Cyclooxygenase-2 expression and relationship to tumour progression in human renal cell carcinoma.

AIMS: Cyclooxygenase (COX), which catalyses the synthesis of prostaglandins from arachidonic acid, has two isoforms; COX-1 and COX-2. There is ample evidence to suggest an important role for COX-2 in cancer. The aim of this study was to evaluate the clinical significance of COX-2 expression and its localization in the development and progression of human renal cell carcinoma (RCC). METHODS AND RESULTS: The expression and localization of COX-2 were evaluated in human RCC tissues from 75 patients by immunohistochemistry. Immunoreactive COX-2 protein was observed in all cases of RCC, and the levels of COX-2 expression were correlated with tumour grade and pathological stage. Expression of COX-2 was higher in the granular cell subtype than in the clear cell subtype of RCC. Immunoelectron microscopy revealed that COX-2 was expressed in the nuclear membrane, rough endoplasmic reticulum, Golgi complex and mitochondrial membrane of RCC cells. CONCLUSION: COX-2 overexpression within these intracellular organelles in RCC may be associated with renal cell carcinogenesis and COX-2 may be a useful biomarker in RCC.

A comparison of mesial molar root canal preparations using two engine-driven instruments and the balanced-force technique.

The purpose of this study was to compare the effects of two engine-driven, nickel-titanium instrument systems with hand files in the final shape of slight and moderately curved canals. A total of 72 mesial roots of extracted human mandibular molars were divided into three groups: ProFile .04 taper, Pow-R rotary systems, and Flex-R hand-filing technique. The roots were mounted and cross-sectioned at two different horizontal levels using a modified Bramante technique. Pre- and postinstrumented cross-sectional roots were imaged, recorded, and computer analyzed. Results showed that, at the middle third, in almost all groups, there was a tendency of cutting more toward the mesial side with only one exception: Pow-R cut more to the distal side (danger zone) (p < 0.02). At the apical third, Flex-R (p < 0.03) and ProFile (0.001) transported to the mesial side (danger zone) when the curvature increased. When the three techniques were compared analyzing each side and considering the two groups of curvature, at the middle third in the moderately curved-canal group, Flex-R cut statistically more than Pow-R toward the lingual side. The other comparisons showed no statistically significant difference. When the techniques were compared in relation with the degree of curvature, in the apical third, ProFile .04 cut statistically more toward the mesial side in the moderately curved canal group than in the slightly curved canal group. The other comparisons showed no statistically significant difference. Canal preparation time was shorter with hand instrumentation (p < .05) in a few instances.

Expression and characterization of hypoxia-inducible factor (HIF)-3alpha in human kidney: suppression of HIF-mediated gene expression by HIF-3alpha.

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors that regulate a number of adaptive responses to low oxygen tension. They are composed of alpha- and beta-subunits that belong to the basic helix-loop-helix-PAS superfamily. Here we examined the expression of HIF-alpha subunit proteins in the human kidney and found that, in addition to HIF-1alpha and -2alpha, HIF-3alpha was also expressed. The sequence analysis revealed that, like mouse HIF-3alpha, human HIF-3alpha has high similarity with HIF-1alpha and -2alpha in the bHLH and PAS domains, but lacks structures for transactivation found in the C-terminus of HIF-1alpha and -2alpha. Furthermore, we performed reporter gene analysis and showed that HIF-3alpha suppresses hypoxia-inducible HIF-mediated gene expression. HIF-3alpha might be a negative regulator of hypoxia-inducible gene expression in the human kidney.

Effects of selenium deficiency on expression of selenoproteins in bovine arterial endothelial cells.

Damage to the vascular endothelium by reactive oxygen species causes many cardiovascular diseases including atherosclerosis. Such damage can be prevented by selenium (Se), which is thought to exert its actions mainly through the expression of selenoproteins. Se deficiency increased the susceptibility to tert-butylhydroperoxide (t-BuOOH) and enhanced lipid peroxidation in bovine arterial endothelial cells (BAEC). We investigated the effects of Se deficiency on the expression of the selenoproteins in BAEC. 75Se metabolic labeling analysis and RT-PCR analysis revealed that BAEC expressed two glutathione peroxidase (GPx) isozymes, cytosolic GPx (cGPx) and phospholipid hydroperoxide GPx (PHGPx), three thioredoxin reductase (TrxR) isozymes, TrxR1, TrxR2 and TrxR3, and selenoprotein P (SelP). Se deficiency reduced both enzyme activity and mRNA level of cGPx, but did not affect those of PHGPx. SelP mRNA level was also reduced by Se deficiency, although the extent of reduction was much smaller than that of cGPx mRNA. We further found that TrxR activity was also decreased by Se deficiency but none of the mRNA levels of TrxR isozymes were reduced. These results indicate that vascular endothelial cells express several selenoproteins including cGPx, PHGPx, TrxR isozymes and SelP which might play important roles in the defense system against oxidative stresses and that the expressions of these selenoproteins are differently regulated by Se status.

Glutathione content is correlated with the sensitivity of lines of PC12 cells to cisplatin without a corresponding change in the accumulation of platinum.

A study of the involvement of glutathione (GSH) in cellular resistance to cisplatin was performed using methylmercury-resistant sublines (PC12/TM series) of the PC12 line of rat pheochromocytoma cells. The seven clonal sublines of PC12 cells (PC12/TM, PC12/TM2, PC12/TM5, PC12/TM11, PC12/TM15, PC12/TM23, PC12/TM26) used in the study had intracellular levels of GSH that ranged from 8.7-39.9 nmol/mg protein. The intracellular level of GSH was significantly correlated (p < 0.01, r = 0.87) with the sensitivity to cisplatin of PC12 cells and the seven sublines. Among the seven sublines, PC12/TM cells contained the highest concentration of GSH and were the most resistant to cisplatin. Treatment of PC12/TM cells with L-buthionine-SR-sulfoximine, which reduced the level of GSH to that in the parental PC12 cells, significantly reduced the resistance of the cells to cisplatin. The amount of platinum accumulated by resistant PC12/TM cells after treatment with cisplatin was higher than that by sensitive PC12 cells. These results suggest that the intracellular level of GSH might be directly involved in the resistance to cisplatin of these cell lines. However, a high intracellular concentration of GSH does not appear to contribute to a decrease in the accumulation of cisplatin in these cells.

Enhancement of androgen-dependent transcription and cell proliferation by tributyltin and triphenyltin in human prostate cancer cells.

Tributyltin (TBT) and triphenyltin (TPT) are known to cause imposex, the superimposing of male genitals on female ones, in some species of gastropods. However, the molecular mechanism of the trialkyltin-induced endocrine dysfunction remains to be elucidated. To clarify the effects of organotin compounds on the activation of androgen receptor (AR)-mediated responses in mammals, a LA16 clone that stably expresses androgen-responsive luciferase reporter gene and proliferates in response to androgen was established from human prostate cancer cell line LNCaP. Stimulation of LA16 cells with 100 nM TBT or 1 nM TPT enhanced both AR-dependent transcription of luciferase gene and cell growth to the same extent as those by 1 nM dihydrotestosterone (DHT). TBT or TPT also enhanced the DNA synthesis and expression of endogenous AR target genes such as prostate specific antigen, but not the expression of AR itself. However, an androgen antagonist, flutamide, did not inhibit the TBT- or TPT-induced AR activation. On the other hand, simultaneous treatment of LA16 cells with DHT and TBT or TPT caused highly enhanced effects on AR activation. These results indicate that trialkyltin compounds have an ability to activate AR-mediated transcription in mammalian cells and suggest that a novel target site other than the ligand-binding site of AR is involved in this activation.

HeLa cell transformants overproducing mouse metallothionein show in vivo resistance to cis-platinum in nude mice.

Plasmid pSV2MT-I encoding mouse metallothionein-I (MT-I) designed to be expressed under the control of an SV40 promoter was introduced into human HeLa S3 cells. Several transformants (HeLa/MTH) carrying multi-copies of mouse MT-I cDNA in their genomes were isolated. These transformants produced 4 to 20-fold larger amounts of MT than their parent cells. The MT levels in HeLa/MTH were well correlated with the extent of resistance to cadmium, but not with that to cis-platinum (cis-DDP) in vitro. To study the role of MT in resistance to cis-DDP in vivo, nude mice were inoculated subcutaneously with two independent HeLa/MTH clones. MT levels in these tumors were about 3-fold higher than those in the parental cells. The growth of tumors derived from either HeLa/MTH clone was not inhibited in the presence of 15 micromol/kg of cis-DDP, which completely inhibited the growth of tumors derived from the parental HeLa cells. These data strongly suggest that the elevated level of MT confers resistance to cis-DDP in vivo but not in vitro. Thus, the results of this study indicate that in vitro determinations of the influence of MT on cis-DDP resistance may underestimate its importance in in vivo situations.

Suppression of a high-affinity transport system for manganese in cadmium-resistant metallothionein-null cells.

Cadmium is a hazardous heavy metal existing ubiquitously in the environment, but the mechanism of cadmium transport into mammalian cells has been poorly understood. Recently, we have established a cadmium-resistant cell line (Cd-rB5) from immortalized metallothionein-null mouse cells, and found that Cd-rB5 cells exhibited a marked decrease in cadmium uptake. To investigate the mechanism of altered uptake of cadmium in Cd-rB5 cells, incorporation of various metals was determined simultaneously using a multitracer technique. Cd-rB5 cells exhibited a marked decrease in manganese incorporation as well as that of cadmium. However, the reduced uptake of manganese was observed only at low concentrations, suggesting that a high-affinity component of the Mn(2+) transport system was suppressed in Cd-rB5 cells. Competition experiments and kinetic analyses revealed that low concentrations of Cd(2+) and Mn(2+) share the same high-affinity pathway for their entry into cells. The mutual competition of Cd(2+) and Mn(2+) uptake was also observed in HeLa, PC12, and Caco-2 cells. The highest uptake of Cd(2+) and Mn(2+) by parental cells occurred at neutral pH, suggesting that this pathway is different from a divalent metal transporter 1 that can transport various divalent metals including Cd(2+) and Mn(2+) under acidic conditions. These results suggest that a high-affinity Mn(2+) transport system is used for mammalian cellular cadmium uptake, and that the suppression of this pathway caused a marked decrease in cadmium accumulation in cadmium-resistant metallothionein-null cells.

Modulation of telomerase activity by zinc in human prostatic and renal cancer cells.

Because the up-regulation of telomerase in most cancer tissues is considered to be responsible for the unlimited proliferation of cancer cells, suppression of telomerase activity is an attractive potential target for cancer therapy. The mechanism for the activation of telomerase in cancer cells, however, is still unclear. In the present study, we demonstrated that Zn induces an enhancement of telomerase activity in the human renal cell carcinoma (NRC-12) and prostatic cancer (DU145) cell lines. The maximum elevation of the activity was observed 6 hr after treatment with 100 microM Zn; it was diminished by the addition of either metal chelator or cycloheximide. Other metals such as Cd and Cu also enhanced telomerase activity but to a lesser extent, and no correlation between the activation of telomerase and the induction of metallothionein was observed. Our findings provide the first evidence that metals, especially Zn, can modulate telomerase activity in cancer cells.

[One-stage surgery of minimally invasive direct coronary artery bypass, abdominal aortic aneurysm repair and carotid endarterectomy].

A 74 year-old man with bladder transitional carcinoma had severe multivascular disease; coronary artery stenosis, abdominal aortic aneurysm and right internal carotid artery stenosis. First, transurethral bladder tumor resection (TUR-Bt) was performed twice but in the second TUR-Bt, no carcinoma cell was found. One stage surgery of minimally invasive direct coronary artery bypass (MIDCAB), abdominal aortic aneurysm (AAA) repair and carotid endarterectomy (CEA) was proposed. MIDCAB was performed first. Inspite of the bradycardia, heart oppression by stabilizer and coronary artery clamping, blood pressure and ST segments were stable. With heparinization and the chest left open, AAA repair was carried out. On aorta clamping and declamping, blood pressure and heart rate were stable. After completion of AAA repair, heparinization was reversed with protamine. Chest and abdominal wounds were closed simultaneously. CEA was performed lastly, because the patient had no cerebral ischemic symptom and no risk of cardiopulmonary bypass. After the operation, no neurologic deficit appeared. This experience of one stage surgery was reported with review of literatures. One stage surgery is a possible approach to the patients with severe multivascular disease.

Effects of methylmercury and inorganic mercury on the growth of nerve fibers in cultured chick dorsal root ganglia.

Inhibition of the growth of nerve fibers by mercurials was quantitatively estimated by measuring the length of fibers in the cultured chick dorsal root ganglion. Morphological changes in nonneuronal cells were also evaluated. The growth rates of nerve fibers were constant for 2 to 6 days after the start of incubation. Methylmercury depressed nerve fiber growth dose- and time-dependently by 50% and completely at 3 x 10(-6) M and 7 x 10(-6) M, respectively. About 10-fold higher concentrations of inorganic mercury were required for the same extent of inhibition. The nerve fibers exposed to inorganic mercury shrank at an early stage of exposure and thereafter grew again within 24 hours. Electron microscopic examination revealed that methylmercury decreased microtubule mass extensively in nerve fibers, while inorganic mercury markedly altered surface membrane structure. These results suggested that microtubule disruption is involved in methylmercury-induced depression of nerve fibers but not in that induced by inorganic mercury. Characteristic effects on the growth of nerve fibers and the proliferation of nonneuronal cells were observed on the treatment with other metals such as cadmium, silver and chromium. Thus, dorsal root ganglion culture seems to be useful for the evaluation of toxic effects of metals in vitro.

A comparison of the relative efficacies of four hand and rotary instrumentation techniques during endodontic retreatment.

AIM: The purpose of this study was to quantify the amount of remaining gutta-percha/scaler on the walls of root canals when two engine-driven instruments (Quantec and ProFile) and two hand instruments (K-file and Hedström file) were used to remove these materials. The amount of apically extruded debris and the time required for treatment were also recorded. METHODOLOGY: One hundred extracted mandibular premolars were prepared using a modified step-back, flare technique and obturated with the lateral condensation technique. After repreparation with the test instruments, the specimens were cut transversally at the cervical, middle and apical thirds with steel discs and the three sections were split longitudinally. The amount of residual debris on the canal walls in each section was examined using a stereomicroscope. RESULTS: In all groups the cervical and middle thirds showed no debris. In the apical third, obturating material was observed in some specimens. No statistically significant difference was found between the two groups for incidence of debris, although the Hedström group showed a greater number of samples with remaining gutta-percha/sealer. When analysing dirty specimens only, there was a statistically significant difference between the four groups (P < 0.01) with the Hedström group having significantly less length of canal wall with remaining obturation material than the Quantec group. There was no significant difference amongst the groups for weight of extruded debris. However, there was a significant difference amongst the groups for mean treatment time with the Hedström file group requiring significantly less time than the Quantec group (P < 0.001); no significant differences were found between the other groups. Six instruments fractured in the Quantec group, four in the ProFile group, two in the Hedström group and two in the K-type group. CONCLUSIONS: The results showed that overall, all instruments may leave filling material inside the root canal. During retreatment there is a risk of instrument breakage, especially rotary instruments.

Modulation of adriamycin toxicity by tissue-specific induction of metallothionein synthesis in mice.

The effect of tissue specific induction of metallothionein (MT) by preadministration of metal compounds on the antitumor activity and adverse effects of adriamycin (ADR) was examined using mice bearing colon 38 adenocarcinoma. Significant increase in MT concentration was observed in the heart and bone marrow but not in the tumor tissue of the mice given bismuth (Bi) compound. Copper (Cu) increased MT in the tumor tissue but did not induce MT either in bone marrow or in the heart, whereas zinc (Zn) increased MT level in the heart and bone marrow as well as in the tumor tissue. ADR exerted cardiotoxicity, indicated by increase in lipid peroxidation in the heart, bone marrow toxicity, indicated by decrease in number of peripheral leukocytes, and antitumor activity, assessed by reduction of tumor weight, in tumor-bearing mice untreated with MT inducing metal compounds. Preadministration of Bi significantly reduced the cardiotoxicity and bone marrow toxicity without compromising the antitumor activity of ADR. Cu pretreatment did not affect the extent of cardiotoxicity and bone marrow toxicity but significantly suppressed the antitumor effect. Pretreatment with Zn markedly reduced not only the adverse side effects but also the antitumor activity. The results described above suggest that ADR toxicity can be attenuated in the tissues in which the MT level was elevated and that the tissue specific induction of MT synthesis may provide a promising regimen for cancer chemotherapy.

Role of antioxidant systems in human androgen-independent prostate cancer cells.

BACKGROUND: Most prostate cancer cells respond to initial hormonal therapy; however, some of them eventually acquire resistance to the hormonal therapy. Hormone-independent prostate cancer usually exhibits resistance to chemotherapy and radiotherapy. Antioxidant systems are known to be involved in the resistance of cancer cells to chemotherapy and radiotherapy. Therefore, it is of significance to examine antioxidant systems of hormone-independent prostate cancer for enhancing the efficacy of cancer therapy. METHODS: Three cell lines of human hormone-independent prostate cancer (PC-3, PC-3 MA2, and HPC36M) were examined for activities of superoxide dismutase, catalase, and glutathione peroxidase, and for levels of protein and nonprotein thiols such as metallothionein, glutathione, and thioredoxin. Sensitivity of these cells to anticancer drugs and inducers of reactive oxygen species such as paraquat, tert-butylhydroperoxide, and hydrogen peroxide was determined by microtiter assay. RESULTS: PC-3 and PC-3 MA2, which were derived from bone metastases, were resistant to paraquat, hydrogen peroxide, and cisplatin compared with HPC36M, which was obtained from the primary prostate cancer. However, HPC36M was resistant to vinblastine compared with PC-3 and PC-3 MA2. Both PC-3 and PC-3 MA2 had higher activities of catalase and glutathione peroxidase and higher levels of glutathione and metallothionein than HPC36M. CONCLUSIONS: These data suggest that enhanced ability in scavenging free radicals by antioxidant enzymes and thiol compounds may, at least in part, contribute to the resistance of bone metastatic prostate cancer during chemotherapy.

The involvement of microtubular disruption in methylmercury-induced apoptosis in neuronal and nonneuronal cell lines.

Methylmercury (MeHg) is known to interfere with cell cycle progression by disruption of microtubules. The relationship between the changes in cell cycle and the induction of apoptosis caused by MeHg was investigated in cultured mammalian cells. MeHg caused nuclear fragmentation and DNA ladder formation in rat pheochromocytoma (PC12) and mouse neuroblastoma cells exposed to MeHg. Flow cytometric analysis revealed that the occurrence of apoptosis was preceded by the accumulation of cells in G2/M after MeHg treatment. Exposure to colchicine, a well-characterized mitotic inhibitor, also caused G2/M-phase arrest followed by the appearance of apoptotic cells. These results suggest that G2/M-phase arrest through the disruption of microtubules is an important event in the development of apoptosis by MeHg. MeHg treatment led to G2/M-phase arrest followed by apoptosis in nonneuronal HeLa cells also. Bcl-2 was phosphorylated by MeHg treatment in HeLa cells but not in PC12 cells; however, p53 expression was not changed in either cell line. Thus, MeHg induces apoptosis via a p53-independent pathway in both cell lines, however, different pathways may be activated after the disruption of microtubules in PC12 and HeLa cells.

Nuclear localization of hypoxia-inducible factor-2alpha in bovine arterial endothelial cells.

Hypoxia-inducible factor (HIF)-2alpha is a recently identified hypoxia-inducible transcription factor abundantly expressed in vascular endothelial cells. As well as HIF-1alpha, HIF-2alpha forms a heterodimeric complex with the aryl hydrocarbon receptor nuclear translocator and upregulates hypoxia-inducible genes such as vascular endothelial growth factor. We found in this study that using green fluorescent protein (GFP) fusion constructs, the subcellular localization of HIF-2alpha was different from that of HIF-1alpha in bovine arterial endothelial cells (BAEC). HIF-1alpha was localized in the cytoplasm under normoxic cells and translocated from the cytoplasm into the nucleus in response to hypoxic induction. In contrast, HIF-2alpha was clearly localized in the nucleus of BAEC even under normoxic conditions. The regulation of HIF-2alpha might differ from that of HIF-1alpha in BAEC. We further showed that nuclear localization of HIF-2alpha was inhibited by either deletion or a single amino acid substitution within the C-terminal end of the protein. The amino acid sequence surrounding Lys737 and Arg738 functions as a nuclear localization signal of HIF-2alpha.

Reduced uptake and enhanced release of cadmium in cadmium-resistant metallothionein null fibroblasts.

Metallothionein (MT) is known to play a predominant role in the protection of cells from cadmium (Cd) toxicity. To investigate other factors involved in Cd resistance, we established Cd-resistant cell lines from simian virus 40-transformed MT null fibroblasts. Cd-resistant MT null cells, Cd-rA7 and Cd-rB5, developed approximately 10-fold resistance to Cd compared to parental cells, but showed no cross-resistance to Zn, Cu, Hg, Ni, As, cisplatin or H2O2. Accumulation of Cd in the resistant cells was 13-18% of that of parental cells after treatment with Cd for 24 h. A short-term experiment revealed that the rate of Cd incorporation into the Cd-resistant cells was suppressed, and the rate of Cd release was enhanced in the resistant cells compared with that of parental cells. These results indicate that the altered transport of Cd, slow uptake and rapid release, may confer resistance to Cd on the Cd-resistant cells established from MT null fibroblasts.

PF1070A, a novel and potent inducer of the synthesis of metallothionein.

Using mouse Ltk(-) cells (L13-17 cells) that had been transfected with a plasmid in which the lacZ gene had been ligated downstream of 1.4 kbp of the sequence of the promoter of the mouse gene for metallothionein-I (MT-I) as a reporter gene, we examined 268 organic compounds for the ability to activate this promoter. We found that PF1070A, an antibiotic produced by Humicola sp., efficiently activated the MT promoter and caused marked enhancement of beta-galactosidase activity in L13-17 cells. The extent of activation by PF1070A was almost equivalent to that by of zinc ions, the most effective known inducer of the synthesis of MT. PF1070A also caused marked elevation of the levels of the mRNA for MT and of MT itself in L13-17 cells. A similar result was obtained in human HeLa-S3 cells. When PF1070A was added to the culture medium simultaneously with cadmium ion or dexamethasone, the level of expression of the reporter gene was markedly elevated, compared to the level of expression induced by each agent independently. The effect of PF1070A was reduced considerably by deletion of nucleotides at positions -150 and -149 from the site of initiation of transcription in the promoter region of the MT gene and also by deletion of the seven bases located at positions -49 to -43. Since no known cis element was found in these two regions, PF1070A might be a new type of inducer of MT synthesis that promotes expression of the gene for MT via a mechanism completely different from those exploited by other known agents. These results also suggest the presence of a system for control of transcription of the gene for MT that has not previously been recognized. Both cadmium ions and bismuth ions induce the synthesis of MT by acting on the metal response element (MRE). Bismuth ions had no significant effect on the promoter activity that had already reached a maximum level in response to treatment with the optimal concentration of cadmium ion. By contrast, PF1070A further and markedly increased the promoter activity. This result suggests that it is possible to increase the concentration of MT in tissue using PF1070A as an inducer even in cases where the MRE-mediated activation of the MT promoter has already been induced by the accumulation of cadmium, as is the case in a clinical setting. PF1070A may prove to be an excellent inducer of MT synthesis that is effective and clinically applicable. Moreover, use of PF1070A in combination with salts of heavy metals might be useful in controlling expression of a transfected gene that is regulated by the MT promoter since PF1070A can activate the MT promoter to an extent that cannot be achieved with heavy metal ions alone, when PF1070A is used in combination with zinc ions at a concentration of the latter considerably below the toxic level.

Regulatory role of metallothionein in NF-kappaB activation.

Metallothionein (MT), a low molecular weight, cysteine-rich metal binding protein, has been associated with cytoprotection from heavy metals and cellular oxidants. As MT has the ability to scavenge hydroxyl radicals, MT may control intracellular redox status. In the present study, we examined whether MT regulates the activity of nuclear factor-kappaB (NF-kappaB), which is one of the redox-regulated transcription factors, using the MT null embryonic cell lines established from MT null mice. We first found that tumor necrosis factor (TNF)-induced activation of the binding of NF-kappaB protein to DNA in wild type MT+/+ cells was lower than that in MT-/- cells. The NF-kappaB activation in MT-expressing cells established from MT-/- cells by the transfection of mouse MT-1 gene was also significantly lower than that in MT-/- cells. In addition, transfection of the MT gene inhibited TNF-induced IkappaB degradation and suppressed NF-kappaB-dependent gene expression induced by TNF. These results demonstrate that MT may function as a negative regulator of NF-kappaB activity.

Metallothionein modulates the carcinogenicity of N-butyl-N-(4-hydroxybutyl)nitrosamine in mice.

We examined the carcinogenicity of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in transgenic mice deficient in the metallothionein (MT) I and II genes and in control (129/Sv) mice. Both strains of mice were given BBN for 8 weeks with or without Zn treatment. All mice were killed at 12 weeks after the cessation of BBN administration. BBN induced bladder tumors in 75% of MT null mice and in 43% of 129/Sv mice. The average number of bladder tumors per mouse was significantly higher in MT null mice (1. 18 +/- 0.27) than in 129/Sv mice (0.43 +/- 0.20). Zn treatment suppressed the carcinogenicity of BBN in 129/Sv mice but not in MT null mice. Histopathological examination of the tumors revealed that the malignant potential of bladder tumors in 129/Sv mice was greater than that in MT null mice. These results indicate that MT is an important modulator of carcinogenicity of BBN in the bladder of mice.