Metabolomics and cytotoxicity of monomethylhydrazine (MMH) and (E)-1,1,4,4-tetramethyl-2-tetrazene (TMTZ), two liquid propellants.

Hydrazine-based liquid propellants are routinely used for space rocket propulsion, in particular monomethylhydrazine (MMH), although such compounds are highly hazardous. For several years, great efforts were devoted to developing a less hazardous molecule. To explore the toxicological effects of an alternative compound, namely (E)-1,1,4,4-tetramethyl-2-tetrazene (TMTZ), we exposed various cellular animal and human models to this compound and to the reference compound MMH. We observed no cytotoxic effects following exposure to TMTZ in animal, as well as human models. However, although the three animal models were unaffected by MMH, exposure of the human hepatic HepaRG cell model revealed that apoptotic cytotoxic effects were only detectable in proliferative human hepatic HepaRG cells and not in differentiated cells, although major biochemical modifications were uncovered in the latter. The present findings indicate that the metabolic mechanisms of MMH toxicity is close to those described for hydrazine with numerous biochemical alterations induced by mitochondrial disruption, production of radical species, and aminotransferase inhibition. The alternative TMTZ molecule had little impact on cellular viability and proliferation of rodent and human dermic and hepatic cell models. TMTZ did not produce any metabolomic effects and appears to be a promising putative industrial alternative to MMH.

Contribution of hydrazines-derived alkyl radicals to cytotoxicity and transformation induced in normal c-myc-overexpressing mouse fibroblasts.

Several hydrazine derivatives (HD) tested so far have pharmacological activities, but many also have toxic side effects, including carcinogenesis. Their toxicity has been ascribed to carbocations (via formation of azoxy intermediates), alkyl radicals or reactive oxygen species. Cytotoxicity and transformation by carbocations is widely accepted, but the role of alkyl radicals is still questioned. We have investigated the cytotoxicity of HD to mouse fibroblasts in three activation systems in which enhanced alkyl radical formation is demonstrated by electron spin resonance/spin-trapping. Cytotoxicity was assayed by inhibition of [3H-methyl]thymidine uptake into DNA of Balb/c 3T3 and/or Myc 9E fibroblasts (normal Balb/c 3T3 cells over-expressing the c-myc proto-oncogene). Based on the results obtained in the cytotoxicity assays we also investigated the transforming potential of procarbazine (PCZ) and methylhydrazine (MeH) activated by horseradish peroxidase (HRP) using the Myc 9E cell line, which aims at the activation of a second cooperating oncogene. Our results show that: (i) cytotoxicity of HD to mouse fibroblasts is increased by HRP activation of MeH, phenelzine and PCZ, which displayed enhanced alkyl radical formation, but not of 1,2-dimethylhydrazine (DMH), which did not produce increased alkyl radical formation under these conditions; (ii) cytotoxicity of neutrophil-activated MeH (producing a 10-fold higher concentration of methyl radicals), is more pronounced than DMH; (iii) MeH and DMH activated by prolonged auto-oxidation in 24-h incubations have comparable cytotoxicity and alkyl radical formation; and (iv) PCZ and MeH activation by HRP to alkyl radicals increased the transformation induced in Myc 9E cells. Taken together, our results strongly support a role for hydrazine-derived alkyl radicals in HD-induced cytotoxicity and cell transformation.

The effect of dietary selenium deficiency on acute colorectal mucosal nucleotoxicity induced by several carcinogens in the rodent.

BACKGROUND: Selenium (SE) has been inversely associated with colon cancer risk. Two potential mechanisms of this effect were examined in a rodent short-term carcinogenesis assay: whether dietary SE deficiency altered the initiation aspect of carcinogenesis in the colon, and whether SE altered carcinogen metabolism. SETTING: Animal laboratory. SUBJECTS: 52 Sprague-Dawley rats, divided into a SE diet deficient group (0.002 parts per million; ppm) and a SE sufficient (0.2 ppm) group. ENDPOINTS: Weight, serum SE concentration, and karryorhectic index (KI), which is a measure of acute carcinogen induced nuclear toxicity in the colonic mucosa. METHODS: After three weeks of acclimation to the diets, eight animals from each dietary group were injected with one of the following: dimethylhydrazine (DMH), a colon specific carcinogen, its metabolite, methylazoxymethanol (MAM), or 0.9% sodium chloride. Twenty-four hours after injection the colons were removed, blood drawn, and the stained colons assayed for nuclear aberrations. RESULTS: No weight differences were generated by the dietary variations. Low-dietary SE resulted in serum SE declining markedly in the study period to 6 ng/ml versus 33 ng/ml in the SE sufficient group. Diet alone, and variations in weight gain, did not alter the KI. Both carcinogens greatly increased the KI in both the left and right colon. A SE-deficient diet was associated with a higher KI in both carcinogen groups in the right colon, with statistical significance for both the left and right colon in the MAM injection group. CONCLUSIONS: Dietary SE deficiency is associated with increased KI of the colon in MAM treated rats. SE, therefore, has a protective effect in the initiation phase of carcinogenesis.

Hydrazine-mediated DNA damage: role of hemoprotein, electron transport, and organic free radicals.

The hydrazines represent an important class of xenobiotic agents encountered in the environment, in industrial settings, and in medical therapeutics. Agents with a hydrazine functionality are metabolized to toxic intermediates capable of damaging cellular macromolecules and stimulating proteolysis. Phenylhydrazine (PH), methylhydrazine (MH), hydrazine (HY), and the therapeutic agents phenelzine (PZ) and hydralazine (HD) were examined for their ability to undergo metabolism via HbO2 and to cause damage to added supercoiled phi x174 RF DNA. The hydrazines, when incubated in hemolysate, caused a time- and concentration-dependent strand scission of DNA as monitored using phi x174 RF DNA. The rank order for hydrazine-mediated damage was phenylhydrazine > phenelzine > hydrazine > hydralazine > methylhydrazine. In addition, hydrazine-mediated damage to DNA increased in proportion to protein concentration (i.e., HbO2 content) of the hemolysate. To examine whether the DNA damage resulted primarily from organic free radicals or reactive oxygen free radical species, a series of mechanistic studies employing antioxidants and a free radical scavenger was initiated. The antioxidants dimethylfuran, dimethyl sulfoxide, and dimethylthiourea failed to inhibit hydrazine-mediated DNA damage in hemolysate. In contrast, the free radical spin trap agent dimethylpyrrolidin-N-oxide effectively inhibited PH-mediated DNA damage, while the free radical scavenger N-acetylcysteine also showed a protective effect against PH-, PZ-, HD-, HY-, and MH-mediated DNA strand scission. Potassium ferricyanide-mediated methemoglobin formation and imidazole, a ligand for the heme moiety of hemoglobin, both inhibited PH-stimulated DNA damage in hemolysate demonstrating the importance of oxyhemoglobin to the process. These results suggest that organic free radicals play a dominant role, relative to oxygen free radical species, in hydrazine-mediated DNA strand scission.

Toxicological studies of the false morel (Gyromitra esculenta): embryotoxicity of monomethylhydrazine in the rat.

The embryotoxic and teratogenic potential of monomethylhydrazine (MMH), a toxic component of the widely consumed false morel (Gyromitra esculenta), was studied in rat. Groups of pregnant Sprague-Dawley rats received MMH as a constant i.v. infusion via implanted osmotic minipumps (1.2, 3.0, 4.2, 6.0, 9.0 or 13.2 mg MMH/kg bw/day) on days 6-13 of pregnancy, or as a single intragastric bolus (1 mg MMH/kg/bw or 5 mg MMH/kg/bw) on day 6 of pregnancy. Controls received corresponding amounts of saline. The average maternal serum concentrations, measured during the infusion treatment with a sensitive HPLC method, ranged from 0.072 micrograms MMH/ml (lowest dose) to 0.60 microgram MMH/ml (highest dose). The average serum levels measured 45 min after the intragastric application (peak levels) were 0.28 microgram MMH/ml and 1.6 microgram MMH/ml, respectively. Serum concentrations of MMH corresponding to those measured in the lower dose groups in this study were seen in pilot studies after a single mushroom meal in human volunteers. A dose-dependent, statistically significant increase in the number of resorptions was seen in all but the lowest dose group after the infusion of MMH. In addition, except for the two lowest doses, there was a dramatic, dose-dependent decrease in the pregnancy rate as compared to controls, with no pregnancies occurring at the two highest dose level groups. The decreased pregnancy rate was probably due to preimplantation loss which was shown to occur after a single intragastric bolus dose of MMH (5 mg/kg bw).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Modulation of monomethylhydrazine-induced seizures by ivermectin.

Exposure to monomethylhydrazine (MMH), a common rocket propellant, can cause dose-related central nervous system (CNS) disturbances ranging from tremors to tonic-clonic convulsions to death. MMH inhibits gamma-aminobutyric acid (GABA) synthesis in the CNS. Diazepam (BZ) acts at the GABA receptor site, and it is also here that ivermectin (AVM) is pharmacologically active. Mice were injected with 30 mg/kg MMH. Groups of 12 mice each were then given varying doses of AVM (5, 10 and 15 mg/kg), or AVM + BZ combinations (5 mg/kg AVM with 5 mg/kg BZ, 10 mg/kg AVM with 5 mg/kg BZ). Time to first convulsion and time to death were recorded over the next 7 h and all groups were monitored over the next 7 days. Times to convulsion were not altered with AVM alone, but death was significantly prevented with AVM dosages. A treatment of 10 mg/kg AVM with 5 mg/kg BZ resulted in no seizures or deaths.

N-methyl-N-formylhydrazine: a toxic and mutagenic inhibitor of the intestinal diamine oxidase.

N-methyl-N-formylhydrazine is the first active intermediate of the poison gyromitrin of the mushroom: false morel. This compound is a non-competitive inhibitor of human intestinal diamine oxidase (ID50 = 1.6 X 10(-5) mol/l). This concentration corresponds to less than 5 g of wet weight of mushroom/l. The diamine oxidases from 5 other sources are inhibited in a similar manner. Semicarbazide and aminoguanidine are 10-respectively 1000-fold more potent inhibitors of the human intestinal diamine oxidase. An involvement of the diamine oxidase inhibitory property of N-methyl-N-formylhydrazine in toxic and mutagenic effects of the substance is considered.

Teratogenic assessment of three methylated hydrazine derivatives in the rat.

The embryotoxicity and teratogenicity of methylhydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine were investigated with pregnant Fischer-344 rats. The compounds were administered ip on d 6-15 of pregnancy. A dose-dependent reduction in maternal weight gains occurred for all three compounds. A dose-related teratogenic effect did not occur for any of the three compounds. Embryotoxicity, manifested as reduced 20-d fetal weights, occurred only in the 1,1-dimethylhydrazine and 1,2-dimethylhydrazine high-dose treatment groups. The results indicate that none of the three methylated hydrazine derivatives are selectively embryotoxic or teratogenic in the rat.

Tumorigenicity of minute dose levels of N-methyl-N-formylhydrazine of Gyromitra esculenta.

Separate administrations of 0.0005 to 0.00025% N-methyl-N-formylhydrazine in drinking water to 6-week-old randomly bred Swiss mice for the remainder of their lifetime induced lung neoplasms. At the high dose level, 64% of the females and 48% of the males developed lung tumors, while the corresponding tumor incidences at the lower dose level were 62% in the females and 54% in the males. In untreated controls, the lung tumor incidences were 29% in the females and 19% in the males. Histopathologically, the lesions were classified as adenomas and adenocarcinomas of the lungs. N-methyl-N-formylhydrazine is a stable constituent of the edible false morel mushroom Gyromitra esculenta. The environmental significance is discussed, in view of the carcinogenicity of minute doses of this chemical.

The tumorigenic effect of low dose levels of N-methyl-N-formylhydrazine in mice.

Solutions of 0.002 and 0.001% N-methyl-N-formylhydrazine (MFH) were given separately daily in drinking water to 6-week-old randomly bred Swiss mice for life. The treatments resulted in induction of tumors of the lungs, blood vessels, liver and gall bladder. The 0.002% solution gave rise to tumors of the above tissues in incidences of 76, 27, 28 and 11%, respectively, while the 0.001% solution induced tumors in incidences of 75, 23, 7 and 6%. Histopathologically, the neoplasms were adenomas and adenocarcinomas of lungs, angiomas and angiosarcomas of blood vessells, benign hepatomas, liver cell carcinomas and adenomas and adenocarcinomas of the gall bladder. MFH is an ingredient of the edible mushroom, the false morel Gyromitra esculenta. The present study proves the carcinogenicity of MFH administered at low dose levels. The possible environmental significance of the findings to the human population is discussed.

False morel mushroom Gyromitra esculenta toxin: N-methyl-N-formylhdrazine carcinogenesis in mice.

N-Methyl-N-formylhydrazine was administered in drinking water as a 0.0039% solution to randomly bred Swiss albino mice for life starting from 6 weeks of age. The compound induced tumors of lungs, livers, blood vessels, gall bladder and bile ducts. The tumor incidences in these five tissues were 77, 46, 21, 10 and 7% while in the untreated controls they were 18, 1, 6, 0 and 0% respectively. Histopathologically, the tumors were classified as adenomas and adenocarcinomas of lungs, benign hepatomas and liver cell carcinomas, angiomas and angiosarcomas of blood vessels, adenomas and adenocarcinomas of gall bladder and cholangiomas. The macroscopic and light microscopic involvement of the tissues with the tumors are described and some of them are illustrated. N-Methyl-N-formylhydrazine is an ingredient of the edible mushroom, the false morel Gyromitra esculenta. The findings are discussed from the viewpoint of a potential human health hazard.

Hepatocarcinogenesis by hydrazine mycotoxins of edible mushrooms.

Two edible mushrooms are known to contain hydrazine analogs. The wild false morel Gyromitra esculenta contains up to 0.3% acetaldehyde methylformylhydrazone and N-methyl-N-formylhydrazine (MFH). The latter chemical, under certain conditions, also yields methylhydrazine. The commonly eaten, cultivated mushroom Agaricus bisporus contains up to 0.04% beta-N-[gamma-L(+)-glutamyl]-4-hydroxymethylphenylhydrazine and 4-hydroxymethylphenylhydrazine. MFH was administered in drinking water continuously for life to Swiss mice and Syrian (golden) hamsters. In both species the compound induced high incidences of benign and malignant hepatocellular neoplasms. Also, methylhydrazine given orally induced a significant incidence of malignant histiocytomas in the livers of hamsters. The N'-acetyl derivative of 4-hydroxymethylphenylhydrazine administered orally to mice gave rise to lung tumors and blood vessel tumors, mainly in the liver. Furthermore, these three compounds produced tumors in various other tissues. Histopathologically, the tumors were classified as benign hepatomas, liver cell carcinomas, angiomas and angiosarcomas of blood vessels, and adenomas and adenocarcinomas of lungs. From representative samples of these neoplasms detailed transmission electron microscopic investigations were also carried out. Since these hydrazine analogs induce tumors in animals and these mushrooms are consumed on a large scale by humans in various parts of the world, their hazardous nature should be considered.

Occupational hazards of missile operations with special regard to the hydrazine propellants.

The second generation of ballistic missiles and boosters, characterized by increased range and quick reaction capability, required the development of new high-energy storage propellants. This exploration led to the introduction of hydrazine (Hz), monomethylhydrazine (MMH), and 1,1-dimethylhydrazine (UDMH) into the USAF inventory. These compounds are all storable, noncryogenic, high-energy fuels which may be used alone or in combination as mixed amine fuels. Early toxicology experiments were to produce data on acute and subacute effects of the propellants in order to set standards for test and operational procedures to protect propellant handlers. The early work indicated that, despite similar chemical characteristics, there were marked differences between the compounds in terms of toxicological mechanisms. Since the propellant systems have been used for some 15 years, recent emphasis on toxicology has been centered on the more chronic effects and on an increasing body of evidence from animal experiments that the compounds may possess oncogenic potential as well as chronic systemic effects. This paper addresses itself to data leading up to current occupational standards.

Tumors induced in mice by N-methyl-N-formylhydrazine of the false morel Gyromitra esculenta.

Continuous administration of 0.0078% N-methyl-N-formylhydrazine (MFH) in drinking water to 6-week-old outbred Swiss mice for life produced tumors of the liver, lung, gallbladder, and bile duct. The incidences of tumors in these four tissues were 33, 50, 9, and 7%, whereas in the untreated controls they were 1, 18, 0, and 0%, respectively. The higher dose (0.0156% MFH) given under identical conditions had no tumorigenic effect, since it proved too toxic for the animals. Histopathologically, the lesions were classified as benign hepatomas, liver cell carcinomas, adenomas and adenocarcinomas of the lungs, adenomas of the gallbladder, cholangiomas, and cholangiocarcinomas. Since the edible false morel Gyromitra esculenta contains a high amount of MFH, the human population should be dissuaded from consumption of this dangerous mushroom.

Tumorigenicity of tetramethylhydrazine hydrochloride in Swiss mice.

Tetramethylhydrazine hydrochloride (TEMH) was administered in drinking water as a 0.125% solution to randomly bred Swiss mice for life beginning at 6 weeks of age. As a result of treatment, the incidence of blood vessel tumors rose from 5 to 96% in females and from 6 to 88% in males, while that of lung tumors increased from 21 to 36% in females and from 23 to 28% in males, as compared with untreated controls. The increased incidence of blood vessel tumors, but not of lung neoplasms, was statistically significant. Histopathologically, the tumors exhibited the characteristics of angiomas and angiosarcomas of blood vessels and adenomas of the lung. The investigation proved for the first time the tumorigenicity of TEMH. The possible role of increased methyl substitution on hydrazine in tumorigenesis was also discussed, as well as hydrazine derivatives as a tumor-producing class.