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PubMed1989

Inhibition of the hepatic O6-alkylguanine-DNA alkyltransferase in vivo by pretreatment with antineoplastic agents.

The mammalian DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AT) is inactivated during the repair process and its activity can only be restored by de novo synthesis. We have made use of this property to determine whether and to what extent various chemotherapeutic agents alkylate DNA in the O6-position of guanine, ie. produce lesions susceptible to AT repair. Adult female Fischer rats received a single i.p. injection of a high dose (LD50) of the respective agent and, 5 hr later, a chasing dose of N-nitroso-[14C]dimethylamine (0.2 mg/kg; 4 hr survival). The amount of 7-[14C]methylguanine formed was approximately 95 mumol/mol guanine and not significantly altered by pretreatment with any of the drugs. The ratio of O6-[14C]methylguanine/7-[14C]methylguanine was 0.019 for control animals, indicating that during the observation period of 4 hr, 83% of the O6-[14C]methylguanine produced had been removed by the hepatic AT. Little or no effect was found in rats that received spirohydantoin mustard, hexamethylmelamine, cis-platinum or mitomycin C. A significant increase in the O6-/7-[14C]methylguanine ratio was found after pretreatment with AZQ (0.026) and cyclophosphamide (0.028), agents for which lesions involving the O6-position of guanine have not yet been identified. N-(2-Hydroxyethyl)-N-nitrosourea and the cytostatic haloethylinitrosoureas, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea (PCNU), and N-chloroethyl-N-hydroxyethylnitrosourea (HECNU) inhibited the hepatic AT, producing a ratio of 0.025-0.035. Considerably higher ratios of 0.059 and 0.101 were observed after administration of the methylating agents procarbazine and 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC), respectively. Complete saturation of the repair system (O6-/7-[14C]methylguanine ratio, 0.11) was only achieved with N-methyl-N-nitrosourea.

Animals↗
PubMed1993

Inhibition of melanogenesis in human melanoma cells by novel analogues of the partial histamine (H2) agonist nordimaprit.

A series of analogues of the weak histamine (H2) agonist S-[2-(N,N-dimethylamine)ethyl] isothiourea (nordimaprit) was produced to investigate the possibility that bulky substituents on the tertiary amine of nordimaprit would enhance potency for depigmentation and killing of melanoma cells. Cell survival studies showed that neither an increase in lipophilicity nor an increase in size of these groups produced selective toxicity, with only the N,N-diisobutyl derivative being more effective than the N,N-diisopropyl derivative in killing the constitutively pigmented human melanoma cell line MM418. The most hydrophobic analogue, the N,N-dibenzyl derivative, was also the most toxic to all cell lines tested. The association of toxicity with lipophilicity was confirmed by the piperidine derivative having greater toxicity than the less hydrophobic morpholine analogue. The ability to decrease tyrosinase activity was lost when lipophilicity and size of the N-terminal groups were increased, but these analogues produced marked depigmentation, even greater than that found with either nordimaprit or the diisopropyl derivative. Surprisingly, an increase in tyrosinase activity was achieved, the most potent agent being the N-ethyl-N-anilino analogue which caused complete depigmentation (0.6% of control) and elevated tyrosinase activity (148%) in MM418 cells after 1 month of treatment. This indicates that nordimaprit and its derivatives possess two different mechanisms of depigmentation, the first being tyrosinase dependent and the second being tyrosinase independent. The latter pathway is yet to be elucidated but appears to require a high degree of hydrophobicity.

Cell Line↗
PubMed1993

Trimethylamine N-oxygenation and N-demethylation in rat liver microsomes.

The in vitro oxidation of trimethylamine (TMA) to TMA N-oxide (TMAO) and dimethylamine (DMA) was studied in rat liver microsomes. Pretreatment of rats with phenobarbital, 3-methylcholanthrene, ethanol or pregnenolone 16 alpha-carbonitrile had little or no effect on the liver microsomal metabolism of TMA to TMAO or DMA. Changing the atmosphere in the incubation vessel from 20% oxygen/80% nitrogen (air) to 100% oxygen had a selective stimulatory effect on the N-oxygenation of TMA but did not affect TMA N-demethylation. In addition, the Km for TMA N-demethylation was 5-fold higher than for the N-oxygenation reaction. The results of these studies suggest that the enzyme systems responsible for N-demethylation and N-oxygenation are different and that they are under different regulatory control. Carbon monoxide (CO/O2 = 80/20) had little or no inhibitory effect on either the N-demethylation or N-oxygenation of TMA by liver microsomes from control or pregnenolone 16 alpha-carbonitrile-treated rats. Additional studies indicated that methimazole, an inhibitor of FAD-containing monooxygenase (FMO), was a potent inhibitor of TMA oxidation. Preincubation of liver microsomes from control or pregnenolone 16 alpha-carbonitrile-treated rats at 37 degrees for 10 min without NADP(H) (a procedure that irreversibly inactivated FMO activity) resulted in > 95% inhibition of TMA N-demethylation and N-oxygenation, and this inhibition was prevented by including a NADPH-generating system in the preincubation medium (a procedure for preventing the thermal inactivation of FMO activity). The data suggest that FMOs are the major enzymes responsible for N-demethylation and N-oxygenation of TMA in rat liver microsomes.

Animals↗
PubMed1993

Induction of brain cytochrome P-450IIE1 by chronic ethanol treatment.

Cytochrome P-450 mediated metabolism is potentially involved in the expression of the pharmacological and/or toxicological effects of a wide variety of drugs and environmental chemicals upon tissues which contain this metabolic system. In the present investigation, the presence of cytochrome P-450IIE1 and associated mono-oxygenase activities in brain and the effect of chronic ethanol treatment on brain cytochrome P-450 (P-450) were studied. Aniline hydroxylase, N-nitroso-dimethylamine N-demethylase and p-nitrophenol hydroxylase activities (known to be mediated by P-450IIE1) were detectable in brain microsomes from untreated rats and were about 5%, 125% and 8.3%, respectively, of the corresponding hepatic levels. Chronic ethanol treatment resulted in induction of the above enzyme activities in brain microsomes by 243%, 496% and 155%, respectively. Intake of ethanol for a prolonged period also resulted in the induction of total P-450 in the brain (150% of the control). Addition of the antisera raised against rat liver cytochrome P-450IIE1 markedly inhibited brain microsomal p-nitrophenol hydroxylase activity. Immunoblot analysis of rat brain microsomes using the above antisera also revealed the induction of brain cytochrome P-450IIE1 following chronic ethanol administration. Immunocytochemical localization of cytochrome P-450IIE1 using the above antisera, revealed the preferential localization of the enzyme in the neuronal cell bodies in the cortex, hippocampus, basal ganglia, hypothalamic nuclei and reticular nuclei in the brainstem of rats treated chronically with ethanol. Based upon these studies, it is conceivable that chronic alcohol ingestion could enhance the sensitivity of certain regions of the brain to environmental chemicals that are metabolized to more toxic derivatives by the P-450 system.

Animals↗
PubMed1988

Synthesis of p-nitrophenyl 6(5)-O-benzyl-alpha-maltopentaoside, a substrate for alpha amylases.

p-Nitrophenyl alpha-maltopentaoside, having a benzyl group on O-6 of the terminal (nonreducing) D-glucosyl group was prepared by use of a reductive ring-opening reaction. Highly regioselective reduction of p-nitrophenyl O-(2,3-di-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl)-(1----4)- tris[O-(2,3,6-tri-O-benzoyl-alpha-D-glucopyranosyl)-(1----4)]-2,3,6-tri- O- benzoyl-alpha-D-glucopyranoside by dimethylamine-borane and p-toluenesulfonic acid, followed by debenzoylation, gave p-nitrophenyl O-(6-O-benzyl-alpha-D-glucopyranosyl)-(1----4)-tris[O-alpha-D-glucopyran osyl- (1----4)]-alpha-D-glucopyranoside. An experiment was done on the mode of action of human pancreatic and salivary alpha amylases on this derivative. The compound is suitable as a substrate for the assay of alpha amylase when used with glucoamylase and alpha-D-glucosidase as coupling enzymes.

Carbohydrate Conformation↗
PubMed1982

Formation of 6-dimethylcarbamyloxy-dGuo, 6-dimethylamino-dGuo and 4-dimethylamino-dThd following in vitro reaction of dimethylcarbamyl chloride with calf thymus DNA and 6-diethylcarbamyloxy-dGuo following in vitro reaction of diethylcarbamyl chloride with calf thymus DNA.

The rodent carcinogens dimethylcarbamyl chloride (DMCC) and diethylcarbamyl chloride (DECC) react with dGuo (pH 7.0-7.5, 37 degrees C, 4 h) to form the O6-acyl derivatives 6-dimethylcarbamyloxy-2'-deoxyguanosine (6-DMC-dGuo) and 6-diethylcarbamyloxy-2'-deoxyguanosine (6-DEC-dGuo), respectively. Reaction of DMCC with dThd under identical conditions yielded 4-dimethylamino-thymidine (4-DMA-dThd). Compounds 6-DMC-dGuo and 6-DEC-dGuo undergo a nucleophilic aromatic substitution reaction with dimethylamine (DMA) to form 6-dimethylamino-2'-deoxyguanosine (6-DMA-dGuo) via displacement of the C-6 dialkylcarbamyloxy moiety. The substitution reaction did not take place when diethylamine or NH3 were substituted for DMA. The structures of the new compounds 6-DMC-dGuo, 6-DEC-dGuo, 4-DMA-dThd and 6-DMA-dGuo were deduced from chemical analyses and syntheses, UV and nuclear magnetic resonance (NMR) spectra and electron impact, isobutane chemical ionization and source insertion isobutane chemical ionization mass spectra. It was postulated that 4-DMA-dThd was formed following reaction of the transient intermediate 4-DMC-dThd with DMA formed by hydrolysis of DMCC. Calf thymus DNA was reacted in vitro with DMCC (pH 7.0-7.5, 37 degrees C, 4 h) and the modified DNA hydrolyzed enzymatically to 2'-deoxynucleosides. Compounds 6-DMC-dGuo, 4-DMA-dThd and 6-DMA-dGuo were identified in the hydrolysate by high-pressure liquid chromatography (HPLC). In an identical manner 6-DEC-dGuo was identified following in vitro reaction of DECC with calf thymus DNA. Compounds 6-DEC-dGuo and 6-DMC-dGuo possess novel structures with respect to the types of adducts known to be formed between carcinogens and bases in DNA. The implications of these findings with respect to chemical mutagenesis and carcinogenesis is discussed. The structural relationship between N4-dimethyl-5-methylcytosine (4-dimethylamino-Thy) formed in DNA following in vitro reaction with DMCC and 5-methylcytosine, the only modified base found in vertebrate DNA is noted.

Animals↗
PubMed1991

Inhibitors of sterol synthesis. Synthesis and spectral properties of 3 beta-hydroxy-24-dimethylamino-5 alpha-chol-8(14)-en-15-one and its effects on HMG-CoA reductase activity in CHO-K1 cells.

A simple, three-step synthesis of the 25-aza analog of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I) is described. Treatment of 3 beta-acetoxy-24-hydroxy-5 alpha-chol-8(14)-en-15-one (III) with 1.75 equivalents of tosyl chloride in pyridine for 24 h at 5 degrees C gave 3 beta-acetoxy-24-tosyloxy-5 alpha-chol-8(14)-en-15-one (IV). In contrast, treatment of III with 3.95 equivalents of tosyl chloride in pyridine for 12 h at 48 degrees C gave 3 beta-acetoxy-24-chloro-5 alpha-chol-8(14)-en-15-one (V). Treatment of IV with dimethylamine in dioxane yielded 3 beta-acetoxy-24-dimethylamino-5 alpha-chol-8(14)-en-15-one (VI). Hydrolysis of VI with LiOH.H2O in methanol gave 3 beta-hydroxy-24-dimethylamino-5 alpha-chol-8(14)-en-15-one (VII). 1H- and 13C-NMR assignments are presented for compounds IV-VII. The 25-aza analog (VII) of the 15-ketosterol (I), at a concentration of 1.0 microM, caused a 47% lowering of the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in Chinese hamster ovary cells.

Animals↗
PubMed1988

Indolealkylamine analogs share 5-HT2 binding characteristics with phenylalkylamine hallucinogens.

Twenty-one indolealkylamines, some of which are known to be psychoactive in man, were examined for their binding interactions with rat brain cortical 5-HT2 receptors labeled with the antagonist radioligand [3H]ketanserin in order to develop structure-activity relationships for binding at these sites. Features investigated included aromatic, alpha-methyl and terminal amine substituents. 4-Methoxy and 5-methoxy substitution impart a higher affinity than 6- or 7-methoxy substitution; a 7-hydroxyl group essentially abolishes affinity whereas a 7-methyl or 7-bromo group enhances affinity. alpha-Methylation has little effect on affinity and, in the one case examined, the S(+) isomer of alpha-methyltryptamine was essentially equipotent with its racemate and twice as potent as its R(-) enantiomer. Terminal amine methylation results in a small but progressive decrease in affinity in the order: primary amine greater than dimethylamine greater than diethylamine. Similarities were noted between these structural requirements for binding and those of the phenalkylamines. Selected compounds (5-methoxytryptamine, N,N-dimethyltryptamine, 5-methoxy-N,N-diethyltryptamine and 5-methoxy-N,N-dimethyltryptamine) were further examined by two-site analysis of displacement studies for [3H]ketanserin specific binding. Hill coefficients were significantly less than unity and computer-assisted analysis indicated that a two-site model better fit the data than a one-site model. In displacement studies using the putative agonist radioligand [3H]DOB to label 5-HT2 receptors affinities were 10-100-fold higher than those using [3H]ketanserin. These results are also consistent with earlier findings using psychoactive phenalkylamines in competition studies for radiolabelled 5-HT2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

DOM 2,5-Dimethoxy-4-Methylamphetamine↗
PubMed1983

Schistosoma mansoni: species-selective response to N-chloroethyl-acetylcholine derivatives.

Three new acetylcholine mustard analogs were tested on schistosome and vertebrate neuromuscular preparations. These compounds extend a previously reported structure-activity series. The new compounds investigated include isopropyl-, cyclohexyl-, and benzyl-2-acetoxyethyl-2'-chloroethylamine (PrM, ChM, and BzM). In schistosome motor activity studies, all of the compounds caused irreversible reductions in the activity of Schistosoma mansoni after 1 hr exposure followed by 19 hr in drug-free medium. Under nonlethal conditions of dosage and exposure time, all compounds blocked carbachol-induced paralysis, indicating a possible action at schistosome cholinergic sites. All the compounds also reduced the labeling of schistosomes by dimethylaminonapthalene-5-sulfonamidoethyl dimethylamine hydrochloride (DDNS), a fluorescent ACh analog. In isolated vertebrate tissue experiments. PrM and ChM had slight agonist activity in the guinea pig ileum, and only PrM had agonist activity in the frog rectus abdominis preparation. PrM and BzM were found to antagonize ACh responses in the guinea pig ileum. Exposure of vertebrate tissues for 1 hr to high concentrations of ChM caused no long-lasting inhibition of ACh, pilocarpine, and serotonin (5HT) responses. Histamine responses were slightly reduced from control. Following 1 hr exposure of vertebrate tissues to PrM, initial reductions in all responses were seen, followed by recoveries to near control. BzM treatment, however, reduced all responses far below control; the tissues did not recover even after washing for 2 hr. It is concluded that ChM and, to a lesser extent, PrM, had a greater effect on schistosomes than on vertebrate tissues. BzM did not display species selectivity in this favorable direction.

Acetylcholine↗
PubMed1993

Amino acid sequence surrounding the retinal-binding site in retinochrome of the squid, Todarodes pacificus.

Squid (Todarodes pacificus) retinochrome was reduced to N-retinyl protein with borane dimethylamine and cleaved by CNBr. The retinyl peptide was then isolated by chromatography while being monitored for absorbances at 215 and 330 nm, and the N-terminal amino acid sequence was determined to be Ser-Lys-Thr-Gly-X-Ala-Leu-Phe-Pro. This sequence was the same that we had observed at the 7th transmembrane domain of retinochrome whose structure was reported previously. During Edman degradation of the retinyl peptide, the yield of the PTH-lysine at the second cycle was lower than those of the other PTH-amino acids, proving that the lysine residue forms a Schiff's base with retinal (Lys-275 in retinochrome). The amino acid sequence surrounding the retinal-binding lysine in retinochrome greatly differed from those in a variety of known visual pigments. This fact would be associated with the difference in the photoisomerization of chromophore between retinochrome and rhodopsin. The protein structure of retinochrome is also compared with that of rhodopsin in Todarodes.

Amino Acid Sequence↗
PubMed1976

Comparative mutagenicity of N-nitrosamines in a semi-solid and in a liquid incubation system in the presence of rat or human tissue fractions.

The rat liver microsome-mediated mutagenicities of a series of N-nitrosodialkylamines and heterocyclic N-nitrosamines were determined in a liquid incubation system using Salmonella typhimurium TA1530. The influence on mutation frequency of the concentration of co-factors for mixed-function oxidase and composition and molarity of the buffer was investigated, using N-nitrosomorpholine as substrate. The mutagenicity of the N-nitroso compounds in the liquid incubation system under optimal reaction conditions at equimolar concentration was compared quantitatively with that obtained in a soft-agar incorporation assay. N-Nitrosodi-n-pentylamine and N-nitrosodi-n-butylamine showed no enzyme-mediated mutagenicity in the liquid incubation system, and metabolically activated N-nitroso-dimethylamine and N-nitroso-diethylamine showed negligible mutagenic activity in the soft-agar assays. In contrast with these results with the N-nitrosodialkylamines, the mutagenic effects of heterocyclic N-nitrosamines were similar in the liquid incubation system and in soft-agar incorporation assays. The heterocyclic N-nitrosamines showed rat-liver microsome-mediated mutagenicity in the following descending order: N-nitrosomorpholine greater than N-nitrosopyrrolidine greater than N-nitrosopiperidine greater than N-nitroso-N'-methylpiperazine. Seven human liver specimens converted all heterocyclic N-nitrosamines into mutagens; this activity was similar to that of rat liver, except that for N-nitroso-N'-methylpiperazine, fractions from three human liver biopsies were three to 30 times more active than those from untreated rats. The specific reversion of S. typhimurium TA1530 to histidine prototrophy provides experimental evidence that all the N-nitrosamines studied were converted by liver microsomal enzymes into monofunctional alkylating agents.

Animals↗
PubMed1976

Inhibition of mouse testicular DNA synthesis by mutagens and carcinogens as a potential sample mammalian assay for mutagenesis.

Since mutagenic substances induce structural changes in DNA, studies were undertaken to determine whether substances would modify DNA replicative activity. DNA synthesis was quantitated 3.5 h after drug administration as uptake into DNA of a 30-min pulse of 10 muCi of [3H] thymidine. Combinations of methylurea (20000 mg/kg) and sodium nitrite (150 mg/kg) administered p.o. resulted in gastric synthesis of nitrosomethylurea and inhibited testicular DNA synthesis by 83%. Combinations of methylurea and sodium nitrite of 1000 and 100 mg/kg respectively inhibited DNA synthesis by 75%. With dimethylamine and sodium nitrite, a combination which results in gastric synthesis of dimethylnitrosamine, inhibitions of 65 and 57% were observed at 2000 mg/kg together with 150 mg/kg and 1000 mg/kg in combination with 100 mg/kg, respectively. In separate experiments, dimethylnitrosamine (50 mg/kg, p.o.) and diethylnitrosamine (100 mg/kg, i.p.) inhibited thymidine uptake by 30 and 89%, respectively. The mutagenic polynucelar hydrocarbon 3-methylcholanthrene (15 mg/kg) inhibited DNA synthesis by 95% and safrole (640 mg/kg), a mutagenic methylenedioxybenzene derivative, inhibited by 60%. Cadmium chloride (10 mg/kg), acetylaminofluorene (160 mg/kg) and dibutylnitrosamine (500 mg/kg) also induced statistically significant effects. Noncarcinogenic analogues of these substances (anthracene, 125 mg/kg; diphenyl-nitrosamine, 500 mg/kg; piperonyl butoxide, 640 mg/kg, and methylurea, 2000 mg/kg in combination with sodium nitrate) were inactive. Highly toxic substances (potassium cyanide, 2.5 mg/kg; 2,4-dinitrophenol, 20 mg/kg; and lead acetate, 150 mg/kg) were also inactive. This index of mutagenicity appears to have considerable sensitivity and therefore may have potential in drug evaluation.

Animals↗
PubMed1978

Screening of safrole, eugenol, their ninhydrin positive metabolites and selected secondary amines for potential mutagenicity.

The mutagenicity of safrole, eugenol, the secondary amines, with which they combine during metabolism, and the ninhydrin positive urinary metabolites of safrole and eugenol was tested. The panel of tests included the direct bacterial assay, a microsomal mutagenesis assay and a host-mediated assay. With the direct bacterial assay employing four mutant strains of Salmonella typhimurium (TA1530, TA1531, TA1532, TA1964), all the compounds gave negative results. In the microsomal mutagenesis assay, employing the same four mutant strains, safrole and safrole metabolite II were mutagenic with strains TA1530 and TA1532. Dimethylamine was also found to be a weak mutagen in the microsomal mutagenesis assay with strain TA1530. Safrole and safrole metabolite II were also mutagenic in the host-mediated assay with strains TA1950 and TA1952. Negative results were observed for safrole metabolites I and III, eugenol, eugenol metabolites I and II, piperidine, pipecolic acid, proline, and pyrrolidine in all three assay systems.

Animals↗
PubMed1987

Effects of UV repair, error-prone repair and critical site of mutation on mutagenesis induced by N-nitrosamines.

Many N-nitrosamines have been assayed for mutagenic activity in bacteria but few have been systematically compared in a series of strains. In this study through the use of several Salmonella tester strains, we have examined the effects of Uvr repair, error-prone repair, and the critical site for mutation (GC or AT base pair) on the mutagenic activities of a diverse group of N-nitrosamines. We have employed the histidine autotrophs, TA1975 (uvrB+), TA1535 (uvrB-) and TA100 (uvrB-/pKM101) which are hisG46 strains, sensitive mainly to G-C base damage, and TA104 (uvrB-/pKM101), a hisG428 strain, which can be reverted at the hisG428 locus by damage to A-T base-pairs, or by suppression at G-C base pairs. The N-nitrosamines studied were, N-nitroso: dimethylamine, diethylamine, dipropylamine, dibutylamine, pyrrolidine, piperidine, morpholine, methylbenzylamine, bis-(2-hydroxypropyl)amine, bis-(2-oxopropyl)amine and 3,4-dichloropyrrolidine. For all of the nitrosamines larger than diethylnitrosamine (except for methylbenzylnitrosamine) mutagenesis was greatly enhanced (3-20 X) by the lack of uvrB activity, indicating that the DNA adducts produced by these nitrosamines can be classified as "bulky adducts". For most nitrosamines the plasmid, pKM101, enhanced mutagenesis in hisG46 strains, several fold, suggesting that error-prone DNA repair plays a role in mutagenesis by these compounds. All of the compounds tested were more mutagenic in TA100 than TA104 except diethylnitrosamine and methylbenzylnitrosamine which were more potent in TA104. Revertants induced by all of the nitrosamines in TA100 were due predominantly to damage at G-C base pairs. Revertants induced by all the nitrosamines except diethylnitrosamine and dibutylnitrosamine resulted mainly from damage to G-C base pairs in TA104.

Base Sequence↗
PubMed1988

Modulating effects of beta-naphthoflavone on the induction of SCEs by model compounds with emphasis on benzo[a]pyrene.

The inducing capability of the synthetic flavonol beta-naphthoflavone (beta-NF) on cytochrome P-450 content was studied in primary chick embryo hepatocytes. In addition, the modulating effects of pretreatment with beta-NF on the induction of sister-chromatid exchanges (SCEs) in V79 cells by mutagens from different chemical classes were investigated in a co-cultivation system consisting of primary chick embryo hepatocytes and V79 Chinese hamster cells. Finally, the effects of pretreatment on benzo[a]pyrene (B(a)P) metabolism were studied in more detail. Pretreatment of cultured primary chick embryo hepatocytes with beta-NF resulted in a large increase in cytochrome P-450 content (a 2.8-fold increase after 31 h). Pretreatment with beta-NF had no effect on the level of SCEs induced by N-nitroso-dimethylamine (NDMA) and 2-aminoanthracene (2AA). Pretreatment with beta-NF resulted in a decrease in B(a)P-induced SCEs. This inhibitory potential was positively related to the beta-NF dose. However, there was an inverse relationship between the inhibitory action of beta-NF and the dose of B(a)P, at higher doses less inhibition was observed. When beta-NF was applied simultaneously with B(a)P the percentage of decrease was about the same as for pretreatment. Pretreatment with beta-NF followed by simultaneous application of beta-NF and B(a)P did not result in larger effects. In addition, subcellular fractions were prepared from chick embryos pretreated with beta-NF in ovo. The use of the S9 fraction resulted in a large decrease (80%) in the induction of SCEs in V79 cells by B(a)P whereas the use of the microsomal fraction resulted in a 70% increase in SCE induction compared with non-pretreated microsomes. Pretreatment with beta-NF in ovo gave rise to a large increase in aryl hydrocarbon hydroxylase (AHH) activity in the hepatic microsomal fraction. Increases were observed in the formation of all B(a)P metabolites. In particular the formation of the proximate carcinogenic and mutagenic metabolite B(a)P-7,8 dihydrodiol was increased 7-fold. The data strongly suggest that the inhibitory effects of pretreatment of cultured primary chick embryo hepatocytes with beta-NF cannot be ascribed to its inducing capabilities but instead seem to be due to the formation of an intracellular pool of beta-NF which acts as a competitive inhibitor for B(a)P metabolism.

Animals↗
PubMed1989

Importance of multiple hydroxylated metabolites in hexamethylphosphoramide (HMPA)-mediated mutagenesis in Drosophila melanogaster.

The mutagenic profiles in Drosophila and the influence of inhibition of metabolism on genotoxic activity were determined for hexamethylphosphoric triamide (HMPA), some synthetically prepared presumed metabolites and ethylated analogs. Demethylated HMPA metabolites are considerably less mutagenic than HMPA, dependent on the degree of demethylation. The mutagenicity of the presumptive primary metabolite, hydroxymethyl pentamethylphosphoramide (HM-Me5-PA), is comparable to HMPA and can be decreased considerably by inhibition of the metabolism by 1-phenylimidazole or iproniazid. This suggests that further oxidative metabolism is required for mutagenic activity. The mutagenicity of the doubly hydroxylated HMPA metabolite, N,N'-bis(hydroxymethyl)-tetramethylphosphoramide (N,N'-(HM)2-Me4-PA) can also be decreased by inhibition of metabolism, whereas the 3-fold hydroxylated N,N',-N"-(HM)3-Me3-PA is not affected by pretreatment with enzyme inhibitors, indicating that no further oxidative metabolism is required for its activation. A second hydroxylation on 1 dimethylamino group, forming N,N-(HM)2-Me4-PA, results in a drastic loss of mutagenic activity. Further oxidation of HM-Me5-PA to formyl pentamethylphosphoramide (formyl-Me5-PA) also leads to a strong reduction of the genotoxic activity. The rearrangement product of N-oxidation, N-[bis(dimethylamino)phosphinyl)-oxy)dimethylamine (HMPOA) is not mutagenic in Drosophila. The very low mutagenicity of hexaethylphosphoramide (Et6-PA) allowed us to study the mutagenicity of some ethyl-hydroxymethyl hybrid compounds. For the ethylated phosphoramides also the presence of only 1 hydroxymethyl group is insufficient for mutagenic activity, whereas the introduction of 2 or 3 hydroxymethyl groups resulted in considerable genotoxicity in the sex-linked recessive lethal (SLRL) test as well as in the ring-X loss test. It is concluded that the bioactivation of HMPA in Drosophila proceeds via multiple metabolic hydroxylations to form multifunctional, cross-linking agents. The presence of an oxygen atom on the phosphorus appears to be a prerequisite for the genotoxic activity of HMPA as hexamethylphosphorus triamide (HMPT), a derivative lacking this oxygen, is only weakly mutagenic in Drosophila. The results presented in this paper do not support the theory that formaldehyde is the active principle of activated HMPA.

Animals↗
PubMed1994

Alkyltransferase transgenic mice: probes of chemical carcinogenesis.

Transgenic mice expressing DNA-repair genes are an instructive model with which to study the protective role of DNA-repair pathways in both spontaneous and chemical carcinogenesis. Of particular interest in chemical carcinogenesis is the DNA-repair protein O6-alkylguanine-DNA alkyltransferase (alkyltransferase) which repairs O6-alkylguanine-DNA adducts. Transgenic mice carrying expression constructs for the alkyltransferase gene--either the human MGMT cDNA or the bacterial ada gene--express increased levels of alkyltransferase and have increased capacity to remove O6-methylguanine-DNA adducts. Protection from the DNA damaging effects of N-nitroso compounds occurs specifically in the cells and tissues in which the alkyltransferase transgene is expressed. For instance, mice carrying the PEPCKada construct have increased alkyltransferase in the liver and more rapid removal of O6methylguanine-DNA adducts. The protective effect is noted in hepatocytes, which express PEPCK-linked genes, not in nonparenchymal cells of the liver, which do not. Other tissues that express the transgene in the various models include the thymus, spleen, testes, muscle, stomach and brain. Mice expressing the human alkyltransferase in the thymus have a reduced incidence of thymic lymphomas following exposure to methyl nitrosourea (MNU), evidence of a role for this DNA-repair protein in protection from carcinogenesis due to N-nitroso compounds. Protection has also been observed in the induction of hepatic tumors by N-nitroso-dimethylamine (NDMA). These models will be used to identify whether overexpression of a single DNA-repair gene can block the carcinogenic process of N-nitroso compounds in many different tissues.

Actins↗
PubMed1984

Potentiation of the hepatotoxicity of N-nitrosodimethylamine by fasting, diabetes, acetone, and isopropanol.

Previous studies indicate that pretreatment with acetone or isopropanol, fasting, and streptozotocin-induced diabetes enhance hepatic microsomal nitroso-dimethylamine (NDMA) demethylase in rats. This study demonstrates that these same treatments also potentiate the hepatotoxicity of NDMA as indicated by plasma glutamic pyruvate transaminase (GPT) levels and histologic data. Pretreatment with acetone or isopropanol (2.5 ml/kg) and 2 days of fasting caused a 2-fold potentiation of NDMA-induced plasma GPT elevation, whereas streptozotocin-induced diabetes caused a 4.6-fold potentiation. The centrilobular necrosis produced by NDMA was more severe after pretreatment with the inducers. NDMA treatment also decreased hepatic microsomal demethylase activity. These results lend support to the concept that a NDMA demethylase is responsible for the activation of NDMA in vivo to a toxic intermediate, and induction of this enzyme activity potentiates NDMA hepatotoxicity.

1-Propanol↗