Structure-activity dependence in some novel ring-substituted 3,3-dimethyl-1-phenyltriazenes. Genetic effects in Drosophila melanogaster and in Saccharomyces cerevisiae by a direct and a host-mediated assay

The structure-activity dependence of ten ring-substituted 3,3-dimethyl-1-phenyltriazenes (DMPT), 3,3-dimethyl-1-(3-pyridyl)-triazene (3-PyDMT) and of 3,3-dimethyl-1-(3-pyridyl-N-oxide)-triazene (3-PyODMT) was investigated by the induction of recessive lethal mutations in Drosophila melanogaster and of mitotic gene conversions in Saccharomyces cerevisiae using both direct and host-mediated assays. Significant differences in genetic effectiveness were detected not only between structurally related compounds but also between the responses of each test system to the same mutagen. Triazenes which are easily cleaved at physological conditions showed the highest genetic activity in the direct yeast test whereas stable triazenes, especially those with ortho and para positions blocked by a halogen, were most active in Drosophila. We have concluded that (1) the released arenediazonium cation is most probably responsible for the convertogenic activity in yeast; (2) metabolites, arising from hydroxylation of the methyl group, are essential for the mutagenic activity in Drosophila. A possible molecular basis which could account for the diversity in genetic effectiveness is discussed in terms of reaction mechanisms which can be predicted from the structural features of the tested triazenes.     

Induction of mitotic gene conversion by 3,3-dimethyl-I-phenyltriazene, I-(3-hydroxyphenyl-)-3,3-dimethyltriazene and by I-(4-hydroxyphenyl)-3,3-dimethyltriazene in Saccharomyces cerevisiae

The induction of mitotic gene conversion by 3,3-dimethyl-1-phenyltriazene (DMPT), 1-(3-hydroxyphenyl)-3,3-dimethyltriazene (3-HO-PDMT) and by 1-(4-hydroxyphenyl)-3,3-dimethyltriazene (4-HO-PDMT) in the diploid strain D4 of Saccharomyces cerevisiae was investigated. The frequencies of the non-reciprocal intragenic recombinations at two unlinked loci ade2 (adenine) and trp5 (tryptophan) were determined. Although all three triazenes showed marked convertogenic activities, significant differences in their genetic effectiveness have been observed. Thus both phenolic triazenes were found to be much stronger convertogens than the unhydroxylated parent compound, DMPT. An attempt is made to account for the established differences in convertogenicity by chemical reactivity that could be expected from the structural features of the tested alkaryltriazenes.     

The effect of N-nitroso carcinogens and triazenes on protein synthesis in cell-free systems

The effect of a series of carcinogenic nitrosamines, alkylnitrosoureas and alkaryltriazenes on enzymatic reactions involved in protein synthesis was studied in cell-free systems from rat liver. The addition of most compounds stimulated the formation of aminoacyl-tRNA complex in test systems from rat liver whereas analogous preparations from Escherichia coli did not show this effect. The polymerization of phenylalanine and the binding of aminoacyl-tRNA to ribosome were only slightly and apparently non-specifically inhibited in the presence of the test compounds. On the other hand, the binding of nRNA to ribosome was markedly stimulated after the addition of most carcinogens tested. It appears that the carcinogens intervene specifically with one of the early steps in peptide initiation. Since the binding of mRNA to ribosome is known to be an important rate-limiting step in protein synthesis, the N-nitroso carcinogens and triazenes may thus control the expression of genetic message at the translation level.     

Genetic characterization of ad-3 mutants induced by chemical carcinogens, I-phenyl-3-monomethyltriazene and I-phenyl-3,3-dimethyltriazene, in Neurospora crassa

180 ad-3 mutants of Neurospora crassa induced by 1-phenyl-3-monomethyl-triazene (PMMT) and 56 ad-3 mutants induced by 1-phenyl-3,3-dimethyltriazene (PDMT) were characterized by dikaryon, trikaryon and complementation tests. Results show that the spectrum of genetic alterations induced by PMMT is different from that of PDMT. This suggests that enzymatic dealkylation of PDMT to PMMT does not occur within Neuropsora crassa conidia, and that the mechanism of mutation induction of PDMT in N. crassa is different from that of PMMT. Hydrolytic breakdown products or its intact molecule or some other converted forms might be responsible for the mutagenic activity of PDMT.Mutation induction of PMMT in N. crassa appears to be via alkylation of DNA by carbonium ions produced by this compound, the same mechanism proposed for its carcinogenic activity. The frequencies of leakiness, allelic complementation and nonpolarized complementation patterns among PMMT-induced ad-3 mutants are similar to those of ad-3 mutants induced by other potent chemical carcinogens, such as MNNG and the aflatoxins.     

Comparison of the photodynamic action of Rose Bengal and tetrachlorosalicylanilide on isolated porcine erythrocyte membranes

The light-mediated effects of Rose Bengal and 3,3',4',5-tetrachlorosalicylanilide (T4CS) on porcine erythrocyte membranes have been investigated. Irradiation in the presence of Rose Bengal and oxygen causes extensive destruction of the unsaturated fatty acids of the erythrocyte membrane. This results in a decrease in the membrane flexibility as measured by a nitroxide spin probe. Irradiation in the presence of T4CS and oxygen had no measurable effect on the unsaturated fatty acids or on the membrane flexibility. Irradiation of erythrocyte membranes both in the presence of Rose Bengal and oxygen and of T4CS gave rise to polymerisation of the membrane proteins. This was evident by polyacrylamide gel electrophoresis and by freeze-fracture electron microscopy. Aggregation of membrane proteins could be observed with low levels of Rose Bengal and short irradiation times at which no loss of unsaturated fatty acids could be detected. Irradiation of the n-butanol-extracted apoprotein of porcine erythrocyte membranes both in the presence of Rose Bengal and of T4CS resulted in polymerisation of the protein as measured by gel electrophoresis and electron microscopy. The results obtained from the two photodynamic compounds are compared in terms of their different mechanisms of action on the membrane. The implications of the results in determining the molecular events leading to photohaemolysis are discussed.     

1,1-Dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS) does not inhibit the in vitro activities of 1-aminocyclopropane-1-carboxylate (ACC) oxidase and ACC synthase obtained from senescing carnation Dianthus caryophyllus L.) petals

The effects of 1,1-dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS) on the in vitro activities of 1-aminocyclopropane-1-carboxylate (ACC) oxidase and ACC synthase isolated from senescing carnation petals were investigated. In contrast to a previous proposal, DPSS at 1 mM did not inhibit the in vitro activity of ACC oxidase. It was confirmed that DPSS does not inhibit ACC synthase activity. DPSS probably does not exert its inhibitory action on ethylene production by a direct action on ACC oxidase and ACC synthase, but by some unknown action.     

Neolignans from Virola elongata

The bark of Virola elongata contains the new 8.8′-neolignan, 1-(3,4-dimethoxyphenyl)2,3-dimethyl-4-piperonylbutan-1-one besides the known 8.8′, 2.7′-neolignans, 4-hydroxy-2,3-dimethyl-6,7-dimethoxy-4-piperonyl-1-tetralone and 4-hydroxy-2,3-dimethyl-5,6-methylenedioxy-4-piperonyl-1-tetralone.     

New Amides from Buckwheat Seeds (Fagopyrum esculentum Moench)

Two new amino acid amides which yield in acid hydrolysis isomeric hydroxybenzylamines and amino acids have been isolated from the achenes of Fagopyrum esculentum Moench. One of them called BN-II is composed of salicylamine and allo-4-hydroxy-l-glutamic acid, and the other, BN-III, p-hydroxybenzylamine and l-glutamic acid. These coupled compounds link one another to form an amide respectively. Finally the structures of BN-II and BN-III were determined to be N⁵-(2′-hydroxybenzyl)-allo-4-hydroxy-l-glutamine and N⁵-(4′-hydroxybenzyl)-l-glutamine respectively from their chemical and spectrometry properties.     

A carbene-generating photoaffinity probe for beta-adrenergic receptors

A new radioiodinated (2.2 Ci/μmol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [¹²⁵I]ICYP-diazirine binds with high affinity (K_d = 60 pM) to β-receptors from turkey erythrocyte membranes. Upon irradiation, [¹²⁵I]ICYP-diazirine is covalently incorporated in a M_r 40 000 protein. Stereoselective inhibition of photolabeling by the (?)enantiomers of alprenolol and isoproterenol indicated that the M_r 40 000 protein contains a β-adrenergic binding site. The yield of specific labeling was up to 8.2% of total β-receptor binding sites. The M_r 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [¹²⁵I]ICYP-diazirine resulted in specific labeling of two proteins with M_r 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximate M_r 67 000) was labeled specifically.     

Participation of peroxisomal beta-oxidation system in the chain-shortening of a xenobiotic acyl compound

A drug, (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoic acid, was metabolized to 4-(1-imidazolylmethyl)benzoic acid in isolated hepatocytes of rats, which was enhanced markedly by the pretreatment of rats with clofibrate. With liver homogenates, the formation of the CoA-ester of this drug and its subsequent chain-shortening were demonstrated. In the series of these reactions, acyl-CoA synthetase, CoA, ATP and NAD were required, whereas cyanide did not inhibit the reaction. These results indicate that peroxisomes are capable of shortening the acyl side-chains of drugs by the beta-oxidation, giving an additional suggestion on the functions of peroxisomes.     

Protective effect of hydroxychavicol, a phenolic component of betel leaf, against the tobacco-specific carcinogens

The phenolic compound, hydroxychavicol (HC), present in betel leaf, was synthesized and tested for its antimutagenic effect against the mutagenicity of the 2 tobacco-specific N-nitrosamines (TSNA), N′-nitrosonornicotine (NNN) and 4-(nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), in 2 different test systems, viz. the Ames Salmonella/microsome assay and the micronucleus test using Swiss male mice. We are reporting the synthesis of HC of a high degree of purity. We observed that HC suppressed the mutagenic effects of NNN and NNK in both test systems used. These results indicate that HC may have a role to play in reducing the risk of oral cancer in betel quid with tobacco chewers.     

Identification of new differentiation inducing factors from Dictyostelium discoideum

Developing Dictyostelium discoideum amoebae form a stalked fruiting body in which individual cells differentiate into either stalk cells or spores. The major known inducer of stalk cell differentiation is the chlorinated polyketide DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one); however a mutant blocked in the terminal step of DIF-1 biosynthesis still produces one of the prestalk cell subtypes – the pstA cells – as well as some mature stalk cells. We therefore searched for additional stalk cell-inducing factors in the medium supporting development of this mutant. These factors were purified by solvent extraction and HPLC and identified by mass spectroscopy and NMR. The mutant lacked detectable DIF-2 and DIF-3 (the pentanone and deschloro homologues of DIF-1) but four major stalk cell-inducing activities were detected, of which three were identified. Two compounds were predicted intermediates in DIF-1 biosynthesis: the desmethyl, and desmethyl-monochloro analogues of DIF-1 (dM-DIF-1 and Cl-THPH, respectively), supporting the previously proposed pathway of DIF-1 biosynthesis. The third compound was a novel factor and was identified as 4-methyl-5-pentylbenzene-1,3-diol (MPBD) with the structure confirmed by chemical synthesis. To investigate the potential roles of these compounds as signal molecules, their effects on morphological stalk and spore differentiation were examined in cell culture. All three induced morphological stalk cell differentiation. We found that synthetic MPBD also stimulated spore cell differentiation. Now that these factors are known to be produced and released during development, their biological roles can be pursued further.     

Mutagenicity of 7,12-dimethyl[a]anthracene and some other aromatic mutagens in Drosophila melanogaster

The optimal conditions for mutagenesis studies with DMBA and some other aromatic carcinogens in Drosophila were investigated in detail. The results presented in this paper indicate the following.The mutagenic effectiveness of DMBA is dependent on the route of administration, injection being far more effective when compared with feeding.The choice of the solvent is a crucial experimental condition. DMBA, when dissolved in oil/DMF, is ineffective whereas a special fat emulsion of DMBA gives high mutation frequencies.There appears to be an extreme strain dependence in the mutagenicity of DMBA. Mutagenic effectiveness in strain Berlin-K was rather low, whereas Oregon-K and Karsnäs-60 proved to be very susceptible to DMBA.Under the conditions of test, DMBA did not induce loss of a ring-X chromosome and did not produce recessive lethal mutations in such a chromosome.DMBA did not produce 2–3 translocations to any significant extent.An increase in DMBA-induced recessive lethal mutations was found upon storage of treated sperm with an optimal storage time of 4–10 days.DMBA is efficient in the production of delayed recessive lethal mutations in strain Berlin-K. Twice as many lethals were recovered with the F3 generation as compared with those in F2. In strain Oregon-K, where the frequency of F2 lethals was much higher than in strain Berlin-K, the ratio of F3/F2 lethals was clearly lower.Enzyme induction with phenobarbital reduces the mutagenic effectiveness of DMBAWith TMBA, similar strain differences in sensitivity were observed as those found for DMBA. Whereas TMBA was not mutagenic in Berlin-K, considerable mutagenicity was observed in Oregon-K and Karsnäs-60.Injection of carcinogenic polycyclic aromatic hydrocarbons and aromatic amines, when dissolved in special fat emulsions, enhances the mutagenic effectiveness of some compounds (DMBA, TMBA, DA and AcO-AAF), but this procedure does not always solve the problems-pertinent to these classes of promutagens in Drosophila.     

Antiprotozoal activity of bicyclic diamines with a N-methylpiperazinyl group at the bridgehead atom

ω-Aminoacyl and -alkyl derivatives of 4-(4-methylpiperazin-1-yl)bicyclo[2.2.2]octan-2-amines and of 5-(4-methylpiperazin-1-yl)-2-azabicyclo[3.2.2]nonanes were prepared and their activities were examined in vitro against the multiresistant K₁ strain of Plasmodium falciparum and against Trypanosoma brucei rhodesiense (STIB 900). Some of the newly synthesized compounds showed very promising antiprotozoal activity and selectivity. A few of the alkylamino-2-azabicyclo[3.2.2]nonanes exhibited high antiplasmodial activity, whereas a single bicyclo[2.2.2]octane derivative was the most potent antitrypanosomal compound. The results of the newly synthesized compounds were compared with the activities of already synthesized compounds and of drugs in use. Structure–activity relationships were discussed.     

Further phloroglucinol derivatives in the fruits of Mallotus japonicus

Two new rottlerin-like phloroglucinol derivatives were detected from the fruits of Mallotus japonicus and identified as 3-(3,3-dimethylallyl)-5-(3-acetyl-2,4-dihydroxy-5-methyl-6-methoxybenzyl)-phlorobutyrophenone and -phloroisobutyrophenone by spectral studies. 2,6-Dihydroxy-3-methyl-4-methoxyacetophenone was also isolated     

High yield purification of soluble guanylate cyclase from bovine lung

Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), is a cytosolic, heme-containing, heterodimeric enzyme that catalyzes the conversion of guanosine 5′-triphosphate (GTP) to 3,5′-cyclic guanosine monophosphate (cGMP) and pyrophosphate (PPi) in the presence of Mg²⁺. Cyclic GMP is then involved in transmitting the NO activating signals to a variety of downstream effectors such as cyclic-nucleotide-gated channels, protein kinases, and phosphodiesterases. In this work, sGC has been purified from bovine lung. The lungs were subjected to grinding and extraction with buffer at physiological pH followed by centrifugation. The resulting solution was subjected to successive column chromatography on DEAE- and Q-Sepharose, Ceramic Hydroxyapatite, Resource Q, and GTP–agarose. The purified enzyme migrated as a two-band protein on SDS–PAGE corresponding to sGC subunits α (M_r = 77,532) and β (M_r = 70,500) and had an A_280nm/A_430nm of 1 indicating one heme per heterodimer. The yield of enzyme was 8–10 mg from 4 to 5 kg bovine lungs. V_max and K_m of non-stimulated sGC were 22 nmol/mg/min and 180 μM, respectively. Upon stimulation with the NO donor 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene, the V_max increased to 1330 nmol/mg/min while the K_m dropped to 43 μM. The quality and quantity of enzyme make it suitable for studies to probe the structure and catalytic mechanism of this enzyme and for research related to drug discovery.     

The effect of ionic strength on a Mg2+-ATPase and its relevance to the determination of (Na++K+)-ATPase

A ouabain-insensitive Mg²⁺-ATPase present in a microsomal fraction prepared from the dog submandibular gland was studied. This Mg²⁺-ATPase was inhibited by increasing concentrations of NaCl, KCl, RbCl and CsCl. The addition of an osmotically equal amount of sucrose was without effect. This inhibition was obtained over a pH range of from 6.3 to 8.8. The Mg²⁺-ATPase present in microsomes treated with NaI showed a similar inhibition. These results indicate that it is advisable to keep the ionic strength constant in solutions used to obtain (Na⁺+K⁺)-ATPase activities.