Acute oral toxicity in rats of 3,7-bis-(4-trifluoromethylphenyl)-1,5,3,7-dioxadiazocane compared with 3,7-bis-(3-trifluoromethylphenyl)-1,5,3,7-dioxadiazocane and N,N'-oxydimethylenebis(2-trifluoromethylaniline).

The acute oral toxicity of 3,7-bis-(4-trifluoromethylphenyl)-1,5,3,7-dioxadiazocane (4-TFMPD) was compared with its 3-substituted isomer, 3,7-bis-(3-trifluoromethylphenyl)-1,5,3,7-dioxadiazocane (3-TFMPD) and with N,N'-oxydimethylenebis(2-trifluoromethylaniline) (N,N'-oxy-DM-bis (2-TFMA)). 4-TFMPD, 3-TFMPD, N,N'-oxy-DM-bis (2-TFMA) and their precursors (4-trifluoromethylaniline (4-TFMA), 3-trifluoromethylaniline (3-TFMA) and 2-trifluoromethylaniline (2-TFMA), respectively) were administered intragastrically to male Wistar rats at a dose of 0.12 mmol/kg body weight/day for three consecutive days and the resulting effects on haematological variables were determined. 4-TFMPD induced the highest methaemoglobinemia as compared with 3-TFMPD and N,N'-oxy-DM-bis (2-TFMA). Haemolytic anaemia with Heinz bodies, neutrophilia, lymphocytosis, enlargement of the spleen and enhanced production of granulocytes/macrophages from multipotential bone marrow cells (as determined by CFU-C test) were observed in animals treated with 4-TFMPD and 4-TFMA, whereas no such effects were observed in the other treatment groups. In conclusion, 4-substituted aniline derivatives exert special haematotoxicity on the red blood cells and induce leucocytosis, which differs from the effects of their 2- and 3-substituted congeners.

Effects of trifluoromethylaniline isomers on enzyme activities in lymphatic organs and hematology of the rat.

Three isomers of trifluoromethylaniline (TFMA) were investigated for their possible different toxic effects on the hematopoietic system in male Wistar rats. The effects of isomeric 2-, 3- and 4-TFMA were compared with those of aniline, the prototypic drug. Strong leukocytosis manifested by considerable increase in the number of all respective white blood elements was observed in the peripheral blood 1 day after the administration of 4-TFMA. In contrast, erythropoiesis, as ascertained by erythrocyte count and hemoglobin concentration, was inhibited by 4-TFMA. The determination of the ED50 revealed lymphocytes to be the most responsive elements towards 4-TFMA administration. Besides hyperemic and proliferative splenomegaly the histological changes in maturation of immunocompetent cells following the 4-TFMA administration were found also in thymus. In accord with an enhanced incorporation of [3H]thymidine, the specific activity of thymidine kinase (TdK) in spleen was increased after a single dose of 4-TFMA. Activities of the catabolic enzymes adenosine deaminase (ADA) and inosine phosphorylase (IP) decreased in both organs with the exception of IP activity in thymus. The effects evoked by the 3-TFMA isomer were regularly less pronounced, and 2-TFMA was nearly inactive.

Trifluoromethylanilines--their effect on DNA synthesis and proliferative activity in parenchymal organs of rats.

Reactive isomeric 3- and 4-trifluoromethylanilines (3-,4-TFMA), and control aniline itself, induced the following effects on biosynthesis of DNA in the liver, kidney, thymus and spleen of rats: (a) The administration of 4-TFMA initially suppressed the utilization of labeled thymidine for splenic DNA synthesis during the early prereplicative stage. However, with progressing time the incorporation of the labeled marker began to increase and in 30 h its level exceeded the controls by more than 200%. As expected, aniline administration resulted in mild depression of incorporation during the whole period studied. (b) 4-TFMA caused a significant increase of incorporation of labeled thymidine into DNA thymine also in the thymus. After administration of aniline the utilization of labeled thymidine for the synthesis of DNA thymine in thymus was suppressed during the first 16 h. (c) The dose-response curve showed a linear increase of incorporation in the spleen within the dose range between 0.125 and 0.500 mmol/kg of 4-TFMA. (d) It appears that enhanced incorporation of labeled thymidine into splenic and thymic DNA is a phenomenon specific for compounds bearing the CF3 group on the 4-position of the phenyl ring, such as 4-TFMA and 4-TFMPD. On the contrary, the analogous 3-CF3 substituted derivatives had no effect. Increased incorporation of labeled thymidine into spleen and thymus DNA apparently represents an increased DNA synthesis and cellular proliferation in lymphatic organs. The proliferative response was possibly evoked by the preceding hemolysis or by other toxic effects caused by the drug.

Effect of 3,7-bis-(4-trifluoromethylphenyl)-1,5,3,7-dioxadiazocane on pyrimidine and DNA synthesis in rat organs.

The administration of the lipophilic 3,7-bis-(4-trifluoromethylphenyl)- 1,5,3,7-dioxadiazocane (TFMPD) to rats induced the following effects on the biosynthesis of DNA in the liver, kidney, thymus and spleen: (a) The utilization of [3H]thymidine for the synthesis of liver DNA thymine was decreased after the administration of a single dose of the drug. The depression of the specific activities of DNA pyrimidines of liver DNA in experimental groups was observed also after an injection of [14C]orotic acid. (b) A decreased incorporation of labeled thymidine had occurred also in the spleen during the prereplicative period. Thereafter the specific activity of DNA thymine was higher than in the control group. (c) The observed mitogenic response in the spleen showed a protracted effect; after the administration of a single dose of the drug the specific activity of DNA thymine as well as the thymidine kinase activity of spleen cytosol have been rising up to the ninth day. The same holds true for DNA thymine of the thymus; the activity of thymidine kinase was not affected. (d) Both the single and repeated doses of TFMPD had no marked effect on the levels of microsomal cytochromes P-450 and b5 in the liver and kidney.

Synthesis and cytotoxic effects of hydroxymethyl-3-pyridyl- and 2-chloro-5-pyridyltriazene derivatives.

3-Hydroxymethyl-3-methyl-1-(3-pyridyl)-triazene (III) and 3-hydroxymethyl-3-methyl-1-(2-chloro-5-pyridyl)-triazene (VI) were synthesized and their cytotoxic effects towards S180 cells compared with those of the corresponding dimethyltriazene and monomethyltriazene derivatives. The hydroxy-methyltriazenes were one to two orders of magnitude more inhibitory than the corresponding dimethyl analogs, as assessed by cell growth, colony forming and macromolecular synthesis assays. Comparable effects were observed with the related monomethyl triazenes indicating that the activity of (III) and (VI) could result, at least in part, from release of monomethyl derivatives. The corresponding dimethyltriazenes were much less toxic to S180 cells and exerted only an unspecific cytotoxic activity at the maximal attainable dose (5 X 10(-3) M). The cytotoxic activities of the tested triazenes were correlated with their chemical half-lives under near physiological conditions (pH 7.5).

Reactions of substituted arenediazonium chlorides with methylamine-formaldehyde premix revisited: reactivity and transformations of methylolamine intermediates and their biological significance.

Modulation of the N-azo coupling between ring-substituted arenediazonium chlorides and premixed methylamine-formaldehyde leads not only to 1-aryl-3-hydroxymethyl-3-methyltriazenes and their dimers, but also to unexpected cyclic and complex products. The syntheses comprise reactions with arenediazonium chlorides bearing both -M and +M substituents at para and ortho/para positions of the phenyl ring. One of the major constituents isolated from a mixture of products is the O-acetate of 3-hydroxymethyl-3-methyl-1-(2,4,6-trichlorophenyl)triazene. This product was obtained from the reaction of 2,4,6-trichlorobenzenediazonium chloride and methylamine-formaldehyde mixture which was then stabilized by acetylation. The structures of the isolated products could be derived from reactive methylolamines and electrophilic intermediates that possible occur in vivo and thereby offer a plausible mechanistic explanation for the carcinogenic and tumour-inhibitory activity associated with the open-chain triazene compounds in the living cell.

In vivo metabolism and reaction with DNA of the cytostatic agent, 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC).

The cytostatic drug dacarbazine [DTIC, 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide] is strongly carcinogenic in rats. Bioactivation of DTIC yields a methylating intermediate but the extent of interaction with cellular macromolecules has not previously been reported. Following a single i.p. injection of [14C-methyl]DTIC, exhalation of 14CO2 occurred with a t1/2 max of approximately 2 hr (0.95 mg/kg) and 2.5 hr (95 mg/kg). Of the total radioactivity administered, 8.5% was exhaled as 14CO2; 54% was excreted via the urine, predominantly as unchanged DTIC. In liver, kidney and lung, formation of 7-[14C]methylguanine in DNA and RNA was directly proportional with dose. DNA methylation by a single dose of DTIC (9.8 mg/kg; 5 hr survival time) was highest in liver (35 mumoles 7-methylguanine/mole guanine), followed by kidney (25 mumoles) and lung (20 mumoles). The remainder tissues showed 7-methylguanine concentrations approximately 50% of those in liver DNA, with the exception of the brain which had a very low extent of DNA modification (approximately 1 mumole/mole guanine). At the specific radioactivity used (48 mCi/mmole), the promutagenic base O6-methylguanine was only detectable in liver, kidney, lung, and stomach DNA (0.6-0.8 mumoles/mole guanine). Autoradiographic studies revealed a diffuse distribution of reaction products in rat liver. In contrast, N-nitrosodimethylamine and related carcinogens known to be bioactivated by the hepatic cytochrome P-450 system show a predominantly centrilobular distribution. This difference may be due to the greater stability of proximate carcinogens generated by alpha-C hydroxylation at one of the methyl groups of DTIC.

Carcinogenicity of cytostatic triazenes.

Introduction of the anticancer drug dacarbazine is the result of an attempt to design antagonists of purine biosynthesis. The mechanism of action of dacarbazine depends mainly on enzymatic transformation into as yet unknown reactive (electrophilic) intermediates. Recent studies have led to the identification and synthesis of 5-(3-hydroxymethyl-3-methyl)imidazole-4-carboxamide (HMTIC), a carbinolamine metabolite with methylating capacity. Although dacarbazine and related cytostatic triazenes are effective in the treatment of malignant melanoma and other human malignancies, dacarbazine has been demonstrated to be a carcinogen in laboratory rodents. Chronic administration of dacarbazine to rats of each sex induced predominantly thymic lymphosarcomas and mammary adenocarcinomas that were transplantable. Intraperitoneally injected 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC), a metabolite of dacarbazine, induced a high incidence of mammary adenofibromas and a low incidence of uterine leiomyosarcomas. Animals treated with 5-diazoimidazole-4-carboxamide developed a low incidence of thymic, stomach, bladder or mammary tumours. Animals receiving 5-aminoimidazole-4-carboxamide developed a variety of tumours. No secondary malignancy has been reported in humans after treatment with dacarbazine alone. Despite their adverse effects, dacarbazine and related cytostatic triazene derivatives are useful clinically since their haematological toxicity is relatively moderate. As a rule, they are not cross-resistant with nitrogen mustard alkylating agents. It is hoped that research and development of second-generation N-(1-hydroxyalkyl)triazene compounds will lead to improvements in their clinical efficacy.

Comparative carcinogenicity of ring-halogenated 3-methyl-1-phenyltriazenes and their 3,3-dimethyl analogs at equimolar dose levels in male Sprague-Dawley rats.

The carcinogenic activity of 3-methyl-1-phenyltriazene and its four 4- and 2-ring halogenated derivatives was similar to the previously reported carcinogenicity of the corresponding 3,3-dimethyl-1-phenyltriazene analogs tested at equimolar dose levels in adult male Sprague-Dawley rats. The strongest carcinogens in each series were 3-methyl-1-phenyltriazene and 3,3-dimethyl-1-phenyltriazene, respectively. The carcinogenic potency of the individual test compounds decreased with progressive 4- and 2-halogenation of the phenyl ring. The administration of either 3-methyl-1-(2,4,6-trichlorophenyl)- or 3-methyl-1-(2,4,6-tribromophenyl)-triazene gave a negligible tumor yield not different from that observed in the control animals. Moreover, 3,5-dichloro-2-(3,3-dimethyl-1-triazeno)-benzoic acid or its ethyl ester, were found to be strong carcinogens that exclusively induced kidney tumors.

Microsome-mediated alkylation of rat liver initiator tRNA by 3,3-dimethyl-1-phenyltriazene and its ring-chlorinated derivatives.

3,3-[3H]Dimethyl-1-phenyltriazene, 1-(4-chlorophenyl)-3,3-[3H]dimethyltriazene and 3,3-[3H]dimethyl-1-(2,4,6-trichlorophenyl)-triazene methylate initiator tRNA in vitro only after pre-incubation with microsomal enzymes and NADPH. The finding confirms that procarcinogenic dialkyl aryltriazenes must be enzymatically converted into reactive metabolites, presumably into the corresponding monoalkyltriazenes, which ultimately react with tRNA. The methylation at 37 degrees C requires 40-60 min and individual triazenes showed differential alkylating capacity if tRNA was the limiting factor. Enzymatic hydrolysis of the modified initiator tRNA, followed by separation of nucleosides on Sephadex G10 or Dowex 50 columns, revealed that 7-methylguanosine was the principal labelled product. The methylated tRNA showed a significantly increased acceptance for L-methionine. It appears that methylation of initiator tRNA at N7 of guanine affected the conformation of initiator tRNA and rendered the nucleic acid more accessible for cognate aminoacyl-tRNA synthetase.

Microsome-mediated stimulation of the aminoacylation of rat liver initiator transfer ribonucleic acid by benzo[alpha]pyrene and 3,3-dimethyl-1-phenyltriazene.

The charging of initiator tRNAmetF with L-methionine was significantly stimulated by pre-incubation of unfractionated tRNA from rat liver with benzo [alpha]pyrne (BP) or 3,3-dimethyl-1-phenyltriazine (DMPT). The presence of microsomal enzymes from rat liver and of NADPH was absolutely required for this effect. Maximum enhancement was obtained after 60 min of incubation with 10(-4)-10(-8) mumol/ml of either compound tested. It appears that either proximate carcinogen, BP or DMPT, must be converted by microsomal enzymes into its direct-acting metabolites (ultimate carcinogens) which interact with initiator tRNA and thereby specifically modulate its aminoacylation.

Comparative metabolism and carcinogenicity of ring-halogenated 3,3-dimethyl-1-phenyltriazenes.

The objective of this study was to compare urinary metabolism of parent 3,3-dimethyl-1-phenyltriazene with that of its ring-substituted 1-(4-chlorophenyl)-3,3-dimethyltriazene and 1-(2,4,6-trichlorophenyl)-3,3-dimethyltriazene congeners, in an attempt to evaluate the molecular requirements for systemic carcinogenic activity. Complementary carcinogenicity assays were conducted at low equimolar dose levels using both 4- und 2,4,6-chlorinated and brominated analogues. Ring halogenation was found to prolong metabolic detoxification and to reduce carcinogenic activity.

5-(3-Hydroxymethyl-3-methyl-1-triazeno imidazole-4-carboxamide is a metabolite of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DIC, DTIC NSC-45388).

The cancer chemotherapeutic drug, 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide (DIC, DTIC, NSC-45388), is metabolised in rats to a structurally related product which was detected by thin-layer chromatography. The novel metabolite has a lower mobility and a colour reaction that is indistinguishable from the parent compound. The metabolite is not retained on an anionic exchanger which is inconsistent with the expected covalent binding of the drug to endogenic anionic substrates (e.g. glucuronic acid). Since both DIC and the metabolite yielded 5-[(4-ethylamino-1-napthyl)-azo]imidazole-4-carboxamide through release of 5-diazoimidazole-4-carboxamide, followed by coupling with N-ethyl-1-napthylamine, no biotransformation (hydroxylation) of the imidazole moiety of the injected DIC had occurred. By corollary, the lowered chromatographic mobility of the metabolite was explicable by the introduction of a polar but non-acidic function into the terminal dimethylamino group of the triazene side-chain. The metabolite was identified as 5-(3-hydroxymethyl-3-methyl-1-triazeno)imidazole-4-carboxamide by co-chromatography with an authentic sample of HMIC and by its methylating capacity for nucleophilic substrates.

Urinary metabolite of 1-(2,4,6-trichlorophenyl)-3,3-dimethyltriazene with an intact diazoamino structure.

The tumour-inhibiting substance 1-(2,4,6-trichlorophenyl)-3,3-dimethyltriazene is metabolised in rats to the corresponding substituted 1-O-(triazenyl-methyl) glucuronic acid. The urinary metabolite was purified by ion exchange chromatography and gel filtration, and isolated from the enriched fractions by freeze-drying. Cold acid cleavage into the 2,4,6-trichlorobenzene-diazonium cation and hydrolysis to glucuronic acid and formaldehyde indicated the presence of an O-glycosidic bond through an enzymically-introduced hydroxymethyl oxygen. This novel type of glucuronoside structure was established by chemical evidence, and confirmed by NMR and field-desorption mass spectrometry. It is conceivable that this metabolite represents a stabilised carrier form of the biologically-active triazene that transports the methylating agent from its site of formation to its ultimate target.

Mechanism of perinatal tumor induction by neuro-oncogenic alkylnitrosoureas and dialkylaryltriazenes.

The methylation by DMPT and MNU of DNA from rat liver and brain was investigated at various developmental stages. Following a single sc injection of [14C]DMPT (100 mg/kg body wt, 15 hr survival time) in pregnant rats (21st day of gestation), the extent of methylation of purine bases was similar in fetal liver and brain. During postnatal growth, this treatment resulted in an increasingly preferential methylation of liver DNA. In 30-day-old BD-IX rats, the concentration of 7-methylguanine in liver was approximately eight times higher than in brain DNA. This suggested that during prenatal development, both liver and brain DNA are transplacentally methylated by a proximate carcinogen produced by maternal organs. After a single ip injection of [3H]MNU (10 mg/kg body wt) to 10-day-old rats, O6-methylguanine was more rapidly removed from hepatic than from cerebral DNA. Within 1 week after the injection, the brain-to-liver ratio for 06-methylguanine increased from 1.4 to 98. These results are compatible with the hypothesis that the deficiency of various organs for repair excision of O6-alkylguanine from DNA correlates with their susceptibility to malignant transformation by monofunctional alkylating carcinogens.

In vivo alkylation of foetal, maternal and normal rat tissue nucleic acids by 3-methyl-1-phenyltriazene.

The carcinogen 3-methyl-1-phenyltriazene (MPT) was administered subcutaneously to normal or pregnant BD VI rats and DNA and RNA were isolated from various tissues after 8 h or 15 h, respectively. Sephadex G-10 chromatography of DNA hydrolysates showed the presence of 7-methylguanine in all tissues examined including that of the brain, one of the target organs for tumour induction. The amounts of the minor product, O6-methylguanine, were characteristic of an SN1 reaction mechanism. Dowex-50 chromatography of RNA hydrolysates showed the presence of 7-methylguanine and of the minor product, 3-methylcytosine. The relative amounts, both of the methylated bases in the individual nucleic acids and of 7-methylguanine in DNA and RNA, were similar to those found previously after administration of 3,3-dimethyl-1-phenyltriazene (DMPT). This suggests the involvment of a common alkylating intermediate. De novo incorporation of radioactivity into purine bases was detected in both DNA and RNA although the levels were not related to the amounts of methylation. The results show that MPT is sufficiently stable to alkylate nucleic acids in vivo and are consistent with the hypothesis that this reaction is a prerequisite for tumour induction. Futhermore, they support the proposal that MPT is the active intermediate in the induction of tumours by DMPT.

Microsomal biphenyl 2-hydroxylation in the presence of some carcinogenic alkylaryltriazenes.

A hepatic mixed-function oxygenase system gave enhanced biphenyl 2-hydroxylation in the presence of known carcinogens in vitro and was therefore suggested as a convenient short-term assay for the detection of potentially dangerous compounds. However, the test did not yield the expected increase of biphenyl 2-hydroxylation with any of 8 selected carcinogenic triazenes. The new data are discussed in relation to the original supposition and to the hydroxylation pattern of various triazene substrates in vivo.

DNA alkylation and neuro-oncogenesis by 3,3-dimethyl-1-phenyltriazene.

The role of DNA alkylation by the neurooncogenic agent 3,3-dimethyl-1-phenyltriazene (DMPT) was investigated perinatally and in adult rats. Following a single subcutaneous injection of 14C-DMPT (100 mg/kg) on the 21 day of gestation, the concentration of methylated purines was similar in both fetal liver and brain whereas during postnatal growth this treatment resulted in an increasingly preferential methylation of liver DNA. In 30-day-old and adult rats the concentration of 7-methylguanine in liver was about 8 times higher in brain DNA, suggesting that during prenatal development both liver and brain DNA are transplacentally methylated by a proximate carcinogen produced by maternal organs. Multiple doses of 14C-DMPT (50 mg/kg) to adult rats led to a preferential accumulation of O6-methylguanine in cerebral DNA. This supports the hypothesis that the deficient repair excision capacity of the hypothesis that the deficient repair excision capacity of the central nervous system is a significant factor in the organ-specific carcinogenicity of DMPT and related carcinogens.