The association between the T309G polymorphism of the MDM2 gene and sensitivity to anticancer drug is dependent on the p53 mutational status in cellular models.

BACKGROUND: We investigated, in the panel of 60 human tumour cell lines of the National Cancer Institute (NCI-60), whether the R72P polymorphism of TP53 and the T309G polymorphism of MDM2 were associated to the in vitro cytotoxicity of anticancer agents, extracted from the NCI database. For validation, the same study was performed independently on a second panel of tumour cell lines, JFCR-45. METHODS: Both SNPs were identified in cell DNA using PCR-RFLP techniques confirmed by direct sequencing and by pyrosequencing. For the analysis of the results, the mutational status of p53 was taken into account. RESULTS: In the NCI-60 panel, the TP53 rare-allele frequency was 32% and the MDM2 rare-allele frequency 39%. The MDM2 alleles were distributed according to Hardy-Weinberg equilibrium whereas this was only found, for the TP53 alleles, in p53 non-mutated cell lines. Comparable results were obtained in the JFCR-45 validation set. The TP53 SNP had low impact on anticancer drug cytotoxicity in either panel. In contrast, the MDM2 gene polymorphism had a major impact on anticancer drug cytotoxicity, essentially in p53 non-mutated cell lines. Presence of the rare allele was associated to significantly higher MDM2 protein expression and to increased sensitivity to DNA-interfering drugs. In the JFCR-45 panel, a similar effect of the MDM2 gene polymorphism was observed, but was less dependent on the p53 mutational status. CONCLUSIONS: We hypothesised that cell lines harbouring the MDM2 G allele presented a lower availability of p53 for DNA repair, translating into higher sensitivity to DNA-damaging agents.

Identification of genes that restrict astrocyte differentiation of midgestational neural precursor cells.

During development of the mammalian CNS, neurons and glial cells (astrocytes and oligodendrocytes) are generated from common neural precursor cells (NPCs). However, neurogenesis precedes gliogenesis, which normally commences at later stages of fetal telencephalic development. Astrocyte differentiation of mouse NPCs at embryonic day (E) 14.5 (relatively late gestation) is induced by activation of the transcription factor signal transducer and activator of transcription (STAT) 3, whereas at E11.5 (mid-gestation) NPCs do not differentiate into astrocytes even when stimulated by STAT3-activating cytokines such as leukemia inhibitory factor (LIF). This can be explained in part by the fact that astrocyte-specific gene promoters are highly methylated in NPCs at E11.5, but other mechanisms are also likely to play a role. We therefore sought to identify genes involved in the inhibition of astrocyte differentiation of NPCs at midgestation. We first examined gene expression profiles in E11.5 and E14.5 NPCs, using Affymetrix GeneChip analysis, applying the Percellome method to normalize gene expression level. We then conducted in situ hybridization analysis for selected genes found to be highly expressed in NPCs at midgestation. Among these genes, we found that N-myc and high mobility group AT-hook 2 (Hmga2) were highly expressed in the E11.5 but not the E14.5 ventricular zone of mouse brain, where NPCs reside. Transduction of N-myc and Hmga2 by retroviruses into E14.5 NPCs, which normally differentiate into astrocytes in response to LIF, resulted in suppression of astrocyte differentiation. However, sustained expression of N-myc and Hmga2 in E11.5 NPCs failed to maintain the hypermethylated status of an astrocyte-specific gene promoter. Taken together, our data suggest that astrocyte differentiation of NPCs is regulated not only by DNA methylation but also by genes whose expression is controlled spatio-temporally during brain development.

Long-term studies on carcinogenicity and promoting effect of phenylbutazone in DONRYU rats.

The carcinogenicity and promoting effect of phenylbutazone were investigated in inbred DONRYU rats. In the carcinogenicity study, both sexes were administered the chemical at dietary levels of 0 (control), 0.125, or 0.25% for 2 years. Toxic lesions were associated with phenylbutazone treatment in the kidney and digestive tract, appearing to have an adverse effect on life expectancy. Various tumors were detected in all groups including the controls. With the exception of pheochromocytoma in the female high-dose group, no statistically significant increase in yield of any tumors, including leukemia, was apparent in the treated groups of either sex when the data were analyzed by Fisher's exact probability and/or chi-square tests. Application of an age-adjusted statistical analysis revealed a slight positive effect regarding the occurrence of pheochromocytomas, neoplastic liver nodules, and leukemias in females. However, these tumors are commonly observed to develop spontaneously in this rat strain, and no such effect was apparent in the male groups. In addition, no differences in incidences of relevant preneoplastic lesions were evident between control and treated groups. Thus phenylbutazone showed no carcinogenic activity in DONRYU rats when given continuously in the diet for 2 years. For the investigation of promoting effect, phenylbutazone was given as a dietary supplement for 2 years subsequent to initiation with N-ethyl-N-nitrosourea or N-propyl-N-nitrosourea. No enhancement of nitrosourea-induced leukemogenesis was apparent, although a slight promoting effect was demonstrated for renal and thyroid tumorigenesis.

Cytokine induction in human epidermal keratinocytes exposed to contact irritants and its relation to chemical-induced inflammation in mouse skin.

In response to exogenous stimuli such as phorbol-12-myristate 13-acetate, ultraviolet B radiation, and lipopolysaccharide, human keratinocytes produce soluble mediators that are important in primary contact irritancy including cytokines that are associated with proinflammatory properties (interleukin-1 alpha [IL-1 alpha], tumor necrosis factor alpha), chemotaxis (IL-8), and growth activation (granulocyte/macrophage colony stimulating factor, IL-6, transforming growth factor alpha). We examined qualitative and quantitative changes in selected intracellular and secreted cytokines in human keratinocyte cultures in response to non-sensitizing contact irritants (croton oil, sodium lauryl sulfate, methyl salicylate, ethyl phenylpropiolate), sensitizing irritants (oxazolone, dinitrofluorobenzene), and ulcerative agents (phenol, benzalkonium chloride, chromium trioxide). The chemicals were also applied to mouse skin to assess whether the chemical-specific pattern of inflammation correlated with the in vitro production of keratinocyte-derived cytokines. Although all agents elicited neutrophils to the site of chemical application, time dependent and chemical-specific patterns of inflammation could be detected. Sodium lauryl sulfate, phenol, and croton oil induced increases in IL-8 production at non-cytotoxic concentrations in semi-confluent human keratinocyte cultures. Phenol and croton oil stimulated tumor necrosis factor alpha production, whereas croton oil was the only agent found to induce granulocyte/macrophage colony-stimulating factor production. Croton oil, phenol, benzalkonium chloride, and dinitrofluorobenzene induced the intracellular production of IL-1 alpha without a concomitant release into the medium. The release of cytokines occurred in parallel with a relative increase in cytokine-specific mRNA transcripts. Studies using neutralizing antibodies to tumor necrosis factor alpha and IL-1 alpha demonstrated that IL-8 induction by croton oil and phenol occurred directly rather than through autocrine circuits. These data suggest that a given pattern of cytokine production is chemical-specific and may predict the contribution of keratinocytes to skin inflammation.

Effect of restraint stress on immune system and experimental B16 melanoma metastasis in aged mice.

An overnight restraint stress was given to young and old mice and its effect was examined in terms of the number and function of T cells and natural killer (NK) cells in spleen and patterns of lung metastasis of B16 melanoma cells. A great decrease was observed in the number and proliferative activity of splenic T cells in old mice after the stress. The decrease in young mice was rather temporary with a quick recovery. The number of NK cells in spleen was not different between young and old mice before giving the stress, but a significant decrease was observed in the old after the stress. NK activity was always much lower in old than in young throughout the experiment. The pattern of metastasis of B16 melanoma cells was different between young and old mice. Metastatic colonies in lungs were larger in number and bigger in size in young mice than in old mice. After the stress, the number increased and the size unchanged in old mice, while the size increased and the number remained unchanged in young mice. It was shown that the same restraint stress resulted in a more serious influence on the immune cells in old than in young mice and gave rise to a differential effect on the pattern of tumor metastasis between young and old mice.

Synergistic effects of phenobarbital and thiourea on proliferative lesions in the rat liver.

To evaluate the effects of phenobarbital (PB) and thiourea (TU), alone or in combination, on proliferative lesions of the liver, thyroid and lung, male F344 rats initiated with 2000 mg/kg body weight N-bis(2-hydroxypropyl) nitrosamine (DHPN) were given diet and/or drinking water containing 0% PB/TU (group 1), 1000 ppm PB (group 2), 0.1% TU (group 3) and 500 ppm PB and 0.05% TU (group 4), from weeks 2 to 20 for 19 weeks. Group 4 showed remarkable increases in the number of hepatocellular altered foci per animal, the values being superior to the averages of groups 2 and 3. The number of thyroid proliferative lesions per animal was highest in group 3 and lowest in group 2. Lung proliferative lesions were induced in all groups, but no modifying influence on their development was evident in the combined group. The present results indicate that combined administration of PB and TU exerts synergistic enhancing effects on hepatocarcinogenesis.

Experimental induction of ovarian Sertoli cell tumors in rats by N-nitrosoureas.

Spontaneous ovarian tumors are very rare in ACI, Wistar, F344 and Donryu rats; the few neoplasms found are of the granulosa/theca cell type. Ovarian tumors were also rare in these strains of rats when given high doses of N-alkyl-N-nitrosoureas continuously in the drinking water for their life-span; however, relatively high incidences of Sertoli cell tumors or Sertoli cell tumors mixed with granulosa cell tumors were induced in Donryu rats after administration of either a 400 ppm N-ethyl-N-nitrosourea solution in the drinking water for 4 weeks or as a single dose of 200 mg N-propyl-N-nitrosourea per kg body weight by stomach tube. Typical Sertoli cell tumors consisted of solid areas showing tubular formation. The tubules were lined by tall, columnar cells, with abundant, faintly eosinophilic, often vacuolated cytoplasm, and basally oriented, round nuclei, resembling seminiferous tubules in the testes. In some cases, Sertoli cell tumor elements were found mixed with areas of granulosa cells. The induction of ovarian Sertoli cell tumors in Donryu rats by low doses of nitrosoureas may provide a useful model for these tumors in man.

Effect of methylene chloride inhalation on replicative DNA synthesis in the lungs of female B6C3F1 mice.

In the National Toxicology Program 2-year inhalation study of dichloromethane (DCM), there was a significant increase in pulmonary neoplasms in female B6C3F1 mice exposed to 2000 ppm (overall rates of 30/48 versus 5/50 in control). Replicative DNA synthesis was examined to evaluate the potential role of treatment-induced lung cell proliferation on pulmonary carcinogenicity. Tritiated thymidine incorporation was assessed in methacrylate plastic sections after 1, 2, 3, or 4 weeks of inhalation exposure to 2000 ppm or 8000 ppm DCM. Similar measurements of labeling indexes were made after 13 and 26 weeks of exposure to 2000 ppm DCM using bromodeoxyuridine as the labeling agent. In all cases the labeling agent was delivered over a 6-day period using osmotic minipumps. The labeling index (LI) of bronchiolar epithelium (two branches proximal to the terminal bronchiole) of mice exposed to 2000 ppm DCM for 2-26 weeks decreased to 40-60% of the control. Terminal bronchioles showed a similar decrease in LI. Mice exposed to 8000 ppm DCM had a less dramatic decrease in LI. No pathological change was found in the exposed lungs. It is concluded that inhalation exposure to DCM for up to 26 weeks reduces cell turnover of bronchiolar cells in female B6C3F1 mice.

The OECD program to validate the rat uterotrophic bioassay to screen compounds for in vivo estrogenic responses: phase 1.

The Organisation for Economic Co-operation and Development has completed the first phase of an international validation program for the rodent uterotrophic bioassay. This uterotrophic bioassay is intended to identify the in vivo activity of compounds that are suspected agonists or antagonists of estrogen. This information could, for example, be used to help prioritize positive compounds for further testing. Using draft protocols, we tested and compared two model systems, the immature female rat and the adult ovariectomized rat. Data from 19 participating laboratories using a high-potency reference agonist, ethinyl estradiol (EE), and an antagonist, ZM 189,154, indicate no substantive performance differences between models. All laboratories and all protocols successfully detected increases in uterine weights using EE in phase 1. These significant uterine weight increases were achieved under a variety of experimental conditions (e.g., strain, diet, housing protocol, bedding, vehicle). For each protocol, there was generally good agreement among laboratories with regard to the actual EE doses both in producing the first significant increase in uterine weights and achieving the maximum uterine response. Furthermore, the Hill equation appears to model the dose response satisfactorily and indicates general agreement based on calculated effective dose (ED)(10) and ED(50) within and among laboratories. The feasibility of an antagonist assay was also successfully demonstrated. Therefore, both models appear robust, reproducible, and transferable across laboratories for high-potency estrogen agonists such as EE. For the next phase of the OECD validation program, both models will be tested against a battery of weak, partial estrogen agonists.

Induction of Sertoli cell tumors in the rat ovary by N-alkyl-N-nitrosoureas.

Relatively high incidences of Sertoli cell tumors of the ovary were induced in Donryu rats given a 400 ppm N-ethyl-N-nitrosourea solution as drinking water for 4 weeks or a single dose of 200 mg/kg N-propyl-N-nitrosourea by stomach tube. Typical Sertoli cell tumors were composed of solid areas showing tubular formation. Tubules were lined by tall, columnar cells having abundant, faintly eosinophilic, often vacuolated cytoplasm, and basally oriented round nuclei. In some cases, Sertoli cell tumors were found to be mixed with granulosa cell tumors.

Carcinogenicity and organ specificity of N-trimethylsilylmethyl-N-nitrosourea (TMS-MNU), N-neopentyl-N-nitrosourea (neoPNU), and N-methyl-N-nitrosourea (MNU) in rats.

The carcinogenicity and organ specificity of TMS-MNU and neoPNU, a carbon-analogue of TMS-MNU, in rats were investigated and compared with those of MNU. Compounds were dissolved in olive oil and rats in the experimental groups received 20 weekly intragastric intubations of 10 mg/kg of MNU or equimolar amounts of TMS-MNU or neoPNU in the same manner. The experiment was terminated when the survivors were sacrificed at the 52nd week after the final administration. In the TMS-MNU and MNU groups, tumors of the forestomach were induced and the incidence was 100% in the groups of both sexes. In addition, tumors of the glandular stomach, nervous system, kidney, and lung were also observed in these groups. Neurogenic tumors were found more frequently in the MNU group than in the TMS-MNU group. The incidence of lung tumors, however, was higher in the TMS-MNU group than in the MNU group. On the other hand, in the control and neoPNU groups, no tumor was found in these organs except the lung, and all tumors observed in these two groups were histologically similar to spontaneous ones in this strain of rats. These results indicate that the carcinogenicity of N-alkyl-N-nitrosoureas is dependent on the chemical structure of their alkyl chain. The result of the present study coincides with the previous result that the species of TMS-MNU in the alkylating step is the same as that of MNU, but different from neoPNU. The difference in the organ specificity between TMS-MNU and MNU demonstrates that the organ specificity is dominantly dependent on the distribution of the chemicals, since TMS-MNU may possibly be distributed differently from MNU because of its different partition property.

FD&C Red No. 105 (Rose Bengal B) neutralizes the thyroid tumor promoting effect of iodine-deficient diet in rats.

The effect of 3,4,5,6-tetrachloro-2',4',5',7'-tetraiodo-fluorescein sodium salt (Rose Bengal B or FD&C Red No. 105, molecular weight 1017.6) on the thyroid of rats treated with N-bis(2-hydroxypropyl)-nitrosamine (DHPN) and iodine-deficient (I-def) diet is studied. Six-week-old male F344 rats were divided into seven groups (20 each) and given a single subcutaneous injection of DHPN (2800 mg/kg body wt.). From week 2 to 20, I-def diet was given in combination with either FR105 (1.25, 5.0, or 20 mg/l) or potassium iodide (KI, 12.5, 50.0 or 200 micrograms/l) in drinking water. As a result, an amount of 1.25 mg/l of FR105 was slightly more effective than 200 micrograms/l of KI in terms of inhibition of the effect of I-def diet on thyroid weight, morphology, thyroid-related hormones and thyroid tumor development. It was calculated that 1 mumol/l of FR105 was slightly more potent than 1 mumol/l of iodide ion. As each FR105 molecule has four iodide residues, at least 25% of total iodide residues were calculated to be utilized by the rats given I-def diet.

Hepatic and pulmonary carcinogenicity of methylene chloride in mice: a search for mechanisms.

An inhalation study utilizing over 1400 female B6C3F1 mice was undertaken to study mechanistic factors associated with liver and lung tumor induction following exposure to 2000 ppm of methylene chloride. Mice were exposed to methylene chloride (treated) or chamber air (controls) 6 h per day, for varying durations up to 104 weeks. Several interim sacrifices and 'stop exposures' were included. Exposure to 2000 ppm methylene chloride caused an increase in liver and lung neoplasia in the absence of overt cytotoxicity. Measurement of replicative DNA synthesis done after 13, 26, 52 and 78 weeks of exposure showed a significant decrease in the hepatocyte labeling index at 13 weeks. Replicative DNA synthesis in pulmonary airways after 1, 2, 3, 4, 13 and 26 weeks of exposure to methylene chloride was significantly lower than in air-exposed controls. Likewise, the increase in tumor induction in treated mice was not associated with increased replicative DNA synthesis in liver foci or in alveolar parenchyma. The frequency and pattern of H-ras gene activation were similar in control and methylene chloride-induced liver neoplasms. Similarly, the frequency and pattern of K-ras activation in lung neoplasms were not altered by exposure to methylene chloride. Early exposure to methylene chloride for only 26 weeks was sufficient to cause an increase in lung tumors by 2 years, suggesting that methylene chloride may cause early and persistent loss of growth control in lung cells. This implies that risk management strategies should be aimed at minimizing or eliminating exposure to methylene chloride. Liver neoplasms continued to increase in incidence and multiplicity as exposure continued, suggesting that methylene chloride-induced hepatocarcinogenesis is facilitated by continuing exposure to methylene chloride. Since methylene chloride is a more potent inducer of lung than liver neoplasia, it is recommended that health risk assessment be based on the lung data. While no novel molecular lesions have been found to explain the induction of lung and liver neoplasia in mice, ongoing studies may identify other molecular changes that are important in the genesis of these neoplasms. Hence, it may be necessary to revise risk assessment and management strategies in light of future research findings.

Lack of carcinogenicity of tartrazine (FD & C Yellow No. 5) in the F344 rat.

The carcinogenicity of tartrazine (C. I. Food Yellow No. 4, FD & C Yellow No. 5), a food, drug and cosmetics colouring, was examined in F344 rats. Tartrazine was dissolved in distilled water at levels of 0, 1 or 2%, and groups of about 50 male and 50 female rats were given one of these solutions ad lib. as their drinking-water for up to 2 yr. No toxic lesions specifically caused by tartrazine were detected in any treated group of either sex. Many tumours developed in all groups including the control group, and the organ distribution of these tumours and their histological characteristics were similar to those of the spontaneous tumours that are known to occur in this strain of rats. Except for mesothelioma in males and endometrial stromal polyp in females, there were no significant increases in the incidences of any tumours over those in the corresponding control group. In males, mesotheliomas were found only in the group given 1% tartrazine and the incidence of this lesion was statistically significant (Fisher's exact test) in comparison with the other two groups (P less than 0.02). The incidence of endometrial stromal polyp was also significantly higher among females given the 1% dose than in the controls (P less than 0.05). However, no positive trend was noted in the occurrence of these two tumours using an age-adjusted statistical analysis. Mesothelioma and endometrial stromal polyp are frequently observed spontaneous tumours in this strain of rats, and their incidences in our historical controls are 4.1 and 21.9%, respectively. However in the present study mesothelioma occurred in none of the male control rats and the incidence of endometrial stromal polyp was only 10.6% in the female control group. Moreover, there was no significant difference between the control and treated groups in hyperplastic or pre-neoplastic changes in the mesothelium or endometrium. From these findings, we concluded that the significant increases in the incidences of mesothelioma and endometrial stromal polyp that occurred in the groups given 1% tartrazine were not attributable to tartrazine administration. Thus, it is concluded that tartrazine was not carcinogenic in F344 rats when administered continuously at doses of up to 2% in the drinking-water for up to 2 yr.

Six-month toxicity study of butylated hydroxyanisole in beagle dogs.

Groups of three or four male and female beagle dogs were maintained for 6 months on diets containing BHA at concentrations of 0 (control group), 0.25, 0.5 and 1.0% (w/w). The average daily intake of BHA was about 60 mg/kg body weight in the 0.25% group, 110 mg/kg in the 0.5% group and 220 mg/kg in the 1.0% group. All animals survived the entire experimental period without showing signs of toxicity other than a dose-related retardation of growth. Serum biochemical examinations conducted at 1, 3 and 6 months revealed a slight decrease in albumin concentration and an elevation of alkaline phosphatase and leucine aminopeptidase activity in the higher dose groups. Histopathological and histometrical examination showed no evidence of mucosal alteration in the stomach, oesophagus or duodenum attributable to the administration of BHA. The mitotic index in the squamous epithelium of the distal oesophagus was comparable in the control and BHA-treated groups. Liver weights were increased in BHA-treated groups, but there were no related histopathological changes. From these data it is concluded that feeding of BHA-supplemented diets at palatable concentrations for 6 months has no pathological effects on the stomach, oesophagus, duodenum or liver of beagle dogs.

Hemopoietic cell kinetics after intraperitoneal single injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widely spread environmental pollutant. Homopoietic system is one of the targets of TCDD in laboratory animals including monkeys. The present study is the hemopoietic cell kinetics in mice, from the severe depression in cellularity of bone marrow and CFU-GM, to their recovery after the intraperitoneal injection of high dosage of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). The bone-marrow cellularity and CFU-GM were severely decreased to 37.8% and 48% of the control, respectively until day 1 after exposure to TCDD. They were, however, soon recovered, even overshot the control value. Subsequently, they tended to show decrease and oscillation again to and under the control value. In conclusion, our cell kinetic study has proven the oscillation in bone-marrow cellularity and CFU-GM during the recovery period, of which the observation seems to be useful to extend our understanding in the hematotoxicity of TCDD.

[Small cell carcinoma of the urinary bladder: a case report and review of the Japanese literature].

A 50-year-old man presented with asymptomatic gross hematuria which he had first noticed 3 months earlier. Clinical examinations revealed a non-papillary, broad-based tumor on the left lateral wall of the urinary bladder with a clinical stage of T3N0M0. The pathological diagnosis of a transurethral biopsy tissue specimen was small cell carcinoma. Neoadjuvant intraarterial infusion chemotherapy using cisplatin and adriamycin was initially administered but proved to be ineffective. Thus, we performed a radical cystectomy. The tumor tissue was apparently homogenous and composed of small cells arranged in sheets and solid patterns, and was staged to be pT3bR1L2V0N0. An electron microscopic study confirmed small cell carcinoma with neurosecretory granules. Postoperatively, 4 courses of adjuvant chemotherapy consisting of cisplatin, etoposide and ifosfamide were administered. The patient is alive without any evidence of tumor recurrence 26 months after the operation.

[In vivo test for endocrine disruptors].

The Endocrine Disruptor Issue has been brought up widely to the public by a book titled "Our stolen future"(Theo Colborn et al., 1996), in which the reproductive impairment of a variety of wild life was documented in relation to chemical exposures. The fear that the similar adverse effects could have been observed in humans is based on the understandings that estrogens and androgens are highly conserved signaling molecules among species. In addition, the estrogen receptors are known to have abilities to bind a variety of chemicals. Although such chemicals bind weakly to the receptor, it is also known that, at least in some assay systems, high concentration of these chemicals can elicit effects that are quite similar to those of the natural hormones. Thus, such "hermonally active agents, HAAs" can be ligands to the hormone receptors in an organism, and may cause adverse effects, namely the "receptor-mediated toxicity", which is a relatively new concept in the field of toxicology. This new aspects of toxicology is briefly discussed in relation to the reversible/irreversible effects in developing organisms and the shape of dose-response curves, both especially in the low-dose ranges, now often referred to as "low-dose effects".