Toxic chemicals in sediments and biota from a creosote-polluted harbor: relationships with hepatic neoplasms and other hepatic lesions in English sole (Parophrys vetulus)

High prevalences of idiopathic hepatic lesions, including neoplasms(e.g., hepatocellular carcinomas, cholangiocellular carcinomas)(27%, 20 of 75 fish) and foci of cellular alteration (putative‘preneoplastic’ lesions) (44%, 33 of 75 fish), werefound in English sole (Parophrys vetulus) exposed to creosote-contaminatedsediments in Eagle Harbor, Puget Sound, WA. Sediments from thecontaminated region of the harbor contained particularly highconcentrations of aromatic hydrocarbons (e.g., benzo[a]pyreneand benz[a]anthracene), and a variety of nitrogen-containingaromatic compounds (e.g., carbazole and acridine). The compositionof the aromatic compounds was characteristic of creosote. Dramaticallylower concentrations of aromatic compounds were found in sedimentsfrom a reference site in which the bottom-dwelling fish examinedwere free of detectable neoplastic or ‘preneoplastic’hepatic lesions. Food organisms in the stomachs of the Englishsole from Eagle Harbor contained substantially higher concentrationsof aromatic hydrocarbons than comparable organisms from thereference site. The concentrations of individual aromatic hydrocarbonsin muscle and liver from the Eagle Harbor fish were low; however,high concentrations of metabolites of aromatic compounds werepresent in the bile. The findings strongly suggest an associationbetween exposure to creosote and the prevalence of hepatic lesions,including neoplasms, in the bottom-dwelling fish, and furthermoresupport the putative role of aromatic hydrocarbons in livercarcinogenesis in fish.     

Lack of significant modulation of the formation and removal of platinum-DNA adducts by aphidicolin glycinate in two logarithmically-growing ovarian tumour cell lines in vitro

Two recently established human ovarian carcinoma cell lines(JA-T and TR175) have been used to study the effects of aphidicolinglycinate (APG), a specific competitive inhibitor of DNA polymerasealpha (Ikegani et al. (1978) Nature, 275, 458–460), onthe formation and removal of four platinum-DNA adducts. Logarithmically-growingcells were exposed to cis-diamminedichloroplatinum(II) (cisplatin)(10 µg/ml, 33.4 µM) in the presence or absence ofAPG (5 or 50 µ/g/ml, 11.6 or 116 µM). Platinum-DNAadducts were quantitated using a competitive ELISA technique.No differences were observed between the initial levels of totalDNA platination and of specific DNA adducts formed in the presenceor absence of APG in either cell line. Following 18 h posttreatmentincubation both lines showed some ability to remove each ofthe three main platinum-DNA lesions (Pt-GMP, Pt-AG and Pt-GG).However, the levels of these specific DNA adducts decreasedover this time period, by similar rates with or without APGaddition. It was also shown that the APG concentrations usedhad minimal inhibitory effects alone on growth or DNA synthesisduring this 18 h posttreatment incubation period. Furthermoreits addition did not significantly modify cisplatin-inducedcyto-toxicity, as judged by Inhibition of growth or DNA synthesisover this time period. We therefore conclude that under theseexperimental conditions APG does not modulate ‘repair’of cisplatin-induced DNA damage in logarithmically-growing culturesof these two apparently ‘repair-proficient’ humanovarian tumour cell lines.     

Association of cigarette smoking and CYP1A1 polymorphisms with adenocarcinoma of the lung by grades of differentiation

We have previously reported on an association of genetic susceptibilityto squamous cell carcinoma of the lung with two polymorphismsof the CYP1A1 gene, an Mspl polymorphism and an isoleucine-valine(Ile-Val) polymorphism. We report here that the two CYP1A1 polymorphismswere associated with current or ex-smokers with adenocarcinomaof the lung. We first compared smoking status of the patientsby differentiation grades of adenocarcinoma. Proportion of currentor ex-smokers and their cigarette consumption was the highestamong the poorly differentiated. Second, the two polymorphismsof CYP1A1 were examined among current or ex-smokers with threedifferentiation grades, and we found that poorly differentiatedadenocarcinoma showed significantly high frequencies of genotypesC and Val/Val, which have been found to be ‘susceptible’in squamous cell carcinoma of the lung. Third, a case-controlstudy was carried out to estimate the genetic risk for CYP1A1genotypes to differentiation grades, selecting only currentor ex-smokers in patients and controls. Current or ex-smokerswith ‘susceptible’ genotype C or Val/Val were atsignificant risk, with an odds ratio of 3.25 or 3.22 (95% confidenceinterval (CI), 1.40–7.56 or 1.00–10.32) for adenocarcinomaas a whole, respectively. The odds ratio increased to 4.51 or4.09 (95% CI, 1.73–11.78 or 1.12–14.91) for poorlydifferentiated, while the odds ratios for other differentiatedgrades were not significant. Finally, a relation of the geneticrisk to cigarette dose levels was evaluated.     

Cyproterone acetate generates DNA adducts in rat liver and in primary rat hepatocyte cultures

Cyproterone acetate (CPA), an active component of certain contraceptiveand antiandrogenic drugs, has been shown recently to induceDNA repair synthesis in rat hepatocytes in vitro. In the presentstudy we examined whether CPA can cause the formation of DNAadducts detectable by the ³²P-postlabeling technique in hepaticcells in vitro and in vivo. Incubation of primary cultures ofhepatocytes from male Wistar rats with CPA resulted in the occurrenceof radioactive spots in the radiochromatograms of ³²P-postlabeledDNA digests indicating the formation of two DNA adducts (‘A’and ‘B’). At 30 µM CPA, the highest concentrationtested,     

High local carcinogenic activity of 1,3-dimethyl-3-phenyl-1-nitrosourea and its inactivation by intravenous application in rats: comparison of in vivo findings with the in vitro direct and a combined in vivo/in vitro sister chromatid exchange assay in V79-E cells

1,3-Dimethyl-3-phenyl-1-nitrosourea (DMPNU) is a very potentlocal carcinogen in rats and induces a 100% frequency of forestomachcarcinomas when applied i.g. in two different dosages (10 applicationsof 0.3 or 0.03 mmol/kg body wt, respectively, at 14-day intervals),but it is inactive upon i.v. administration (10 applicationsof 0.03 mmol/kg body Wt at 14-day intervals). By means of thedirect sister chromatid exchange (SCE) assay in V79-E cellsin the presence of rat blood, serum or plasma, respectively,as well as by a ‘host-mediated’ SCE assay (in whichthe agent was given i.v. to rats, and blood taken from the animalwas checked for the recovery of genotoxic activity in cell cultures),we tried to elucidate the unexpected lack of carcinogenic activityof i.v. DMPNU. The direct SCE assay revealed a drastic reductionof DMPNU genotoxicity by rat blood, serum or plasma, respectively,which is abolished by the esterase inhibitor di-isopropylfluorophosphate.In the ‘host-mediated’ SCE assay a genotoxic activityof DMPNU was only recoverable after a very high i.v. dose andwhen the blood added to the cell cultures had been taken fromthe rat heart within 1 mm after DMPNU administration in vivo.1-Methyl-1-nitrosourea (MNU) and 1-methyl-3-phenyl-1-nitrosourea(MPNU) were used as positive controls in these experiments andalso gave a lower response than theoretically expected, butthe relative loss of activity with the latter compounds wasmuch lower than with DMPNU. It is assumed that an esterase inrat blood effectively decomposes this trisubstituted nitrosourea.Problems of the novel ‘host-mediated’ SCE assayare discussed.     

Biological potential of basophilic hepatocellular foci and hepatic adenoma induced by the peroxisome proliferator, Wy-14,643

The biological potential of hepatic foci and tumors inducedby peroxisome proliferators such as Wy-14,643 has been poorlycharacterized. In this study, male F-344 rats (n = 20/group/timepoint) were fed Wy-14,643 (0.1%) for 22, 37 or 52 weeks (‘W-22’,‘W-37’ or ‘W-52’ respectively). At eachtime point some rats were killed and additional Wy-14,643-fedrats were switched to basal diet (Wy-14,643/‘stopped’)for up to 104 weeks (referred to as ‘W-22/S’, ‘W-37/S’and ‘W-52/S’). Homogeneous basophilic foci, butnot clear cell foci, increased in number and size in W-37 andW-52 rats. In W-37/S rats, clear cell foci replaced basophilicfoci as the most frequent phenotype. In serial section overlays,adenosine triphosphatase deficient foci accounted for only 16%of basophillc foci in W-52 rats and 16% of clear cell foci inW-37/S rats at 52 weeks. The replication of basophilic fociof W-37 rats was markedly Increased (focal labeling index, ELI= 61.8% versus non-focal labeling index, LI = 11.4%; controlLI = 0.8%). Clear cell foci from W-37/S rats at 52 weeks hada ELI of 1.6% (non-focal LI = 0.6%). Hepato cellular adenomaswere increased in W-37 (11/20 rats and 0.8 tumors/rat) and W-52groups (19/20 rats and 2.8 tumors/rat). Prevalence of hepatocellularcarcinomas was elevated in W-52 rats (6/20 rats) but not inW-22 or W-37 rats. Following removal of Wy-14,643, prevalenceof animals with malignant, metastatic hepatocellular carcinomasin W-52/S rats was similar to the prevalence in W-52 rats. However,Wy-14,643-induced adenomas completely regressed in W-37/S andW-52/S groups. In summary, significant morphological continuitybetween highly proliferative basophilic foci and hepatocellulartumors was identified, emphasizing the superiority of basophiliaas a marker for lesious leading to development of hepatocellularneoplasia in rats fed Wy-14,643. An important biological distinctionwas noted between regressive hepatic adenomas and progressivehepatocellular carcinomas induced by a peroxisome proliferator.     

Evaluation of chlordecone in a two-stage model of hepatocarcinogenesis: a significant sex difference in the hepatocellular carcinoma incidence

Chlordecone (Kepone) has been extensively studied for its toxicityin male production workers who were exposed to large quantitiesof this organochlorine pesticide. Concern that these workersmight be at an increased risk of developing liver cancer promptedus to test chlordecone in a two-stage rat model of hepatocarcinogenesis.Chlordecone acted largely as a liver tumor promoter rather thanas a complete hepatic carcinogen in both male and female Sprague—Dawleyrats. Dose—response experiments showed that the hepatocarcinogeniceffects of long-term chlordecone administration became undetectableat concentrations in non-initiated rat liver in the same rangeas those measured in human biopsies taken from exposed workerswho exhibited no liver effects. Although the toxicity of chlordeconein women has never been studied, we found a dramatic sex differencein the incidence of malignant liver tumors caused by chlordeconepromotion in rats. Frank hepatocellular carcinomas were observedin up to 63% of female rats whose livers were previously ‘initiated’with a subcarcinogenic dose of diethylnitrosamine given 24 hafter partial hepatectomy, and then ‘promoted’ by27 weeks of chlordecone administration. In contrast, none ofcomparably treated males had malignant liver tumors, even after44 weeks of ‘promotion’ with chlordecone. Femalesin the diethylnitrosamine-initiated/chlordecone-promotion groupsalso contained -glutamyltranspeptidase-positive ‘preneoplastic’hepatocellular foci that were more abundant and larger thanthose observed in comparably-treated males. Moreover, becausesimilar levels of chlordecone were measured in the livers ofboth sexes at the end of the experimental period, the developmentof hepatocellular carcinomas in the diethylnitrosamine-initiatedfemale rats appeared to be due to their increased sensitivityto the promotion treatment.     

Implications for oxidative and nitrative stress in the pathogenesis of AIDS-related Kaposi's sarcoma

AIDS-related Kaposi's sarcoma (AIDS-KS), which is the most prevalentAIDS related cancer, arises in a unique environment characterizedby profound immunosuppression in conjunction with sustainedimmunostimulation. Persistent inflammation and the accompanyingincreased production of reactive species can promote carcinogenesisby numerous routes including sustained cell proliferation, initiationof nuclear and mitochondrial DNA mutations and induction ofa proangiogenic environment. Furthermore, during conditionsof continuous inflammation, protein nitration can result inirreversible inactivation of enzymes including the cytoprotectiveand reactive species degrading enzyme, mitochondrial superoxidedismutase (MnSOD). Because MnSOD serves as a putative tumorsuppressor gene in addition to its reactive species inactivatingcapacities, the loss of MnSOD's cytoprotective functions couldmarkedly facilitate malignant transformation. The purpose ofthis study was to investigate biochemical and molecular pathwaysby which reactive species facilitate AIDS-KS pathogenesis. Immunohistochemicalstudies of AIDS-KS tumors showed intense AIDS-KS lesional cellstaining for MnSOD, inducible nitric oxide synthase (NOS 2)and the presence of a cellular ‘fingerprint’ ofnitrative stress, 3-nitrotyrosine. Collectively, these resultsthat imply reactive species stress occurs in situ. Similarly,cultured AIDS-KS cells derived from the AIDS-KS tumors containedboth MnSOD protein and the ‘high output’ isoform,NOS 2. Co-localization studies established that the mitochondriaare a primary site for 3-nitrotyrosine localization and immunoprecipitation/immunoblottingexperiments confirmed that MnSOD tyrosine nitration occurs inAIDS-KS cells. Functional SOD assays showed that AIDS-KS cellspossess significantly lower MnSOD activity relative to matchedcontrol cells; findings which correspond with ongoing MnSODtyrosine nitration and subsequent inactivation within AIDS-KScells. These results, which show in situ evidence of reactivespecies stress within AIDS-KS tumors and functional deficitsattributable to nitrative stress in tumor-derived AIDS-KS lesionalcells, imply that reactive species are intimately associatedwith AIDS-KS pathogenesis and provide insights for developmentof novel strategies for AIDS-KS clinical treatments.     

'Tomudex' (ZD1694): A novel thymidylate synthase inhibitor with clinical antitumour activity in a range of solid tumours

Background: Anti-metabolites such as methotrexate (MTX) and5-fluorouracil (5-FU) have been used clinically for many years.Although their effects are partly due to thymidylate synthase(TS) inhibition, they also have non-specific, non TS effectson RNA and purine synthesis. Direct and specific TS inhibitorstherefore presented an attractive research target Collaborativeresearch between the Institute of Cancer Research and ZenecaPharmaceuticals led to the design of specific folate based quinazolineTS inhibitors. ZD1694 ('Tomu-dex'), the first of these drugsreaching advanced clinical development, is currently completingphase III studies. Design: Eight phase II trials were carried out using ‘Tomudex’,3.0 mg/m², given as a short 15-minute infusion 3-weekly. Results: ‘Tomudex’ demonstrates activity in a rangeof tumour types, most notably advanced colorectal and breastcancer (objective response rate 26%) and has acceptable toxicity:the most common WHO grade 3 and 4 adverse events were self-limitingreversible increases in liver transaminases, transient leucopenia,diarrhoea, nausea and vomiting and tiredness or malaise. Mucositis/stomatitis,alopecia and skin toxicity were notable for their low incidenceand mild intensity. Conclusions: ‘Tomudex’ represents the successfulculmination of a rational drug design programme, and shows promiseas a new cytotoxic for the treatment of colorectal cancer. Furtherstudies in other tumour types are planned. colorectal cancer (advanced), thymidylate synthase inhibitor, ‘Tomudex’ (ZD1694)     

The induction of transformed-like morphology and enhanced growth in Syrian hamster embryo cells grown at acidic pH

The effect of the pH, Na⁺ concentration and osmolality of theculture medium on early passage Syrian hamster embryo (SHE)clonal cell proliferation was examined. The pH of the mediumwas adjusted from 6.49 to 7.45 by addition of different amountsof NaHCO₃ to the medium and incubating the cell cultures ina fixed atmosphere of 10% CO₂/90% air. Our results indicatethat clonal SHE cell proliferation is optimal at pH 6.65–6.80 while plating efficiency is independent of pH between 6.65and 7.45. Adjustment of Na⁺ to that concentration in the medium(3450 p.p.m., 0.15 M) of the greatest NaHCO₃addition causeda moderate depression of cell proliferation over the entirepH series. Adjusting the osmolality of the culture medium toa constant value of 338 mOsm/kg did not alter the pH effecton cell proliferation. The pH of the medium also affected cellularand colony morphology. Below pH 6.90 there was an increase inthe number of colonies which exhibited a transformed-like morphology(‘altered’ colonies). The ‘altered’phenotype was characterized by a multilayered, criss-cross patternof growth throughout the colony. This phenotype was stable uponsub-cloning into pH 6.65 medium but was reversible if sub-clonedinto pH 736 medium. The induction of ‘altered’ coloniesat low pH could be partially suppressed by Na⁺ or osmolalityadjustment. These results are discussed in terms of optimizinggrowth conditions for SHE cells in order to enhance their usefulnessfor cell transformation studies. The induction of ‘altered’colonies by low pH is also discussed relative to the involvementof pH regulation in tumor-promoter and growth-factor actionon cells in culture.     

Sister chromatid exchanges and chromosomal aberrations in mouse cells infected with the Abelson and Moloney leukemia viruses

‘Spontaneous’ and mitomycin C (MMC)-induced sisterchromatid exchanges (SCE) and chromatid breaks were scored inANN-1 fibroblasts, a non-producer mouse cell line transformedby the Abelson murine leukemia virus (AMuLV), a replicationdefective retrovirus whose genome contains the v-abl oncogene.Normal, non-transformed NIH3T3 fibroblasts were used as control.SCE and chromatid break frequencies in untreated or MMC-treatedANN-1 and NIH3T3 cells were compared with those observed inthe same cells after infection with the helper murine Moloneyleukemia virus (M-MuLV), which rescues the ability of A-MuLVto replicate in ANN-1 cells. The frequency of spontaneous andMMC-induced SCE were not significantly different in both ANN-1and NIH3T3 cells, independently of M-MuLV infection. After M-MuLVinfection, however, increased ‘spontaneous’ frequencyof SCE and altered susceptibilty to the induction of SCE byMMC was observed in both cell lines compared to M-MuLV-uninfectedcells. In the case of chromatid breaks, the baseline frequencywas not significantly different between the two cell lines bothin the presence or in the absence of M-MuLV infection, nor wasit significantly increased by M-MuLV, with respect to the valueobserved in uninfected cells. These results indicate that, atvariance with what occurs with SCE, viral replication is notneeded to increase the frequency of chromosomal aberrationsand that the portion of A-MuLV genome alone is sufficient to increase chromatid breaks but not SCE in ANN-1 cells. Thus,in mouse cells carrying retroviruses, SCE and chromosomal aberrationsseem to be independently generated, and influenced by differentviral genes.     

The conditions for the modification of radiation transformation in vitro by a tumor promoter and protease inhibitors

These experiments were designed to define the conditions necessaryfor the modification of radiation-induced transformation inC3H/10T1/2 cells by TPA and protease inhibitors. The resultsshow that: (i) the lowest effective dose of various proteaseinhibitors to suppress transformation in vitro varies over severalorders of magnitude; on a molar basis, the inhibitors of chymotrypsinappear to be the most effective protease inhibitors at suppressionof radiation-induced transformation in vitro, (ii) the proteaseinhibitors antipain and the Bowman-Birk (soybean) protease inhibitorhave no effect on radiation transformation when present onlyduring irradiation, (iii) the protease inhibitor antipain cansuppress radiation transformation in vitro when applied to proliferating‘initiated’ cells as late as 10 days and 13 celldivisions post-irradiation, and (iv) TPA treatment followinga 10-day protease inhibitor (antipain) exposure of X-irradiated‘initiated’ cells does not lead to promotion invitro. These results suggest that protease inhibitor treatmentof the initiated cells has irreversibly reverted cells to theiroriginal or ‘uninitiated’ condition which existedbefore irradiation.     

Mutations in the p53 tumor suppressor gene in rat esophageal papillomas induced by N-nitrosomethylbenzylamine

Mutations in thep53 tumor suppressor gene have been implicatedin the pathogenesis of a wide variety of human neoplasms. Thelocation and type of p53 gene mutation can reflect exposureof humans to certain types of carcinogenic agents. Much lessis known about the role of p53 mutational inactivation in rodenttumors. Using both ‘Hot’ (radioactive) and ‘Cold’(non-radioactive) single strand conformation polymorphism (SSCP)analyses, the present study analyzed exons 5–8 and theexon-intron junction of the p53 gene from rat esophageal papillomasinduced by N-nitrosomethylamine (NMBA) for mutations. Nine of30 (30%) esophageal papillomas contained SSCP mobility shifts,principally within exons 5 and 7. These positive SSCP findingswere further validated by direct DNA sequencing analysis. Eightof the nine mutations were G: C     

Awareness of disease among Italian cancer patients: Is there a need for further improvement in patient information?

Background: Studies are available showing that cancer patientsin southern Europe may be less well informed about their diseasethan patients in northern Europe and North America. Patients and methods: In the framework of a survey aimed atexploring the meaning of quality of life for the Italian cancerpatient, carried out all over Italy in a one-week time spanon 6098 consecutive patients, two visual analogue scales evaluatingseverity and curability of disease were also submitted to thepatients. Four patterns of patients' answers were defined: veryeasy/difficult-to-cure disease, and not-severe/severe disease.Multifactorial analyses were performed using logistic modelsfor each of the four responses, assuming patient characteristics,time since diagnosis and disease extent as explanatory variables. Results: Only 26% of 2088 patients with disseminated diseasebelieved it to be ‘difficult to cure’, while 39%felt it to be ‘easy to cure’. In the same subgroupof patients, only 47% found their disease ‘severe’. Conclusions: Authors were impressed by these unexpected results,which are therefore reported separately from the overall analysisof data, aimed at exploring the quality of life domains forthe Italian cancer patient. In fact, they would suggest a greatlack of awareness of the severity and curability of their diseasein a large group of unselected Italian cancer patients. Thismay depend on various factors, including cross-cultural ones,but could also be partly related to inadequacies in the processby which the Italian patient is informed, and this should befurther investigated. awareness, neoplasms/psychology, quality of life, questionnaires, truth disclosure     

Dose and time dependence of the cellular phenotype in rat hepatic preneoplasia and neoplasia induced by continuous oral exposure to N-nitrosomorpholine

The dose and time dependence of the cellular phenotype in preneoplasticand neoplastic liver lesions was evaluated quantitatively ingroups of male Sprague–Dawley rats continuously exposedto 0, 6, 12 and 24 mg/kg body wt of N-nitrosomorpholine (NNM)and studied at different time points up to 80,50,37 and 20 weeksrespectively. Continuous oral administration of NNM resultedin a dose- and time- dependent increase in the total numberand volume of preneoplastic foci of altered hepatocytes (FAH)and in the incidence of hepatocellular adenomas (HCA) and carcinomas(HCC) at all dose levels studied. In contrast to earlier stopexperiments with 24 mg NNM/kg body wt, there was no reversion-linkedphenotypic Instability but a rapid progression of FAH of themixed cell type to a high incidence of hepatocyte nodules andHCC after continuous treatment with NNM at this dose level.At the two lower dose levels of NNM (12 and 6 mg/kg) the well-knownsequence of cellular changes leading from glycogenotic (clear,combined clear/acidophllic and acidophilic) to mixed and diffuselybasophilic cell populations, in which HCC prevailed. A considerablepart of the glycogenotic foci contained exclusively acidophiliccells, and HCA consisting of a mixture of acidophilic and basophiliccells were the most common benign tumour type In these groups.At the end of the observation period there was also a high incidence(>50%) of HCC at both dose levels, indicating the potentialof persistent nodules (HCA) containing acidophilic cells toprogress to HCC. From and HCA exhibiting a tigroid cell patternappeared only at the two lower dose levels, but for this typeof HCA it remained open whether It may progress to HCC. Froma comparison of the different dosing regimens of NNM studiedin this and previous experiments we conclude that the rapid,potentially reversible shift from glycogenotic to mixed cellpopulations at the highest dose level of continuously appliedNNM (24 mg/kg) and the high proportion of pure acidophilic glycogenstorage foci observed after continuous administration of NNMat the two lower dose levels (6 and 12 mg/kg) represent differentphenotypic expressions of promoting effects exerted by the ongoinginfluence of the carcinogen on FAH initiated by the same compound.The metabolic and molecular changes underlying these ‘initiating’and ‘promoting’ effects of NNM seem to differ interms of quantity rather than quality.     

Enhancement of the skin tumor-initiating activity of polycyclic aromatic hydrocarbons by methyl-substitution at non-benzo 'bay-region' positions

The effect of substituting a methyl group at the non-benzo ‘bay-region’site of several polycyclic aromatic hydrocarbons on skin tumor-initiatingactivity was determined. A methyl group at this position enhancedthe tumor-initiating activity of dibenz[a,h]anthracene, 3-methylcholanthreneand 7-methyldibenz[a,j]anthracene but not of dibenz[a,c]anthracene.2,3,7,8-Tetrachlorodibenzo-p-dioxin was an effective inhibitorof skin tumor initiation by 7, 14-dimethyldibenz-[a,h]anthraceneand 3,6-dimethyicholanthrene. There appears to be a generalrule regarding methyl-substituted hydrocarbons: where a ‘bay-region’exists in a polycyclic aromatic hydrocarbon molecule, methyl-substitutionat the non-benzo ‘bay-region’ site results in enhancedtumor-initiating activity. The effect of methyl substitutionin this position can be most simply explained as due to enhancementof the reactivity of the benzo ring through distortion of thearomatic ring system from planarity. The consequences of thiseffect are discussed.     

Arteriosclerotic plaque development is 'promoted' by polynuclear aromatic hydrocarbons

In previous work we found that weekly injections of the polynucleararomatic hydrocarbon (PAH) carcinogen 7,12-dimethylbenz[a]anthracene(DMBA) induced spontaneous aortic plaques in cockerels to growto a larger size and at a faster rate than plaques in controlanimals. To determine whether plaque-stimulating ability isrelated to carcinogenic potency or mutagenicity we have nowtested a variety of agents, including PAH carcinogens, non-PAHcarcinogens and weakly carcinogenic PAHs. Cockerels were injectedweekly (from 4–20 weeks of age) with one of the followingcompounds: benzo[a]pyrene (B[a]P), benzo[e]pyrene (B[e]P), dibenz[a,h]anthracene (AH), dibenz [a,c]anthracene (AC), 3-methylcholanthrene(MCA), acetylaminofluorene (AAF), N-methyl-N,N'-nitro-nitrosoguanidine(MNNG) or anthracene (ANT). Plaques were present in the abdominalaortas of all animals. Plaque volumes were 8–14 timesgreater in AC-, B[a]P-, B[e]P-, MCA- and AH-treated cockerelsthan in controls. Plaques were slightly larger in the AAF-treatedgroup than in control animals, and in the ANT- and MNNG treatedgroups were indistinguishable in size from plaques in controlanimals. The largest plaque volumes were in AH treated cockerelsand were comparable in size to those elicited by DMBA treatment.The accelerated development of plaques is consistent with a‘promotional’role for these agents. There was a poor correlation betweenmutagenicity or carcinogenicity and plaque ‘promotion’,which may reflect a role for different metabolites in theseprocesses.     

Multi-step metabolism of the carcinogen dibenzo[a,e]fluoranthene. II. Metabolic pathways

The structural identification of nineteen metabolites of dibenzo[a,e]fluoranthene(DBF) obtained by incubation in rat and mouse liver microsomes,allows one to establish a qualitative and semi-quantitativemetabolic chart, involving up to three distinct oxidative attacks.The primary steps lead to dihydrodiols on rings A and D andphenols on rings A and E. Secondary vicinal epoxidation of dihydrodiolsis a minor route as compared to attack at a second peripheralring. Even after a third oxidation, one of the peripheral ringsA, D and E remains unsubstituted. A model for cytochrome P-450enzymatic activity which takes into account most of the observalionsis proposed. It requires that the catalytic site for monooxygenationis 0.6 nm apart from the center of an hydrophobic protein siteaccommodating one of the unsubstituted peripheral beuzenoidrings. Both trans diequatorial dihydrodiols of ring A and Dcorresponding to the ‘bay’ and ‘pseudo bayregion’ of DBF appear in the activation pathways for thein vivo carcinogenesis. The ultimate metabolite reacting withDNA is thus, most probably, a vicinal dihydrodiol epoxide ofring A or D. The great complexity of the metabolic chart ofDBF as compared to other carcinogenic polycyclic aromatic hydrocarbonsleaves also the possibility of sequential reactions at thesetwo distinct sites of the molecule.     

Cytogenetic effects caused by phorbol ester tumor promoters in HeLa cells: mechanistic aspects

The effect of the convertogenic (‘first-stage’)tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate) andthe nonconvertogenic (‘second-stage’) tumor promoterRPA (12-O-retinoylphorbol-13-acetate) on chromosomes was investigatedin HeLa cells which have previously been shown to exhibit aradiomimetic response to TPA. As in the case of mouse keratinocytes,only TPA had a significant clastogenic activity at non-cytotoxicconcentrations ranging from 10^–8 to 10^–6 M measuredafter 24 and 48 h. The values observed with RPA did not differsignificantly from values observed in the presence of the solvent(acetone, 0.2%). The response to TPA was saturable with respectto the dose of TPA. The chromosomal aberrations (mostly gapsand breaks) were predominantly of the chromatid type. Isochromatidaberrations were caused by a 24 or 48 h treatment with 10^–6M TPA. The aberrations appear as early as 6–8 h afterTPA application, i.e. as soon as the cells have recovered fromTPA-induced inhibition in G₂-phase. Even a 30 min exposure to10^–7 M TPA gives the same yield of aberrations as longertreatment, i.e. the response to TPA is ‘saturable’with respect to time. Both TPA and the non-clastogenic RPA causea temporal G₂-delay thus indicating that the G₂-inhibition isnot related to induction of chromosomal aberrations by TPA.The data are consistent with the hypothesis that TPA induceschromosomal aberrations via a receptor-mediated pathway.     

Human tumor cell strains defective in the repair of alkylation damage

We have previously identified four human astrocytoma cell strainsas defective in the repair of N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) damaged adenovirus 5. We now show that two of these strains(the only two tested), in comparison to other tumor strainsor normal human skin fibroblasts, are very sensitive to MNNG-producedkilling as measured by colony forming ability, but are normallysensitive to ultraviolet light. Further, such repair deficientcells may be cultured from tumors of the colon, lung, skin,and neck. The phenotype of deficient repair of MNNG-treatedadenovirus 5 has now been found in a subgroup of 9 of the 39human tumor strains tested. We propose to call this phenotypethe Mer^– phenotype. None of the 22 strains of normal humanskin fibroblasts tested showed deficient repair of MNNG damage.MNNG treatment (80 µM) causes a decrease in semi-conservativeDNA synthesis from which Mer^– tumor cells do not recover,but from which cells capable of normal repair of MNNG damage(Mer⁺) do. Somewhat paradoxically, Mer^–cells show moreMNNG-stimulated DNA synthesis (‘repair synthesis’)than do Mer⁺ cells. Besides being deficient in the repair ofMNNG-damaged adeno-viruses Mer– cells also have difficultyin repairing viruses damaged either by other N-alkyl-N'-nitro-N-nitrosoguanidines,or by N-methyl- or N-ethyl-N-nitrosoureas.