ABO genotyping of various hematopoietic cell lines to select model cells for research purposes.

Various cells from humans and animals have been established as cell lines, and their features, characteristics, and origins have been reported. Many laboratories use cell lines as model cells, which are selected to suit research purposes. We attempted to identify the ABO genotypes of 31 human leukemia and lymphoma cell lines stored in our laboratory using three methods: the PCR amplification of specific alleles (PASA), PCR-restriction fragment length polymorphism (RFLP), and the direct DNA sequencing of PCR products. We distinguished 31 human leukemia and lymphoma cell lines examined into six major ABO genotypes: A/O (A101/O01: n = 1, A101/O12: n = 4, A101/O26: n = 1, A101/O49: n = 1, A102/O01: n = 3), A/A (A101/A101: n = 1, A102/A102: n = 2), B/O (Bw29/O01: n = 1), B/B (B101/B101: n = 2), O/O (O01/O01: n = 9, O01/O02: n = 1, O01/O26: n = 1, O02/O03: n = 1), and A/B (A102/B101: n = 3). To the best of our knowledge, this is the first study to identify the ABO genotypes of various cell lines. The ABO genotypes of cell lines are important when selecting an experimental model cell for an ABO blood group study, and are essential information for cell lines. These results may be employed by research and clinical laboratories as well as in the forensic field.

Increased cellular distribution of vimentin and Ret in the cingulum induced by developmental hypothyroidism in rat offspring maternally exposed to anti-thyroid agents.

To elucidate target molecules of white matter development responding to hypothyroidism, global gene expression profiling of cerebral white matter from male rat offspring was performed after maternal exposure to anti-thyroid agents, 6-propyl-2-thiouracil and methimazole, on postnatal day 20. Genes involved in central nervous system development commonly up- or down-regulated among groups treated with anti-thyroid agents. Immunohistochemical distributions of vimentin, Ret proto-oncogene (Ret), deleted in colorectal cancer protein (DCC), and Claudin11 (Cld11) were examined based on the gene expression profile. Immunoreactive cells for vimentin and Ret in the cingulum, and the immunoreactive intensity of Cld11 and DCC in whole white matter were increased by treatment with anti-thyroid agents. Immunoreactive cells for vimentin and Ret were immature astrocytes and oligodendrocytes, respectively. Thus, immunoreactive cells for vimentin and Ret may be quantitatively measurable targets of developmental hypothyroidism in white matter.

Prostaglandin synthases influence thyroid follicular cell proliferation but not carcinogenesis in rats initiated with N-bis(2-hydroxypropyl)nitrosamine.

To clarify roles of prostaglandin synthases in rat thyroid follicular carcinogenesis, effects of an antithyroid agent, sulfadimethoxine (SDM), and two prostaglandin H synthase (COX) inhibitors, indomethacin and nimesulide, on prostaglandin synthase expression, follicular cell proliferation, and tumor induction in thyroids of rats with or without N-bis(2-hydroxypropyl)nitrosamine (DHPN) initiation were examined. In experiment 1, F344 male rats were allowed free access to drinking water containing SDM (0.1%), SDM + indomethacin (0.0025% in diet), or SDM + nimesulide (0.04% in diet) for 4 weeks. Both COX inhibitors suppressed goitrogenic activity of SDM, but they did not significantly affect microsomal prostaglandin E synthase-2 (mPGES-2) expression levels enhanced by SDM. In experiment 2, all rats received an injection of DHPN (2800 mg/kg body weight), and starting 1 week later, they were treated as in experiment 1 for 4 or 10 weeks. Cell proliferation was suppressed or showed a tendency for suppression by the COX inhibitors in the follicular preneoplastic/neoplastic lesions and surrounding parenchyma, and this was obviously thyroid stimulating hormone independent at least at week 4. However, neither of the COX inhibitors altered the incidence or multiplicity of preneoplastic/neoplastic lesions. Immunohistochemistry revealed significant reduction and elevation of COX-2 and mPGES-2 expression, respectively, in the lesions, but these were also not changed by the COX inhibitors. These results suggest that COX-2 and PGES, and in turn PGE(2), might play important roles in follicular cell proliferation but do not affect tumor induction in this rat thyroid carcinogenesis model. Further studies are needed to clarify the significance of the reduction of COX-2 expression in preneoplastic/neoplastic lesions.

Juvenile rats do not exhibit elevated sensitivity to acrylamide toxicity after oral administration for 12 weeks.

Acrylamide (AA), a neurotoxic, testicular toxic, genotoxic and carcinogenic chemical, has been reported to be formed in processed food, and sensitivity to AA intoxication in childhood is a concern. In the present study, to clarify the general toxicological profile of AA in juvenile rats, subchronic toxicity was evaluated in F344 rats administered AA in the drinking water at 0 (control), 10, 20 and 40 ppm, presented to the dams (three per group) immediately after the birth of their litters, through lactation (3 weeks), and directly to the offspring in their drinking water after weaning for a further 9 weeks (12 weeks total). Treatment with AA caused a decrease in body weights in 20 and 40 ppm F(1) females, compared with the controls. Average AA intake throughout the treatment period for the 10, 20 and 40 ppm groups after weaning was equivalent to 1.0, 2.1 and 4.4 mg kg(-1) body weight per day, respectively, in males and 1.2, 2.5 and 4.9 mg kg(-1) body weight per day, respectively, in females. No toxicologically significant organ weight changes were observed. AA-induced histopathological changes were limited to focal degeneration and necrosis of the seminiferous epithelium in the testes and desquamated epithelium in the ducts of epididymides, noted only in 40 ppm males. Taken together with previous reports, juvenile rats are not necessarily more susceptible to AA-induced toxicity as compared with young adults.

Preventive effects of calcitriol on the development of capsular invasive carcinomas in a rat two-stage thyroid carcinogenesis model.

We have shown phosphoinositide 3-kinase (PI3K)/Akt signaling activation in thyroid capsular invasive carcinomas (CICs), which are highly induced by promotion with sulfadimethoxine (SDM) in a rat 2-stage thyroid carcinogenesis model. To examine the potency of calcitriol, a synthetic vitamin D3 analog, on the development or progression of CICs, male F344 rats were injected with calcitriol (0.1 µg/kg body weight) three times a week intraperitoneally, during an entire period of SDM-promotion for 13 weeks (Experiment 1) or during the last 2 weeks of a 15-week SDM-promotion (Experiment 2). Initiation with N-bis(2-hydroxypropyl)nitrosamine preceded all treatments. In Experiment 1, long-term calcitriol treatment reduced the multiplicity of CICs, while cell proliferation activity, estimated by Ki-67 cell index in the induced CICs, was unchanged with SDM-promotion alone. Considering the strong dependency of promotion with SDM during the early stages on thyroid-stimulating hormone, the reduced multiplicity in Experiment 1 may be due to the effect on an early stage of neoplastic proliferation. Although the magnitude was mild, cell proliferation activity was decreased in existing CICs after short-term calcitriol treatment in Experiment 2, which was associated with a mild decrease in cyclin-dependent kinase-2-positive cells, cytoplasmic immunolocalization of phosphorylated, inactive, Rb protein and a mild increase in nucleocytoplasmic expression of p27(kip1). Although the effect was mild at the late stage of SDM-promotion in this hypothyroidism-related thyroid carcinogenesis model, our results suggest that calcitriol targets cell proliferation via inhibition of a molecular cascade downstream of PI3K/Akt signaling, controlling G1/S transition.

Impaired oligodendroglial development by decabromodiphenyl ether in rat offspring after maternal exposure from mid-gestation through lactation.

Pregnant Sprague-Dawley rats were given diet containing decabromodiphenyl ether (DBDE) either at 0, 10, 100, or 1000 ppm from gestation day (GD) 10 until day 20 after delivery (PND 20). No significant alterations were observed in maternal and offspring reproductive parameters. At PND 20, serum triiodothyronine concentrations examined in males were slightly reduced at 1000 ppm (84.2% of the control value), and incidence of thyroid follicular cell hypertrophy was increased in both sexes with significant difference in males at 1000 ppm. Diffuse liver cell hypertrophy accompanying increased relative liver weight and increased cytoplasmic eosinophilia of the renal proximal tubules were observed in both sexes with significant difference from 10 ppm in males and females, respectively. At postnatal week 11, serum thyroxine concentrations examined in males were slightly reduced at 1000 ppm (85.9% of the control value), and the incidence of thyroid follicular cell hypertrophy was non-significantly increased from 10 ppm in males. There were reductions in the corpus callosum area and density of 2',3'-cyclic nucleotide 3'-phosphodiesterase-immunoreactive oligodendrocytes in the cingulate deep cortex in males from 100 ppm. Conversely, NeuN-immunoreactive neuronal distribution in the hippocampal CA1 was unchanged. This suggests that developmental DBDE-exposure caused irreversible white matter hypoplasia targeting oligodendrocytes from 100 ppm, accompanied with developmental hypothyroidism. The lowest-observed-adverse-effect level of DBDE was determined to be 10 ppm (0.7-2.4 mg/kg-body weight-d).

Involvement of PTEN/Akt signaling in capsular invasive carcinomas developed in a rat two-stage thyroid carcinogenesis model after promotion with sulfadimethoxine.

PURPOSE: Rat thyroid follicular cell carcinomas invading into the thyroid capsule are highly produced by promotion with sulfadimethoxine (SDM) in a rat two-stage thyroid carcinogenesis model. In this study, we investigated the participation of phosphoinositide 3-kinase (PI3K) signaling pathway that is associated with malignant phenotypes of many cancers on the development of SDM-induced capsular invasive carcinomas. METHODS: Thyroid proliferative lesions developed 10 or 15 weeks after promotion with SDM in male F344 rats initiated with N-bis(2-hydroxypropyl)nitrosamine were immunohistochemically analyzed with regard to cellular distribution of phosphatase and tensin homolog (PTEN) and Akt isoforms, as well as their downstream molecules. RESULTS: Increased expression of PI3K signaling molecules was evident in association with the development of lesion stages from the early focal hyperplasia to the late carcinomas. Capsular carcinomas, and the less frequent parenchymal carcinomas, exclusively expressed phosphorylated, inactive PTEN, and active Akt isoforms, as did their downstream molecules. Among the Akt isoforms, enhanced expression of Akt1 was more prominent than that of Akt2 in both capsular and parenchymal carcinomas. CONCLUSIONS: Activation of the PI3K pathway through phosphorylation of PTEN promotes the high production of capsular carcinomas as well as the development of less frequent parenchymal carcinomas.

Low-dose carcinogenicity of 2-amino-3-methylimidazo[4,5-f ]quinoline in rats: Evidence for the existence of no-effect levels and a mechanism involving p21(Cip / WAF1).

The carcinogenicity of the low amounts of genotoxic carcinogens present in food is of pressing concern. The purpose of the present study was to determine the carcinogenicity of low doses of the dietary genotoxic carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and to investigate mechanisms by which IQ exerts its carcinogenic effects. A total of 1595 male F344 rats were divided into seven groups and administered with IQ at doses of 0, 0.001, 0.01, 0.1, 1, 10 and 100 p.p.m. in the diet for 16 weeks. We found that IQ doses of 1 p.p.m. and below did not induce preneoplastic lesions in either the liver or the colon, while IQ doses of 10 and 100 p.p.m. induced preneoplastic lesions in both of these organs. These results demonstrate the presence of no-effect levels of IQ for both liver and colon carcinogenicity in rats. The finding that p21(Cip/WAF1) was significantly induced in the liver at doses well below those required for IQ mediated carcinogenic effects suggests that induction of p21(Cip/WAF1) is one of the mechanisms responsible for the observed no-effect of low doses of IQ. Furthermore, IQ administration caused significant induction of CYP1A2 at doses of 0.01-10 p.p.m., but administration of 100 p.p.m. IQ induced CYP1A1 rather than CYP1A2. This result indicates the importance of dosage when interpreting data on the carcinogenicity and metabolic activation of IQ. Overall, our results suggest the existence of no-effect levels for the carcinogenicity of this genotoxic compound.

Effect of cigarette smoke on mutagenic activation of environmental carcinogens by cytochrome P450 2A8 and inactivation by glucuronidation in hamster liver.

To elucidate the mechanism underlying suppression of N-nitrosobis(2-oxopropyl)amine (BOP)-induced hamster pancreatic carcinogenesis by cigarette smoke (CS), hepatic levels of microsomal cytochrome P450 (CYP) enzymes, mutagenic activation of environmental carcinogens and three types of uridine diphosphate-glucuronyltransferase (UDPGT) and sulphotransferase (ST) activities were assayed in male Syrian golden hamsters and F344 rats exposed to CS. Immunoblot analyses of microsomal CYP proteins revealed induction of constitutive CYP1A2 (2.6-fold increase) and 2A8 (4.0-fold increase) and induction of CYP1A1 and constitutive CYP1A2 (3.9-fold increase) in rats following exposure to CS for 4 weeks using a Hamburg type II smoking machine. CS exposure enhanced mutagenicities of four heterocyclic amines in the presence of liver S9 in both species, whereas the mutagenicities of aflatoxin B(1) (AFB(1)), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were significantly increased by CS in hamsters but not in rats. However, no CS-induced alterations in the mutagenic activities of other carcinogens, including BOP and other pancreatic carcinogens, were observed in either species. Application of several CYP inhibitors revealed that the mutagenic activities of MeAαC, AFB(1) and NNK in the hamster liver S9 were partly associated with CYP2A8, whereas those of the three pancreatic carcinogens were selectively associated with CYP2B. CS enhanced UDPGT activities towards 4-nitrophenol (4-NP) (1.9- to 2.0-fold) but did not affect those of bilirubin, testosterone UDPGTs and three STs in both species. Together with the previous findings that BOP does not induce tumourigenesis in rats and that the glucuronidation of ß-oxypropylnitrosamines is higher in rats than in hamsters, suppression of BOP-induced pancreatic carcinogenesis by CS might be attributed to increased detoxification by 4-NP UDPGT and not decreased CYP2B activation. This is the first demonstration of the induction of CYP2A protein by CS; CYP2A protein polymorphisms have been associated with oral and pulmonary carcinogenesis in smokers.

Lack of modifying effects of prepubertal exposure to acrylamide (AA) on N-methyl-N-nitrosourea (MNU)-induced multi-organ carcinogenesis in F344 rats.

Acrylamide (AA) has been reported to be formed in fried and baked foods with various concentrations, and exposure levels to AA from cooked foods in children are estimated to be higher than those in adults. In order to evaluate the carcinogenicity of AA exposure during childhood, we conducted a medium-term carcinogenicity study with prepubertal administration of AA followed by treatments of a multi-organ-targeted genotoxic carcinogen and a promoting agent for thyroid carcinogenesis in rats. A total of 36 postpartum F344 rats were given drinking water containing AA at 0, 20, 40 or 80 ppm for 3 weeks during the lactation period, and their weaned offspring received the same AA-containing water for 3 more weeks. Offspring were then injected with N-methyl-N-nitrosourea (MNU; 40 mg/kg body weight, i.p.) once at week 7 after birth. Half the animals of the 0 and 40 ppm groups were additionally treated with the anti-thyroid agent sulfadimethoxine (SDM; 125 ppm) in the drinking water thereafter. Offspring were subjected to complete necropsy at week 50. All the major organs and macroscopic abnormalities were excised and examined histopathologically. There was no significant difference in the incidences of hyperplastic and neoplastic lesions in the target organs of AA and/or MNU, such as the brain, spinal cord, pituitary gland, thyroid, adrenal glands, uterus, mammary glands, clitoral gland and tunica vaginalis. In conclusion, no significant modifying actions of AA on MNU-induced multi-organ carcinogenesis were exhibited in any organs of rats when exposed prepubertally under the present experimental conditions.

Carcinogenic potential of alizarin and rubiadin, components of madder color, in a rat medium-term multi-organ bioassay.

Madder color (MC), a food coloring extracted from roots of Rubia tinctorum L., has been proven to exert carcinogenicity in the rat kidney and liver. Furthermore, it induces DNA adducts in the kidney, liver, and colon. MC is in fact composed of anthraquinones such as lucidin-3-O-primeveroside and alizarin. To clarify which of these might be responsible for the carcinogenicity, a rat medium-term multi-organ carcinogenesis bioassay was performed focusing on the kidney, liver, and colon. Male 6-week-old F344 rats after receiving five different carcinogens were fed a diet containing either 0.008% or 0.04% of alizarin or rubiadin, a metabolite of lucidin-3-O-primeveroside, for 23 weeks. Treatment with 0.04% rubiadin significantly increased atypical renal tubules/hyperplasias and induced renal cell adenomas and carcinomas. Renal cell tumors were also increased with 0.04% alizarin, although at lower incidence than with rubiadin. In addition, glutathione S-transferase placental form-positive liver cell foci and large intestinal dysplasias were significantly increased with 0.04% rubiadin. These results indicate that both rubiadin and alizarin can increase renal preneoplastic lesions, the potential of the latter being weaker. Rubiadin may also target the liver and large intestine, suggesting a major role in madder color-induced carcinogenicity.

Increased H-ras mutation frequency in mammary tumors of rats initiated with N-methyl-N-nitrosourea (MNU) and treated with acrylamide.

We recently demonstrated the incidence and multiplicity of N-methyl-N-nitrosourea (MNU)-induced mammary tumors to be increased by administration of acrylamide (AA) in post-initiation in rats. In the present study, to clarify the mechanisms of enhancement, H-ras gene mutations in mammary tumors induced in MNU-initiated rats with or without subsequent AA administration were investigated. Frequencies of mutations in codon 12 from GGA to GAA were significantly (p < 0.05) higher in rats with AA administration (82%, 23 out of 28 tumors) as compared to those without AA (50%, 9 out of 18 tumors), but the latency and volume of H-ras mutation-harboring tumors were similar to those of the mutation-lacking tumors. No mutations in codons 13 or 61 were detected in either treatment groups. The results thus indicate that H-ras gene mutations in codon 12 play a pivotal role in initiation of carcinogenesis and it appears possible that AA administration may selectively co-stimulate and/or maintain initiated cells via other genomic or non-genomic events in MNU-treated rats.

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.

A 55-week chronic toxicity study of dietary administered kojic acid (KA) in male F344 rats.

A chronic toxicity study of kojic acid (KA) was performed using male F344 rats by dietary administration at concentrations of 0 (control), 0.5 and 2.0% for 55 weeks. Body weight gain was suppressed in the 2.0% group. The major hematological findings were decreased red blood cell (RBC) count and hematocrit (Ht) values at both 0.5 and 2.0%. In serum biochemistry, increased aspartate transaminase (AsT), alanine transaminase (AlT), alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (gamma-GTP) levels were detected in the 0.5 and 2.0% groups. Histopathologically, single cell necrosis of hepatocytes and proliferation of bile ductules in both treatment groups, and hypertrophy of hepatocytes, granulomas and proliferation of bile ducts in the 2.0% group were increased in incidence, and numbers and areas of glutathione-S-transferase placental-form (GST-P) positive foci were increased in the liver of the 2.0% group. In the thyroids, diffuse follicular cell hyperplasia at 0.5 and 2.0% and focal follicular cell hyperplasia and follicular adenoma at 2.0% were increased. A thyroid follicular carcinoma was also observed at 2.0%. Additionally, increased incidences of hyaline casts and basophilic tubules in the kidneys at 2.0% and microgranulomas containing crystals in the lung in both treatment groups were noted. At 2.0%, hypertrophy of cortical cells in zona fasciculata was also increased in the adrenals. In conclusion, no observed adverse effect level of KA was below 0.5%, which is equivalent to 227 mg/kg body weight/day in male rats.

The process behind the expression of mdr-1/P-gp and mrp/MRP in human leukemia/lymphoma.

There is a controversy over the link between phenotypes of multidrug resistance (MDR) and clinical outcome in leukemia/lymphoma patients. This may be because the process behind the induction and loss of expression of genotypes and phenotypes by which MDR develops and the role of MDR in fresh cells of human leukemia/lymphoma are not clearly defined. P-glycoprotein (P-gp) increased and decreased along with mdr-1 expression in three cell lines out of five vincristine (VCR)-resistant cell lines. MRP appeared with increased mrp expression in the other two cell lines. After the drug was removed from the culture system, mdr-1/P-gp changed in parallel with the level of VCR resistance, although mrp and MRP did not. It was concluded that P-gp is directly derived from mdr-1 and that mdr-1/P-gp supports the VCR-resistance but mrp/MRP is not directly linked to the VCR-resistance. These results should contribute to a better understanding of MDR phenomenon in cancer.

Identification and metastatic potential of tumor-initiating cells in malignant rhabdoid tumor of the kidney.

PURPOSE: Malignant rhabdoid tumor of the kidney (MRTK) is a rare and highly aggressive malignancy of infanthood. In an effort to delineate MRTK progression, we investigated the metastatic fate of some MRTK cells using xenotransplantation animal models and the tumor-initiating potential of CD133(+) MRTK cells. EXPERIMENTAL DESIGN: We established two MRTK cell lines (JMU-RTK-1 and JMU-RTK-2) from patients with MRTK. We generated five luciferase-expressing MRTK cells for in vivo luminescent imaging and evaluated the metastatic fate in an orthotopic xenotransplantation model. Capacities of MRTK-initiating cells were examined in nonobese diabetic/severe combined immunodeficient mice after antibody-mediated magnetic bead sorting. Use of chemokine receptor CXCR4 expression as a metastatic marker was evaluated by flow cytometry and Western blotting. RESULTS: MRTK cell lines showed distant organ metastasis. JMU-RTK-1, JMU-RTK-2, and G401 cells showed considerable aggressiveness compared with SWT-1 and SWT-2 cells (P < 0.05). Moreover, as few as 1,000 CD133(+) MRTK cells initiated tumor development in nonobese diabetic/severe combined immunodeficient mice by 21 days (60-100%) in all examined cell lines, although the same number of CD133(-) MRTK cells could not form tumors (0%). Interestingly, the metastatic potential of the CD133(+) population remained unaffected compared with a nonenriched population. The potential metastatic marker CXCR4 was expressed in CD133(+) and CD133(-) MRTK cells, and CD133(-) cells seemed to play a cooperative role in terms of tumorigenicity and metastasis. CONCLUSIONS: These results suggest that CD133(+) cells may determine the metastatic fate of MRTK cells and that CD133(-) cells may play an auxiliary role in tumor progression and metastasis.

Cellular distributions of molecules with altered expression specific to thyroid proliferative lesions developing in a rat thyroid carcinogenesis model.

To identify differentially regulated molecules related to early and late stages of tumor promotion in a rat two-stage thyroid carcinogenesis model by an antithyroid agent, sulfadimethoxine, microarray-based microdissected lesion-specific gene expression profiling was carried out. Proliferative lesions for profiling were divided into two categories: (i) focal follicular cell hyperplasias (FFCH) and adenomas (Ad) as early lesions; and (ii) carcinomas (Ca) as more advanced. In both cases, gene expression was compared with that in surrounding non-tumor follicular cells. Characteristically, upregulation of cell cycle-related genes in FFCH + Ad, downregulation of genes related to tumor suppression and transcription inhibitors of inhibitor of DNA binding (Id) family proteins in Ca, and upregulation of genes related to cell proliferation and tumor progression in common in FFCH + Ad and Ca, were detected. The immunohistochemical distributions of molecules included in the altered expression profiles were further examined. In parallel with microarray data, increased localization of ceruloplasmin, cyclin B1, and cell division cycle 2 homolog A, and decreased localization of poliovirus receptor-related 3 and Id3 were observed in all types of lesion. Although inconsistent with the microarray data, thyroglobulin immunoreactivity appeared to reduce in Ca. The results thus suggest cell cycling facilitation by induction of M-phase-promoting factor consisting of cyclin B1 and cell division cycle 2 homolog A and generation of oxidative responses as evidenced by ceruloplasmin accumulation from an early stage, as well as suppression of cell adhesion involving poliovirus receptor-related 3 and inhibition of cellular differentiation regulated by Id3. Decrease of thyroglobulin in Ca may reflect dedifferentiation with progression.

Possible contribution of rubiadin, a metabolite of madder color, to renal carcinogenesis in rats.

Madder color (MC) has been shown to exert carcinogenic potential in the rat kidney in association with degeneration, karyomegaly, increased cell proliferation of renal tubule cells and increased renal 8-OHdG levels. To clarify the causal relationship of components and metabolites of MC to renal carcinogenesis, male F344 rats were fed lucidin-3-O-primeveroside (LuP) or alizarin (Alz), and the genotoxic LuP metabolites lucidin (Luc) or rubiadin (Rub) for up to 26 weeks. After one week and four weeks, Luc did not induce any renal changes. In contrast, after one week, cortical tubule degeneration was apparent in the Alz and LuP groups, and cytoplasmic swelling with basophilic change and karyomegaly in the outer medulla was observed only in the Rub group. LuP and Rub increased the proliferative activity of tubule cells in the outer medulla, and Alz and LuP increased renal 8-OHdG levels. After 26 weeks, Rub but not Alz induced atypical tubules, a putative preneoplastic lesion, and karyomegaly in the outer medulla. These results indicate that Rub may be a potent carcinogenic metabolite of MC, targeting proximal tubule cells in the outer medulla, although oxidative stress increased by Alz or LuP might also be involved in renal carcinogenesis by MC.

Modifying effects of prepubertal exposure to potassium perchlorate and tetrabromobisphenol A on susceptibility to N-bis(2-hydroxypropyl)nitrosamine- and 7,12-dimethylbenz(a)anthracene-induced carcinogenesis in rats.

Early life exposure to certain kinds of chemicals is of concern because of a possible increase in cancer risk, but relevant data are limited. In the present experiment, modifying effects of prepubertal administration of potassium perchlorate (KClO(4)) and tetrabromobisphenol A (TBBPA) on susceptibility to multi-organ carcinogenesis were evaluated. F344 dam rats were administered 0% (control), 0.01%, 0.1% or 1% TBBPA in diet or 0.01% KClO(4) in drinking water after parturition. Their weaned offspring in each group were treated for 2 weeks in the same manner. From 6 weeks of age, all offspring were treated with N-bis(2-hydroxypropyl)nitrosamine in drinking water for 4 weeks. In addition the females at 7 weeks of age were gavaged once with 7,12-dimethylbenz(a)anthracene. At weeks 39 and 47 of age, the males and females, respectively, were euthanized and the liver, kidney, lung, esophagus, thyroid, urinary bladder, testis, epididymis, ovary and mammary gland were histopathologically examined. The incidences of thyroid follicular adenomas in 1% TBBPA females (p<0.05) and of transitional cell papillomas in the urinary bladder of 0.01%, 0.1% and 1% TBBPA females were increased (p<0.05) as compared to the controls. These results indicate that prepubertal exposure to TBBPA raises susceptibility to thyroid and urinary bladder tumorigenesis in rats. Although causes of the effect on thyroid carcinogenesis might be direct and/or indirect hormonal actions, further studies are needed for confirmation.

Induction of kidney and liver cancers by the natural food additive madder color in a two-year rat carcinogenicity study.

Madder color (MC) extracted from the roots of Rubia tinctorum (madder root) has been used as a food coloring in Japan. Our previous studies revealed MC to have obvious subchronic and chronic toxicity and potent carcinogenicity targeting rat liver and kidney. In the present two-year carcinogenicity study, conducted to further elucidate the long-term effects of MC and its target organs, male and female F344 rats were fed diet containing 0%, 2.5%, and 5.0% MC for 104 weeks. Body weights were significantly decreased in treated groups of both sexes throughout the feeding period. However, survival rates at week 104 were higher in treated groups of both sexes than in controls. Relative weights of the kidneys and liver were significantly increased in treated groups of both sexes. Histopathologically, karyomegaly and atypical tubules/hyperplasias, as well as renal cell adenomas and carcinomas were significantly increased in treated groups of both sexes with dose-dependence. Moreover, the incidence of hepatocellular adenomas and/or carcinomas was increased significantly with a dose-relation in treated groups of both sexes. These data provide clear evidence that MC exerts unequivocal carcinogenicity against renal tubule cells and hepatocytes in rats.