Alpha,beta-unsaturated carbonyl compounds: induction of oxidative DNA damage in mammalian cells.

Alpha,beta-unsaturated carbonyl compounds occur in food and other environmental media. Due to their reactivity with cellular nucleophiles (e.g. Michael adduct formation with DNA bases and with glutathione) they might represent a potential health risk. In this study, induction of oxidative DNA damage was investigated in mammalian cells, as a consequence of glutathione depletion induced by selected food relevant 2-alkenals, including E-(2)-hexenal (HEX), (2E,4E)-2,4-hexadienal (HEXDI) and (E)-2-cinnamaldehyde (CA) and the cyclic analogue 2-cyclohexen-1-one (CHX). Oxidative DNA breakage was monitored with the Comet assay, using treatment with formamidopyrimidine-DNA glycosylase (FPG). Total cellular glutathione (tGSH) was determined in a kinetic, photometric assay. After 1 h incubation of V79 cells with HEX (100 microM) and CHX (300 microM), HEXDI and CA (300 microM each), tGSH was depleted down to <20% of control (viability >85%). Under these conditions, FPG-sensitive sites were not observed; moderate direct DNA breakage, however, was detectable. During 3 h post-incubation (without test compound) distinct oxidative DNA breakage occurred in HEX- and CA-, but not in CHX- and HEXDI-pretreated cells. Direct DNA breakage was markedly diminished, most probably by repair processes, and tGSH concentrations were observed to increase again within 3 h post-treatment. The results give strong evidence for alkenal-mediated oxidative stress contributing to cytotoxic/genotoxic cell damage. The extent of oxidative stress appears to be influenced by structure-specific properties of the alkenals.

Mutagenic and cytotoxic effectiveness of diisopropyl xanthogen polysulphide in human lymphocyte cultures.

The mutagenic and cytotoxic effectiveness of the new rubber vulcanisation accelerator diisopropyl xanthogen polysulphide (Robac AS 100) was tested in human lymphocyte cultures of four healthy probands. The concentrations of Robac AS 100 were 0.57, 5.7 and 57.0 microg/ml. Higher concentrations showed too high cytotoxicity to be evaluable. Without external activation, incubation time with Robac AS 100 was 21 h. In the presence of rat liver microsomes from aroclor-induced rats (2mg microsomal protein/ml), incubation of the test compound was 2h. Mutagenicity testing was performed by analysis of micronuclei (MN), structural chromosome aberrations (CAs) and sister chromatid exchanges (SCEs). The MN-rate was determined using the cytochalasin B (cyt B) block method. For evaluation of cytotoxicity, mitotic index (MI) and nuclear division index (NDI) were determined. The validity of the test methods was ascertained by positive controls: mitomycin C (MMC) and bleomycin (BLM) were used in experiments without exogenous activation and cyclophosphamide (CP) in experiments with exogenous activation. The presence of rat liver microsomes increased the mutagenic effect of Robac AS 100 in the SCE- and MN-test. But only the highest Robac AS 100-concentration (57.0 microg/ml) showed significantly increased mutagenic activity in all tests. However, cytotoxicity at this concentration was already substantial. Therefore, we consider the evidence for mutagenicity of Robac AS 100 as limited.

Mutagenic and cytotoxic effectiveness of zinc dimethyl and zinc diisononyldithiocarbamate in human lymphocyte cultures.

The mutagenic and cytotoxic effectiveness of the vulcanisation accelerators zinc dimethyldithiocarbamate (ZDMC; ziram) and zinc diisononyldithiocarbamate (ZDINDC; arbestab Z) was tested in lymphocyte cultures of five healthy probands. ZDMC and ZDINDC (c=0.1, 1.0 and 10.0microg/ml) were studied in lymphocyte cultures without external metabolic activation. Additionally, incubation of the compounds (c=10.0microg/ml) was performed in the presence of liver microsomes from aroclor-induced rats (1 and 2h, 1 and 2mg microsomal protein). Genotoxicity testing was performed by analysis of chromosomal aberrations (CA), sister chromatid exchanges (SCEs) and micronuclei (MN). For evaluation of antiproliferative effects, mitotic index (MI) and cell cycle kinetics (CCK) were determined. In contrast to earlier investigations we found no significantly increased mutagenic or cytotoxic activity of ZDMC; ZDINDC also was inactive under these conditions.

DNA-damaging potential and glutathione depletion of 2-cyclohexene-1-one in mammalian cells, compared to food relevant 2-alkenals.

2-Cyclohexene-1-one (CHX) occurs as a natural ingredient in some tropical fruits and has been detected as a contaminant in certain artificially sweetened soft drinks. To elucidate its cytotoxic/genotoxic effectiveness, CHX was tested in mammalian cell lines (V79 and Caco-2) and in primary human colon cells in comparison to structurally related 2-alkenals. Inhibition of cell growth (IC(50)) and cytotoxicity (LC(50)) were determined by protein staining with sulforhodamin B (SRB) and by trypan blue exclusion, respectively. DNA damage--both strand breaks and oxidised purines--was quantified by comet assay. Depletion of glutathione was measured in a kinetic assay, based on 5-thio-2-nitrobenzoate (TNB) formation. For CHX, a moderate cytotoxicity was observed after 1h incubation in V79 cells (LC(50): 4.75mM). The 2-alkenals ((E)-2-octenal (OCTE), (2E,4Z)-2,4-hexadienal (HEXDI), (E)-2-nonenal (NONE), (2E,6Z)-2,6-nonadienal (NONDI)) exhibited a distinctly higher cytotoxicity, except for (E)-2-hexenal (HEX) (LC(50): 3.67mM) and cinnamaldehyde (CA) (LC(50): 4.45mM). If the incubation time was prolonged to 24h, an IC(50) of 15microM was obtained for CHX which is well within the range obtained for the 2-alkenals (4 and 17microM). Concentration-dependent DNA damage was observed after 1h incubation with CHX. The respective DC(50) values (concentration inducing DNA damage in 50% of cells) were 272microM (V79) and 455microM (Caco-2). All 2-alkenals were more active under these conditions, except for CA. In primary human colon cells, CHX (800microM, 30min) exhibited a weak, but still significant DNA-damaging potential. Glutathione levels in V79 cells were effectively depleted (down to approximately 20%) by CHX concentrations not yet inducing DNA damage (c < or = 50microM). Incubation with CHX or 2-alkenals (50 and 100microM, 1h), followed by H2O2 treatment (5min, 25microM) resulted in increased levels of oxidised purines in the modified comet assay. CHX and HEX, additionally tested in primary human colon cells, depleted glutathione and increased the sensitivity towards oxidative stress.

5-Hydroxymethylfurfural: assessment of mutagenicity, DNA-damaging potential and reactivity towards cellular glutathione.

5-(hydroxymethyl)-2-furfural (HMF), a common product of the Maillard reaction, occurs in many foods in high concentrations, sometimes exceeding 1 g/kg (in certain dried fruits and caramel products). The toxicological relevance of this exposure has not yet been clarified. Induction of aberrant colonic crypt foci had been reported for HMF, in vitro studies on genotoxicity/mutagenicity have given controversial results. To elucidate the toxic potential of HMF, cytotoxicity (trypan blue exclusion), growth inhibition (SRB assay), mutagenicity (HPRT assay), DNA damage (single-cell gel electrophoresis) and depletion of cellular glutathione were investigated in mammalian cells. Genotoxicity (SOS repair) was monitored in Salmonella typhimurium (umu assay). HMF induced moderate cytotoxicity in V79 cells (LC(50): 115 mM, 1 hr incubation) and in Caco-2 cells (LC(50): 118 mM, 1 hr incubation). Growth inhibition was monitored following 24 hr of incubation (V79, IC(50): 6.4 mM). DNA damage was detectable neither in these cell lines nor in primary rat hepatocytes up to the cytotoxic threshold concentration (75% absolute viability). Likewise, in primary human colon cells, obtained from biopsy material, DNA damage was not measurable. At 120 mM, already exhibiting some reduction in cell viability, HMF was weakly mutagenic at the hprt-locus in V79 cells (mutants/10(6) cells: HMF 120 mM: 16 vs control: 3). Intracelluar glutathione was depleted by HMF (>/=50 mM) in V79 cells, in the human colon adenocarcinoma cell line Caco-2 and in primary rat hepatocytes down to approximately 30% of control (120 mM). Genotoxicity was observed with HMF in the umu assay without external activation (16 mM: 185 rel. umu units, %, P<0.001). The genotoxic potential was not altered by addition of rat liver microsomes. By comparison, the natural flavour constituent (E)-2-hexenal (HEX) was already cytotoxic, mutagenic and depleted glutathione at about 1000-fold lower concentrations. It induced DNA damage in mammalian cells (200-400 microM). These results suggest that HMF does not pose a serious health risk, even though the highest concentrations in specific foods approach the biologically effective concentration range in cell systems.

Probing the mechanism of the carcinogenic activation of N-nitrosodiethanolamine with deuterium isotope effects: in vivo induction of DNA single-strand breaks and related in vitro assays.

A series of bioassays, including in vivo induction of DNA single-strand breaks (SSB) and cytotoxicity in cytochrome P450 2E1-transfected cells, were utilized with N-nitrosodiethanolamine (NDELA), its deuterated isotopomers (alpha-D4NDELA and beta-D4NDELA), N-nitroso-2-hydroxymorpholine (NHMOR), and two of its deuterated isotopomers (2-D-NHMOR and 5,5-D2-NHMOR) to probe the mechanism of carcinogenic activation of NDELA and the role of its metabolite NHMOR. DNA samples, taken from the livers of male Wistar rats 4 h after the administration of NDELA, exhibited dose-dependent DNA SSB levels over the range of 0.08-0.75 mmol/kg (body weight), with the greatest SSB level at the highest dose. Deuterium isotope effects on DNA SSB levels were inversely dependent on dose: alpha-D4NDELA, 3. 22-1.37; and beta-D4NDELA, 1.38-0.79. At the lowest dose of 0.15 mmol/kg (body weight), 5,5-D2-NHMOR gave an isotope effect for DNA SSB of 2.8 while that for 2-D-NHMOR was 0.7. NDELA and beta-D4NDELA were equally cytotoxic to human P450 2E1-transfected V79 Chinese hamster cells, while alpha-D4NDELA was not. Significant DNA SSB levels were observed in these cells for NDELA and beta-D4NDELA but not for alpha-D4NDELA. A kinetic deuterium isotope effect of 2.6 for Vmax/Km was observed for the horse liver alcohol dehydrogenase-mediated oxidation of beta-D4NDELA to NHMOR, while kH/kD for alpha-D4NDELA was 1.05. These data provide the first definitive evidence for the activation of NDELA by a pathway involving the scission of the alpha-CH bond and are consistent with P450 2E1-mediated alpha-hydroxylation of NDELA producing the corresponding reactive alpha-hydroxynitrosamine.

Potential nitrosamine formation and its prevention during biological denitrification of red beet juice.

High nitrate intake has been shown to result in an increased risk of endogenous formation of N-nitroso compounds. Certain vegetables and vegetable juices contain high concentrations of nitrate. Biological denitrification using strains of Paracoccus denitrificans (P.d.) has been proposed as effective means to reduce nitrate contents in such vegetable juices. During this bacterial denitrification process, substantial nitrite concentrations are transiently formed. This study investigated whether N-nitrosation reactions might occur. The easily nitrosatable amine morpholine was added to red beet juice at high concentration (100 ppm) during denitrification 10 different batches of red beet juice served as raw material. Each batch was submitted to denitrification in the presence and absence of ascorbic acid. In the absence of ascorbic acid, formation of N-nitrosomorpholine (NMOR) was observed in the low ppb range (0.5-8 ppb). Addition of ascorbic acid (500 mg/litre) inhibited the formation of NMOR, except for those instances where the pH was less than 6 and/or nitrate turnover was low (< 200 mg NO3-/litre/hr). Under conditions leading to high rates of nitrate turnover (> 200 mg NO3-/litre/hr), nitrosamine formation can reliably be prevented by ascorbic acid. The results show that bacterial denitrification of red beet juice high in nitrate can be accomplished without the risk of nitrosamine formation.

(E)-2-hexenal-induced DNA damage and formation of cyclic 1,N2-(1,3-propano)-2'-deoxyguanosine adducts in mammalian cells.

(E)-2-Hexenal (hexenal), a natural flavor compound, acts as directly genotoxic agent and forms cyclic 1,N2-propano adducts with deoxyguanosine. Formation of this adduct in isolated DNA and in cells was studied with a modified 32P-postlabeling procedure including HPLC separation, nuclease P1 enrichment, two-dimensional TLC of adducted nucleotide bisphosphates on PEI-cellulose, and quantification of adduct spots by liquid scintillation counting. Adduct formation with the more reactive crotonaldehyde was included for comparison. Synthesized adducted dG-3'-phosphates served as external standards for identification and quantification. In calf thymus DNA, hexenal (0.2 mM) shows a time dependent formation of adducts, yielding 1.55 pmol/mumol of DNA at 5 h incubation. With crotonaldehyde (0.2 mM) the adduct rate was about 10-fold higher. Hexenal also generated 1,N2-propano-dG adducts in the human lymphoblastoid Namalva cell line (0.2 mM, 1 h, 86 fmol/mumol of DNA) and in primary rat colon mucosa cells (0.4 mM, 30 min, 50 fmol/mumol of DNA). In primary colon mucosa cells from rats and humans, hexenal and crotonaldehyde (0.4 mM, 30 min) induced DNA damage, detected by single cell microgel electrophoresis (comet assay). In primary rat gastric mucosa cells, hexenal was only weakly active, inducing detectable DNA damage in 20% of cells at 0.8 mM concentration. In contrast, primary mucosa cells from rat esophagus were as sensitive as colon cells. After single oral application of hexenal to rats (up to 320 mg/kg body wt) DNA damage was not detectable in gastrointestinal mucosa. Analysis of hexenal in selected flavored foods revealed concentrations up to 14 ppm (0.14 mM) that are comparable to its natural occurrence in some fruits and vegetables (up to 30 ppm). Thus, the concentration range selected for the toxicological studies described here clearly is relevant: Hexenal, at concentrations found in food, exerts genotoxic effects in cells from rat and human gastrointestinal tract.

[Nitrite formation and nitrosation potential of heterotrophic biological denitrification of drinking water in laboratory conditions]. / Untersuchungen zur Nitritbildung und zum Nitrosierungspotential bei der heterotrophen biologischen Denitrifizierung von Trinkwasser unter Laborbedingungen.

In a laboratory construction for heterotrophic biological denitrification of drinking water treatment, the formation of nitrite, the potency of nitrosation and the genotoxic activity were tested. Parameter as nitrate concentration, the water flow rate in the system, nitrite and morpholine addition and the pH-value were checked. For testing the potency of nitrosation and formation of nitrite in the reactor we took morpholine, a fast nitrosing amine. The results show for the break down rate of nitrate, there is no influence of the initial nitrate concentration (80-195 mg/L), the nitrite addition (5-20 mg/L) and the water flow rate (45-100 min) in the system. pH-values below 5.5 showed a little break down rate of nitrate. There was no correlation between the starting point of nitrate concentration and the formation of nitrite, although there was a positive correlation between the length of stay and the formation of nitrite. Nitrite concentrations of 5 mg/L with morpholine concentrations of 5 and 10 mg/L didn't show detectable formation of nitrosomorpholine. The analyses of different watertests in the construction didn't show significant results for DNA damage by the sister-chromatid exchange (SCE). The results of the Salmonella microsome assay (tester strain TA1535) didn't show any mutagenic effects relating to the potency of nitrosation. According to our experiments the potency of generalising nitrosamides or nitrosamines by drinking water denitrification seems to be low. There is no final assessment of detriment to health by denitrifying drinking water.

Stable expression of human cytochrome P450 2E1 in V79 Chinese hamster cells.

A V79 Chinese hamster cell line was constructed for stable expression of human cytochrome P450 2E1 (CYP2E1) by integration of a SV40 Early promoter recombinant CYP2E1 cDNA into the chromosomal DNA. The cDNA encoded CYP2E1 was effectively expressed and enzymatically active, as shown by hydroxylation of chlorzoxazone and of p-nitrophenol, at rates of about 70 pmol x mg-1 total protein x min-1. CYP2E1 content and activity was increased upon cultivation in the presence of ethanol indicating a substrate mediated stabilization effect. A similar stabilizing effect was also observed for inhibitors of CYP2E1, e.g. imidazole, 4-methylpyrazole, and isoniazid. The feasibility of the newly established cell line V79MZh2E1 for toxicological studies was shown by CYP2E1-mediated activation of N-nitrosodimethylamine and p-nitrophenol and a dose-dependent cytotoxic and mutagenic effect.

Mitochondrial formation of beta-oxopropyl metabolites from bladder carcinogenic omega-carboxyalkylnitrosamines.

Certain environmentally relevant nitrosamines specifically induce malignant tumors in the urinary bladder in several animal species. For this organotropic effect, formation of omega-carboxylated proximal metabolites has been found to be obligatory. The mechanism of action of these intermediates, however, is not yet clear. We investigated biotransformation of butyl-3-carboxypropylnitrosamine (CAS: 38252-74-3), methyl-3-carboxypropylnitrosamine (CAS: 61445-55-4) and methyl-5-carboxypentylnitrosamine by mitochondrial fractions from rat liver and renal cortex. On incubation with mitochondrial fractions, the respective beta-oxidized metabolites butyl-2-oxopropylnitrosamine (CAS: 51938-15-9) or methyl-2-oxopropylnitrosamine (CAS: 55984-51-51) were formed. This biotransformation was ATP dependent, associated with the presence of mitochondrial marker enzyme (cytochrome c oxidase) in 7000 x g subfractions and was inhibited by octanoic acid. Highest metabolic rates were observed with rat liver fractions. These results demonstrate that omega-carboxylated nitrosamines are substrates for mitochondrial enzymes of fatty acid degradation, most probably following the degradation pathway of medium-chain fatty acids. By this reaction, water-soluble carboxylated nitrosamines of low genotoxic potential are converted into rather lipophilic 2-oxopropyl metabolites with high genotoxic and carcinogenic potency. In contrast to carboxylated metabolites, 2-oxopropyl derivatives are good substrates for cytochrome P-450 dependent mono-oxygenases. Therefore, mitochondrial beta-oxidation appears to be an important step in metabolic activation of nitrosamines tumorigenic in the urinary bladder.

Mechanism of action of the urinary bladder carcinogen N-nitrosobutyl-3-carboxypropylamine.

The carcinogenic action of N-nitrosodibutylamine in the urinary bladder is related to omega-oxidation of a butyl chain. N-Nitrosobutyl-4-hydroxybutylamine and its proximate metabolite N-nitrosobutyl-3-carboxypropylamine (NBCPA) selectively induce urinary bladder tumours in different animal species. The mechanism by which NBCPA exert its carcinogenic action is not known. We found a small but significant dealkylation of NBCPA with microsomes from rat liver or pig urinary bladder, which could be inhibited by SKF 525A. NBCPA was not mutagenic to Salmonella typhimurium (with or without external metabolizing systems from rat liver or pig urinary bladder) and did not induce DNA strand breaks in tumour cell lines (with or without external activation) or primary cells (rat hepatocytes, pig urinary bladder epithelia). Significant induction of sister chromatid exchange and micronuclei, however, was observed in human tumour cells. N-Nitrosoureas that generate the same electrophiles as NBCPA after alpha- or via beta-oxidation (N-butyl-N-nitrosourea, N-3-carboxypropyl-N-nitrosourea and N-2-oxopropyl-N-nitrosourea) induced single-strand breaks in Namalva cells, the oxopropyl compound being more potent than the butyl or carboxypropyl compounds. Our data suggest that NBCPA is activated via alpha-oxidation in the urinary bladder, even though the activation rate in vitro is so low that a positive response is not detectable by classical short-term tests. Provided that beta-oxidation to a highly genotoxic agent proceeds at an adequate rate, it might also be a relevant activation pathway.

Investigations on organ-specific metabolism and genotoxic effects of the urinary bladder carcinogen N-nitrosobutyl-3-carboxypropylamine (BCPN) and its analogs N-nitrosodibutylamine (NDBA) and N-nitrosobutyl-4-hydroxybutylamine (4-OH-NDBA).

N-Nitrosodibutylamine (NDBA) and its omega-oxidized metabolites N-nitrosobutyl-4-hydroxybutylamine (4-OH-NDBA) and N-nitrosobutyl-3-carboxypropylamine (BCPN) are potent urinary bladder carcinogens. To study putative organ specific activation of BCPN, its alpha-oxidation by liver and urinary bladder microsomal fractions was investigated in comparison to NDBA and 4-OH-NDBA. Additionally, induction of DNA single strand breaks (SSB) was monitored in hepatocytes and in a human lymphoblastoid cell line (Namalva) in the presence and absence of external metabolic activation, including N-nitroso-t-butyl-n-butylamine as a negative control. BCPN was alpha-hydroxylated and dealkylated at both alkyl chains in small rates (about 1 nmol x mg protein-1 x 60 min-1) by microsomes from rat liver and pig urinary bladder epithelium. NDBA and 4-OH-NDBA were dealkylated at similarly low rates by pig urinary bladder microsomes, in strong contrast to the high debutylation rates observed for rat liver microsomes. Correspondingly, SSB induction by NDBA and 4-OH-NDBA was observed in Namalva cells with NDBA and 4-OH-NDBA in the presence of PB-induced rat liver microsomes but not with urinary bladder microsomes or without external activation. BCPN did not induce DNA-damage in Namalva cells (with or without external activation) or in rat hepatocytes. Significant induction of sister chromatid exchanges (SCEs) and micronuclei, however, was observed in Namalva cells after incubation with NDBA and BCPN. Our data suggest activation of BCPN via alpha-oxidation in the urinary bladder, even though activation rate in-vitro is so low that a positive response is not detectable by several short-term tests.

Fluoro-substituted N-nitrosamines. 8. N-Nitrosodibutylamine and omega-fluorinated analogues: in vivo metabolism in relation to the induction of urinary bladder cancer in the rat.

Urinary excretion of N-nitrosodibutylamine (NDBA) and of two omega-fluorinated analogues [N-nitroso-4,4,4-trifluorobutyl-butylamine, NDBA-F3; N-nitroso-bis(4,4,4-trifluorobutyl)-amine, NDBA-F6] was studied in male Sprague-Dawley (SD) rats. After oral application of equimolar doses (0.44 and 1.32 mmol/kg body wt.) urines were collected (48 h) and analyzed for parent compounds, and for nitrosamine metabolites by gas chromatography/Thermal Energy Analyzer (GC/TEA) and gas chromatography/mass spectrometry (GC/MS). After administration of NDBA the known major metabolites N-nitroso-3- hydroxybutylbutylamine (3-OH-NDBA) and N-nitroso-3-carboxypropylbutylamine (BCPN) were excreted in urine. After application of the omega-fluorinated analogue NDBA-F6, however, urinary and biliary nitrosamine metabolites were detected only in trace amounts. This finding demonstrates a strong inhibitory effect of fluorine substitution on oxidations at omega, (omega-1) and beta-positions. Confirmation of this inhibitory effect of omega-fluorine substitution is given from the excretion profiles of NDBA-F3 which shows metabolic oxidations only at the nonfluorinated chain: N-nitroso-3-hydroxybutyl-4,4,4-trifluorobutylamine (3-OH-NDBA-F3) and N-nitroso-3-carboxypropyl-4,4,4-trifluorobutylamine (BCPN-F3) were excreted as main metabolites. Our results on metabolism together with the available data on carcinogenicity of the compounds in the rat strongly support the hypothesis that omega-oxidation of one butyl-chain is a prerequisite for the induction of urinary bladder tumors with NDBA. For the induction of liver tumors, alpha-C-hydroxylation appears to be the crucial event.

Fluoro-substituted N-nitrosamines. 7. Non-genotoxic N-nitroso-bis(2,2,2-trifluoroethyl)amine and N-nitroso-bis(2,2,3,3,4,4,4-heptafluorobutyl)amine: binding to cytochrome P-450, acidity of alpha-protons and pharmacokinetic investigations.

The biologically inactive fluorinated nitrosamines N-nitroso-bis(2,2,2-trifluoroethyl)amine (NDEA-F6) and N-nitroso-bis-(2,2,3,3,4,4,4-heptafluorobutyl)amine (NDBA-F14) were investigated for binding affinity to cytochrome P-450 and for ease of alkali-induced proton abstraction at the alpha-C atom, in comparison with biologically active analogues (N-nitroso-diethylamine, NDEA; N-nitroso-2,2,2-trifluoroethylethylamine, NDEA-F3; N-nitrosodibutylamine, NDBA; N-nitroso-4,4,4-trifluorobutylbutylamine, NDBA-F3; N-nitroso-bis(4,4,4-trifluorobutyl)amine, NDBA-F6). Binding to cytochrome P-450 was studied by spectroscopic measurements (optical difference spectra with microsomal fractions); base-catalyzed deuterium exchange of alpha-hydrogen atoms was followed by 1H n.m.r. measurements. Additionally the excretion of NDEA-F6 and NDBA-F14 in expired air, urine and faeces was studied after oral application to the rat. Compared with the biologically active nitrosamine analogues, NDEA-F6 and NDBA-F14 showed higher binding affinity to cytochrome P-450. N.m.r. spectroscopy showed that NDEA-F6, NDBA-F14 and NDEA-F3 (at the fluorinated alkyl chain) were rapidly deprotonated at the alpha-C-position in sodium perdeutero methylate, in contrast to the other analogues tested. In vivo, NDEA-F6 and NDBA-F14 were excreted unchanged, mainly via exhalation. The biological inactivity of NDEA-F6 and NDBA-F14, together with the observed blocking of their microsomal activation can be reconciled with the experimental findings which indicate that homolytic alpha-C-H bond fission is more likely to be involved in alpha-C-hydroxylation of dialkylnitrosamines, than alpha-proton abstraction.

Effects of fluorination on in-vitro metabolism and biological activity of N-nitrosodialkylamines.

Substitution of N-nitrosodialkylamines with fluorine at specific sites inhibits oxidative metabolism at the respective carbon atoms. The results of in-vitro metabolism studies with N-nitrosodiethylamine (NDEA), N-nitrosodibutylamine (NDBA) and their fluorinated analogues, N-nitroso-2,2,2-trifluoroethyl-ethylamine (NDEA-F3), N-nitroso-bis(2,2,2-trifluoroethyl)amine (NDEA-F6), N-nitroso-4,4,4-trifluorobutyl-butylamine (NDBA-F3), N-nitroso-bis(4,4,4-trifluo-robutyl)amine (NDBA-F6) and N-nitroso-bis(2,2,3,3,4,4,4-heptafluorobutyl)amine (NDBA-F14), showed effects of fluorination on biotransformation which can explain results of carcinogenicity and mutagenicity experiments; NDEA-F6 and NDBA-F14 were practically not metabolized by microsomal fractions, even though no decrease in binding affinity to cytochrome P450 was observed. Both compounds were not biologically active and were exhaled unchanged in high proportions after oral administration to the rat. Biologically active analogues, NDEA, NDBA, NDBA-F3 and NDBA-F6, were found to be dealkylated at the unfluorinated alkyl chains and, to a lesser extent, at the omega-fluorinated alkyl chains. Detection of corresponding alcohols as hydrolysis products confirmed the generation of electrophilic intermediates by alpha-C-hydroxylation. However, trifluorethanol was detected only in very small proportions from dealkylation of NDEA-F3, although dealkylation occurred almost exclusively at the unfluorinated site.