Reduction of drug toxicity using dendrimers based on melamine.

Dendrimers based on melamine can reduce the organ toxicity of solubilized cancer drugs administered by intraperitoneal injection. Methotrexate and 6-mercaptopurine, both FDA approved anticancer drugs, are known hepatotoxins. The solubility of these molecules can be increased by mixing them with a dendrimer based on melamine. C3H mice were administered subchronic doses of methotrexate or 6-mercaptopurine with and without a solubilizing dendrimer. Forty-eight hours after dosing, the mice were sacrificed and serum was collected for biochemical analyses. The levels of alanine transaminase, ALT, were used to probe liver damage. When the drugs are encapsulated by the dendrimer, a significant reduction in hepatotoxicity is observed: ALT levels from the rescued groups (drug + dendrimer) were 27% (methotrexate) and 36% (6-mercaptopurine) lower than those of animals treated with the drug alone.

[Nitroxyl radical Tempol as a modulator of toxic and antineoplastic effect of 6-mercaptopurine]. / Nitroksil'nyi radikal tempol kak moduliator toksicheskogo i protivoopukholevogo deistviia 6-merkaptopurina.

Both intact mice and those with transplantable adenocarcinoma 755 were used in the investigation. The nitroxyl radical Tempol was shown to cut down the toxicity of 6-mercaptopurine and potentiate its antitumor effect to a certain degree. The study results suggest on the basis of an investigation of cytochrome P450 and some other evidence that said effect of Tempol might be due, at least, in part to antioxidant activity.

Reversal of methylmercaptopurine ribonucleoside cytotoxicity by purine ribonucleosides and adenine.

6-Methylmercaptopurine ribonucleoside-5'-phosphate (MeSPuRMP), the sole metabolite of 6-methylmercaptopurine ribonucleoside (MeSPuRib), is a strong inhibitor of purine de novo synthesis, inducing depletion of intracellular purine nucleotides and subsequent cell death in several tumor cell lines. In this study prevention of MeSPuRib cytotoxicity by compounds of the purine salvage pathway was studied in Molt F4 human malignant T-lymphoblasts. Adenosine, adenine and inosine were able to prevent depletion of the adenine nucleotide pool when used in combination with 0.5 microM MeSPuRib, but had virtually no effect on depletion of guanine nucleotides. Nevertheless, these three purine compounds were able to reduce the cytotoxic effects induced by MeSPuRib. Addition of guanosine to cells treated with 0.5 microM MeSPuRib normalized the guanine nucleotide pool, but adenine nucleotides remained depleted. Under these conditions, inhibition of cell growth was significantly decreased. With the combination of guanosine and 10 microM MeSPuRib, cytotoxicity was increased compared to 10 microM MeSPuRib alone, associated with a depletion of adenine nucleotides to 9% of untreated cells. Since cell growth and cell viability of Molt F4 cells are less inhibited by MeSPuRib under conditions where adenine nucleotide depletion is prevented by purine compounds (and where the other nucleotides are depleted) we conclude that depletion of adenine nucleotides is an important factor in MeSPuRib cytotoxicity.

Reversal of 6-mercaptopurine and 6-methylmercaptopurine ribonucleoside cytotoxicity by amidoimidazole carboxamide ribonucleoside in Molt F4 human malignant T-lymphoblasts.

Cytotoxicity of 6-mercaptopurine (6MP) and 6-methylmercaptopurine ribonucleoside (Me-MPR) was studied in Molt F4 human malignant lymphoblasts. Both drugs are converted into methylthioIMP (Me-tIMP), which inhibits purine de novo synthesis. Addition of amidoimidazole carboxamide ribonucleoside (AICAR) circumvented inhibition of purine de novo synthesis, and thus partly prevented 6MP and Me-MPR cytotoxicity. Purine nucleotides, and especially adenine nucleotides, were recovered by addition of AICAR. Under these conditions, Me-tIMP formation decreased. The results of this study indicate that formation of Me-tIMP may be important for 6MP cytotoxicity in Molt F4 cells. These data suggest that depletion of adenine nucleotides is the main cause for Me-tIMP cytotoxicity.

[The mutagenic effect of the recombinant plasmid pBR322ins carrying a native and a modified human insulin gene]. / Mutagennyi éffekt rekombinantnoi plazmidy pBR322ins, soderzhashchei nativnyi i izmenennyi gen insulina cheloveka.

The mutagenic activity of pBR322 bacterial plasmid DNA and of pBR322ins and pBR322insN recombinant plasmid DNAs has been investigated in Blld-ii-FAF 28 line cultivated fibroblasts of Chinese hamster. The pBR322 bacterial plasmid DNA is shown to induce no resistant mutations to 6-mercaptopurine; the pBR322insN (the frameshift mutation in human insulin gene; this gene is supposed to have no expression in the cells because of frameshift mutation) induces neither such mutations as well. The pBR322ins recombinant plasmid carrying the native human insulin gene induces the gene mutations in this system. The influence of the insulin gene structure on the gene mutations induction is taken into account. The results obtained and the data of other authors illustrate a great interest of recombinant DNA molecules carrying transgenes in the field of mutation research.

[The induction of mutations in 6-mercaptopurine resistance in Chinese hamster cells under the action of the recombinant plasmid pAins containing the human insulin gene]. / Induktsiia mutatsii rezistentnosti k 6-merkaptopurinu v kletkakh kitaiskogo khomiachka pod deistviem rekombinantnoi plazmidy pAins, soderzhashchei gen insulina cheloveka.

The mutagenic activity of the pUC19 bacterial plasmid DNA and the pAins recombinant plasmid DNA carrying human insulin gene has been investigated. Both pUC19 and pAins plasmid DNAs have been shown to induce the gene mutations in hprt locus of Chinese hamster cell line. The high level of the gene mutations (similar to the indices of the gene mutations induced by the chemical mutagens) has been in the focus of attention. The conclusion has been made concerning impossibility to use pAins plasmid DNA in the diabetes mellitus gene therapy. It is necessary to test the mutagenic properties of the DNA molecules produced for gene therapy of human inherited diseases.

Modification of the clastogenic activity of X-ray and 6-mercaptopurine in mice by prefeeding with vitamins C and E.

The effect of a 30-d pretreatment with vitamin C (L-ascorbic acid) in the drinking water and vitamin E (all-rac-alpha-tocopherol) in the diet on the clastogenic activity induced by X-rays and 6-mercaptopurine was investigated in female ICR/Jcl mice by the bone-marrow micronucleus test. Prefeeding with vitamin E-deficient diets led to a significant decrease in serum vitamin E concentration and to an enhancement of micronucleus formation by X-rays in bone marrow cells. Although dietary supplementation with vitamin E significantly increased the vitamin E concentration in serum, it did not affect the frequency of X-ray-induced micronuclei. Treatment with a high level of vitamin C in drinking water was effective in protecting against micronucleus formation by X-rays. The increase in micronucleus frequency in the vitamin E-deficient mice compared with the mice fed vitamin E-normal diets was no longer observed when a high level of vitamin C in drinking water was given simultaneously. The most efficient protective action against X-rays was observed when vitamin E-supplemented diets and a high level of vitamin C in drinking water were used together as a pretreatment. Any combination of the vitamins did not affect the micronucleus induction by 6-mercaptopurine.

Biochemical basis of the prevention of 6-thiopurine toxicity by the nucleobases, hypoxanthine and adenine.

Co-incubation of human leukemia cell lines with naturally occurring nucleobases (hypoxanthine or adenine) significantly prevented the cytotoxic activity of 6-thiopurines. Extracellular hypoxanthine decreased the transport of 6-mercaptopurine into cells, but adenine had no significant effect. However, intracellular thioinosine monophosphate accumulation in the presence of 10 microM, 6-mercaptopurine was reduced to below 1% or 10% of that of the controls when 50 microM hypoxanthine or adenine was added, respectively. Finally, in adenine phosphoribosyl transferase deficient mutants, adenine provided no protective effect against 6-thiopurines, whereas hypoxanthine retained its modulating activity. These data suggest that the nucleobases compete with 6-thiopurines for the ribose-phosphate donor, 5'-phosphoribosyl-1-pyrophosphate, thus preventing the formation of active metabolites of 6-thiopurines.

[The mutagenic action of the DNA fragments of bovine adenovirus type 3 (BAV-3) containing early E1 (oncogene) and E4 regions]. / Izuchenie mutagennogo deistviia fragmentov DNK bych'ego adenovirusa tipa 3 (BAV-3), soderzhashchikh rannie oblasti E1 (onkogen) i E4.

A study of mutagenic action of DNA fragments of bovine adenovirus type 3 (BAV-3), containing early E1 (oncogenic) and E4 regions has been carried out. Viral DNA was digested with Xba 1 restriction endonuclease. The mutagenic effect revealed itself by induction of resistance to 6-mercaptopurine in Chinese hamster cells. The data obtained suggest that the early regions E1 and E4 can cause mutagenic effect in mammalian cells.

[Mutagenic activity of native and inactivated herpes simplex virus]. / Mutagennaia aktivnost' nativnogo i inaktivirovannogo virusa prostogo gerpesa.

The mutagenic activity of native and inactivated herpes simplex virus (HSV-1) has been studied. The native HSV-1 strain has been shown to induce the gene mutations of 6-mercaptopurine resistance in Chinese hamster cells. The 1.5- to 2-fold decrease of mutagenic activity is due to HSV inactivation by thiophosphamide and formalin treatment. These data indicate the possibility of the given agents application for decreasing of the genetic danger of antiviral vaccine.

[RAMT cell line as a substrate for the propagation of vaccinal viral strains]. / Liniia kletok RAMT kak substrat dlia razmnozheniia vaktsinnykh shtammov virusov.

Properties of the PAMT cell line which are deficient in hypoxanthine guanyl phosphoribosiltransferase were studied. The PAMT cell line derived from African green monkey kidney tissue may be identified by selective nutrient media, contains no contaminants, is not tumorigenic, is susceptible to a wide range of viruses. Titres of poliomyelitis, tick-borne encephalitis, and carnivore plague viruses were similar in cell cultures grown in the medium with 10% serum and in those adapted to growth in the medium containing 1% serum. The properties of PAMT cell line allow it to be used in manufacture of killed virus vaccines.

[Mutagens and the tumor promoter]. / Mutageny i opukholevyi promotor.

We studied the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) and saccharin on the frequency of induced mutations of resistance to 6-mercaptopurine and ouabain in Chinese hamster and mouse cells. UV-rays, bovine adenovirus-3 (BAV-3) and 5-bromdeoxyuridine (BrdU) were used as mutagens. In the case of BAV-3 and BrdU, we investigated, apart from the mutagenic effect, the tumor-inducing activity of these mutagens in mice, BrdU proved to have no carcinogenic effect. The data about the influence of TPA on the mutagenic effect of the three different mutagens indicate that TPA increases the frequency of the gene mutations induced by UV-rays and BAV-3. The results of the study of BrdU and TPA combined action revealed the fact that TPA does not increase the mutagenic effect of BrdU. We demonstrated that saccharin also possesses the promoter activity; it increases the mutagenic effect of BAV-3. The results described above lead to the assumption that TPA influence on the mutagenic effect only takes place when carcinogenic mutagens are used.

[Genetic nature of one of the traits of malignant cell transformation in vitro]. / Geneticheskaia priroda odnogo iz priznakov zlokachestvennoi transformatsii kletok in vitro.

The genetic events controlling the ability of transformed cells to grow in a medium with a low serum content (ser+) were studied. A hypodiploid clone of Chinese hamster cells with normal serum requirements (49a5ser-) was used as starting material. The results of the fluctuation tests have shown that serum-independence is a random spontaneous event. Its rate of occurrence is 1-2 . 10(-5). The concomitant study of a gene mutation (resistance to 6-mercaptopurine) revealed similar characteristics with respect to the distribution of the number of mutants in replicate cultures and the mutation rate. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the oncogenic SV40 virus significantly increased the frequency of ser+ colonies. In the majority of clones isolated in a medium with 1% serum (11 spontaneous and 7 induced by MNNG), the ser+ character proved to be stable after different periods of cultivation without selective pressure. The degree of serum-independence varied in different clones. The results suggest that the ability to grow in a medium with a low serum content originates, in most cases, from a mutation event.

[Mutagenic action of the DNA of the highly oncogenic bovine adenovirus type 3 (BAV-3)]. / Mutagennoe deistvie DNK vysokoonkogennogo bych'ego adenovirusa tipa 3 (BAV-3).

The induction of mutations to the 6-mercaptopurine resistance has been studied in Chinese hamster cells after transfection with highly oncogenic bovine adenovirus type 3 DNA (BAV-3). A statistically significant increase in the yield of resistant mutants is induced by viral DNA after two days expression time. The value of induction varies from 56,12 X 10(-5) to 70,02 X 10(-5). The comparison of the mutagenic activity of DNA and the intact viral particles has revealed that the number of DNA molecules must be higher than after infection with the whole virus to obtain a similar effect.

Biochemical approaches to enhancement of antitumor drug selectivity: selective protection of cells from 6-thioguanine and 6-mercaptopurine by adenosine.

The cytotoxicity of 6-thioguanine and 6-mercaptopurine to cultured lymphoblasts and fibroblasts was strongly antagonized by pretreatment of the cells with 100 microM adenosine. Administration of adenosine 2 hours after the antipurine agent did not cause antagonism. In two rat hepatoma cell lines, adenosine pretreatment did not protect cells from the antipurines. Treatment of lymphoblasts or fibroblasts with 100 microM adenosine gave increases up to 150% in cellular ATP and ADP and decreases greater than 80% in UTP and UDP. In the hepatoma lines, adenine nucleotides did not increase by greater than 45%, and uridine nucleotides did not decrease by greater than 40% following adenosine treatment. The selective protection of the normal cells from 6-thioguanine and 6-mercaptopurine was probably the consequence of phosphoribosylpyrophosphate (PRPP) depletion, since adenosine pretreatment decreased PRPP pools by greater than 90% in the normal cells but by only 30% in the malignant hepatoma cells. In the absence of PRPP the antipurines would not be metabolically activated. The selectivity of the adenosine and antipurine combinations was probably attributable to the low activity of adenosine kinase and high activities of adenosine deaminase and PRPP synthetase characteristic of malignant hepatomas.

Attenuating effect of zinc on abnormal placental morphology in 6-mercaptopurine treated rats.

Pregnant Sprague-Dawley rats were fed diets containing 9 (marginal level), 100 (control level), or 1,000 (very high level) ppm zinc and were given a single intraperitoneal injection of 6-mercaptopurine (6-MP, 55 mg/kg) or an equivalent volume of carboxymethylcellulose on day 11 of gestation. Live young and their placentas were recovered at surgery on day 21 of gestation: they were weighed, measured, and placentas were examined histologically. Placentas from drug treated animals were smaller in diameter and lighter in weight than controls; however, the placentas of animals fed the 1,000 ppm zinc diet were significantly heavier than those of the other drug-treated groups. Histologically, the placentas of the 6-MP treated dams showed a highly disproportionate reduction in the labyrinthine layer with larger, less subdivided maternal sinuses than in controls, reduction of fetal vasculature, vesiculation of trophoblast nuclei, deposition of PAS positive material in the septal wall, and fibrinous degeneration of trophoblast cells. These morphological changes were reduced in placental tissues of drug treated rats given 1,000 ppm of zinc. In contrast, no placental abnormalities were observed in rats not treated with 6-MP.

[Antimutagenic and antiteratogenic properties of dimexide (DMSO)]. / Issledovanie antimutagennykh i antiteratogennykh svoistv dimeksida (DMSO).

The influence of dimexide (dimethylsulphoxide, DMSO) on the manifestation of teratogenic and embryolethal properties and mutagenic effect of pyrimethamine (antimalarial drug) and 6-mercaptopurine (antitumoral compound) has been examined. Under the conditions of preliminary action of dimexide the embryolethal and teratogenic activity of the drugs studied reduced on the 13th day of rat embryos; the level of cytogenic effect of these compounds on rat males bone marrow cells also reduced.

[Influence of phenobarbital on the immunodepressive action of antitumor substances]. / Vliianie fenobarbitala na immunodepressivnoe deistvie protivoopukholevykh sredstv

In the experiments on mice, immunized by mutton erythrocytes, the authors have studied the effect of phenobarbital, in the doses activating drug-metabolizing hepatic systems, on immunodepressive action of imurane, 6-mercaptopurine and cyclophosphane. It has been found that phenobarbital eliminates the immunodepression and reduces considerably the toxicity of the mentioned cytostatics. Under these conditions imurane and 6-mercaptopurine in certain doses render an immunostimulative instead of immunodepressive effect.