Initial fibroblast attachment to Erbium:YAG laser-irradiated dentine.

AIMS: To evaluate the effects of Erbium (Er):YAG laser irradiation on the morphology of resected dentine surfaces, and to investigate fibroblast attachment to laser-irradiated dentine surfaces. METHODOLOGY: Dentine blocks obtained from single-rooted human teeth were divided into the following groups after sterilization in an autoclave: (i) Laser group treated with Er:YAG laser irradiation (30 mJ per pulse, 10 pps, 60 s); (ii) L-MTAD group treated with laser irradiation as in (i) plus a mixture of doxycycline, tetracycline isomer and citric acid; (iii) RC-Prep group treated with EDTA gel or cream (RC-Prep) and (iv) Control group left untreated. After each treatment, the dentine blocks were incubated with NIH/3T3 fibroblasts cultured to subconfluency in Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum and antibiotics. The number of attached cells amongst the groups was analysed statistically at the 5% significance level. The dentine surface morphologies and cell attachments were evaluated by counting assays, histological observations and scanning electron microscopy (SEM). RESULTS: The number of attached cells was significantly higher (P < 0.05) in the Laser group than in the RC-Prep and Control groups at 16 h. Dendritic cell extension of the fibroblasts was only observed in the Laser group at 8 h by SEM. In the histological analyses, significantly more attached cells were found on the dentine surfaces treated with laser irradiation. CONCLUSIONS: Er:YAG laser irradiation induced morphological alterations in dentine surfaces, which may improve the attachment of fibroblasts to dentine.

Concurrent generation of methylamine and nitrite during denitrosation of N-nitrosodimethylamine by rat liver microsomes.

With the goal of identifying the organic amine product(s) of enzymatic N-nitrosodimethylamine (NDMA) denitrosation, 4 mM NDMA was incubated with liver microsomes from ethanol-treated rats. The concentrations of dimethylamine and methylamine were determined by derivatization with 2,4-dinitrofluorobenzene followed by gas chromatography-mass spectrometry. There was no net increase in the concentration of dimethylamine during incubation, but the yield of methylamine was equimolar with that of nitrite. Additional incubations of NDMA using acetone-induced microsomes, 1 mM and 0.1 mM substrate, gave methylamine/nitrite ratios of 0.9 and 0.7, respectively, confirming the quantitative linkage between these two products. Control incubations conducted with pure methylamine or dimethylamine indicated that the secondary amine is not a significant intermediate in the metabolic generation of the primary amine. Experiments with 15N-labeled NDMA showed that the methylamine nitrogen came from the amino moiety of the nitrosamine. The results suggest that NDMA metabolism is best viewed as a competition between at least two important pathways, demethylation and the presumably deactivating denitrosation route, a formulation which seems to account for the previously reported detection of methylamine as a urinary metabolite of NDMA and for the production of less than theoretical yields of labeled dinitrogen gas during NDMA metabolism.

Synthesis and mutagenicity of 4-(N-butylnitrosamino)-4-hydroxybutyric acid lactone, a possible activated metabolite of the proximate bladder carcinogen N-butyl-N-(3-carboxypropyl)nitrosamine.

4-(N-Butylnitrosamino)-4-hydroxybutyric acid lactone (BBAL) was synthesized as a possible intermediate produced by metabolic activation of a selective bladder carcinogen, N-butyl-N-(3-carboxypropyl)nitrosamine. BBAL was stable in neutral sodium phosphate buffer (ionic strength, 0.2), having a half-life of more than 30 hr at 25 degrees. The mutagenic effects of BBAL were tested with the use of Salmonella typhimurium TA1535 and Escherichia coli B/rWP2-try-, WP2-try-hcr-, and Sd4. The gene-damaging effects were assayed by repair tests with Bacillus subtilis H17 (rec+) and M45 (rec-). BBAL showed potent effects in the mutation and repair tests on all the strains tested without activation. A possibility is suggested for the metabolic activation of N-butyl-N-(3-carboxypropyl)nitrosamine to BBAL by alpha-hydroxylation at the site of the 3-carboxypropyl chain followed by lactonization in target tissues prior to interaction with macromolecules to lead to carcinogenesis.

Deuterium isotope effect on denitrosation and demethylation of N-nitrosodimethylamine by rat liver microsomes.

In an attempt to elucidate the molecular basis for the decrease in rat liver carcinogenicity and DNA-alkylating ability that accompanies deuteration of N-nitrosodimethylamine (NDMA), NDMA and its fully deuterated analogue ([2H6]NDMA) were incubated with acetone-induced rat liver microsomes. Rates for the competing metabolic routes, denitrosation and demethylation, were determined from colorimetric data on nitrite and formaldehyde generation, respectively. The Vmax calculated for demethylation of NDMA was 7.9 nmol/min/mg, while that for denitrosation was 0.83 nmol/min/mg. Deuteration of NDMA did not significantly change the Vmax for either pathway, but it did increase the Km for demethylation from 0.06 to 0.3 mM. The Km for denitrosation was also increased from 0.06 to 0.3 mM on deuteration, as determined by incubating an equimolar mixture of amino-15N-labeled NDMA with [2H6]NDMA and measuring the methyl[15N]amine:[2H3]methylamine ratio by derivatization-gas chromatography-mass spectrometry. The fact that the Km values for denitrosation were so similar to those for demethylation suggested that the two pathways were catalyzed by the same enzyme. The isotope effects calculated from these data [VmaxH/VmaxD approximately 1 and (Vmax/Km)H/(Vmax/Km)D approximately 5] show that microsomal metabolism of NDMA is not significantly shifted from demethylation to denitrosation on deuteration of substrate and may indicate a low commitment to catalysis for the enzyme. The results are consistent with the view that the metabolism of NDMA is initiated by formation of an alpha-nitrosamino radical which either combines with a hydroxyl radical to form the alpha-hydroxynitrosamine as the initial product of the demethylation pathway or fragments to nitric oxide and N-methylformaldimine as the first products of denitrosation.

Inactivity of fecapentaene-12 as a rodent carcinogen or tumor initiator.

The possible carcinogenic activity of synthetic fecapentaene-12 (FP-12) was studied in several mammalian test systems: (a) for carcinogenicity by intrarectal instillation in male F344/NCr rats as well as by intrarectal and subcutaneous application in male B6C3F1 mice; (b) for initiation by skin painting in female SENCAR mice followed by repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA), with 7,12-dimethylbenz[a]anthracene (DMBA) followed by TPA as positive control; (c) in a rat subcutaneous granuloma pouch assay in which mutagenicity was measured by induction of 6-thioguanine (6-TG) resistance and carcinogenicity was determined by induction of subcutaneous tumors in the pouch. There was no significant increase in tumor incidence after 72-78 weeks in test (a), although 2 rats receiving FP-12 intrarectally developed colon polyps. FP-12 did not initiate any skin tumors in test (b), nor did it significantly convert DMBA-initiated papillomas into carcinomas when 8 of the positive control mice were given FP-12 weekly for 10 weeks after 10 weeks on the DMBA-TPA regimen. Although FP-12 and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were comparably mutagenic in test (c), FP-12 induced no tumors after more than a year in 133 rats at risk while MNNG induced 7 tumors in 107 rats. These rodent assays provide no evidence that FP-12 is a strong carcinogen, although the possibility remains that it may possess weak carcinogenic activity not revealed by these experiments.

Mutagenicity of alpha-hydroxy N-nitrosamines in V79 Chinese hamster cells.

Carcinogenic and mutagenic N-nitrosodialkylamines are metabolically activated through alpha-hydroxylation. The synthesis, chemical properties, and microbial mutagenicity of alpha-hydroxy N-nitrosamines have been reported previously. Potent mutagenicity of four N-nitroso-N-(hydroxymethyl)-alkylamines (alkyl = methyl, ethyl, propyl, and butyl) was demonstrated in the present study in V79 Chinese hamster cells, ouabain resistance being used as an indicator. All the compounds were strong mutagens in the absence of metabolic activation systems. The mutagenic and cytotoxic potencies correlated well with each other, and depended on the alkyl group, decreasing in potency in the following order: methyl greater than ethyl greater than propyl = butyl. Their alkylating reactivity was measured by alkylation of thiophenol, and a good linear relationship was observed between the mutagenic and cytotoxic potencies and their alkylating reactivity. The mutagenic and cytotoxic potencies of the alpha-hydroxy N-nitrosamines in V79 cells were well correlated with those of alpha-acetoxy and alpha-hydroperoxy N-nitrosamines with respect to the effect of alkyl group. The results obtained here supported further that alpha-hydroxy N-nitrosamine is the active species in the metabolic activation of N-nitrosodialkylamine.

Carcinogenicity of N-alkyl-N-(acetoxymethyl)nitrosamines after subcutaneous injections in F-344 rats.

As model compounds for metabolically activated N,N-dialkylnitrosamines, five N-alkyl-N-(acetoxymethyl)nitrosamines were synthesized and their carcinogenicity was tested in F-344 rats of both sexes. Compounds used in this study are N-methyl-(MAMN), N-ethyl-(EAMN), N-propyl-(PAMN), N-butyl-(BAMN), and N-isobutyl-N-(acetoxymethyl)nitrosamines (i-BAMN). All chemicals were dissolved in olive oil and rats received 10 weekly subcutaneous injections of these chemicals (10 X 5 mg MAMN or equimolar amounts of other chemicals) at the interscapular region. Subcutaneous tumors were detected in many rats of all groups treated with the chemicals, although no tumor was detected in the control group. Lung and/or thyroid tumors were also observed in many rats in the experimental groups. The incidence of subcutaneous tumors was highest in EAMN, followed in order by MAMN, PAMN, BAMN, and i-BAMN. On the contrary, the incidence of lung and thyroid tumors was highest in MAMN and decreased as the length of the alkyl chain of the chemicals increased. Histologically, almost all subcutaneous tumors were malignant fibrous histiocytomas. The results indicate that the chemicals possess systemic as well as local carcinogenicity in F-344 rats. The potent carcinogenic effects at the injection site of the alpha-acetoxy nitrosamines, coupled with their direct mutagenic activity reported previously, support the notion that these derivatives are useful as models for the ultimate form in the metabolic activation of N,N-dialkylnitrosamines.

Enzymatic O-methylation of catechol estrogens in red blood cells: differences in animal species and strains.

Enzymatic O-methylation of catechol estrogens in red blood cells has been investigated with respect to species difference. In the presence of S-adenosylmethionine, 2- or 4-hydroxyestradiol (2-OHE2 or 4-OHE2) was incubated with blood lysate obtained from rats (five strains), guinea pigs, mice, rabbits, dogs, monkeys, and humans, respectively. The yielded guaiacols and unchanged substrate were determined by gas chromatography/mass spectrometry in a selected ion monitoring mode employing the corresponding 2H4-labeled compounds as internal standards. The total amounts of guaiacols formed from 2-OHE2 and 4-OHE2 were different, being the highest (79.6% and 38.1%) in monkeys and the lowest (5.1% and 1.9%) in humans. The ratios of isomeric guaiacols formed from 4-OHE2 (4Me/3Me) were 7.6-71, while those from 2-OHE2 (2Me/3Me) were 1.4-3.2. Thus, marked differences in O-methylation of catechol estrogens were observed among animal species, but no significant strain difference was detected in rats.

Mutagenicity of N-alkyl-N-(alpha-hydroperoxyalkyl) nitrosamines in V79 Chinese hamster cells in relation to alkylating activity.

Mutagenicity and cytotoxicity of a series of N-alkyl-N-(alpha-hydroperoxyalkyl)-nitrosamines (alkyl = methyl, ethyl, propyl, butyl and tert-butyl) were tested in V79 Chinese hamster cells to examine the effects of alkyl chain length and the mode of substitution at the alpha-carbon bearing the hydroperoxy group on the biological activities. Among a series of hydroperoxymethyl compounds whose carbon bearing the hydroperoxy group is primary, the N-methyl compound was the most mutagenic and cytotoxic, and the biological activities of the compounds decreased in the following order: methyl much greater than ethyl greater than propyl greater than or equal to butyl greater than tert-butyl, the last one being nonmutagenic at the concentration tested. In a series of alpha-hydroperoxyalkyl compounds whose alpha-carbon bearing the hydroperoxy group is secondary, the N-methyl compound was also found to be the most mutagenic and cytotoxic, and the biological activities of the compounds decreased in the following order: methyl greater than ethyl much greater than propyl greater than butyl. A comparison of the biological activities of the corresponding compounds having the hydroperoxy group at primary or secondary alpha-carbon showed that the latter compounds were far more active than the former. A plot of the alkylating activity of the compounds toward 4-(rho-nitrobenzyl) pyridine after deoxygenation with NaHSO3 versus their mutagenic potency in V79 cells was linear, indicating that the chemical reactivity of the compounds plays an important role in inducing mutation in V79 cells.

Mutagenicity of N-alkyl-N-(alpha-acetoxyalkyl) nitrosamines in V79 Chinese hamster cells in relation to alkylating activity.

Mutation to ouabain resistance and cytotoxicity were tested in V79 Chinese hamster cells after the cells had been treated for 2.5 hr with a series of N,N-dialkylnitrosamines (alkyl=methyl, ethyl, propyl, butyl or tert-butyl) monosubstituted at the alpha-carbon with an acetoxy group. The effects of the length of alkyl chain and the mode of substitution with the acetoxy group on the cytotoxicity and mutagenicity were examined. In the series of N-alkyl-N-(acetoxy-methyl) nitrosamines with an acetoxy group at the primary alpha-carbon, the methylating compound N-methyl-N-(acetoxymethyl) nitrosamine was the most cytotoxic and mutagenic, and the biological activities decreased in the order of ethyl, butyl and propyl homologs. N-tert-Butyl-N-(acetoxymethyl) nitrosamine was not mutagenic at the concentration tested. At equitoxic concentrations, N-ethyl-N-(acetoxymethyl) nitrosamine was found to be the most mutagenic. Of the two N-alkyl-N-(alpha-acetoxybutyl) nitrosamines having an acetoxy group at the secondary alpha-carbon, N-methyl-N-(alpha-acetoxybutyl) nitrosamine was more cytotoxic and mutagenic than N-butyl-N-(alpha acetoxybutyl) nitrosamine. A comparison of the corresponding N-alkyl-N-(acetoxymethyl) nitrosamines and N-alkyl-N-(alpha-acetoxybutyl) nitrosamines showed that the latter had stronger activities. A plot of the mutation frequency versus the ability of the alpha-acetoxy compounds to alkylate 4-(p-nitrobenzyl) pyridine was linear. This indicates that the chemical reactivity of the compounds plays an important role in inducing mutation in V79 Chinese hamster cells.

Species variations in the metabolism of N-butyl-N-(4-hydroxybutyl) nitrosamine and related compounds in relation to urinary bladder carcinogenesis.

Species variations in response to urinary bladder carcinogens, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN), and N,N-dibutylnitrosamine (DBN), were investigated in several animal species from the metabolic point of view. Since N-butyl-N-(3-carboxypropyl) nitrosamine (BCPN) and N-ethyl-N-(3-carboxypropyl) nitrosamine (ECPN) had been found to be the principal urinary metabolites which are responsible for the induction of bladder tumors by BBN or DBN and EHBN, respectively, in rats, acidic urinary metabolites with the N-nitroso moiety were isolated and determined by a colorimetric method after oral administration of these nitrosamines to rats, mice, hamsters, guinea pigs, and dogs. Qualitatively almost no species differences were observed among these animals in regard to the urinary metabolites except in the case of mice, in which the glycine conjugate of BCPN was isolated from the urine and identified as the principal metabolite of BBN and DBN. However, appreciable quantitative differences in the urinary excretion of BCPN or ECPN were found among these animal species, indicating that the differences in the susceptibilities of different animal species to urinary bladder carcinogenesis induced by BBN, DBN and EHBN may be closely related to the different extents of urinary excretion of the active metabolites of these nitrosamines.

Potential for metabolic deactivation of carcinogenic N-nitrosodimethylamine in vivo.

Enzymatic cleavage of N-nitrosodimethylamine (NDMA) to nitrite (normally representing about 10% of the total metabolism in vitro) also produces methylamine in yields roughly equimolar to those of nitrite, suggesting that the 'denitrosation' pathway may be responsible for the previously unexplained detection of methylamine as a urinary metabolite of NDMA and, at least in part, for the recovery of less than stoichiometric amounts of dinitrogen in 15N-labelling experiments. We have now followed excretion of labelled methylamine by rats receiving 14C-NDMA as a possible index of the extent of in-vivo denitrosation. Correcting for the proportion of labelled methylamine recovered in the urine following its administration under the conditions used for NDMA, 2.5-10% of the NDMA metabolism in Fischer rats appeared to proceed by a methylamine-forming route. The results are consistent with the conclusion that the metabolism of NDMA is best viewed as a competition between two pathways, with denitrosation diverting a significant proportion of the clearance to a presumably deactivating metabolic route at the expense of the activating alkylation pathway responsible for carcinogenesis.

Kinetic isotope effect on the demethylation and denitrosation of N-nitrosodimethylamine in vitro.

Deuteration of N-nitrosodimethylamine (NDMA) has been shown to decrease the carcinogenicity of this compound. This result is believed to be due to a kinetic isotope effect on the metabolic activation of this carcinogen, but conflicting views exist concerning whether the isotope substitution affects the Km or Vmax of the reaction. In order to elucidate the molecular basis of these observations, as well as the mechanisms of the demethylation and denitrosation reactions, the metabolism of NDMA and deuterated NDMA (NDMA-d6) was studied using acetone-induced rat-liver microsomes. The demethylation of NDMA displayed a Km of 0.06 mM and a Vmax of 7.9 nmol/min per mg protein. Deuteration of NDMA increased the Km value by five fold but did not appreciably affect the Vmax. The denitrosation of NDMA also displayed a Km of 0.06 mM, but the Vmax was 0.83 nmol/min per mg; deuteration again increased the Kmax several fold but had no effect on the Vmax. The results indicate that deuteration inhibits the metabolism of NDMA by increasing the Km but not the Vmax and suggest that there is a close relationship between the demethylation and denitrosation reactions.

Chemical and mutagenic properties of alpha-phosphonooxynitrosamines.

The chemical and mutagenic properties of the products of solvolysis of alpha-acetoxynitrosamines in phosphate buffer were investigated. alpha-Acetoxynitrosamines decomposed in two ways: O-acyl fission yielded alpha-hydroxynitrosamines, which decomposed into aldehydes and alcohols, while O-alkyl fission gave a resonance hybrid of alpha-N-nitrosocarbonium and -iminium ions, which was trapped with phosphate and afforded alpha-phosphonooxynitrosamine. Formation of alpha-phosphonooxynitrosamines was dependent on the structure of alpha-acetoxynitrosamines; those with a secondary alpha-phosphonooxy group, including cyclic nitrosamines, were easily formed, while among those with a primary phosphonooxymethyl group, only those with an alkyl group containing a branched alpha-carbon as isopropyl, sec-butyl and tert-butyl were isolated. They were good substrates of alkaline phosphatase and showed a nuclear magnetic resonance spectrum due to the presence of a phosphorus atom. They were decomposed by acid catalysis, and the rate was dependent on the structure. They were directly mutagenic in bacterial tester strains, except for a compound with a tert-butyl group. The activity was similar or stronger in Salmonella typhimurium TA1535 and much weaker in Escherichia coli WP2 and WP2 hcr- than those of alpha-acetoxynitrosamines. Stability in neutral aqueous solution and the strong mutagenicity of alpha-phosphonooxynitrosamines suggested their possible involvement in metabolic activation as a precursor of alpha-hydroxynitrosamines, and also in the organotropic carcinogenicity of N-nitrosodialkylamines as a transport form.

Mutagenicity of alpha-hydroxy N-nitrosamines in V79 Chinese hamster cells.

N-Nitrosodialkylamines are activated metabolically by alpha-hydroxylation. Chemical properties and bacterial mutagenicity of alpha-hydroxy N-nitrosamines have been reported previously. This paper describes potent and direct mutagenicity of four N-nitroso-N-(hydroxymethyl)alkylamines in V79 Chinese hamster cells, using ouabain resistance as an indicator. The mutagenic potency depended on the alkyl group, decreasing in the following order: methyl greater than ethyl greater than propyl, butyl. A similar order was observed for cytotoxicity. Mutagenic and cytotoxic potencies of these alpha-hydroxy N-nitrosamines in V79 cells were well correlated not only with those of model compounds (alpha-acetoxy and alpha-hydroperoxy N-nitrosamines), but also with their alkylating ability, measured by alkylation of thiophenol. The mutagenic activity of the alpha-hydroxy N-nitrosamines in V79 cells was shown to be parallel to that in Salmonella typhimurium TA1535 and to that of N-nitrosodialkylamines in V79 cells, after metabolic activation by rat hepatocytes. The results obtained here further support the conclusion that the alpha-hydroxy N-nitrosamine is the active species in the metabolic activation of carcinogenic and mutagenic N-nitrosodialkylamines.

Formation, deoxygenation and mutagenicity of alpha-hydroperoxydialkylnitrosamines.

N-Nitroso-N-alkyl-alpha-hydroperoxyalkylamines were prepared either by treatment of N-nitroso-N-alkyl-alpha-acetoxyalkylamines with hydrogen peroxide in acetic acid or by oxygenation of lithiated N-nitrosodialkylamines with oxygen. They were strongly mutagenic towards S. typhimurium TA1535 and E. coli WP2 strains without metabolic activation. Treatment of the alpha-hydroperoxy compounds with deoxygenating agents, such as sodium bisulfite, gave the alpha-hydroxynitrosamine, which could be isolated in aprotic solvents and characterized. The alpha-hydroperoxy compounds can possibly be formed from N-nitrosodialkylamines by biochemical and environmental routes and can serve as precursors for the alpha-hydroxynitrosamines.

Mutagenic and DNA-damaging effects of N-alkyl-N-(alpha-acetoxyalkyl)nitrosamines, models for metabolically activated N,N-dialkylnitrosamines.

Mutagenic and DNA-damaging effects of a series of N,N-dialkylnitrosamines monosubstituted at the alpha-carbon with an acetoxyl group were tested in Salmonella typhimurium, Escherichia coli, and Bacilus subtilis in the absence of metabolic activation system. The compounds comprised 8 N-alkyl-N-(acetoxymethyl)nitrosamines (alkyl=methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, or tert-butyl) and N-butyl-N-(1-acetoxybutyl)nitrosamine. All the compounds, except one with a tert-butyl group, gave positive results in these mutagenicity and repair tests. Presumed release of alkyl cations from the corresponding alpha-acetoxy derivatives by hydrolysis and heterolysis caused mutagenic and DNA-damaging effects in the bacteria. Structure-activity correlation of the compounds was noted in these tests and discussed in regard to the mutagenicity with metabolic activation and carcinogenicity of N,N-dialkylnitrosamines. The results support the hypothesis that alpha-carbon hydroxylation is one probable mechanism involved in the metabolic activation of N,N-dialkylnitrosamines.

Mutagenicity of alpha-acetoxy derivatives of N-methyl-N-benzylnitrosamine and N,N-dibenzylnitrosamine in V79 cells: a comparison between methylating and benzylating mutagens.

The mutagenicity and cytotoxicity of N-methyl-N-(alpha-acetoxybenzyl) nitrosamine, N-benzyl-N-(acetoxymethyl) nitrosamine, N-benzyl-N-(alpha-acetoxybenzyl)-nitrosamine and related compounds were examined in V79 Chinese hamster cells. These three alpha-acetoxy nitrosamines were all mutagenic, the first one (a compound ultimately yielding a methylating species) being far more strongly mutagenic than the other two (compounds ultimately giving benzylating species). The alkylating or aralkylating activity of these compounds was determined by assessing their reactivity toward 4-(rho-nitrobenzyl) pyridine. The chemical reactivity-mutagenicity correlation among these alpha-acetoxy nitrosamines and related compounds is discussed in relation to the carcinogenicity of their mother compounds.

Determination of catechol and guaiacol estrogens in urine by capillary gas chromatography/mass spectrometry.

A gas chromatographic (GC)/mass spectrometric method for the simultaneous determination in urine of 2- and 4-hydroxyestrones and hydroxyestradiols, and their monomethyl ethers, is described. Separation of these catechol and guaiacol estrogens was achieved by derivatization into their trimethylsilyl and tert-butyldimethylsilyl ethers, followed by capillary GC on a DB-1 column. The calibration graphs were satisfactorily constructed for these estrogen metabolites by selected ion monitoring at the respective molecular ions using 2-bromoestrone and 4-hydroxyestradiol-d3 3-methyl ether as internal standards. The extraction and purification of the desired estrogens in biological fluids were effected by the combined use of Extrelut-3 and ion exchange columns. The sensitivity and reliability obtained by the newly developed method has proved to be satisfactory for the quantitation of catechol and guaiacol estrogens in human urine.

Mutagenic and DNA-damaging effects of N-(omega-acetoxyalkyl and omega-methoxycarbonylalkyl)-N-(alpha-acetoxyalkyl)nitrosamines, models for metabolically activated N,N-dialkylnitrosamines with an omega-functional group.

Mutagenic and DNA-damaging effects of a series of N,N-dialkylnitrosamines having an alpha-acetoxy group together with an alpha-acetoxy or an omega-methoxycarbonyl group were tested in Salmonella typhimurium, Escherichia coli, and Bacillus subtilis without metabolic activation. The compounds comprised each of 3 N-(omega-acetoxyalkyl)-N-(acetoxymethyl)nitrosamines, N-(omega-methoxycarbonylalkyl)-N-(acetoxymethyl)nitrosamines, and N-(omega-methoxycarbonylalkyl)-N-(alpha-acetoxybutyl)nitrosamines. All the compounds gave positive results in these mutagenicity and repair tests. No definite differences were observed in the mutagenic and DNA-damaging effects between the model compounds derived from the carcinogenic compounds and those derived from the non-carcinogenic counterparts. The strongest mutagenic activity was observed so far with N-(2-methoxycarbonylethyl)-N-(l-acetoxybutyl)nitrosamine in all of the four bacterial strains.