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.     

Specific binding of the tumor promoter TPA in various mouse organs as measured by a 'cold acetone-filter assay'

Rapid, specific, saturable and partially reversible bindingof the tumor promoter 12-O-tetradecanoylphorbol-13-acetate ([³H]TPA)to a particulate fraction of mouse brain can be demonstratedby means of a ‘cold acetone-filter assay’; by washingwith cold acetone at -20°C, nonspecific binding of the highlylipophilk [³H]TPA to membranes can be reduced to 10% of thetotal binding. A comparative study of homogenates of severalorgans with this test revealed specific [³H]TPA binding/ µgDNA in the order brain > lung spleen Over kidney thymus ovaries. Specific binding sites were also detected hi 600 xg supernatant fractions of homogenates from epidermis, forestomach,glandular stomach and small intestine. Competition experimentsshowed displacement of [³H]TPA by TPA > phorbol-12,13-didecanoate> 12-deoxyphorbol-13-tetradecano-ate > phorbol-12,13-dibutyrate(PDBu) > phorbol-12,13-diacetate > 4-O-methyl-TPA >4-phorbol-12,13-didecanoate in the brain particulate fraction.Specific [³H]TPA binding is sensitive to heat, periodate anddithiothreitol, but is relatively insensitive to urea or to1–5% solutions of several common detergents. It is estimatedthat the present binding test is 500 times more sensitive thanthe widely-accepted [³H]PDBu assay; perhaps more important,the present method employs TPA, which is extremely effectiveboth as a tumor promoter in vivo and as a pleiotropic effectorof cells in vivo and in vitro.     

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.     

Supplemental administration of 1{alpha}-hydroxyvitamin D3 inhibits promotion by intrarectal instillation of lithocholic acid in N-methyl-N-nitrosourea-induced colonic tumorigenesis in rats

The effect of 1-hydroxyvitamin D₃ [1(OH)D₃) on promotion byintrarectal instillation of lithocholic acid (LC) in N-methyl-N-nitrosourea(MNU)-induced colonic tumorigenesis was studied in a rodentmodel. Ninety-two female F344 rats received intrarectal injectionof 2.5 mg of MNU twice in one week followed by 1 mg of LC orits vehicle alone three times weekly for 48 weeks. Those whichreceived LC were given a concomitant intragastric administrationof 0.04 µg of 1(OH)D₃ or its vehicle alone three timesweekly. In the group receiving MNU alone (n=30) five rats borecolomc tumors; in the MNU + LC group (n=32) 15 and in the MNU+ LC + 1(OH)D₃ group (n=30) six rats bore colonic tumors (MNU+ LC versus MNU + LC + 1(OH)D₃ group, P<0.05). These resultsindicated that promotion of MNU-induced colonic tumorigenesisby LC was suppressed by supplemental administration of 1(OH)D₃.     

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.     

Induction of hepatic glutathione S-transferase activity by butylated hydroxyanisole and conjugation of benzo[a]pyrene diol-epoxide

Dietary administration of 2(3)-tert-butyl-4-hydroxyanisole (BHA)to mice caused an increase in the hepatic soluble glutathioneS-transferase activity towards (±)-7ß, 8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) of 5-fold whereasthat towards 1-chloro-2,4-dinitrobenzene (CDNB) was increasedby 14-fold. Whereas with either substrate the catalytic capacityof the enzyme was elevated by BHA treatment, there was littleeffect on the K_m for CDNB but an increase in the K_m for BPDEas substrates. The results thus suggest that BHA-induced GSHS-transferase activity may be of limited importance for protectionfrom certain reactive intermediates of polycyclic aromatic hydrocarbons.     

Eicosanoids and multistage carcinogenesis in NMRI mouse skin: role of prostaglandins E and F in conversion (first stage of tumor promotion) and promotion (second stage of tumor promotion)

When applied to NMRI mouse skin in vivo, phorbol esters suchas 12-O-tetradecanoylphorbol-13-acetate (TPA) and 12-O-retinoylphorbol-13-acetate(RPA) have been found to induce two early waves of prostaglandinE₂ (PGE₂) synthesis after 15 and 90 min and a delayed accumulationof prostaglandin F₂ (PGF₂) after 2 h. With respect to PGF₂ formationdifferent activities of both agents were observed, in that TPAbut not RPA induced additional PGF waves after 4 and 17 h. Functionally,PGE₂ was previously shown to be an endogenous mediator of theTPA- or RPA-induced epidermal hyperproliferation and hyperplasia.A functional role of PGF could be attributed to the post-initiationstages of tumor development in initiated mouse skin, i.e. theconversion stage (stage I of tumor promotion) elicited by twoTPA applications and the promotion stage (stage II of promotion)brought about repetitive RPA treatments. PGF₂, appearing asone distinct biosynthetic wave 3–4 h after TPA applicationseems to be critically involved in the conversion steps since(i) inhibition of its accumulation by indomethacin led to aninhibition of tumor formation, (ii) the inhibitory effect ofindomethacin could be reversed by PGF₂ and (iii) RPA was notable to give rise to the accumulation of PGF₂ 4 h after applicationas obtained by TPA treatment. Moreover, RPA-induced promotionof DMBA- and TPA-treated mouse skin was inhibited by indomethacin.The inhibitory effect of indomethacin on papilloma formationwas again reversed by PGF treatment concomitant with RPA.     

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.     

Inhibitory effects of glutathione level-raising agents and D-{alpha}-tocopherol on ornithine decarboxylase induction and mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate

The constituent amino acids of reduced glutathione (GSH), GSHitself, and D--tocopherol inhibited 12-O-tetradecanoylphorbol-13-acetate(TPA)-induced ornithine decarboxylase (ODC, L-omithine carboxy-lyase,EC 4.1.1.17 [EC] ) activity in mouse epidermis in vivo and in vitro.The inhibitory effects of cysteine (Cys), GSH and D--tocopherolon ODC induction were proportional to their abilities to decreasethe incidence of skin tumors in the initiation-promotion protocol.Moreover, the ability of the constituent amino acids of GSHand GSH to inhibit TPA-induced ODC activity correlated wellwith their ability to increase the ratio of GSW/oxidized glutathione(GSSG) in isolated epidermal cells. In vitro, various treatmentswith 1 mM GSH, 1 mM glutamic acid (Glu), 1 mM glycine (Gly),0.4 mM Cys and/or 0.2 mM cystine (CysCys) inhibited dramaticallythe sharp decline in the intracellular ratio of GSH/GSSG causedby 0.1 µM TPA. Since the inhibitory effects of Cys onboth the decrease in the ratio of GSH/GSSG and the inductionof ODC activity by TPA were greatly reduced by the inhibitorsof -glutamyl transpeptidase and -glutamylcysteine synthetase,it is suggested that some of the inhibitory effects of Glu,Cys and Gly on tumor promotion could result from their interferencewith the metabolism of the tripeptide GSH, a natural antioxidantwhich inhibits chemical carcinogenesis. The free radical scavengerD--tocopherol, which did not alter directly the intracellularratio of GSH/GSSG, also prevented completely the decrease inthe ratio of GSH/GSSG caused by TPA. These results, therefore,suggest that GSH level-raising agents and other antioxidantsmight inhibit by diverse means the effects of TPA on GSH metabolismand skin tumor promotion.     

Typical signature of DNA damage in white blood cells: a pilot study on etheno adducts in Danish mother-newborn child pairs

The impact of DNA damage commonly thought to be involved inchronic degenerative disease causation is particularly detrimentalduring fetal development. Within a multicenter study, we analyzed77 white blood cell (WBC) samples from mother–newbornchild pairs to see if imprinting of DNA damage in mother andnewborn shows a similar pattern. Two adducts 1,N⁶-ethenodeoxyadenosine(dA) and 3,N⁴-ethenodeoxycytidine (dC) were measured by ourultrasensitive immunoaffinity ³²P-post-labeling method. Thesemiscoding etheno-DNA adducts are generated by the reaction oflipid peroxidation (LPO) end products such as 4-hydroxy-2-nonenalwith DNA bases. Mean dA and dC levels when expressed per 10⁹parent nucleotides in WBC-DNA from cord blood were 138 and 354,respectively; in maternal WBC-DNA, the respective values were317 and 916. Thus, the DNA-etheno adduct levels were reliablydetectable and about two times lower in child cord blood, thedifference being significant at P < 0.0004. Analysis of dAand dC levels in cord versus maternal blood WBC showed strongpositive correlations (R² 0.9, P < 0.00001). In conclusion,LPO-induced DNA damage arising from endogenous reactive aldehydesin WBC of both mother and newborn can be reliably assessed bydA and dC as biomarkers. The high correlation of etheno adductlevels in mother and child WBC suggests that a typical signatureof DNA damage is induced similarly in fetus and mother. Prospectivecohort studies have to reveal whether these two WBC-DNA adductscould serve as risk indicator for developing hematopoietic cancersand other disorders later in life. Abbreviations: dA, 1,N⁶-ethenodeoxyadenosine; dC, 3,N⁴-ethenodeoxycytidine; LPO, lipid peroxidation; M₁dG, 3-(2-deoxy-D-erythro-pentofuranosyl)pyrimido[1,2-]purin-10(3H)-one; WBC, white blood cell Received September 19, 2008; revised November 13, 2008; accepted November 18, 2008.     

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.     

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-.     

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.     

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.     

Development of monoclonal antibodies specific for 1,N2-ethenodeoxyguanosine and N2,3-ethenodeoxyguanosine and their use for quantitation of adducts in G12 cells exposed to chloroacetaldehyde

Monoclonal antibodies specific for N²3-ethenodeoxyguanosine(N²,3-dGuo) and 1,N²-ethenodeoxyguanosine (1,N²-dGuo) were developed.In a competitive ELISA, 50% inhibition of binding of the N²,3-dGuospecific antibody (ETH1) was achieved with 18 fmol of N²,3-dGuo.Fifty per cent inhibition of the 1,N²-dGuo-specific antibody(ETH2) required 11 pmol 1,N²-dGuo. Immunoassays for N²,3-dGuoand 1,N²-dGuo in single-stranded DNA were developed using theseantibodies. The immunoassays could detect as little as 48 fmolof N²,3-dGuo or 340 fmol 1,N²-dGuo in 25 µg of singlestranded DNA. These assays and previously developed immunoassaysfor 1,N⁶-ethenodeoxyadenosine (1,N⁶-dAdo) and 3,N⁴-ethenodeoxycytidine(3,N⁴-dCyd) were used to measure etheno adduct levels in DNAof cells exposed to chloroacetaldehyde. The cells used wereV79 cells with an inactivated hprt gene and a single copy ofthe bacterial gpt gene (G12 cells). The most abundant ethenoadduct was 1,N⁶-dAdo, followed by 3,N⁴-dCyd and N²,3-dGuo. 1,N²-dGuowas not detected in chloroacetaldehyde-treated G12 cells. Chloroacetaldehydewas also shown to be mutagenic in these same cells.     

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

Immunohistochemical staining using anti-rat glutathione S-transferaseplacental form (GST-P) rabbit antibody and enzyme histochemicalstaining for -glutamyltranspeptidase (-GT) were investigatedin putative preneoplastic lesions and adenocarcinomas in thepancreas of Syrian golden hamsters treated with N-nitrosobis(2-hydroxypropyl)amine(BHP). Areas with ductular proliferation, ductal hyperplasia,and intraductal carcinoma were strongly positive for GST-P bindingand negative for -GT. Cystic adenoma, microcarcinoma, and carcinomaswere constantly positively stained by GST-P and partially positivefor -GT. GST-P appears to be useful as a positive marker forputative preneoplastic lesions in pancreatic carcinogenesis.Since normal acinar cells are strongly positive for -GT, thefindings might suggest that acinar cells constribute to thedevelopment of cystic adenoma, microcar-cinoma, and carcinomas.     

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.     

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.     

Investigations on the mutagenicity of primary and secondary {alpha}-acetoxynitrosamines with Salmonella typhimurium: activation and deactivation of structurally related compounds by S-9

-Acetoxynitrosamines may serve as model compounds to study mechanismsof action of N-nitrosamines. They are readily cleaved throughhydrolysis, or by esterases, to yield the same ultimate, reactivespecies presumably also arising after metabolic activation ofN-nitrosamines. Structure-activity investigations on -acetoxynitrosaminespromise to aid in elucidating mechanisms involved during theactivation of N-nitrosamines. A series of -acetoxyalkylnitrosamineswas therefore tested for mutagenicity with Salmonella typhimuriumTA 1535. The compounds were readily cleaved, by hydrolysis,to mutagenic intermediates. When comparing compounds accordingto their proposed alkylating properties, unstable secondarya-acetates were considerably more mutagenic than the correspondingrelatively stable primary -acetates. Addition of S-9 mix causedboth activation as well as deactivation in an unexpected structure-relatedpattern. This was so because an exactly opposite influence ofS-9 components on the mutagenicity was observed for each pairof primary and secondary compounds containing the same alkylatingspecies. Furthermore, pairs of compounds with both methylatingand ethylating properties were differently influenced by S-9addition than those with propylating or butylating effects.This dearly demonstrates how different chemical properties ofintermediate forms may strongly influence the biological activityof otherwise quite similar compounds.