A preliminary assessment of the potential environmental and human health impact of unsymmetrical dimethylhydrazine as a result of space activities.

A preliminary assessment of the potential environmental and human health impact of UDMH as a result of space activities has been carried out applying a theoretical approach in comparison with selected experimental data. The theoretical framework includes QSAR, ADME and PASS modelling as well as studies on the possible atmospheric dispersion of UDMH as calculated applying the OML-Multi model. The possible impact on the environment and the human health has been elucidated and it has been concluded that UDMH especially inside the fall region of burned-out rocket stages constitute a significant threat to both environmental and human health, the latter as a results of the carcinogenic, mutagenic, convulsant, teratogenic and embryotoxic characteristics of UDMH in addition to the general toxic characteristics of the compound.

Distribution of preneoplastic lesions and tumors, and beta-catenin gene mutations in colon carcinomas induced by 1,2-dimethylhydrazine plus dextran sulfate sodium.

Mucin-depleted foci (MDF) are considered as useful biomarkers in rat colon carcinogenesis. The purpose of the present study was to examine the mechanism(s) underlying rat colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) plus 1% Dextran Sulfate Sodium (DSS). Twelve male F344 rats were given subcutaneous injections (40mg/kg body) of DMH twice a week. They received DSS in the drinking water for 1 week after the first injection of DMH and then were maintained on tap water. The rats were sacrificed at 10 and 14 weeks after the first injection of DMH. Colon tissues were divided into 10 segments from anus to cecum (A/J) and stained with Alcian blue (AB) to identify MDF. We found that MDF and tumors were induced in the rat colon after treatment with DMH plus DSS and that the number of MDF in each segment of the colon was significantly correlated with that of tumors (p=0.006). In addition, we found that the beta-catenin protein was accumulated in cytoplasm and nuclei of MDF and the frequent beta-catenin gene mutations in the colon tumors. These results suggest that MDF is closely related to rat colon carcinogenesis induced by DMH plus DSS.

[Microbiological degradation of asymmetrical dimethylhydrazine--a toxic component of rocket fuel]. / Mikdrobiologicheskaia degradatsiia nesimmetrichnogo dimetilgidrazina--toksichnogo komponenta raketnogo topliva.

A possibility of microbiological cleaning of water and soil polluted with asymmetric dimethylhydrazine (ADMH), a highly toxic rocket fuel ingredient (RFI), was studied. Several isolates (bacteria, yeast, and micromycetes) capable of utilizing ADMH as the only source of nitrogen, carbon, and energy were isolated from RFI-polluted tundra soil. Acceleration of RFI biodegradation was achieved using a biosorbent that involved cells of the degrader strain immobilized on granulated activated carbon. Biological testing in Escherichia coli and cereals (wheat and barley) demonstrated that biodegradation significantly decreased the integral toxicity of solutions containing ADMH, suggesting its utility for microbiological cleaning of polluted territories.

Proliferation of mouse fibroblasts induced by 1,2-dimethylhydrazine auto-oxidation: role of iron and free radicals.

Activation of 1,2-dimethylhydrazine (DMH) by prolonged auto-oxidation (24-h) induced proliferation of mouse fibroblasts at low hydrazine concentrations (0.1-1.0 mM) as determined by [3H-methyl]-thymidine uptake of confluent quiescent cells. Incubations were performed under conditions in which alkyl radicals are slowly formed by DMH auto-oxidation. The proliferative stimulus induced by DMH auto-oxidation complements that induced by insulin, PMA, and EGF. Inhibition by the iron chelators, o-phenanthroline and desferrioxamine, demonstrates that the induction of the proliferative effect is dependent on simple iron complexes. Proliferation was also inhibited by superoxide dismutase, catalase, and mannitol, implicating reactive oxygen species, although superoxide dismutase and catalase also inhibited alkyl radical formation, as determined by spin-trapping. These results suggest that cell proliferation induced by DMH auto-oxidation is mediated by reactive oxygen species, mainly the hydroxyl radical, and is dependent on simple iron complexes, possibly involving the Fenton reaction.

Chemopreventive effect of S-allylcysteine and its relationship to the detoxification enzyme glutathione S-transferase.

Sulfur-containing substances derived from garlic and onion have been shown to prevent experimental carcinogenesis. One of the hypotheses explaining the mechanisms of the chemopreventive activity of these substances is that they activate detoxification systems such as glutathione S-transferase (GST). In this study the effects of S-allylcysteine (SAC), a water-soluble organosulfur compound derived from garlic, on GST activities in the liver, small intestine and colon were investigated. Additionally, we examined SAC for chemopreventive effects on aberrant crypt foci, which are the most likely precursors of colon cancers. In the rat colonic aberrant crypt assay administration of SAC during the initiation period decreased the number of aberrant crypt foci by 33 and 54% in groups given 40 or 80% maximum tolerated dose (MTD) of SAC respectively. The number of aberrant crypt foci, however, was not changed when SAC was given during the promotion period. GST activity in the liver was increased significantly by 41% 12 h after a single oral administration of 3.5 mmol/kg SAC and this elevated GST level was maintained over a 72 h period. GST levels were increased significantly by the administration of SAC (1.8 mmol/kg/ day for 3 days) not only in the liver but also in the proximal and middle small bowel. Isozyme levels of GST after administration of SAC were also determined using Western blotting. Hepatic GST-alpha and GST-mu were significantly increased by 35 and 42% respectively after oral administration of SAC. GST-pi levels were lower than the detection limit (130 ng/mg/protein) in both the control and SAC-treated groups. These results strongly support the previous working hypothesis that SAC exhibits chemopreventive activity by exerting specific effects on carcinogen detoxification systems.

Methylation of RNA purine bases by methyl radicals.

During the metabolism of 1,2-dimethylhydrazine (a colon carcinogen) in rats, methyl radicals (.CH3) are produced. To gain an insight into the nature of .CH3 interactions with biomolecules in vivo, we conducted in vitro studies using RNA as a model compound. RNA was incubated in a system where .CH3 was generated from the oxidation of dimethyl sulfoxide by hydroxyl radicals (.OH); .OH was produced from a Fenton-type reaction between Fe(2+)-EDTA and H2O2. Four new products were detected from acid hydrolysates of .CH3-treated RNA: 8-methylguanine, 2-methyladenine, 8-methyladenine, and a highly unstable compound of unknown structure. The production was significantly affected by pH. The concentrations of Fe2+ and H2O2 influenced the production of methylated purine residues in a dose-dependent manner. Catalase, ethanol, alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, and O2 inhibited the production of methylated nucleobases.

Carbon-centered free radical formation during the metabolism of hydrazine derivatives by neutrophils.

The neutrophil-catalyzed metabolism of hydrazine derivatives to carbon-centered radicals was investigated by the spin-trapping technique using alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). Oxidation of methylhydrazine (MeH), dimethylhydrazine (DMH), phenylethylhydrazine or procarbazine by neutrophils from rat peritoneal exudates led to the formation of alkyl radicals. The monosubstituted hydrazine oxidation by phorbol ester (PMA)- or Zymocel-activated neutrophils generated, on average, 2- to 4-fold more POBN-alkyl adducts than di-substituted hydrazines. Supernatant from sonicated neutrophils generated similar yields of radicals. Azide, an inhibitor of myeloperoxidase, effectively reduced the neutrophil-catalyzed radical yield from the oxidation of MeH but not DMH. On the other hand, superoxide dismutase and catalase effectively inhibited radical formation in DMH metabolism by PMA-activated neutrophils, in contrast to MeH metabolism. Our results show that neutrophils are able to metabolize hydrazine derivatives, the pathway depending on the hydrazine substitution. Alkyl radical production during the oxidation of mono-substituted derivatives, such as MeH, was mediated mainly by myeloperoxidase, and that of di-substituted derivatives, such as DMH, was mediated mainly by active oxygen species.

Oxidation of methylhydrazines to mutagenic methylating derivatives and inducers of the adaptive response of Escherichia coli to alkylation damage.

The methylhydrazines, monomethylhydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine, are known carcinogens but only weak mutagens in the Ames test. Chemical oxidation of these compounds by potassium ferricyanide greatly enhanced their mutagenicity to an Escherichia coli ada mutant and converted them into inducers of the adaptive response of E. coli to alkylation damage. Enzymatic oxidation of monomethylhydrazine by horseradish peroxidase-H2O2 also yielded products which induced the adaptive response. Thus, methylhydrazines can be oxidized to active DNA-methylating derivatives which generate methylphosphotriesters (the inducing signal of the adaptive response), O6-methylguanine and/or O4-methylthymine (the miscoding bases repaired by the Ada protein) in DNA. These observations support the suggestion that metabolic oxidation of methylhydrazines in mammalian systems may be required to generate the mutagenic/carcinogenic derivatives.

Characterization of cytochrome P450-dependent dimethylhydrazine metabolism in human colon microsomes.

Polyclonal antibodies to components of the rat liver cytochrome P450 system were used to examine the composition and function of the microsomal cytochrome P450-dependent monooxygenase system of human colonic mucosal cells. Anticytochrome P450 reductase antibody gave a strong band of immunocross-reactivity in human colon microsomes at the same molecular weight level as purified cytochrome P450 reductase from rat liver, as well as hepatic microsomes isolated from untreated or phenobarbital-treated rats. These results demonstrate the presence of cytochrome P450 reductase in human colon cells. Similarly, cytochromes P450 IIB1 and IIA1 also appear to be present in Western blots of human colon microsomes. These antibodies, as well as antibodies to reductase and cytochrome b5, inhibit dimethylhydrazine metabolism in human colon microsomes to varying degrees. These data argue for a functional P450-dependent drug metabolism system in colon capable of activating/metabolizing the colon-specific model carcinogen, 1,2-dimethylhydrazine.

Effects of dietary fat on hepatic microsomal metabolism of 1,2-dimethylhydrazine.

The present study was designed to examine the effects of different high fat diets on the liver microsomal metabolism of aminopyrine (AMP) and 1,2-dimethylhydrazine (DMH). Male Sprague-Dawley rats were fed either a low fat (5% corn oil) or high fat (20%) diets containing either corn oil, menhaden oil or beef tallow for a period of up to 9 months. Liver microsomes were assayed for N-demethylase activity for both AMP and DMH substrates at 2 weeks, 1, 6 and 9 months of diet only, and also after 1, 2, 5 and 10 DMH treatments (20 mg/kg body weight). The menhaden oil-fed group had consistently higher AMP demethylase activity, which increased up to 6 months and then declined. Beef tallow-fed rats had the highest DMH demethylase activity following DMH, but this decreased by 10 treatments. These data indicate that type and amount of dietary fat affects microsomal metabolism of carcinogens, which may enhance tumor initiation.

Lack of modifying effects of linolic acid hydroperoxides and their secondary oxidative products on combined 7,12-dimethylbenz[a]anthracene and 1,2-dimethylhydrazine-initiated mammary gland, ear duct and colon carcinogenesis in female Sprague-Dawley rats.

Effects of hydroperoxides, autoxidation products of linolic acid (HPO) and secondary oxidative products of HPO (SOP) (5% each in diet) were examined in female Sprague-Dawley rats. HPO and SOP administration was carried out during or subsequent to two injections of dimethylhydrazine (DMH) (40 mg/kg body wt s.c.), and a single i.g. dose of 7,12-dimethylbenz[a]anthracene (DMBA) (50 mg/kg body wt). No significant differences in the incidences of tumors in the mammary gland, colon, ear duct and hematopoietic system associated with HPO or SOP treatment were evident, during or after carcinogen exposure. The present results therefore indicate that the environmental contaminants, HPO and SOP, lack any potential for modification of mammary gland or colon carcinogenesis under the conditions of the investigation.

Carcinogenicity of deuterium-labeled 1,2-dimethylhydrazine in mice.

To study the effect of deuterium substitution on the carcinogenicity of 1,2-dimethylhydrazine (DMH) in mice, two comparisons were made between DMH and its fully methyl-deuterated analogue, [2H6]DMH. In a lifetime study with the CBA strain, groups of 19-30 animals of each sex were dosed s.c. weekly with 8 mg/kg of either DMH or [2H6]DMH for 8, 16, or 32 weeks. In the second study, female CF-1 mice were given DMH or [2H6]DMH in 10 weekly s.c. doses of 12 mg/kg each (13.2 mg/kg for [2H6]DMH) and examined for colon tumors 36 weeks after the first dose. Deuteration significantly decreased tumor incidence in the colon of males (P less than 0.01) and the anal tissue of both sexes (P less than 0.05) but increased that of hepatomas and lung tumors in males (P less than 0.01). Substrate deuteration did not significantly affect overall incidence of any other tumor type, however, including hemangioendotheliomas and kidney tumors in both sexes, as well as colon, uterine, ovarian, liver, and lung tumors in females. The results indicate that C--H bond breakage is kinetically important in the activation of DMH to its ultimately carcinogenic form in organs such as the male colon (relative risk in DMH-versus [2H6]DMH-treated animals approximately equal to 6), and that inhibition of this process by substrate deuteration allows a diversionary mechanism having a smaller isotope effect to become relatively more extensive. The qualitatively different effect in other organs (e.g., kidney, relative risk approximately equal to 1) supports recent suggestions that the net mechanism of activation can differ from one target tissue to another, possibly by striking a different balance between parallel metabolic pathways. The lack of a significant isotope effect on overall colon tumor incidence in females of either strain suggests that differences in relative importance among competing enzymes may also be responsible for sexual dimorphism in tumor induction by DMH.

Magnetic polyethyleneimine (PEI) microcapsules as retrievable traps for carcinogen electrophiles formed in the gastrointestinal tract.

Semi-permeable magnetic microcapsules containing polyethyleneimine (PEI) have been developed as retrievable carcinogen traps. In vitro, the soluble core PEI and membrane both bound reactive substances of limited aqueous stability, such as from [14C]N-methyl-N-nitrosourea ([14C]NMU), and aqueous stable dyes of molecular weight up to 1000. The core/membrane location ratio of binding was dependent upon membrane characteristics of the microcapsule batch used. Microcapsules administered intragastrically to rats bound up to 0.006% of [14C]dimethylhydrazine ([14C]DMH) and 1.4% of [14C]NMU administered i.p. or intrarectally, respectively. Time-dependency of [14C]DMH binding was consistent with labelling of microcapsules within the small intestine. There were no detectable metabolites from [14C]DMH trapped within the colon, whereas binding of [14C]NMU indicated that microcapsules could bind transient species present within the colon in competition with the faecal bulk. These results indicate that this approach could be used to detect highly unstable and possibly genotoxic substances in situ, hitherto unknown, formed within the intestinal lumen.

Organ-specific effects of 1,2-dimethylhydrazine in hamster.

Uptake and metabolism of the carcinogen 1,2-dimethylhydrazine (DMH) were compared in isolated epithelial cells from the colon and the small intestine. A new method was developed to separate colonic epithelial cells into surface columnar cells and crypt cells without the use of any proteolytic enzymes. Colonic columnar cell-enriched fraction exhibited DMH metabolism two to three times higher than that of crypt cells. The carcinogen binding was much lower in the small intestine as compared to the colon. In the small intestine, the crypt cell-enriched fraction showed higher carcinogen binding as compared to villus cells. Pyrazole was found to inhibit DMH binding by isolated small intestinal and colonic epithelial cells. The extent of inhibition was maximum in cells showing the greatest ability to incorporate DMH.

Effect of dietary selenium on biotransformation and excretion of mutagenic metabolites of N-nitrosodimethylamine and 1,1-dimethylhydrazine in the liver perfusion/cell culture system.

The mutagenicity of N-nitrosodimethylamine (NDMA) and 1,1-dimethylhydrazine (UDMH) has been studied in an isolated liver perfusion/cell culture system. The liver donors, male Wistar rats, were either selenium (Se)-deficient or had a physiologically adequate Se status (Se-supplemented). Mutagenicity was measured in perfusate and bile with Chinese hamster V79 cells as the genetic target. Se deficiency increased the mutagenic effect of NDMA in the perfusate, whereas no mutagenicity was detected in the bile of either Se-deficient or Se-supplemented livers. No significant increase in the mutagenicity of UDMH was seen in the perfusate with Se deficiency, but the bile became mutagenic. Se deficiency thus increased the mutagenicity of both NDMA and UDMH: with NDMA, the effect was observed in the perfusate, and with UDMH, in the bile.

Enzymatic activation of the carcinogens 2-phenylethylhydrazine and 1,2-dimethylhydrazine to carbon-centered radicals.

Spin-trapping experiments demonstrate that oxidation of 1,2-dimethylhydrazine and 2-phenylethylhydrazine generates a comparable yield of carbon-centered radicals when catalyzed by horseradish peroxidase - H2O2. Using oxyhemoglobin as the catalyst, 2-phenylethylhydrazine oxidation generates ten times more carbon-centered radicals than 1,2-dimethylhydrazine oxidation. This result is in agreement with oxygen consumption studies from which the apparent KM values of 8.0 mM and 72 mM were calculated for the oxyhemoglobin-catalyzed oxidation of 2-phenylethylhydrazine and 1,2-dimethylhydrazine, respectively. These differences in metabolic activation of mono- and disubstituted hydrazines may be of importance regarding the carcinogenic properties of these derivatives.

Enzymatic activation of the carcinogens 2 phenylethylhydrazine and 1,2 dimethylhydrazine to carbon centered radicals

Spin-trapping experiments demonstrate that oxidation of 1,2-dimethylhydrazine and 2-phenylethylgydrazine generates a comparable yield of carbon-centered radicals when catalyzed by horseradish peroxidase-H2O2. Using oxyhemoglobin as the catalyst, 2-phenylethylgydrazine oxidation generates ten times carbon-centered radicals than 1,2-dimethylhidrazine oxidation. This results is in agreement with oxygen consumption studies from which the apparent KM values of 8.0 mM and 72 mM were calculated for the oxyhemoglobin-catalyzed oxidation of 2-phenylethylhydrazine and 1,2-dimethylhydrazine, respectively. These differences in metabolic activation of mono- and disubstituted hydrazines may be of importance regarding the carcinogenic properties of these derivatives

Activation of the colon carcinogen 1,2-dimethylhydrazine in a rat colon cell-mediated mutagenesis assay.

Suspensions of rat colon epithelial cells metabolized the potent colon carcinogen, 1,2-[14C]dimethylhydrazine (DMH), into 14C-labeled, alkali-soluble volatile products, presumably CO2. The colon cell suspensions, however, were less effective than rat hepatocyte suspensions. In addition, we used a cell-mediated mutagenesis assay to test rat colon epithelial cells grown from tissue explants for their ability to metabolize DMH into products mutagenic for human P3 teratoma cells. Mutagenesis in the P3 cells was indicated by an acquired resistance to 6-thioguanine. Cocultivation of the colon cells with the P3 cells in the cell-mediated assay resulted in mutagenesis, whereas in the absence of the colon cells, no mutagenesis by DMH was observed. Similar results were obtained in a hepatocyte-mediated mutagenesis assay. Colon cells were also able to activate another carcinogen, benzo(a)pyrene, into products mutagenic for the P3 cells. Individual epithelial clonal populations isolated from the colon cultures grown from tissue explants, however, expressed different capacities to activate DMH and benzo(a)pyrene into mutagens, and a high degree of DMH activation by cells from a colon clone was not necessarily associated with a similar degree of benzo(a)pyrene activation. Our results indicate that the colon itself contains epithelial cell types capable of effectively converting DMH into mutagenic (and presumably carcinogenic) products without necessarily involving intermediary metabolism by hepatocytes as previously thought.

In vitro DNA and nuclear proteins alkylation by 1,2-dimethylhydrazine.

The methylating carcinogen 1,2-dimethylhydrazine (DMH) CAS 540.73.8 is highly organ-specific and, under certain experimental conditions, produces a high incidence of adenocarcinoma in the colon of rodents. We have tried to assess the possibility that part of the organ-specifity in the carcinogenic effect of DMH could be attributed to its metabolism by specific microsomal enzymes. In particular, we compared the in vitro effects of DMH in the presence of either colon or liver microsomes from animals that had been treated with microsomal enzyme inducers. V79 Chinese hamster cells were used as the target to evaluate the damage to the genetic material, as judged by (1) formation of adducts of DNA bases and (2) amino acid modifications in nuclear proteins using [Me-14C]DMH and appropriate analytical detection systems. Our results tend to support the above postulated hypothesis.