Expression of glutathione S-transferase isoenzymes in human small cell lung cancer cell lines

A glutathione S-transferase (GST) isoenzyme having common antigenicityto rat placental form (GST-P) and human placental form (GST-)has recently been suggested may be a marker of carcinogenesis.In the present study we have investigated the expression ofthis isoenzyme in three small cell lung cancer cell lines inorder to determine whether or not this isoenzyme can be usedas a general marker of carcinogenesis. GST activity towards1-chloro-2,4-dinitrobenzene in two of the cell lines (NES andNOC-361) was moderately higher than that in normal human lung,but this activity was markedly suppressed in one of the celllines (NCI-H69). Quantitation of the GST isoenzymes in the tumorsgrown in nude mice by injecting these cell lines also revealeda moderate increase of GST--type isoenzyme in NES and NOC-361and its suppression in NCI-H69. Immunocytochemical localizationstudies with these tumors using antibodies raised against GST-also indicated a drastic decrease of GST--type isoenzyme inNCI-H69 and this finding was confirmed by Western biot studies.These results suggest that GST- or the isoenzyme(s) having similarimmunological nature to GST-, cannot be used as the generalmarkers of neoplastic states.     

Utilization of 1, N6-Etheno-2'-deoxyadenosine 5'-triphosphate during DNA synthesis on natural templates, catalyzed by DNA polymerase I of Escherichia coli

To test whether vinyl chloride-induced mutagenesis might involveambiguous base pairing of 1, N⁶-etheno-adenine (A) during DNAsynthesis, we examined the base pairing potential of dATP duringDNA synthesis catalyzed by Escherichia coli DNA polymerase I(Klenow fragment). An electrophoretic assay of chain elongationwas used to assess the degree to which dATP could substitutefor each of the normal dNTPs during elongation of a primer annealedto a bacteriophage template. Despite the fact that the ethenobridge completely blocks normal Watson-Crick pairing of A withT, we observed that dATP could substitute for dATP during primerelongation (although inefficiently). In addition, detectablesubstitution of dATP for dGTP and dCTP occurred, indicatingthat A exhibits ambiguous base pairing properties. The relativeease of dAMP incorporation (opposite template T, C and G) appearedto vary considerably at different positions along the template.The major, form of eA incorporation (replacement of A) was confirmedby measurements of dATP-dAMP turnover (a commonly used methodfor detecting misincorporation), and also by the demonstrationthat A was present in enzymatic hydrolysates prepared from DNAthat was synthesized with dATP replacing dATP. A model for ambiguousbase pairing of dATP is proposed, in which incorporation occursvia the protonated, syn form of dATP.     

Expression of the glutathione S-transferase placental form in human lung carcinomas

Expression of glutathione S-transferase placental form (GST-)in human lung carcinoma tissue taken at autopsy or biopsy wasinvestigated immunohistochemically. All of 34 cases of squamouscell carcinomas, including poorly, moderatelyand well-differentiatedexamples were shown to stain positively for GST-. Poorly differentiatedadenocarcinomas were, however, negatively stained (0/5 cases),while moderately and well differentiated adenocarcinomas werefound tostain with GST- at rates of 69% (9/13 cases) and 71%(5/7 cases), respectively. Six cases of small cell carcinomasexamined were all negative. The results indicate that GST- maybe a useful marker fornon-small cell type lung cancer, especiallysquamous cell carcinoma which is in agreement with findingsfor rat lung neoplastic lesions reported previously.     

Involvement of transforming growth factor-{alpha} and its receptor in a model of DES-induced renal carcinogenesis in the Syrian hamster

This study explores the role played by TGF in estrogen-inducedrenal tumors. Tumors were induced in male Syrian hamster bychronic administration of diethylstilbestrol (DES). Six experimentalgroups (n = 5–9) were chronically exposed to DES and sacrificedafter 1, 2, 4, 6, 9 and 11 months, respectively. In the courseof treatment, the nephrons were the site of an important increaseof cell turnover, which was characterized by a process of hyperplasia/dysplasiain proximal tubules preceding the neoplastic transformation.In treated animals and in controls, the analysis of renal tissueby Western blot revealed the presence of a 6 kDa polypeptidecrossreacting with anti-TGF antibody. In controls, TGF immunoreactivitywas localized in proximal and in distal tubules. Before tumordevelopment (1–4 months), TGF RIA showed an increase ofTGF concentration in renal tissue, in parallel with the increasedcell proliferation observed in proximal tubules. In addition,Western blot analysis also demonstrated in kidney tissue thepresence of a 165 kDa protein displaying the immunoreactivityof EGF/TGF receptor. The receptor immunoreactivity was localizedin proximal tubular cells suggesting an involvement of TGF intubular epithelial growth through autocrine or paracrine pathways.In large neoplasms, immunocytochemistry revealed only clustersof transformed cells intensely stained by the anti-TGF antibody.These cells displayed the appearance of stellate or polyhedriccells infiltrating adjacent neoplastic tissues. Antisera raisedagainst intra-or extracytoplasmic domains of the EGF/TGF receptorwere assayed to localize this receptor in the tumors. In contrastwith tubular structures, immunoreactivity to EGF/TGF receptorwas never detected in tumoral tissue. The apparent absence ofEGF/TGF receptor immunoreactivity in malignant cells seems toexclude an involvement of this growth factor in the tumorigenicprocess, although it could be involved in tumor neovascularization.     

1,N6-etheno-2' -deoxyadenosine and 3,N4-etheno-2'-deoxycytidine detected by monoclonal antibodies in lung and liver DNA of rats exposed to vinyl chloride

1,N⁶-Etheno-2'-deoxyadenosine (dAdo) and 3,N⁴-Etheno-2'-deoxycytidine(dCyd) are formed in vitro by reaction of DNA with the electrophilicmetabolites of vinyl chloride (VC), chloroethylene oxide andchloroacetaldehyde. To detect and quantitate these DNA adductsin vivo, we have raised a series of specific monoclonal antibodies(Mab). Among those, Mab EM-A-1 and Mab EM-C-1, respectively,were used for detection of dAdo and dCyd by competitive radioimmunoassay(RIA), following pre-separation of the etheno adducts from DNAhydrolysates by high perfonnance liquid chromatography. At 50%inhibition of tracer-antibody binding, both Mab had a detectionlimit of 187 fmol and antibody affinity constants (K) of 2 x10⁹ l/mol. The levels of dAdo and dyd were quantitated in theDNA of lung and liver tissue of young Sprague-Dawley rats exposedto 2000 p.p.m. of VC for 10 days. The dAdo/2'-deoxyadenosineand dCyd/2'-deoxycytidine molar ratios were 1.3 x 10^–7and 3.3 x 10^–7 respectively, in lung DNA, and 5.0 x 10^–8and 1.6 x 10^–7 in liver DNA. When hydrolysates of 3 mgof DNA were analyzed by RIA at 25% inhibition of tracer-antibodybinding, dAdo and dCyd were not detected in liver DNA from untreatedrats above the limiting dAdo/2'-deoxyadenosine and dCyd/2'-deoxycytidinemolar ratios of 2.2 x 10^–8 and 3.1 x 10^–8, respectively.     

Intercellular communication in primary cultures of putative preneoplastic and 'normal' hepatocytes

Gap junctional intercellular communication (IC) was studiedin -glutamyltranspeptidase (-GT)-positive and -negative hepatocytesisolated from carcinogen-treated rats. Putative preneoplastic-GT-positive hepatocytes were visualized in monolayer culturesby indirect immunofluorescence using anti--GT-antibodies. ICwas evaluated by studying dye coupling of the cells. -GT-positivehepatocytes showed a significantly lower dye coupling than did-GT-negative liver cells. Spread of the dye Lucifer Yellow CHto neighboring cells was decreased further by the tumor-promotingchemical phenobarbital in both cell types in vitro. Also treatmentin vivo with the barbiturate significantly reduced dye couplingof hepatocytes. The findings suggest that as a result of theirdecreased ability to communicate, preneoplastic hepatocytesmay escape from growth control and differentiation signals givenout by surrounding ‘normal’ cells.     

UVR induction of TGF{alpha}: a possible autocrine mechanism for the epidermal melanocytic response and for promotion of epidermal carcinogenesis

The occurrence of the epidermal growth factor homologue, transforminggrowth factor (TGF), in embryonic and neoplastic tissues suggeststhat it may he an oncofetal version of epidermal growth factor.A strong case is developing for TGF to have an autocrine modeof action in sustaining the autonomous growth of several typesof tumour. We propose that TGF normally has an autocrine rolenot only in stimulating the growth of some fetal tissues butalso with postnatal epidermal cells in response to local stimuli—inparticular ultraviolet radiation (UVR). As a first step to testthis hypothesis we have checked whether UVR will induce theproduction of TGF, measured by radioimmunoassay, using a highlyspecific monodonal antibody which recognizes native, biologicallyactive human TGF. We found that cultures of normal foreskinmelanocytes do not produce detectable amounts of TGF when grownunder routine conditions, but, within 12 h of exposure to lowdoses of short-wavelength UVR, significant quantities of TGFare produced. The UVR-induced TGF is both cell associated andreleased into the medium of these cultures. Also, UVR has apromoting action on epidermal cells which have been initiatedby carcinogenic activity. A significant part of the promotingactivity may be due to autocrine stimulation of multiplicationof partially transformed epidermal cells. In this regard wefound that UVR induced TGF in HeLa cells and all human melanomalines so far tested. Induction was complete within 24 h of asingle exposure. Dose-response curves of TGF induction in amalignant melanoma cell line showed a distinctive peak of factorinduced by low (2 J/m²) doses of UVR. Higher doses which inhibit[³H]thymidine incorporation resulted in lower levels of inducedTGF. These findings are consistent with the participation ofTGF as an autocrine mediator of UVR-induced tumour promotion,as well as cell multiplication, in sun-exposed skin.     

Existence of multiple forms of microsomal epoxide hydrolases with radically different substrate specificities

Evidence for the existence in rat and rabbit liver of two microsomalepoxide hydrolases with radically different substrate specificitieswas obtained, one with a broad specificity (EH_b), whilst theother catalyzed the hydrolysis of cholesterol 5,6-oxide (EH_ch),a reaction taken as diagnostic since it was not observed withpure fractions of EH_b. The two enzymes were physically separatedby immunoprecipitation using antibodies which had been raisedagainst EH_b purified to apparent homogeneity. The substratespecificity of the two enzymes is radically different and mutuallycomplementary. Cholesterol 5, 6-oxide has a trisubstituted oxiranering. All epoxides of this nature tested to date were not, orvery poor, substrates of EH_b. The two enzymes can also effectivelybe discriminated by inhibitors, in that 5, 6-imino-5-cholestane-3ß-olpotently inhibits EH_ch but not EH_b whilst 1, 1, 1-trichloropropeneoxide has the opposite specificity. The cytosolic EH did notsignificantly contribute to the catalysis of the hydrolysisof cholesterol 5, 6-oxide.     

Isolation of {gamma}-glutamyl transpeptidase positive hepatocytes during the early stages of hepatocarcinogenesis in the rat

-Glutamyl transpeptidase (GT) positive hepatocytes were isolatedfrom F344 male rats fed 2-acetylaminofluorene. The isolationprocedure is rapid and highly selective for cells exhibitingGT on their surface. Suspensions of liver cells obtained fromperfusion in situ with a collagenase solution were incubatedon Petri dishes coated with affinity purified rabbit anti-GTantibody. GT-positive cells bound to the dish within fifteenminutes and could be recovered as viable cells. Approximately15% of the GT-positive cells are isolated using this procedure.Novikoff hepatoma cells, a GT-positive cell line, were usedto define the parameters of the assay. The binding was bothtime and temperature dependent. Binding of cells to the anti-GTantibody coated dishes was 50% inhibited by 2.8 mM sodium azideand 86% inhibited by 4.6 mM.     

Autocrine production of TGF-{alpha} and TGF-{beta} during tumour progression of rat oral keratinocytes

This study describes a new technique to separate transforminggrowth factor- (TGF-) and transforming growth factor-ß(TGF-ß) from culture supernatants using ion exchangechromatography; assays of competitive inhibition of ligand bindingwere used to quantify the amount of growth factor. The methodwas simple, inexpensive and did not require large volumes ofculture medium. The autocrine production of TGF- and TGF-ßwas examined in oral keratinocyte cell lines derived from thepalatal and lingual mucosa of rats painted with the carcinogen4-nitroquinoline N-oxide (4NQO). Escape from cellular senescence(immortality) was associated with a marked increase in TGF-production (cell line R2P) but tumour progression, as reflectedby the development of anchorage independence in agarose gelsand tumorigenicity in athymic mice, did not result in a consistentincrease or decrease of TGF- production compared to normals.Four cell lines(R8AP, R1T, R3T, R1P), with different functionalcellular phenotypes, produced two to three times more TGF- thannormals. TGF- production was inversely correlated to epidermalgrowth factor cell surface receptor expression. The autocrineproduction of TGF-ß was variable with the majorityof cell lines producing markedly little TGF-ß threecell lines (R4T, R8BP, R9T) produced more TGF-ß thannormals. The production of TGF-ß was unrelated totumour progression, the expression of TGF-ß cell surfacereceptors or TGF- production. The results indicate that theautocrine production of TGF- and TGF-ß are not accuratemarkers of tumour progression in the rat 4NQO model of oralcarcinogenesis.     

Transforming growth factor-{alpha} and epidermal growth factor expression in the exocrine pancreas of azaserine-treated rats: modulation by cholecystokinin or a low fat, high fiber (caloric restricted) diet

Expression of transforming growth factor- (TGF-) and epidermalgrowth factor (EGF) was studied in normal pancreatic tissueand in (pre)neoplastic pancreatic lesions of azaserine-treatedrats. They were given either a low fat, high fiber (low caloric)diet, to inhibit carcinogenesis, or a low fat diet combinedwith injections of the cholecystokinin analog caerulein to enhancecarcinogenesis. The control groups, maintained on a low fatdiet, were injected with azaserine or were not treated at all.Autopsy was performed at 6 and 15 months after the last azaserineinjection. After both 6 and 15 months immunohistochemistry revealeda weak expression of EGF and TGF- peptides in the acinar cellsand a stronger expression in the ductular and centroacinar cells.TGF- peptide expression was reduced in both putative preneoplasticand neoplastic acinar cell lesions, but no differences in EGFpeptide expression were observed between the various stagesof exocrine pancreatic carcinogenesis. After 16 months an increasein TGF- mRNA due to treatment with azaserine was detected bysemi-quantitative PCR in total pancreatic homogenates, whereasEGF mRNA expression had decreased. TGF- mRNA levels in macroscopicallyisolated tumors were significantly lower, but EGF mRNA levelswere significantly higher, than in total pancreatic homogenatesfrom azaserine treated rats. Furthermore, EGF and TGF- mRNAlevels in isolated tumors did not differ significantly frommRNA levels in non-carcinogen-treated rats. Neither with immunohistochemistrynor with PCR were differences in EGF or TGF- expression observeddue to either inhibition or stimulation of carcinogenesis. Itis concluded that putative preneoplastic acinar cell lesionsinduced in rat pancreas by azaserine may develop into acinaradenocarcinomas independently of TGF- and EGF. The results suggestinvolvement of these growth factors at the early stage of thecarcinogenic process, during the initiation of normal acinarcells into putative preneoplastic cells. However, modulationof azaserine-induced pancreatic carcinogenesis by cholecystokininor a low fat, high fiber (caloric restricted) diet appearednot to be regulated by EGF or TGF-.     

Spectroscopic characterizations and comparisons of the structures of the covalent adducts derived from the reactions of 7,8-dihydroxy-7,8,9,10-tetrahydrobenzo [a]pyrene-9,10-oxide, and the 9,10-epoxides of 7,8,9,10-tetrahydrobenzo[a]pyrene and 9,10,11,12-tetrahydrobenzo[e]pyrene with DNA

The conformation of covalent adducts derived from the reactionsof racemic 7ß,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BaPDE), 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPE),and 9,10-epoxy-9,10,11,12-tetrahydrobenzo[e]pyrene (BePE) withcalf thymus DNA in aqueous buffer solution (25°C, pH 7.0)were investigated and compared by means of absorption, fluorescenceand electric linear dichroism techniques. Two types of conformationsare recognized. Site I is characterized by a red shift (10 nm)in the absorption maximum of the pyrene nucleus, a significantlyreduced fluorescence yield, and a negative electric linear dichroismsignal (A); this site is presumed to involve a near-parallel(within 25°) orientation of the planar pyrene residue withthe planes of the DNA bases, and a relatively strong interactionbetween the electrons of the nucleic acid bases and the pyreneresidue. In site II, there is only a small red-shift in theabsorption maximum (2 nm), a non-zero fluorescence yield, anda positive A throughout the absorption region of the pyreneresidue; in this conformation the pyrene residue is presumedto lie on the outside of the DNA molecule, possibly in one ofthe grooves. The BaPDE-DNA complex displays predominantly asite II-type conformation while the BaPE- and BePE-DNA complexesdisplay both site I and site II adducts, with site I, conformationspredominating. The lack of hydroxyl groups in BaPE and BePElead to a loss in stereospecificity in covalent adduct formation.The 7 and 8 hydroxyl groups in BaPDE appear to reduce the probabilityof formation of site I-type of covalent adducts, and appearto be, at least in part, responsible for the enantiomeric stereospecificityin the covalent reaction between BaPDE and DNA.     

Effects of single doses of irradiation on the expression of resistance-related proteins in murine NIH 3T3 and human lung carcinoma cells

In this report the effects of single doses of ionizing radiationon the mRNA expression of several proteins involved in multipledrug resistance were analyzed. Murine NIH 3T3 cells treatedwith single doses of 5, 10 and 20 Gy during the time intervalfrom 1.5 to 72 h after irradiation were compared with theircorresponding controls at the same points of time. The glutathioneS-transferase- (GST) level was elevated in cells treated with10 or 20 Gy from 24 to 72 h after irradiation compared withthe control. Topoisomerase II and thymidylate synthase weredecreased in irradiated cells 24–72 h after exposure.These down-regulations were associated with cellular proliferation,determined by mRNA expression of the proliferation marker histone3. Irradiated cells exhibited no alteration in the P-glycoproteinor glutathione peroxidase mRNA content. The finding that GSTmRNA was overexpressed after irradiation was validated by investigationson a human lung carcinoma cell line (LXF 289) on the mRNA andprotein level. Thus, our results indicate that irradiation altersthe expression of proteins involved in multidrug resistanceand may, therefore, play a role in clinical drug response.     

Glutathione conjugation and DNA-binding of ({+/-})-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and ({+/-})-7{beta},8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in isolated rat hepatocytes

Isolated rat liver hepatocytes, previously depleted of glutathione(GSH) by treatment with diethylmaleate, were allowed to incorporate[³H]glycine into their GSH. Incubation of ³H-labelled cellswith ¹⁴C-labelled (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene((±)-BP-7,8-dihydrodiol) or (±)7ß,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene(()-BPDE) revealed the formation of double labelled products.This together with evidence from amino acid analysis indicatesformation of GSH-conjugates of the highly carcinogenic BP-derivatives.Incubation of hepatocytes isolated from 3-methylcholanthrene(MC) treated rats with ³H-labelled (±)-BP-7,8-dihydrodiolor (±)-BPDE resulted in binding of radioactivity to DNA.Reduction of the intracellular level of GSH to 40% of the normallevel resulted in an approximate 2-fold increase in the DNA-bindingof either substrate. In addition there was a concurrent decreasein the amount of GSH-conjugates formed. These data clearly demonstratethat GSH participates in conjugation reactions with carcinogenic(±)-BP-7,8-dihydrodiol and (±)-BPDE and that theintracelluilar level of GSH is important in preventing reactiveintermediates from reacting with the DNA in intact cells.     

The influence of cytochrome P-450 induction on the disposition of carcinogenic benzo[a]pyrene 7, 8-dihydrodiol 9, 10-epoxide in isolated cells

The disposition of the carcinogenic (+)-7ß, 8-dihydroxy-9,10-epoxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE]has been studied in isolated hepatocytes obtained from 3-methylcholanthrene-pretreatedrats. In these cells different routes are acting in concertand contribute to diol-epoxide elimination. Conjugation of (+)-anti-BPDEwith glutathione (GSH) and cytochrome P-450c-mediated metabolismof the diol-epoxide to 1- and 3-hydroxy-anti-BPDE (triol-epoxides)appears to be equally important. The reactive triol-epoxidesundergo a number of secondary reactions, including covalentbinding to cellular constituents, e.g. protein and GSH, andhydrolysis to pentahydroxyderivatives. The effective intracellularlifetime of (+)-anti-BPDE is 1 min and comparable to that previouslyobserved in hepatocytes obtained from uninduced animals.     

Comparative histochemical investigation of the glutathione S-transferase placental form and {gamma}-glutamyltranspeptidase during N-nitrosobis(2-hydroxypropyl)amine-induced lung carcinogenesis in rats

Immunohistochemical staining using anti-rat glutathione S-transferaseplacental form (GST-P) rabbit antibody and enzyme histochemicalstaining for -glutamyltranspeptidase (-GT) were investigatedin lesions appearing during lung carcinogenesis induced by N-nitrosobis(2-hydroxypropyl)amine(BHP) in rats. Rats were given BHP at a concentration of 2000p.p.m. in drinking water, and were killed after 12 weeks ofBHP intake, after 12 weeks of BHP intake followed by 12 weeksof tap water intake or after 20 weeks of continuous BHP intake.It was found that bronchiolo-alveolar hyperplasias, adenomas,adenocarcinomas, squamous metaplasias and squamous cell carcinomashad been induced by BHP. All of the squamous metaplasias andsquamous cell carcinomas were shown to stain with GST-P butnot with -GT. On the other hand, the hyperplasias, adenomasand adenocarcinomas stained with -GT to various degrees andin different areas, but did not stain with GST-P. The incidenceof -GT phenotype and the average percentage of -GT positiveareas in hyperplasias and adenomas suggested that adenocarcinomasmight develop from hyperplasias and adenomas. These resultssuggest that GST-P is a marker for squamous lesions while -GTis a marker for adenomatous lesionsin rat lung carcinogenesis.Furthermore, squamous metaplasias appear to be preneoplasticlesions of squamous cell carcinomas while -GT-positive hyperplasiasor adenomas are preneoplastic lesions of peripheral adenocarcinomas.     

Relative merits of immunohistochemical demonstrations of placental, A, B and C forms of glutathione S-transferase and histochemical demonstration of {gamma}-glutamyl transferase as markers of altered foci during liver carcinogenesis in rats

The values of the immunohistochemical demonstrations of glutathioneS-transferases (GSTs) A, B, C and P and histo-chemical demonstrationsof -glutamyl transpeptidase (-GT) for detection of enzyme alteredfoci in F344 rat liver were compared. Rats were given a singlei.p. injection of 200 mg/kg body weight of diethylnitrosamine(DENA), from 2 weeks later they were given 0.02% N-2-fluorenylacetamide(2-FAA), phenobarbital (PB), butylated hydroxyanisole (BHA)or butyl-ated hydroxytoluene (BHT) in their diet for 6 weeksand then they were given basal diet and tap water for 4 weeks.They were subjected to partial hepatectomy at the end of week3. Results showed that immunohistochemical demonstration ofGSTs A, B and C for detection of foci were only effective whenthe administration of 2-FAA, PB, BHA or BHT in the diet wasdiscontinued, because these GSTs were induced in surroundinghepatocytes by these compounds in the diet. -GT was inducedin periportal hepatocytes strongly by BHA and BHT and slightlyby PB, and -GT positive foci in periportal areas were not distinguishablefrom -GT positive peri-portal hepatocytes. GST-P was also inducedmoderately by BHA and slightly by BHT in periportal hepatocytes,but all GST-P positive foci were clearly distinguishable. Inaddition, almost all -GT positive foci gave a positive reactionfor GST-P, but 5–10% of the GST-P positive foci were not-GT positive.     

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-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 applicationof equimolar doses (0.44 and 1.32 mmol/kg body wt.) urines werecollected (48 h) and analyzed for parent compounds, and fornitrosamine metabolites by gas chromatography/Thermal EnergyAnalyzer (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 -fluorinatedanalogue NDBA-F₆, however, urinary and biliary nitrosamine metaboliteswere detected only in trace amounts. This finding demonstratesa strong inhibitory effect of fluorine substitution on oxidationsat , (-1) and ß-positions. Confirmation of this inhibitoryeffect of -fluorine substitution is given from the excretionprofiles of NDBA-F₃ which shows metabolic oxidations only atthe nonfluorinated chain: N-nitroso-3-hydroxybutyl-4, 4, 4,-trifluorobutylamine(3-OH-NDBA-F₃) and N-nitroso-3-carboxypropyl-4, 4, 4-trifluoro-butylamine(BCPN-F₃) were excreted as main metabolites. Our results onmetabolism together with the available data on carcinogenicityof the compounds in the rat strongly support the hypothesisthat -oxidation of one butyl-chain is a prerequisite for theinduction of urinary bladder tumors with NDBA. For the inductionof liver tumors, -C-hydroxylation appears to be the crucialevent.     

Inhibitory effect of {alpha}-tocopherol on the formation of nitrosomorpholine in mice treated with morpholine and exposed to nitrogen dioxide

The possibility that -tocopherol (vitamin E) inhibits the formationof nitrosomorpholine (NMOR) in vivo was investigated in miceorally pretreated with -tocopherol (2.5–100 mg/kg bodywt) once daily for 6 days. Twenty-four hours later, the animalswere injected i.p. with 2 mg of morpholine (MOR) per animalfollowed by exposure txo 47 p.p.m. of NO₂ for 2 h. Under theseconditions, low oral doses of -tocopherol (2.5–5 mg/kgbody wt) significantly decreased NMOR formation in vivo. Astotal body -tocopherol levels increased, in vivo NMOR formationdecreased, and a maximal 50–70% inhibition of NMOR formationoccurred at -tocopherol levels of 5 µg/g body wt. Additionalresults showed that NMOR was rapidly eliminated in mice, sothat studies which measure the levels of NMOR found in animalstreated with MOR and then exposed to NO₂ may underestimate theamount of NMOR that is actually formed. Finally, oral pretreatmentof up to 100 mg of -tocopherol/kg body wt had no effect on NMORelimination.