Surgical repositioning of intruded immature permanent incisor: an updated treatment concept.

Intrusion of immature permanent anterior teeth presents a great dilemma due to variety of treatment options. The ideal treatment option is the one with least probability of developing complications like external root resorption, obliteration of pulp canal, marginal bone loss etc. This paper presents a case report with treatment strategy of repositioning, splinting, successfully attempted apexification and obturation of a completely intruded immature permanent central incisor. Excellent healing with no post-operative complications even after 10 months of follow up.

Induction of thymidine kinase in mouse skin exposed to 12-O-tetradecanoylphorbol-13-acetate.

Topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) exhibited an increase in the epidermal thymidine kinase (TK) activity in a dose dependent manner. Maximum induction of TK was observed at a TPA concentration of 2.5 microg per animal. The induction of TK by TPA appeared to be a function of time with the maximum TK induction between 4 and 16 h after TPA application. Repeated applications of TPA every 24 h did not show any cumulative effect rather TK activity appeared to be normal after two applications. However, repeated applications of TPA at an interval of 48 h exhibited increased TK activity even after 16 applications. Cycloheximide and actinomycin D, the inhibitors of protein and RNA synthesis, inhibited the TPA induced activation of TK. Our results demonstrated that TPA induced the TK activity may be, by increasing de novo synthesis of enzyme protein and this induction might lead to increased de novo DNA synthesis after TPA application. DMBA was used as a reference compound. As far as the authors are aware, this is the first report on TK induction by topical application of TPA.

Subchronic oral toxicity of a combination of insecticide (HCH) and herbicide (ISP) in male rats.

Rats were treated orally with technical hexachlorocyclohexane (HCH, 12.5, 25 and 50 mg kg-1 day(-1)) and technical isoproturon (ISP 22.5. 45 and 90 mg kg-1 day(-1)) daily for a period of 90 days alone and in combination. Treatment with HCH alone showed mild to severe toxicity and death. Significant changes occurred in liver weight, clinical enzyme profiles, haematological parameters and pathomorphological changes. Treatment with ISP alone did not produce such changes. The combination of HCH and ISP produced changes not suggestive of synergism.

Effect of nicotinamide on 12-O-tetradecanoyl-phorbol-13-acetate exposed mouse skin endonuclease activity and DNA synthesis.

Nicotinamide (NA), a relatively nontoxic compound, has been shown to inhibit tumor development, induce differentiation, increase the sensitization of the anticancer drug resistant cancer cells and is being used in different skin ailments. But there are not many reports on its mechanism of action. Here we report that NA induced endonuclease activity. This endonuclease induction by NA appeared to be dose dependent and a function of time. As evident by the use of modifiers of DNase I, this endonuclease appeared to be like DNase type I. Increased [3H] thymidine incorporation in DNA in the presence of NA is possibly a consequence of increased 3-OH' nicks due to increased DNA fragmentation by increased endonuclease activity. The present results would be of help in the better understanding of the mechanism of NA action and its improved use in cancer control.

Effects of nicotinamide on mouse skin tumor development and its mode of action.

Nicotinamide (NA), a naturally occurring vitamin and a protease inhibitor, has been shown to be effective in treating some skin ailments. It inhibits cell proliferation and induces cell differentiation. This report shows the effects of NA on mouse skin tumor development and on the critical events involved in this process. NA reduced tumor growth, inhibited the 12-O-tetradecanoylphorbol-13-acetate (TPA) induced ornithine decarboxylase activity, but induced the transglutaminase activity which was inhibited by TPA under different experimental conditions. The effects of NA on ornithine decarboxylase (ODC) and transglutaminase (TG) indicated that nicotinamide (NA) probably programmed the cells for their death in the natural course of time, i.e. programmed cell death. This observation indicates that NA might be a better agent for the detailed study and for the better use in prevention of cancer alone or in combination with other drugs.

Potent induction of human colon cancer cell uptake of chemotherapeutic drugs by N-myristoylated protein kinase C-alpha (PKC-alpha) pseudosubstrate peptides through a P-glycoprotein-independent mechanism.

Phorbol ester protein kinase C (PKC) activators and PKC isozyme over-expression have been shown to significantly reduce intracellular accumulation of chemotherapeutic drugs, in association with the induction of multidrug resistance (MDR) in drug-sensitive cancer cells and enhancement of drug resistance in MDR cancer cells. These observations constitute solid evidence that PKC plays a significant role in the MDR phenotype of cancer cells. PKC-catalyzed phosphorylation of the drug-efflux pump P-glycoprotein was recently ruled out as a contributing factor in MDR. At present, the sole drug transport-related event that has been identified as a component of the role of PKC in MDR is PKC-induced expression of the P-glycoprotein-encoding gene mdr1. The objective of this study was to test the hypothesis that PKC can modulate the uptake of chemotherapeutic drugs in cancer cells independently of P-glycoprotein. We analyzed the effects of selective PKC activators/inhibitors on the uptake of radiolabelled cytotoxic drugs by cultured human colon cancer cells that lacked P-glycoprotein activity and did not express the drug efflux pump at the level of message (mdr1) or protein. We found that the selective PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) significantly reduced uptake of [14C] Adriamycin and [3H] vincristine in human colon cancer cells devoid of P-glycoprotein activity, and that PKC-inhibitory N-myristoylated PKC-alpha pseudosubstrate synthetic peptides potently and selectively induced uptake of the cytotoxic drugs in the phorbol ester-treated and non-treated colon cancer cells. TPA treatment of the cells did not induce expression of either P-glycoprotein or its message mdr1. In contrast with [14C]Adriamycin and [3H] vincristine uptake, [3H] 5-fluorouracil uptake by the cells was unaffected by TPA and reduced by the PKC-inhibitory peptides. These results indicate that PKC activation can significantly reduce the uptake of multiple cytotoxic drugs by cancer cells independently of P-glycoprotein, and that N-myristoylated PKC-alpha pseudosubstrate peptides potently and selectively induce uptake of multiple cytotoxic drugs in cultured human colon cancer cells by a novel mechanism that does not involve P-glycoprotein and may involve PKC isozyme inhibition. Thus, N-myristoylated PKC-alpha pseudosubstrate peptides may offer a basis for the development of agents that reverse intrinsic drug resistance in human colon cancer.

Differential effects of butyric acid on mouse skin tumorigenesis.

We studied the effects of butyric acid (BA) on mouse skin tumorigenesis using chronic animal bioassays. Topical application of BA immediately after each treatment with 12-0-tetradecanoylphorbol-13-acetate (TPA) promoter-inhibited skin tumors. The effect was dependent on the dose of BA applied. BA showed no marked inhibitory effect on either skin tumor initiation or complete tumorigenesis induced by dimethylbenzanthracene (DMBA). Since tumor promotion reportedly involves epigenetic events whereas tumor initiation or complete tumorigenesis takes place through genetic pathways, it is possible that BA exerts its antitumorigenic effects mainly by altering the epigenetic events responsible for tumor promotion. The results of the study could further be used to study the mechanism of action and modification of antitumorigenic effects of BA in combination with other substances.

Partial reversal of multidrug resistance in human breast cancer cells by an N-myristoylated protein kinase C-alpha pseudosubstrate peptide.

The predominant characteristics of multidrug resistant (MDR) cancer cells are broad spectrum resistance to chemotherapeutic agents and a pronounced defect in intracellular accumulation of the drugs, in association with overexpression of the drug efflux pump P-glycoprotein. Protein kinase C (PKC) phosphorylates the linker region of P-glycoprotein. Evidence has been presented that the isozyme PKC-alpha may contribute to the drug resistance phenotype of human breast cancer MCF7-MDR cells, PKC-alpha is markedly overexpressed in MCF7-MDR cells, and artificial overexpression of PKC-alpha in MCF7 constructs that overexpress P-glycoprotein significantly enhances the MDR phenotype of the cells in association with increased P-glycoprotein phosphorylation. Verapamil, cyclosporin A, and a number of other agents that compete with cytotoxic drugs for binding sites on P-glycoprotein can potently reverse MDR, but this is accompanied by severe toxicity in vivo. In this report, we demonstrate that an N-myristoylated peptide that contains a sequence corresponding to the pseudosubstrate region of PKC-alpha (P1) partially reverses multidrug resistance in MCF7-MDR cells by a novel mechanism that involves inhibition of PKC-alpha. P1 and two related PKC inhibitory N-myristoylated peptides restored intracellular accumulation of chemotherapeutic drugs in association with inhibition of the phosphorylation of three PKC-alpha substrates in MCF7-MDR cells: PKC-alpha, Raf-1 kinase, and P-glycoprotein. A fourth N-myristoylated peptide substrate analog of PKC, P7, did not affect drug accumulation in the MCF7-MDR cells and failed to inhibit the phosphorylation of the PKC-alpha substrates. The effects of P1 and verapamil on drug accumulation in MCF7-MDR cells were additive. P1 did not affect P-glycoprotein expression. MCF7-MDR cells were not cross-resistant to P1, which suggest that the peptide was not transported by P-glycoprotein. Furthermore, P1 was distinguished from MDR reversal agents such as verapamil and cyclosporin A by its inability to inhibit [3H]azidopine photoaffinity labeling of P-glycoprotein. P1 actually increased [3H] azidopine photoaffinity labeling of P-glycoprotein in MCF7-MDR cells, providing evidence that the effects of P1 on P-glycoprotein in MCF7-MDR cells are not restricted to inhibition of the phosphorylation of the pump. P1 may provide a basis for developing a new generation of MDR reversal agents that function by a novel mechanism that involves inhibition of PKC-alpha-catalyzed P-glycoprotein phosphorylation.

The tumor promoter receptor protein kinase C: a novel target for chemoprevention and therapy of human colon cancer.

The high affinity receptor of phorbol-ester and related tumor promoters is the isozyme family protein kinase C (PKC). Activation of PKC by the phorbol esters is a pivotal event in phorbol ester-mediated tumor promotion. PKC activation is also implicated in tumor promotion of colonic epithelial cells by endogenous and dietary factors such as bile acids, free fatty acids, and diacylglycerols, suggesting that suppression of the inappropriate activation of colonic epithelial PKC by these factors could be an effective strategy of chemoprevention. Phorbol-ester tumor promoters induce pleiotropic resistance against anti-cancer drugs in cultured human colon cancer cells. By characterizing PKC isozyme expression in the cells and the induction of resistance by isozyme-selective PKC activators, we have obtained evidence that the induction of resistance is triggered by phorbol-ester activation of cPKC-alpha. We have also found that cPKC-alpha is expressed abundantly in surgical specimens of human colon cancer, indicating that cPKC-alpha-mediated drug resistance may contribute to the intrinsic drug resistance of clinical colon cancer. The intrinsic resistance of human colon cancer to multiple anticancer drugs precludes effective therapy of disseminated disease with available chemotherapeutic regimens. The development of reversal agents of intrinsic drug resistance that function by selective inhibition of cPKC-alpha might allow successful management of the disease with standard cytotoxic chemotherapeutic regimens.

Tumour-promoting activity of ninhydrin on mouse skin.

Ninhydrin (2,2-dihydroxy-1,3-indanedione; CAS No. 485-47-2) is widely used as a reagent for the detection of free amino and carboxyl groups in proteins and peptides. It is an irritant to mammalian skin. Various toxic effects of ninhydrin have been reported in laboratory animals; however, so far there has been no evaluation of its carcinogenic and co-carcinogenic potential in laboratory animals by long-term in vivo bioassay. Ninhydrin was found to induce the activity of gamma-glutamyl transpeptidase (GGT) in mouse skin but it failed to alter the activity of the enzyme ornithine decarboxylase when compared with animals treated with standard tumour promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). In the present investigations, the tumour-promoting activity of ninhydrin (including both stage I and stage II of tumour promotion) was tested on Swiss albino mice in a multistage mouse skin model of carcinogenesis. The animals were initiated with a single topical application of 7,12-dimethylbenz-anthracene followed by four topical applications of ninhydrin biweekly as stage I promoter for 2 wk. Stage II promotion was twice weekly through topical application of mezerein. The results revealed that ninhydrin is a strong stage I tumour promoter and its efficacy was comparable with that of TPA at the dose level used in the experiment. However, ninhydrin failed to produce tumours when tested as a stage II or complete tumour promoter on mouse skin.

Dermal toxicity of hexachlorocyclohexane and pirimiphos-methyl in female rats.

Technical hexachlorocyclohexane (100 mg/kg/d) and pirimiphosmethyl EC 50 (250 mg/kg/d) given individually and in combination to female rats for 7, 15 or 30 d by skin application caused poisoning, pathomorphological changes in vital organs, and significant enzymatic changes in liver and serum. The changes produced by the 2 compounds in combination did not suggest potentiation at the tested dose levels.

Formation and persistence of safrole-DNA adducts over a 10,000-fold dose range in mouse liver.

The spice constituent safrole (1-allyl-3,4-methylenedioxybenzene) and related allylbenzenes form DNA adducts and are rodent carcinogens. This study examined both dose and time dependence of hepatic safrole-DNA adduct formation over a 10,000-fold dose range up to 30 days after single administration. Female CD-1 mice were treated with safrole i.p. at 0.001, 0.01, 0.1, 1.0, and 10.0 mg/mouse in 0.2 ml tricaprylin or with vehicle alone. Liver DNA was analyzed at 0.5, 1, 2, 3, 7, 15 and 30 days via the dinucleotide/monophosphate version of the 32P-postlabeling assay. An approximately 10-fold increase in total safrole adduct levels with each successive 10-fold increase in dose was observed, giving relative adduct labeling (RAL) values of 10(-9)-10(-5). Each dose elicited identical kinetics of adduct formation, showing peak levels at 2 days and only slight decreases thereafter. The time course of adduct persistence was independent of the dose (0.01-10 mg/mouse). An in vitro experiment established that the assay responded in strictly linear fashion to adduct concentration over a 10,000-fold range, and thus was suitable for in vivo dosimetry. DNA synthesis, as measured by [3H]thymidine incorporation, was enhanced only for the 10.0 mg dose at 2, 3 and 7 days. These results indicate a linear response of safrole-DNA adduct formation and persistence in mouse liver following administration of minute (0.001 mg/mouse) to high (10.0 mg/mouse) doses of the carcinogen.

Altered fidelity of a nucleic acid modifying enzyme, T4 polynucleotide kinase, by safrole-induced DNA damage.

Mouse liver DNA adducted with metabolites of the spice constituent safrole (1-allyl-3,4-methylenedioxybenzene), when analyzed via the bisphosphate version of the 32P-postlabeling assay, exhibits two major adducts, which had been previously identified as N2-(trans-isosafrol-3'-yl)2'-deoxyguanosine 3',5'-bisphosphate (adduct 1) and N2-(safrol-1'-yl)2'-deoxyguanosine 3',5'-bisphosphate (adduct 2). However, analysis of the same DNA preparation by the dinucleotide/monophosphate version of the assay gave two additional spots on PEI-cellulose TLC whose nature was clarified in the present study. Several enzymes (T4 polynucleotide kinase, nuclease P1, venom phosphodiesterase and spleen phosphodiesterase) were utilized to hydrolyze these compounds, and the products co-chromatographed on PEI-cellulose thin layers with radiolabeled and non-radioactive nucleotides of known structure. The additional spots were found to be adducted dinucleotides carrying 32P-label at both the 5'- and 3'-hydroxyls. T4 polynucleotide kinase-catalyzed 3'-phosphorylation was highly specific in that only dinucleoside monophosphate derivatives of adduct 1, with an unmodified purine in the 3'-position, were susceptible to both 5'- and 3'-phosphorylation by the enzyme. Thus, the structures of the two additional 32P-labeled safrole derivatives were pX1pAp and pX1pGp where X1 denotes N2-(trans-isosafrol-3'-yl)2'-deoxyguanosine. The official name of T4 polynucleotide kinase, ATP:5'-dephosphopolynucleotide 5'-phosphotransferase (EC 2.7.1.78), denotes the specific action of this enzyme as a 5'-phosphokinase. Although the enzyme has 3'-phosphatase activity at acidic pH, no 3'-kinase reaction has been previously reported. Possible implications for chemical carcinogenesis of the finding that carcinogen-DNA adducts can specifically alter the fidelity of protein-nucleotide interactions are discussed.

Ornithine decarboxylase inactivation by putrescine: involvement of lipid peroxidation.

Effect of polyamines on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced reduction of lipid peroxidation was studied. Putrescine protected this lowering of lipid peroxidation in a concentration-dependent manner, but spermidine or spermine could not do so. Putrescine also inhibited the TPA-induced ornithine decarboxylase (ODC) activity and lowered the free sulfhydryl content of TPA exposed mouse skin. These observations indicate that putrescine inactivates ODC probably by lowering SH groups through lipid peroxidation.

Acute and subchronic oral toxicity of technical quinalphos in rats.

The organophosphate insecticide quinalphos is extensively used in agriculture. Information on the mammalian toxicity of quinalphos is limited. The acute po LD50 of technical quinalphos was 19.95 mg/kg in males and 13.78 mg/kg in female rats. Administration of 0.75, 1.50 or 3.0 mg/kg/d technical quinalphos for 90 d po to rats produced poisoning and death. Male rats were more susceptible to quinalphos than female rats in the subchronic toxicity studies based on mortality, enzyme profiles and cholinesterase inhibition.

Interaction of technical hexachlorocyclohexane and oxydemeton methyl 25 EC to female rats after dermal application.

Technical hexachlorocyclohexane (HCH, 100 mg/kg/day) and oxydemeton methyl 25 EC (125 mg/kg/day) to female rats for 7, 15 and 30 days individually and in combination through skin application caused pathomorphological changes in vital organs and significant enzymatic changes in liver and serum. However changes produced by the two compounds in combination were not suggestive of potentiation effect at the tested dose level in female rats.

Status of ornithine decarboxylase activity and DNA synthesis in mancozeb-exposed mouse skin.

The effect of mancozeb, a fungicide, on mouse skin ornithine decarboxylase (ODC) activity and DNA synthesis was studied. ODC activity was induced after topical application of mancozeb and exhibited a peak level at 5 h. This ODC induction was dependent on the dose of mancozeb applied. Cycloheximide, an inhibitor of protein synthesis, inhibited the mancozeb-caused ODC induction, indicating the effect on enzyme protein synthesis. The rate of DNA synthesis was also increased by mancozeb, as indicated by increased [3H]thymidine incorporation into skin DNA. Induction of ODC activity and DNA synthesis are among the events probably involved in the tumorigenic action of mancozeb on mouse skin.

Inhibition of mouse skin tumor promotion by tenuazonic acid.

Tenuazonic acid (TA) was topically applied to the interscapular region of Swiss albino mice at different doses before the application of 12-O-tetradecanoyl phorbol-13-acetate (TPA). Skin from the painted area was examined for ornithine decarboxylase (ODC) enzyme estimation. It was observed that TA inhibited TPA induced ODC activity. The inhibitory effect of TA was also found in mouse skin tumor promotion in the two stage initiation promotion protocol. There was a remarkable delay in the latency period and decrease in the number of tumors developed and the percentage of tumor bearing animals after TA treatment.