. The first involves chemicals that exert a direct carcinogenic effect in the thyroid gland and the other involves chemicals which, through a variety of mechanisms, disrupt thyroid function and produce thyroid gland neoplasia secondary to hormone imbalance. These secondary mechanisms predominantly involve effects on thyroid hormone synthesis or peripheral hormone disposition. There are important species differences in thyroid gland physiology between rodents and humans that may account for a marked species difference in the inherent susceptibility for neoplasia to hormone imbalance. Thyroid gland neoplasia, secondary to chemically induced hormone imbalance, is mediated by thyroid-stimulating hormone (TSH) in response to altered thyroid gland function. The effect of TSH on cell proliferation and other aspects of thyroid gland function is a receptor mediated process and the plasma membrane surface of the follicular cell has receptors for TSH and other growth factors. Small organic molecules are not known to be direct TSH receptor agonists or antagonists; however, various antibodies found in autoimmune disease such as Graves' disease can directly stimulate or inhibit the TSH receptor. Certain chemicals can modulate the TSH response for autoregulation of follicular cell function and thereby increase or decrease the response of the follicular cell to TSH. It is thus important to consider mechanisms for the evaluation of potential cancer risks. There would be little if any risk for non-genotoxic chemicals that act secondary to hormone imbalance at exposure levels that do not disrupt thyroid function. Furthermore, the degree of thyroid dysfunction produced by a chemical would present a significant toxicological problem before such exposure would increase the risk for neoplasia in humans.

The significance of hepatic microsomal enzyme induction and altered thyroid function in rats: implications for thyroid gland neoplasia.

Hepatic microsomal enzymes play an important role in thyroid hormone homeostasis. Glucuronidation of thyroxine is the rate limiting step in the biliary excretion of thyroxine in the rat; the monodeiodinases are important in the conversion of T4 to T3 and reverse T3 and subsequent deiodinations. Phenobarbital is known to affect thyroid function in rats due to an alteration of hormone disposition. We have further characterized these effects and have demonstrated that phenobarbital increases the biliary excretion of thyroxine glucuronide primarily as a result of an induction of hepatic thyroxine glucuronyltransferase. Studies on the mode of action for phenobarbital promotion of thyroid follicular neoplasia were conducted using an initiation-promotion model established by Hiasa et al (35). In this model, we demonstrated that supplemental administration of thyroxine blocked the promoting effect of phenobarbital and furthermore, using various dosages of thyroxine, we observed that the tumor promoting effect of phenobarbital was directly proportional to the level of plasma TSH. The results of these studies support the hypothesis that the tumor promoting effect of phenobarbital in the thyroid gland is mediated via increased secretion of pituitary TSH as a compensatory response to the known effects of phenobarbital on peripheral thyroid hormone disposition. Since a number of microsomal enzyme inducing agents have increased the incidence of thyroid follicular neoplasia in rat carcinogenicity studies, thyroid function should be assessed and a secondary mechanism of hormone imbalance should be considered in the interpretation of the significance of these findings in rodents.

Potentiation of the teratogenic effects and altered disposition of diphenylhydantoin in mice fed a purified diet.

The effect of a standardized purified diet (AIN-76) on the teratogenic response to diphenylhydantoin (DPH) was studied in mice. Mice were fed either the purified diet or Purina Rodent Laboratory Chow for 2-4 weeks prior to mating, and were treated with either saline or 50 mg/kg of DPH on Days 12, 13, and 14 of gestation (copulatory plug = Day 0). The teratogenic response to DPH was found to be markedly potentiated in mice fed the purified diet (75% cleft palate) as compared to mice fed rodent chow (21% cleft palate). The potentiated teratogenic response to DPH correlated with markedly higher plasma DPH levels in pregnant mice fed the purified diet, indicating that the disposition of DPH was impaired. These effects were attributed to a decreased basal level of drug-metabolizing enzymes in mice fed the purified diet, as indicated by markedly prolonged hexobarbital sleeping times. Modifications of the purified diet, which included the replacement of soluble carbohydrate (50% sucrose) in the purified diet with either cornstarch or casein, did not alter the high incidence of cleft palate. A reduction in the incidence of cleft palate was observed, however, when corn oil in the purified diet was replaced with linseed oil. The replacement of corn oil with linseed oil in the purified diet also restored the hexobarbital sleeping times to those observed in mice fed rodent chow. It is concluded that mice fed purified diets have decreased basal levels of drug-metabolizing activity that alter the disposition of DPH and, as a consequence, potentiate its teratogenic effects.

Reproduction studies in rats treated with ornidazole.

Reproduction studies were performed with ornidazole, a compound with trichomonacidal activity. Male rats were treated for 61 days prior to mating and female rats were treated for 2 weeks prior to mating and throughout gestation and lactation at doses of 0 (control), 25, 100, and 400 mg of ornidazole/kg/day. A decrease in the pregnancy rate was observed in high-dose rats without altered mating performance. Crossover matings between high-dose treated and control male and female rats showed that male but not female fertility was affected and that the effect on fertility was reversible within several days after the cessation of treatment. Testicular and epididymal weights were not altered in treated male rats. Histopathological examination revealed that spermatogenesis and the testes were normal and that the epididymides of treated male rats contained normal appearing sperm. It is concluded that ornidazole, at high dosages, produces infertility in the male rat; however, unlike many other 5-nitroimidazole compounds which are reported to inhibit spermatogenesis, no effect on spermatogenesis was observed under the conditions of these studies. This in conjunction with the rapid reversibility of infertility suggests that the mode of action of ornidazole involves a rapidly reversible effect on epididymal sperm function.

The effect of ornidazole on fertility and epididymal sperm function in rats.

A comprehensive study of male fertility and sperm production and function was performed in 20 control and 20 rats treated with ornidazole, a compound with trichomonacidal activity. Rats were treated for 4 weeks at dosages of 0 (control) and 400 mg/kg/day of ornidazole during which fertility was assessed by weekly matings. Testicular sperm production and epididymal sperm function were assessed in one-half of the rats while the reversibility of effects after a 2-week recovery period was assessed in the remaining half. Male rats treated with ornidazole were infertile during the second week of treatment. After 4 weeks of treatment, testicular and epididymal weights, testicular spermatid counts, epididymal sperm reserves, sperm morphology, and sperm viability were similar in treated and control rats. A quantitative assessment of epididymal sperm motility using a dark-field photomicroscope with a stroboscopic light source revealed that ornidazole markedly inhibited sperm motility. Although the percentage of nonmotile sperm was not substantially increased in treated rats, the vigor of tail movement was markedly decreased which resulted in decreased sperm velocity. Restoration of fertility and normal sperm motility and velocity were observed in the group of recovery rats assessed 2 weeks after the cessation of ornidazole treatment. It is concluded that ornidazole, at a high dosage of 400 mg/kg/day, produces infertility in male rats by inhibiting epididymal sperm motility in terms of decreased sperm velocity. These effects are rapidly reversible after the cessation of treatment.

Comparison of effect of tumor promoter treatments on DNA methylation status and gene expression in B6C3F1 and C57BL/6 mouse liver and in B6C3F1 mouse liver tumors.

The effects of different liver tumor-promoting treatments (i.e., a choline-devoid, methionine-deficient (CMD) diet, phenobarbital (PB), or both) on Ha-ras and raf methylation status and expression were determined in mouse strains with different susceptibilities to liver tumor formation: the relatively sensitive B6C3F1 and the relatively resistant C57BL/6. Additionally, B6C3F1 mouse liver tumors, spontaneous or PB induced, were assessed for alterations in global DNA methylation status and expression of Ha-ras and raf. The CMD diet led to hypomethylation of Ha-ras and raf after 12 wk of administration in B6C3F1 and C57BL/6 mice. At this early phase of tumor promotion, the frequency of increased expression of both Ha-ras and raf mRNAs was higher in the B6C3F1 but not the C57BL/6 mice. This is a mechanism that may, in part, underlie the heightened sensitivity of the B6C3F1 mouse to liver tumorigenesis. Subpopulations of B6C3F1 mouse liver tumors displayed altered global methylation status, with both hypomethylation and hypermethylation evident. Carcinomas were significantly more hypomethylated than adenomas. The level of raf mRNA was not changed in spontaneous or PB-induced B6C3F1 mouse liver tumors. Increased expression of Ha-ras was evident in some spontaneous B6C3F1 liver tumors and in most of the PB-induced liver tumors. These experiments support the concept that altered DNA methylation plays a key role in tumorigenesis and indicate that the high propensity of the B6C3F1 mice to liver tumorigenesis may be due, in part, to a decreased ability to maintain normal methylation status.

Potentiation of methotrexate embryolethality by aspirin in rats.

The augmentation of methotrexate-induced embryotoxicity by aspirin was studied. Pregnant Charles River CD rats were given methotrexate or aspirin alone or in combination on gestation day 9 or 12. The frequency of fetal abnormalities was not affected and fetal body weight loss was not additive in the combined treatment. However, pretreatment with aspirin (200 mg/kg) significantly enhanced the embryolethality of methotrexate given at soes of 0.2 mg/kg on day 9 and 1.5 mg/kg on day 12. Studies with tritiated methotrexate in pregnant rats demonstrated that aspirin delayed the renal excretion of methotrexate and increased the concentrations of methotrexate in maternal plasma and the embryos. It is suggested that these effects are responsible for the observed potentiation of embryolethality.

Effect of metronidazole on fertility and testicular function in male rats.

The reproductive toxicology of metronidazole was studied in rats. Male Charles River Crl:CD(SD)BR rats (10/group) were treated with metronidazole as a dietary admixture at doses of 0 (control), 25, 100, and 400 mg/kg/day for 8 weeks. The reversibility of effects after a 3 1/2-month recovery period was determined in separate groups of 10 control and 10 rats treated with 400 mg/kg/day of metronidazole. After 2 and 4 weeks of metronidazole treatment, mating performance and fertility in treated and control animals were comparable. After 6 weeks of treatment, all high-dose rats were infertile; however, fertility in low- and middose rats was not affected. High-dose male rats killed after 8 weeks of treatment showed markedly decreased testicular and epididymal weights, and markedly decreased testicular spermatid counts and epididymal sperm counts. Most of the few epididymal sperm present in high-dose rats were viable, but morphologically abnormal. Histologically, severe degeneration of the seminiferous epithelium was observed in the testes of high-dose rats; the tubules were generally devoid of primary or secondary spermatocytes and spermatids. Rats treated with the low and middle doses of metronidazole exhibited normal testicular and epididymal weights, and normal testicular spermatid counts and epididymal sperm reserves. Epididymal sperm viability and morphology of treated and control animals were comparable. Minimal histologic changes were observed in the testes of middose rats, including degenerative fragmentation of spermatozoa and spermatids. In high-dose recovery rats, fertility was restored in most rats by 8 weeks after the cessation of treatment; however, the testicular and epididymal weights and sperm counts of rats killed after 3 1/2 months of recovery had increased but were still significantly decreased in treated rats as compared with controls. Histologically, spermatogenesis was observed in most tubules; however, a portion of atrophic tubules persisted. It is concluded that high doses of metronidazole produce infertility in the male rat through inhibition of spermatogenesis as early as the stage of the primary spermatocyte. This effect is partially reversible.

The effect of phenobarbital on the metabolism and excretion of thyroxine in rats.

The effect of phenobarbital on thyroid function and the metabolism and biliary excretion of thyroxine in rats was determined. Phenobarbital, administered for 2 weeks at a dose of 100 mg/kg/day, resulted in an increase in hepatic and thyroid gland weights, decreased circulating levels of T4, T3 and rT3, and increased TSH levels in male and female rats. After 3 months of treatment liver and thyroid weights were still increased; however, hormone values were not as markedly affected indicating that the rats had partially compensated for the effect on thyroid function. In thyroidectomized rats the plasma clearance of thyroxine was increased with phenobarbital. In bile duct cannulated phenobarbital-treated male rats the hepatic uptake at 4 hr was markedly increased. Bile flow was increased and the 4-hr cumulative biliary excretion of administered radioactivity was increased by 42%. Most of the increase in the excretion (76%) was accounted for by an increase in the excretion of thyroxine-glucuronide in phenobarbital-treated rats. Hepatic thyroxine-glucuronyltransferase activity in phenobarbital-treated rats expressed as picomoles per milligram of protein was increased by 40%; enzyme activity per gram of liver was increased by about twofold which, coupled with increased hepatic weight, resulted in about a threefold increase in total hepatic thyroxine-glucuronyltransferase activity in phenobarbital-treated rats as compared to that of controls. Qualitatively similar effects on metabolism, excretion, and enzyme induction were noted in female rats; however, the magnitude of increase was less than that observed in male rats. It is concluded that the effect of phenobarbital on thyroid function in rats is primarily a result of its effects on the hepatic disposition of thyroid hormone. The induction of thyroxine-glucuronyltransferase appears to play an important role in the increased metabolism and excretion of thyroxine in phenobarbital-treated rats.

Neurogenic signals regulate chromaffin cell proliferation and mediate the mitogenic effect of reserpine in the adult rat adrenal medulla.

The adrenal medulla is innervated by nerve fibers from several sources, which synapse on chromaffin cells and stimulate the secretion of catecholamines. The antihypertensive agent reserpine is known to reflexively increase this neurogenic stimulation by depleting catecholamine stores, and long-term administration of reserpine is associated with adrenal medullary hyperplasia and neoplasia. To determine the role of neurogenic signals in regulating normal and reserpine-stimulated proliferation of chromaffin cells, the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into replicating nuclei was assessed in the adrenal medulla of adult rats. Unilateral adrenal denervation caused a 4-5 fold decrease in chromaffin cell labeling by 5-bromo-2'-deoxyuridine during a 2-week labeling period. Denervation also prevented stimulation of labeling in animals receiving reserpine in their diet. These findings suggest that neurogenic control of cell proliferation may play an important role in the pathogenesis of adrenal medullary hyperplasia and neoplasia, and in the normal development of the peripheral and central nervous systems.

Sustained stimulation of rat adrenal chromaffin cell proliferation by reserpine.

Chronic administration of reserpine is associated with the development of pheochromocytomas in rats. Short-term administration of reserpine to rats has been shown to stimulate chromaffin cell proliferation, leading to the hypothesis that reserpine causes pheochromocytomas indirectly by providing a proliferative backdrop on which genetic damage may occur. However, it is not known whether the proliferative effects of reserpine persist long enough for this model to be tenable. In the present investigation, the effects of reserpine on bromodeoxyuridine (BrdU) incorporation into epinephrine (E)- and norepinephrine (NE)-type chromaffin cells were studied after 1, 4, and 12 weeks of reserpine administration. Reserpine administered in the diet at 10 or 50 ppm was shown to result in a persistent mitogenic stimulation of the rat adrenal medulla. Cells that incorporated BrdU at all time points appeared to be typical E- and NE-type chromaffin cells, and the ratio of BrdU-labeled E cells to BrdU-labeled NE cells was not altered by reserpine. An additional observation was that the ratio of all E cells to all NE cells declined after Week 1 and that the decline could be accelerated by administration of reserpine. This finding suggests that neural stimulation of chromaffin cells might play a role in age-related functional changes of the adrenal medulla during early adult life. The present observations support the hypothesis that reserpine induces pheochromocytomas indirectly by increasing chromaffin cell proliferation. They also decrease the likelihood that rat pheochromocytomas arise from preferential stimulation of proliferation of a particular cell type.

5-methylcytosine is present in the 5' flanking region of Ha-ras in mouse liver and increases with ageing.

Modifications to DNA-5-methylcytosine (5MeC) content (i.e., alterations in the level of 5MeC) constitute epigenetic events. In general, hypomethylation of a gene is necessary but not sufficient for expression, while methylated genes typically are quiescent. Ha-ras is an oncogene commonly implicated in murine liver tumorigenesis, often, though not always, involving mutation. A PCR-based approach using pre-PCR digestion with methylation-sensitive enzymes was employed to determine the 5MeC content of the 5' flanking region of this gene in (i) B6C3F1 and C57BL/6 mouse liver from young animals (4 months old) and (ii) B6C3F1 mouse liver from aged animals (24 months old). Two segments of the 5' flanking region of Ha-ras were examined. We demonstrate the presence of 5MeC in a portion of the 5' flanking region of Ha-ras that does not share characteristics of a CpG island, while a region that shares CpG island characteristics is primarily unmethylated. Differences in methylation status in these areas of Ha-ras were not observed between B6C3F1 and C57BL/6 mouse livers. Increases in methylation status were observed with ageing in B6C3F1 mouse liver. These data provide a role for methylation in regulating Ha-ras expression in mouse liver. Ha-ras in human liver has been reported to be unmethylated. There are substantial sequence differences in a key region of the 5' flanking region of Ha-ras in mice as compared to humans. These differences in DNA methylation and sequence may, in part, provide a basis for the frequent involvement of Ha-ras in mouse liver tumors and its virtual lack of involvement in human tumors.

Alterations in the methylation status and expression of the raf oncogene in phenobarbital-induced and spontaneous B6C3F1 mouse liver tumors.

The liver tumor-prone B6C3F1 mouse (C57Bl/6 female x C3H/He male), in conjunction with the more susceptible C3H/He paternal strain and the resistant C57BL/6 maternal strain, is an excellent model for studying the mechanisms involved in carcinogenesis. The study reported here indicated that the B6C3F1 mouse inherited a maternal raf allele containing a methylated site not present in the paternal allele. Seven days after partial hepatectomy or after administration of a promoting dose of phenobarbital (PB) for 14 d; raf in B6C3F1 mouse liver was hypomethylated. The additional methylated site in the allele inherited from C57BL/6 was not maintained. The methylation status of raf in the liver of the C57BL/6 mouse was not affected by PB treatment. This indicates that the B6C3F1 mouse is less capable of maintaining methylation of raf than the C57BL/6 strain is. In both PB-induced and spontaneous B6C3F1 liver tumors, raf was hypomethylated in a nonrandom fashion. The level of raf mRNA increased in seven of 10 PB-induced tumors but in only one of five spontaneous tumors, whereas the level of Ha-ras mRNA increased in nine of 10 PB-induced tumors and in four of five spontaneous tumors. The results of our investigation (a) support the hypothesis that hypomethylation of DNA is a nongenotoxic mechanism involved in tumorigenesis, (b) support the notion that PB promotes liver tumors that develop along a pathway different from that leading to spontaneous tumors, and (c) indicate that differences in DNA methylation between C57BL/6 and B6C3F1 mice could, in part, account for the unusually high tendency of the latter strain to develop liver tumors.

Studies on the mode of action for thyroid gland tumor promotion in rats by phenobarbital.

A study was conducted to determine the mode of action for phenobarbital promotion of thyroid follicular cell neoplasia in rats using an initiation-promotion model established by Hiasa et al. (Y. Hiasa, Y. Kitahori, M. Ohshima, T. Fujita, T. Yuasa, N. Konishi, and A. Miyashiro, 1982a, Carcinogenesis 3, 1187-1190). Seven groups of Charles River Crl: CD(SD)BR rats (20/sex/group) were treated with either saline or 700 mg/kg DHPN [N-bis(2-hydroxypropyl)nitrosamine] administered subcutaneously once a week for 5 weeks (Initiation Phase) followed by 15 weeks of treatment with control diet or diets containing 500 ppm of phenobarbital (Promotion Phase). Groups of rats were also treated with L-thyroxine (50 micrograms/kg/day) in the diet to determine its effect on thyroid gland tumor promotion by phenobarbital. The incidence of thyroid follicular adenomas in DHPN male rats treated with phenobarbital was markedly increased [83% (15/18 rats)] as compared to rats receiving DHPN alone [37% (6/16 rats)]. Thyroxine treatment completely blocked the tumor promoting effect of phenobarbital in that the tumor incidence [25% (5/20 rats)] was reduced back to or somewhat less than that observed with DHPN alone. In female rats no tumors were observed with DHPN nor was a promoting effect of phenobarbital observed. These results demonstrate the potential for a microsomal enzyme inducer such as phenobarbital to alter the incidence of thyroid gland neoplasia in the male rat. The inhibitory effect of L-thyroxine on tumor promotion by phenobarbital supports the hypothesis that the promoting effect of phenobarbital is mediated via increased pituitary secretion of thyroid stimulating hormone as a compensatory response to the known effects of phenobarbital on peripheral thyroxine metabolism and excretion.

Neonatal mouse model: review of methods and results.

The neonatal mouse model, in various forms, has been used experimentally since 1959 and a large number of chemicals have been tested. The neonatal model is known to be very sensitive for the detection of carcinogens that operate via a genotoxic mode of action. In contrast, it is known not to respond to chemicals that act via epigenetic mechanisms, commonly observed in the two-year carcinogenicity studies. As such, the model has a high sensitivity and specificity in its response. Dose selection for the neonatal model is based on the maximum tolerated or feasible dose. Traditionally, compounds have been tested via the IP route of administration in this model. In some cases, this has limited the amount of material that can be administered because of the low dosing volumes (10 to 20 microL) that can be administered IP. For the ILSI project, the neonatal model was adapted for oral administration, which has the advantages of being the same route for which most pharmaceuticals are administered. In addition, a 10-fold increase in the volume of administration (100 to 200 microL) and the ability to dose drugs in suspension, permits much higher doses to be used as compared to the IP route of administration. The spontaneous tumors in the neonatal model occurred mainly in the liver of male mice and lung of male and female mice with a few tumors observed in the Harderian gland. The positive control, DEN produced a robust, uniform, and reproducible tumor response with the target organs essentially limited to liver and lung. A total of 13 compounds out of the 21 ILSI ACT compounds were evaluated in the neonatal model involving 18 studies with duplicate studies for some compounds. The genotoxic carcinogens including those used as positive controls were clearly positive (cyclophosphamide, diethylnitrosamine, 6-nitrochrysene). The non-genotoxic rodent carcinogens were clearly negative (chlorpromazine, sulfisoxazole, sulfamethoxazole, clofibrate, DEHP, haloperidol, metaproteranol, and phenobarbital). The non-genotoxic human carcinogen (cyclosporin) was clearly negative. The two other human carcinogens phenacetin and DES were negative and interestingly estradiol was negative in one of the two oral studies, but was clearly positive in the other. Considering the mode of action for three of the human carcinogens (DES, cyclosporin and phenacetin), which were negative in this model, the mode of action in humans is likely to be epigenetic. Overall, for the 3 clearly genotoxic chemicals, all were positive. For the 9 clearly non-genotoxic chemicals, all 9 were negative. The two human carcinogens for which genotoxicity may or may not play a role (DES and phenacetin) were negative and estradiol was positive in I of the two oral studies. Overall, the extensive database for compounds tested in the neonatal mouse model would support its use as an alternative model for the assessment of the carcinogenic potential of a chemical. The model responds to chemicals that act via a genotoxic mode of action that represent a greater concern for human cancer risk.