Enhancement of BHA-induced proliferative rat forestomach lesion development by simultaneous treatment with other antioxidants

Synergistic effects of butylated hydroxyanisole (BHA) and otherantioxidants on induction of rat forestomach lesions were investigated.Groups of F344 male rats were treated with 1% BHA phis 0.7%butylated hydroxytoluene (BHT), 1% BHA phis 1% propyl gallate(PG), 1% BHA plus 1% sodium L-ascorbate (SA), 1% BHA phis 1%DL--tocopherol (-TP), 0.4% BHT phis 0.4% BHA plus 0.4% PG plus0.4% SA plus 0.4% -TP, 1% BHA or 2% BHA. Further groups of 10rats each received antioxidants without BHA as controls. Histo-togfcalexamination revealed significantly increased incidences of hyperplasiain the groups given BHA together with SA or PG at the prefundicregion or at the mid region respectively. The forestomach changesinduced by BHA together with SA were equal to those inducedby 2% BHA. On the other hand, simultaneous treatment with BHAand PG or -TP reduced the incidence of hyperplasia at the prefundkregion. It is concluded that mixed treatment with BHA and otherantioxidants exerted enhancing or inhibitory effects on theinduction of hyperplasia at different sites of the forestomachepithelium.     

Promoting activities of butylated hydroxyanisole and butylated hydroxytoluene on 2-stage urinary bladder carcinogenesis and inhibition of {gamma}-glutamyl transpeptidase-positive foci development in the liver of rats

Butylated hydroxyanisole (BHA) and butylated hydroxy-toluene(BHT) were evaluated for possible promoting activity for urinarybladder carcinogenesis in male F344 rats initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine(BBN). The rats were treated with 0.01 or 0.05% BBN in the drinkingwater for 4 weeks and then administered 2% BHA or 1% BHT inthe diet for 32 weeks. Surviving rats were killed at the endof week 36 of the experiment. The incidences of cancer and papillomaand the average number of cancers, papillomas and papillaryor nodular hyperplasias (PN hyperplasias) per 10 cm of basementmembrane were significantly increased in the group receivingBHA following initiation by 0.05% BBN compared with the groupgiven BBN only. BHT also significantly increased these lesionsof the bladder, but not the average number of cancers, in ratstreated with 0.05% BBN. The ability of four antioxidants, BHA,BHT, sodium L-ascorbate (ascorbate) and ethoxyquin, to promotethe induction of -glutamyllranspeplidase (-GT)-positive fociinitiated by diethylnitrosamine (DENA) in the liver of F344rats was tested. Rats were given a single i.p. injection of200 mg/kg body weight of DENA, and 2 weeks later the animalswere exposed to 2% BRA, 1% BHT, 5% ascorbate or 1% ethoxyquin,respectively, in the diet for 6 weeks. All animals were subjectedto partial hepatectomy at the end of week 3. The number of -GT-positivefoci in the groups fed either BHA, BBT or ethoxyquin after DENAwere significantly decreased compared with the control group.These findings show that BRA and BHT are promoters for the urinarybladder carcinogenesis initiated by BBN, but that these andother antioxidants significantly inhibit the induction of -GTpositive foci in the liver.     

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.     

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.     

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.     

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.     

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-.     

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.     

Modifying effects of butylated hydroxyanisole, ethoxyquin and acetaminophen on induction of neoplastic lesions in rat liver and kidney initiated by N-ethyl-N-hydroxyethylnitrosamine

Studies were made on the effects of butylated hydroxyanisole(BHA), ethoxyquin (EQ) and acetaminophen (AAP) on the inductionof neoplastic lesions in the liver and kidney of rats initiatedby N-ethyl-N-hydroxyethylnitrosamine (EHEN). The number andarea of histochemical -glutamyltrans-peptidase-positive (-GT⁺)foci per unit area of liver section in rats given BHA, EQ orAAP were significantly less than in rats given EHEN alone. Similarly,the number of hyperplastic nodules (HN) in groups given BHAor AAP and their area in groups given BHA, EQ or AAP were significantlyless than in control groups. Induction of hepatocellular carcinoma(HCC) was also clearly inhibited by these three chemicals. Noliver lesions were found in any animals given BHA, EQ or AAPorally without EHEN. In contrast, the incidence and quantitativevalues of preneoplastic lesions and renal cell adenoma weresignificantly increased in groups given BHA, EQ or AAP. Theresults clearly demonstrated that BHA, EQ and AAP inhibitedthe development of -GT⁺ foci, HN and HCC, whereas they enhancedthe appearance of preneoplastic and neoplastic lesions in thekidney.     

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.     

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.     

Differential regulation of two microsomal epoxide hydrolases in hyperplastic nodules from rat liver

Two microsomal epoxide hydrolases of the rat liver were foundto be differentially regulated in hyperplastic nodules. Whilstthe activity for substrates of the well-known microsomal epoxidehydrolase with a broad substrate specificity (EHb), benzo[a]pyrene4,5-oxide and androstene oxide (16,17-epoxyandrosten-3-one),was greatly (5-fold) increased in the nodule microsomes andmoderately (2-fold) increased in the surrounding tissue, thatfor the substrate of the novel microsomal epoxide hydrolase,cholesterol 5, 6-oxide (EH_ch) remained unchanged. Since bothenzymes convert endogenous steroid epoxides but with distinctstructural features, this differential regulation may indicatea role of endogenous steroid epoxide(s) of a defined structureduring hepatocarcinogenesis. Alternatively, this differentialregulation may serve as a marker during hepatocarcinogenesis.     

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.     

Induction by butylated hydroxyanisole of specific molecular forms of glutathione S-transferase and UDP-glucuronyltransferase and inhibition of development of {gamma}-glutamyl transpeptidase-positive foci in rat liver

Effects of an antioxidant, butylated (3-tert-butyl-4-) hydroxyanisole(BHA) on the induction of specific molecular forms of glutathioneS-transferase (GST), UDP-glucuronyltransferase (UDP-GT) andother glutathione-related enzymes in rat liver were investigated.The development of -glutamyl transpeptidase (-GTP)-positivefoci and hyperplastic nodules induced by diethylnitrosamine,200 mg/kg i.p., followed by 0.02% N-2-fluorenylacetamide (FAA)in diet plus partial hepatectomy was inhibited by the administrationof 0.75% BHA in the FAA-containing diet. Inhibition was reflectedin decreased area of -GTP-positive foci which correlated witha decrease in -GTP activity measured biochemically. Under thepresent experimental conditions, total activities of GSTs, especiallythat of GST-A form, and of UDP-GTs, especially that of the latefetal form (o-GT), were markedly increased, together with glutathionelevels in the whole liver, within one week after BHA administration.Without BHA administration the activities of GST-A and o-GT,as well as glutathione levels, were also increased by FAA treatment,primarily localized within -GTP-positive foci. These resultssuggest that the induction of specific molecular forms of detoxicatingenzymes either in enzyme-altered foci or in the whole livermay play an important role in determining the extent of developmentof preneoplastic nodules from initiated foci under the shortterm induction conditions used.     

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.     

The metabolism of nitrosodi-n-propylamine, nitrosodiallylamine and nitrosodiethanolamine

Nitrosodiallylamine has been reported to be non-carcinogenicin rats while nitrosodipropylamine and nitrosodiethanolamineare liver carcinogens. That nitrosodipropyiamine is metabolizedat the -position by liver microsomes from Fischer-344 rats supportsthe widely held contention that such metabolism is responsiblefor the carcinogenicity of nitrosamines. Nitrosodiallylamineis also metabolized at the a-position by the same microsomalpreparations. Thus, although -oxidation may be responsible forthe carcinogenicity of some nitrosamines, this mechanism alonecannot account for tumorigenicity. Nitrosodiethanolamine isnot metabolized by rat liver microsomes, but is metabolizedby hepatocytes for Fischer-344 rats. In this case, a mechanismother than the oxidation at the -position may be responsiblefor the carcinogenic action.     

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.