Effects of nickel compounds on incorporation of [3H] thymidine into DNA in rat liver and kidney

In vivo incorporation of [³H] thymidine into DNA was determinedin rats at 28 h after partial hepatectomy. Administration ofnickel carbonyl (Ni(CO)₄) at 2 or 4 h before sacrifice inhibited[³H] thymidine uptake into liver and kidney DNA. For example,in rats killed 4 h after i.v. injection of Ni(CO)₄ (2 mg Ni/100g), [³H]-labelling of liver DNA averaged 54 (SE ± 10)%of controls (p<0.05), and [³H]-labelling of kidney DNA averaged53 (SE ± 6)% of controls (p<0.01). Injection of NiCI₂(2 mg Ni/100 g, i.m.) 4 h before death did not significantlyaffect [³H] thymidine uptake into liver DNA, but did inhibit[³H] thymidine uptake into kidney DNA (65 ± 6%, p<0.02).Binding of ⁶³Ni to DNA in liver and kidney of rats killed 4h after injection of ⁶³Ni(CO)⁴or ⁶³NiCl² ranged from 0.3 to2.2 mol ⁶³Ni/mol of DNA nucleotides. Ultracentrifugation ofDNA on alkaline sucrose gradients did not reveal any differencesbetween sedimentation profiles of hepatic DNA from Ni(CO)⁴-treatedrats versus paired control rats.     

The effect of N-nitrosodimethylamine and N-nitroso-N-benzylmethylamine on [3H]thymidine incorporation into the DNA of target and non-target tissues in the zinc-deficient rat

The influence of dimethylnitrosamine (NDMA), a liver carcinogen and nitrosobenzylmethylamine (NBMA) as esophageal carcinogen on [3H]thymidine incorporation into DNA was studied in the esophagus, liver, forestomach and gastric-stomach of fasted zinc-deficient and pair-fed zinc-sufficient rats, measured 1 h after the thymidine injection. In the untreated animals, dietary zinc deficiency significantly depressed [3H]thymidine incorporation (89%) into the DNA of forestomach only. NDMA, administered 4 h before death at 30 mg/kg, produced 50-55% inhibition in [3H]thymidine incorporation in the esophagus of rats of both dietary groups. This inhibition became more pronounced in the forestomach, reaching 90-94% in the zinc-deficient forestomach and 63-86% in their zinc-sufficient counterparts at NDMA levels ranging from 5 to 20 mg/kg. NBMA at 2 mg/kg produced 60% inhibition in the DNA synthesis of zinc-deficient esophagus and 40% in the corresponding zinc-sufficient ones, this difference being significant at P less than 0.01. On the other hand, [3H]thymidine incorporation in the forestomach DNA was markedly lowered in the presence of NBMA. Recovery of DNA synthesis in the 4 tissues from a single dose of NDMA or NBMA was monitored up to 12 days. Following NDMA injection, [3H]thymidine incorporation in the forestomach of both dietary groups remained inhibited (3% of untreated control) for 5 days, a significant recovery (45% of untreated control) was observed only in the zinc-sufficient animals. Following NBMA injection, [3H]thymidine incorporation was also inhibited in the zinc-deficient esophagus for a longer time than in the zinc-sufficient ones. In autoradiographic studies, the percentage of cells showing 30 or more grains/nucleus was significantly decreased (P less than 0.001) in the NBMA-treated and marginally decreased (P less than 0.05) in the NDMA- or NBMA-treated zinc-deficient and zinc-sufficient rats as compared with the saline-treated zinc-sufficient controls. These results were discussed in the light of our previous findings that NBMA enhanced esophageal tumorigenesis in the zinc-deficient rats and that NDMA, a liver carcinogen produced forestomach tumors in the zinc-deficient but not in the zinc-sufficient rats.     

Soluble factors from liver and hepatomas which inhibit [3H]thymidine incorporation into DNA of Novikoff hepatoma cells.

The nature of soluble factors from liver and hepatomas which inhibit [3H]thymidine incorporation into DNA was studied in Novikoff hepatoma cells. The decreased activity in hepatoma preparations was due to loss of a high-molecular-weight heat-labile factor. Although this factor cochromatographed with arginase activity on Sephadex G-150, it does not appear to result from this activity as judged by the failure of arginine to prevent the inhibitory effect on [3H]thymidine incorporation. Both liver and hepatomas contained a heat-stable factor with inhibitory activity. Studies with ethanol-soluble material suggested that the action was not solely attributable to the presence of unlabeled thymidine, since the apparent molecular weight was too high and since the factor(s) inhibited [3H]leucine incorporation into protein in addition to inhibiting [3H]thymidine incorporation in DNA.     

Asbestos and benzo[a]pyrene act synergistically to induce squamous metaplasia and incorporation of [3H]thymidine in hamster tracheal epithelium

When exposed to either crocidolite asbestos (single 1-h exposureto 0.4 mg/ml medium) or the polycyclic aromatic hydrocarbon,benzo [a]pyrene (BaP) (2.5 µg/ml medium, 1x weekly for4 weeks), the epithelium of hamster tracheal explants exhibitsinsignificant amounts of squamous metaplasia, an atypical lesion,in comparison to amounts observed in untreated tissues. Incorporationof [³H]thymidine, an indication of DNA synthesis by epithelialcells, likewise is unchanged. However, the extent of squamousmetaplasia and numbers of labeled basal and suprabasal cellsare increased substantially when BaP and asbestos are addedin combination. These results suggest an important mechanismof co-carcinogenesis involving chemical and physical carcinogensand support epidemiologic observations documenting an increasedrisk of bronchogenic carcinoma in asbestos workers who smoke.     

Effect of copper administration on the incorporation of [3H]-thymidine into the liver DNA of rats stimulated by dimethyl-nitrosamine and diethylnitrosamine

The incorporation of [³H]thymidine into liver DNA of rats increased6–8 times 48 h after a single injection of dimethylnitrosamine(DMN, 30 mg/kg) and diethyl-nitrosamine (DEN, 100 mg/kg). Totest the suppressive effect of copper, the incorporation of[³H]thymidine in to liver DNA in the DMN groups or DEN groupspretreated with copper was measured 48 h after the administrationof DMN or DEN. The incorporation of [³H]thymidine into liverDNA of rats stimulated by the injection of DEN was strikinglysuppressed by the injection of cupric acetate (20 mg Ci/kg),but that of rats simultated by the injection of DEN was notsuppressed by the injection of copper. Some other metal salts,silver nitrate (20 mg Ag/kg), nickel acetate (20 mg Ni/kg) andbasic lead acetate (20 mg Pb/kg) did not significantly suppressthe incorporation of [³H]thymidine stimulated by DMN or DEN.The accumulation of copper was much higher in the liver of copper-administeredrats than that of nickel or lead in the liver of nickel-administeredrats or lead-administered rats. The accumulation of silver wascomparatively high in the liver of silver-administered rats.     

Fractional incorporation of [3H]thymidine and DNA specific activity as assays of inhibition of tumour growth.

The Fractional Incorporation (FI) of [3H] thymidine ([3H]TdR) has been examined in small lung tumours after cyclophosphamide (CY) treatment in vivo and compared to the DNA specific activity (SA) at different times after treatment. FI was found to correlate with the incidence of labelled cells after treatment, whereas SA did not, due to the loss of DNA from drug-killed cells 72 h after treatment. The FI is independent of the precursor concentration in the tissue, and therefore may give a better index of DNA synthesis in irregularly perfused tissues than SA. Following either CY or 60Co radiation treatment, the time necessary for FI to reach the pretreatment level is quite similar to the growth delay measured for the FI depression 45 h after treatment and growth delay has been established in the Lewis lung tumour, which would allow the prediction of growth delay induced by another agent to be made within 2 days of treatment.     

Effect of bleomycin on [3H]Thymidine 5'-Triphosphate incorporation into host liver and hepatoma nuclei.

The effect of bleomycin on [3H]thymidine 5'-triphosphate ([3H]TTP) incorporation into isolated sucrose nuclei from host liver and Morris hepatomas has been compared. Bleomycin stimulates [3H]TTP incorporation 13-fold in host liver and hepatoma 16 nuclei, 8-fold in hepatoma 7800 nuclei, and 3-fold in hepatoma 7777 nuclei. Differences in the nuclear membranes are not responsible for the different response of the nuclei. Nuclei, denuded of their membranes by Triton X-100 treatment, give similar results to sucrose nuclei. Analysis of DNA extracted from liver or hepatoma nuclei incubated with bleomycin indicates that bleomycin produces scissions in the nuclear DNA and that some repair synthesis takes place. Incubation of nuclei with 111indium-labeled bleomycin shows an equal binding capacity of liver and hepatoma nuclei for bleomycin. Bleomycin also stimulates incorporation of [3H]TTP in a system using chromatin or calf thymus DNA as primer. Host liver or hepatoma chromatin incubated with a DNA polymerase extracted from normal rat liver nuclei is stimulated approximately to the same extent by bleomycin. When DNA polymerase extracts from host liver and hepatoma nuclei are assayed with calf thymus DNA as primer, bleomycin has a greater stimulatory effect on [3H]TTP incorporation with host liver DNA polymerase than with hepatoma DNA polymerase in the system. We suggest that a defect in the repair system in hepatoma nuclei is responsible for the relatively lower response to bleomycin.     

Food restriction inhibits [3H] 7,12-dimethylbenz(a)anthracene binding to mouse skin DNA and tetradecanoylphorbol-13-acetate stimulation of epidermal [3H] thymidine incorporation

It has been known for many years that reducing the food intake of laboratory mice and rats inhibits the development of a broad spectrum of chemically induced and spontaneous tumors, but the mechanism of this effect is poorly understood. Food restriction of A/J mice for two weeks is now shown to inhibit the binding of topically applied [3H]7,12-dimethylbenz(a)anthracene (DMBA) to skin DNA by 50% and to abolish the stimulation of [3H]-thymidine incorporation in the epidermis produced by topical application of the tumor promoter tetradecanoylphorbol-13-acetate (TPA). Similar effects on the actions of DMBA and TPA are observed following topical application of the adrenal steroid, dehydroepiandrosterone (DHEA), a potent glucose-6-phosphate dehydrogenase (G6PDH) inhibitor, while food restriction for two weeks depresses epidermal G6PDH activity by 60%. It is suggested that both the inhibition of [3H]DMBA binding to skin DNA and the TPA stimulation in epidermal [3H]thymidine incorporation result from a reduction in the NADPH cellular pool as a result of G6PDH inhibition.     

Enhanced incorporation of thymidine into DNA in the liver of intact and partially hepatectomized rats pretreated with 5-azacytidine.

The administration of 5-azacytidine to rats resulted in enhancement of thymidine incorporation into liver DNA. Repeated doses of the drug caused a greater than 10-fold increase of thymidine incorporation. In 5-azacytidine-treated intact rats no changes in the activity of thymidine and thymidylate kinases were observed, whereas a marked depression of thymidine phosphorylase activity occurred. In intact animals the mitotic activity in the drug-treated livers changed slightly; however, 5-azacytidine administration before partial hepatectomy resulted in a dramatic increase of mitotic activity in 24-hr regenerating livers. In this case the observed increase in incorporation of thymidine was paralleled both by increased activity of thymidine and thymidylate kinases and by a decline in thymidine phosphorylase activity. The higher incorporation of radioactivity into liver DNA in intact rats pretreated with the drug can be accounted for, at least partially, by the lower cellular degradation of the injected radioactive thymidine used for labeling. In addition, alterations in the labeling and in the mitotic activity of hepatocytes and nonparenchymal cells caused by 5-azacytidine should be taken into consideration.     

Differentially expressed genes execute zinc-induced apoptosis in precancerous esophageal epithelium of zinc-deficient rats

Zinc deficiency (ZD) in rats increases esophageal cell proliferation and the incidence of N-nitrosomethylbenzylamine-induced esophageal tumors. Conversely, zinc replenishment (ZR) rapidly induces apoptosis in esophageal epithelia and reverses cancer development. We investigated gene expression changes in ZR versus ZD esophageal epithelia to identify differentially expressed genes associated with the antitumor effect of ZR. Weanling rats were fed a ZD diet for 6 weeks to establish esophageal cell proliferation or a zinc-sufficient (ZS) diet. Then, 10 ZD rats were treated with zinc gluconate intragastrically and switched to ZS diet; the remaining 10 ZD and ZS animals were treated with saline. All animals were killed 26-28 h later. Using cDNA microarrays, real-time polymerase chain reaction amplification and RNA hybridization techniques, we identified novel differentially expressed genes, including a RNA-binding protein with two RNA recognition motifs and a zinc knuckle (ZD7), and a DNA/RNA helicase with a DEAD box (ZD10) with two splice variants, ZD10a and ZD10b. In situ hybridization detected increased mRNA expression of ZD7, ZD10a and ZD10b in ZR esophageal epithelia, which displayed markedly increased occurrence of apoptotic cells, relative to ZD epithelia. Overexpression of ZD7 in human esophageal cancer cells resulted in induction of apoptosis and activation of caspase-3 and -7, activities that were inhibited by caspase-specific inhibitors. In addition, ZD7 mRNA levels and zinc-induced apoptosis in rat squamous carcinoma cells were reduced by specific small interfering ribonucleic acids. Thus, ZR rapidly induces ZD7 and ZD10 expression, which in turn stimulates apoptosis. These results provide the beginnings of a molecular pathway for zinc-induced apoptosis under conditions that reverse esophageal tumor initiation.     

Inhibitory effects of molybdenum on esophageal and forestomach carcinogenesis in rats

Male weanling inbred SD rats were given ad libitum a nutritionally adequate semipurified diet and demineralized drinking water without or with 100 or 200 ppm tungsten (W) or 2 or 20 ppm molybdenum (Mo) added to the drinking water. The animals were gastrically intubated with a solution of N-nitrososarcosine ethyl ester (NSEE) from the 4th week twice weekly for 2-8 consecutive weeks. The addition of Mo at either the 2- or 20-ppm level significantly inhibited NSEE-induced esophageal and forestomach carcinogenesis. The 200 ppm W significantly countered the inhibitory effect of a low level of Mo naturally occurring in the diet.     

DNA adduct formation in liver following the administration of [3H]2-nitrofluorene to rats in vivo.

The formation of RNA and DNA adducts by the environmental pollutant 2-nitrofluorene (2-NF) has been investigated in rat liver in vivo. The adduct pattern was studied after trifluoroacetic acid hydrolysis of DNA or RNA, followed by analysis of the adducts by HPLC. This was also done by enzymatic hydrolysis of DNA, followed by 32P-postlabeling. Both after oral and i.v. administration of [3H]2-NF, one major adduct was found. This adduct did not co-migrate with one of the known adducts of 2-(acetyl)-aminofluorene, N-deoxyguanosin-8-yl-2-aminofluorene (dG-C8-AF), which could have been formed after nitroreduction of 2-NF. 32P-Postlabeling revealed that two minor adducts were also formed, one of which was dG-C8-AF. The observation that the major adduct was also formed after i.v. administration of 2-NF to bile duct-catheterized rats makes a role for the intestinal microflora in the formation of this adduct very unlikely. In vitro experiments with inhibitors of the enzyme epoxide hydrolase indicated that epoxidation of 2-NF may play a role in the microsomal bioactivation of this compound.     

Alpha-difluoromethylornithine induction of apoptosis: a mechanism which reverses pre-established cell proliferation and cancer initiation in esophageal carcinogenesis in zinc-deficient rats.

Alpha-difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase, the first enzyme in polyamine synthesis. Previous work showed simultaneous administration of DFMO and a zinc-deficient (ZD) diet to weanling rats from the beginning inhibited the onset of zinc-deficiency-induced esophageal cell proliferation by activating apoptosis and reduced the incidence of N-nitrosomethylbenzylamine (NMBA)-induced esophageal cancer. Because esophageal cancer initiation by NMBA is very rapid in ZD rats, this study determined whether DFMO is effective in preventing esophageal carcinogenesis when administered after the establishment of a carcinogenic environment. Weanling rats were given a ZD diet for 5 weeks to establish sustained increased esophageal cell proliferation and then an intragastric dose of NMBA. Thereafter, 20 rats were switched to DFMO-containing water while nine control ZD animals remained on deionized water; all of the animals continued on the ZD diet. Esophagi were collected 15 weeks later. The upper portion was processed for immunohistochemical analysis of cell proliferation, apoptosis, and expression of related genes, and the lower was processed for polyamine content. DFMO substantially reduces the levels of esophageal putrescine and spermidine and esophageal tumor incidence from 89 to 10% in ZD rats. Importantly, DFMO-treated ZD esophagi display increased rate of apoptosis accompanied by intense bax expression and greatly reduced cell proliferation by proliferating cell nuclear antigen expression. In addition, the p16(ink4a)/retinoblastoma control at G1 to S, deregulated in ZD esophagi, is restored after DFMO treatment. These results demonstrate that DFMO, a highly effective chemopreventive agent in esophageal carcinogenesis, reverses and counteracts esophageal cell proliferation/cancer initiation in ZD animals by way of stimulating apoptosis.     

Enhancement of esophageal carcinogenesis in male F344 rats by dietary phenylhexyl isothiocyanate

The purpose of this study was to evaluate the potential effectsof dietary 6-phenylhexyl isothiocyanate (PHITC) on N-nitrosomethylbenzylamine(NMBA)-induced esophageal carcinogenesis in rats. Groups of15 male F344 rats received weekly s.c. injections of NMBA in20% dimethylsulfoxide or the vehicle alone for 15 consecutiveweeks. Two weeks prior to initiation of carcinogen or vehicleinjections rats were provided with modified AIN-76A diet ormodified AIN-76A diet containing PHITC at levels of 0.4, 1.0or 2.5 µmol/g diet. Experimental controls consisted ofgroups that received only the vehicle (vehicle controls), NMBA(carcinogen controls) or PHITC at the high dose level of 2.5µmol/g diet. No esophageal tumors or preneoplastic lesionswere detected in rats that received the vehicle or PHITC alone.In contrast, all rats treated with NMBA alone or PHITC + NMBAexhibited esophageal tumors and preneoplastic esophageal lesions.In groups that received PHITC + NMBA tumor multiplicity wasincreased by 21–69% when compared with rats treated withNMBA alone, indicating that PHITC enhanced esophageal tumorigenesisin this model system. These results, in conjunction with ourprevious work, demonstrate that arylalkyl isothiocyanates mayinhibit or enhance esophageal tumorigenesis in the NMBA-treatedrat. The ability of isothiocyanates to inhibit or enhance experimentaltumorigenesis may depend on alkyl chain length of the isothiocyanate,the animal species examined and the specific carcinogen employed.     

食管上皮癌变过程中端粒长度、hTERT含量的定量研究

目的:研究端粒DNA长度、hTERT蛋白定量表达与食管上皮细胞癌变的关系。方法:对84例食管上皮脱落细胞分别应用流式荧光原位杂交检测端粒DNA长度,细胞免疫荧光流式定量法检测hTERT蛋白含量。结果:①正常食管上皮脱落细胞组、重度增生组和癌组增殖指数(PI)分别为11.51±4.08,19.04±5.39和29.46±5.50,癌组细胞增殖活性显著高于重度增生组(P<0.01),重度增生组高于正常组(P<0.01),PI值与细胞学分级呈正相关(r=0.80,P<0.01)。②正常食管上皮脱落细胞组、重度增生组和癌组的端粒长度Q-FISH值分别为50.83±8.86、36.96±8.02和27.81±6.59,癌组端粒长度明显短于重度增生组(P<0.01),重度增生组短于正常组(P<0.01)。端粒长度与细胞学分级呈负相关(r=-0.73,P<0.01)。③正常组、重度增生组和癌组hTERT蛋白荧光指数(FI)分别为0.95±0.14、1.47±0.22和1.75±0.19,癌组hTERT蛋白含量高于重度增生组(P<0.01),重度增生组高于正常组(P<0.01)。hTERT含量与细胞学分级呈正相关(r=0.81,P<0.01)。重度增生组和癌组hTERT蛋白阳性表达率分别为91.43%(32/35)和96.77%(30/31),二者都明显高于正常组(P<0.01)。结论:端粒DNA长度缩短、hTERT蛋白含量升高与食管上皮癌变密切相关。