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.     

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.     

TGF-{alpha} and EGF-receptor mRNAs in human oral cancer

Transforming growth factor alpha (TGF-) and epidermal growthfactor receptor (EGFR) have been shown to be present in mostsquamous cell carcinomas. Using the Syrian hamster oral cancermodel, we have recently demonstrated the consistent presenceof TGF- and EGFR mRNAs in chemically transformed hamster oralkeratmocytes. We now present evidence that in human oral cancer(in vivo and in vitro), TGF- and EGFR mRNAs can also be consistentlydetected. No TGF- mRNA can be detected in normal human oralepithelium by Northern blot analysis. These findings reinforcethe use of the hamster cheek pouch as an experimental modelfor the study of oral cancer development, at least in referenceto the possible participation of TGF- in the malignant transformationprocess.     

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.     

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.     

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.     

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.     

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.     

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in human kidney epithelial cells transfected with rat CYP2B1 cDNA

In all species where it has been tested, the tobacco-specificnitrosamine 4-(methylnitrosainino)-l-(3-pyridyl)-l-butanone(NNK) has been shown to be a potent carcinogen, and NNK andother nitrosamines may play a role in human tobacco-relatedcarcinogenesis. Purified rat CYP2B1 has been shown to metabolizeNNK, and the CYP2B1 gene is expressed constitutively in ratlung. The objectives of this study were to test the capacityof CYP2B1, synthesized from a rat hepatic cDNA in Ad293 cells,to metabolize NNK, and to define the type and the proportionsof the final metabolites produced. Ad293 cells were transfectedwith a CYP2B1 expression vector (pMT2-2Bl), or with a controlvector and incubated in culture medium containing [³H]NNK, afterwhich -carbon hydroxylation and pyridine N-oxidation metaboliteswere identified by HPLC analysis and quantitated by scintillationcounting. pMT2-2Bl-transfected cells were capable of catalyzing-carbon hydroxylation and pyridine N-oxidation of NNK, althoughthe reduction product 4-(methylnitrosamino)-l-(3-pyridyIH)-butan-l-ol(NNAL)was the major metabolite formed in cells regardless of transfectiontreatment. The total amount of -carbon hydroxylation metabolitesproduced by pMT2-2Bl-transfected cells was greater than thatof pyridine N-oxidation metabolites. However, pMT2-2Bl transfectedcells produced sim; ten-fold more pyridine N-oxidation metabolitesand only two-fold more -carbon hydroxylation metabolites thancontrol cells. Furthermore, the amount of NNAL-N-oxide was muchlower than that of NNK-N-oxide in the medium of pMT2–2Bl-transfectedcells, even though the amount of available NNAL, resulting fromcarbonyl reduction of NNK, was very high; this suggests thatNNAL is poorly N-oxidized by CYP2B1 compared to NNK. These resultsshow that within living cells NNK was metabolized by CYP2B1via both the pyridine N-oxidation and -carbon hydroxylationpathways. However, CYP2B1 preferentially catalyzed pyridineN-oxidation, which is considered to be a deactivation reaction.     

A combination of palytoxin with 1-oleoyl-2-acetyl-glycerol (OAG) or insulin or interleukin-1 synergistically stimulates arachidonic acid metabolism, but combinations of 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-type tumor promoters with OAG do not

The combination of palytoxin and 1-oleoyl-2-acetyl-glycerol(OAG) synergistically stimulates production of 6-keto-PGF₁ andPGF₂ by rat liver cells (the C-9 cell line). In contrast, thecombination of 12-O-tetradecanoylphorbol-13-acetate (TPA)-typetumor promoters (TPA, dihydroteleocidin B, aplysiatoxin, phorbol-12,13-didecanoate)and OAG does not. Production of 6-keto-PGF₁ by palytoxin addedwith recombinant murine interleukin-1 (IL-1) or with insulinis also greater than the sum of the two effects taken independently.Palytoxin and OAG individually stimulate the release of radio-labeledcompounds from the rat liver cells pre-labeled with [³H]arachidonicacid and also act synergistically to release labeled metabolites.After separation by h.p.l.c., these materials co-chromatographwith authentic 6-keto-PGF₁ and arachidonic acid. The synergisticstimulation by palytoxin and OAG is biphasic; a rapid synergisticproduction of 6-keto-PGF₁ or release of radiolabel from [³H]arachidonicacid prelabeled cells is followed, after 2 –4 h, by aprolonged synergistic response.     

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.     

Initiating activity in a two-stage mouse skin model of nine mutagenic pyrolysates of amino acids, soybean globulin and proteinaceous food

Trp-P-1, Trp-P-2, MeAC, AC, Glu-P-1, Glu-P-2, Lys-P-1, IQ andPhe-P-1 were tested for tumour initiating activity in a two-stageskin carcinogenesis model using 12-O-tetradecanoylphorbol-13-acetate(TPA) as the promoter. The total initiating doses were 20 mgfor Trp-P-1, Trp-P-2, Glu-P-1 and Glu-P-2, 40 mg for MeAC andAC, 5 mg for Lys-P-1, 7.5 mg for IQ and 100 mg for Phe-P-1.7,12-Dimethylbenz[a]anthracene was used as a positive controlcompound at a total dose of 100 µg. All compounds weretopically applied twice weekly for 5 weeks on the dorsal skin,and then followed by similar TPA administration for 47 weeks.Trp-P-2 induced skin tumours in 30% of the mice (0.35 tumours/mouse),and Trp-P-1, MeAC and Phe-P-1 in 10–20% (0.20–0.25tumours/mouse). The smaller amounts of Lys-P-1 and IQ appliedinduced tumours at an incidence of 10 and 5% respectively. Notumours appeared in the groups treated with test chemicals aloneor TPA alone. Statistical analysis according to either the Fisherexact test or Peto trend test revealed significant differencesfor tumour appearance in the Trp-P-1, Trp-P-2, MeAC and Phe-P-1followed by TPA groups as compared with that given TPA alone.The data were used to generate ID₅₀ (50% initiating dose) valuesfor each of the compounds.     

Effects of manganese compounds on carcinogenicity of nickel sub-sulfide in rats

The effects of manganese compounds upon the carcinogenicityof Ni₃S₂ were tested in male Fischer rats. In Experiment I,rats were given i.m. injections of Ni₃S₂ (2.5 mg) and Mn dust(2.0 mg), singly or in combination. By 100 weeks, sarcomas occurredat the injection site in 0 of 24 rats in the vehicle controlgroup, in 0 of 24 rats that received Mn dust alone, and in 23of 24 rats that received Ni₃S₂ alone. Combined administrationof Ni₃S₂ plus Mn dust as a single i.m. injection resulted insarcomas in 14 of 23 rats (p <0.05 versus Ni₃S₂ alone). Inrats that received injections of Ni₃S₂ in one thigh and Mn dustin the opposite thigh, the sarcoma incidence at the site ofNi₃S₂ injection was 24 of 24 rats. In Experiment II, rats weregiven i.m. injections of Ni₃S₂ (1.2 mg) and Mn compounds (MnS,Mn₂O₃, MnO₂ or MN₂(CO)₁₀, in dosages equivalent to 1.0 mg ofMn), singly or in combination. No sarcomas occurred at the injectionsite in rats that received the vehicle or any of the manganesecompounds alone. Sarcomas occurred in 13 of 27 rats that receivedNi₃S₂ alone; this sarcoma incidence was not reduced by admixtureof any of the Mn compounds. The median tumor latent period andthe me- Wan survival period were significantly longer (p <0.05)in rats that received MnS plus Ni₃S₂, compared with rats thatreceived Ni₃S₂ alone, suggesting that MnS may have weak anticarcinogeniceffect. These experiments demonstrate that inhibition of Ni₃S₂-carcinogenesisby Mn dust is a local rather than a systemic effect, and that,with the possible exception of MnS, the other manganese compoundsthat were tested are ineffective as inhibitors of Ni₃S₂-carcinogenesis.     

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.     

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.     

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.     

Studies by fluorescence and n.m.r. spectroscopy of the major product formed after further metabolism of ({+/-})-7{beta}, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

Racemic 7ß, 8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene(anti-BPDE) is further metabolized by liver microsomes obtainedfrom 3-methylcholanthrene pretreated rats in the presence ofNADPH to at least four products as revealed by h.p.l.c. Dataobtained from measurements by fluorescence spectroscopy underneutral and alkaline conditions and high resolution two-dimensional¹H n.m.r. spectroscopy on the major metabolite derived fromanti-BPDE are consistent with aromatic hydroxylation at the3-position either directly or indirectly via transient epoxideintermediates.     

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-.