Growth characteristics and Ha-ras mutations of cell cultures isolated from chemically induced mouse liver tumours

Cells have been isolated from liver tumours that have arisenin control C3H/He mice, in mice given 10 µg diethylnitrosamine(DEN) during the neonatal period or in mice given a diet containingphenobarbitone (PB) to allow a daily intake of 85 mg/kg/day.The cells were grown to the 8° subculture when their growthcharacteristics were investigated in monolayer culture and followingsuspension in soft agar and on transplantation into nude mice.In addition, DNA was isolated from the cultures and from tumoursthat grew in nude mice and analysed for mutations at codon 61of the Ha-ras oncogene. All cells derived from DEN-induced hepatocellularcarcinomas (HCC) demonstrated a lack of density inhibition ofgrowth in monolayer culture, grew in soft agar and formed tumoursin nude mice with an average mean latency of 29 days. Threeof the seven lines showed mutations in Ha-ras: two were CAA     

Biological and biochemical characterization of cell lines derived from initiation-promotion transformed C3H/1OT1/2 cells

A two-stage transformation protocol was used to chemically transformthe mouse embryo fibroblasts, C3H/10T1/2 Cl 8. To initiate thecells 0.37 µM 20-methyicholanthrene was used and 0.17µM of the tumor promoter 12-O-tetradecanoylphorbol-13-acetatewas employed to complete the transformation process. Six weekslater transformed foci were identified and isolated by the ring-cloningtechnique. Altogether eight different foci were trypsinizedresulting in a total of 12 morphologically transformed subclones.Three of these clones, designated TPA 41, TPA 42 and TPA 482,have been characterized in detail. Their growth morphologieswere different. The TPA 482 cells grew in a criss-cross patternwith piled up foci, thus showing a characteristic type III morphology.The TPA 482 clone did not show cell-density growth inhibitionand grew in soft agar. The TPA 41 and TPA 42 clones exhibitedcell-density growth inhibition, grew as monolayers and formedonly few colonies in soft agar. Late passages of the TPA 42clone acquired growth characteristics similar to TPA 482. TheC3H/1OT1/2 Cl 8 and the TPA 41 cells were not tumorigenic whentransplanted into syngeneic mice. TPA 482 cells were stronglytumorigenic, producing tumors in 6/6 mice in 21 days. The TPA42 cells were also tumorigenic, the first tumors appearing after4 weeks; all animals injected with TPA 42 cells had tumors after8 weeks. All tumors observed appeared to be fibrosarcomas. Flowcytometric analysis indicated differences in DNA distributionsbetween tumor cells grown in vitro and the tumors in vivo. Two-dimensionalgel analysis of the total cellular and the nuclear proteinsshowed an increase in the TPA 42 and TPA 482 cells of an acidic48 000 and a basic 83 000 mol. wt polypeptides, and a decreaseof a neutral polypeptide of mol. wt 46 000, located in the nucleusof TPA 482 cells.     

Chemoprevention of bicyclol against hepatic preneoplastic lesions

Oral administration of 100 and 200 mg/kg body weight/day of 4,4-dimethoxy-5,6,5', 6'-dimethylene-dioxy-2-hydroxymethyl-2'-carbonyl biphenyl, Bicyclol, inhibited rat hepatic preneoplastic lesions induced by diethylnitrosamine (DEN). Bicyclol reduced densities of number and area of gamma-glutamyltransferase positive foci, indexes for neoplastic hyperplasia; and also suppressed protein expressions for glutathione S transferase P isoform (GST-P) and alpha-fetal protein and mRNA for N-ras, c-myc and PKCalpha genes. With increases of total microsomal P450 and specific CYP2B1 activities in normal rat liver, Bicyclol enhanced particularly the denitrosation of DEN, a low toxic pathway of metabolism. There is a minor effect of Bicyclol on the deethylation of DEN to produce highly mutagenic metabolites. These results suggest that Bicyclol exists the ability of protecting hepatocytes from the mutagenicity of DEN. Such hypothesis was validated by the observation that Bicyclol inhibited DEN-induced unscheduled DNA synthesis, a DNA damage index, in primary cultured rat hepatocytes. More, in virto Bicyclol inhibited two-stages transformation of mice fibroblastic Balb/c 3T3 cells induced by 3-methylcholanthrene and tetradecanoyl-phorbol 13-acetate (TPA), and blocked the anchorage-independent growth of transformed cells in soft agar. Bicyclol also suppressed TPA-stimulated Balb/c 3T3 cell proliferation in both cell number and 3H-thymidine incorporation. Dot blot indicated that Bicyclol inhibited mRNA expressions of H-ras, c-myc and PKCalpha genes by TPA-stimulation. These data demonstrate that Bicyclol prevents carcinogens-induced animal neoplasm and cell malignant transformation via mechanisms at stages of initiation and promotion. It substantiates those evidences that Bicyclol would be used as potential a chemopreventive agent for hepatocarcinogenesis along with its major therapy against chronic anti-hepatitis.     

Carcinogen-induced unscheduled DNA synthesis in xenotransplanted human tracheobronchial epithelium: comparison with rat tracheal epithelium

Extrapolation from rodent genotoxicity data to humans is complicatedby variables such as interspecies differences in carcinogenmetabolism and DNA repair. A xenograft system containing humanbronchial epithelial cells was used to assess the inductionof unscheduled DNA synthesis (UDS) by carcinogens and to comparethe response with that of rat tracheal epithelium. Cells fromhuman bronchus were grown in explant culture, inoculated intode-epithelialized rat tracheas and implanted subcutaneouslyinto nude mice. Within six weeks, a differentiated mucociliaryepithelium lined the xenografted tracheas. Fresh rat tracheasand human xenografts were cut into rings and incubated in mediacontaining [³H]thymidine and either the direct-acting car cinogen,N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 3–1000 µM),or a carcinogen requiring metabolic activation, 4-nitroquinoline-1-oxide(4-NQO, 3–100 pM). Tissues were then fixed, sectioned,processed for autoradiography and the number of nuclear grains(NG) determined for 100 epithelial cells lining the tracheain each section. A time- and concentration-dependent increasein NG was observed in both human xenografts and rat tracheasafter treatment with MNNG or 4-NQO, indicating induction ofUDS by these agents. The UDS response to MNNG in the human xenograftswas similar to that observed in the rat tracheas, whereas theresponse to 4-NQO was greater in rat tracheas. These studiesindicate that the human xenograft system should have applicationsfor the study of carcinogen-induced damage in normal human bronchialepithelial cells.     

In vitro exposure to cadmium in rat L6 myoblasts can result in both enhancement and suppression of malignant progression in vivo

Cadmium (Cd), a carcinogenic metal in humans and rodents, hasbeen shown to transform cells in vitro. Cd in certain instancescan also be anti-carcinogenic. The effects of Cd have been studiedin different mammalian cell culture systems, where it has beenshown to increase expression of several proto-oncogenes. Inthe present study the ability of Cd to affect malignant transformationwas systematically investigated in L6 cells. Cells were grownin monolayer culture with concentrations of either 0 or 0.5µM CdCl₂ in the medium. Cell cultures treated with Cdfor 9 weeks showed growth of large colonies in soft agar, whileuntreated control cells did not. When injected s.c. into athymicnude mice the 9 week Cd-treated cells gave rise to large, highlymalignant sarcomas, resulting in high host mortality (9 dead/9injected, 100%) by 7 weeks. Mice injected with untreated controlcells also developed tumors, but of significantly smaller sizeand growth rate and associated with a lower host mortality (4/10,40% P     

Genotoxicity of fecapentaene-12 in bacterial and mammalian cell assay systems

Fecapentaene-12 (Fec-12), a compound thought to be responsiblefor much of the mutagenicity in fecal extracts from groups athigh risk for colon cancer, was assayed for genotoxic potentialin a battery of bacterial and mammalian cell short-term assays.This compound demonstrated significant mutagenic activity withSalmonellatyphimurium tester strains TA104, TA100 and TA98, inducing approximately1400, 700 and 100 revertants/µg, respectively. Fec-12caused dose-dependent increases in unscheduled DNA synthesisin both rat hepatocytes and human fibroblasts, indicating itspotential genotoxicity to mammalian as well as bacterial cells.Finally, Fec-12 had the ability to induce neoplastic transformationin mouse BALB/c 3T3 cells in the absence of exogenous metabolicactivation.     

17{beta}-Estradiol-induced cell transformation and aneuploidy of Syrian hamster embryo cells in culture

The ability of 17ß-estradiol to induce morphologicaltransformation of Syrian hamster embryo cells was examined anddose-dependent increases were observed over the concentrationrange of 1—10 µg/ml. However, treatment of the cellswith 17ß-estradiol failed to induce any detectableincreases in gene mutations, chromosome aberrations, sisterchromatid exchanges or unscheduled DNA synthesis. In contrast,over the dose range that was effective in inducing cell transformation,17ß-estradiol induced numerical chromosome changes(both chromosome gains and losses). These findings are similarto the reported observations with the synthetic estrogen, diethylstilbestrol,and support the hypothesis that aneuploidy induction is importantin cell transformation and possibly carcinogenesis induced byestrogens.     

Exposure of mammalian cell cultures to benzo[a] and light results in oxidative DNA damage as measured by 8-hydroxydeoxyguanosine formation

Carcinogenic polycyclic aromatic hydrocarbons induce DNA damagethrough direct covalent interactions with nucleotides of theDNA in cells in which they are activated to ‘ultimatecarcinogenic metabolites’. To determine whether they alsoinduce oxidative damage to DNA under the same circumstances,early passage Syrian hamster embryo and human mammary carcinomacell line MCF-7 cultures were treated for 24 h with 0–5µg/ml benzo[a]pyrene (BaP) or for 1 h with 0–100µM methylene blue (as a positive control for oxidativedamage). The cells were then exposed to fluorescent light for1 or 4 h or retained in darkness. After cell harvest, DNA isolationand enzymatic digestion of the DNA to deoxyribonucleosides,the amounts of 8-hydroxy- 2'-deoxyguanosine (8-OH-dGuo) andunmodified deoxy guanosine present were determined by reverse-phaseHPLC with electrochemical and UV detection respectively. Culturestreated with methylene blue for 1 h followed by light exposurefor 1 h contained 5-fold (10 µM) and 8- to 28-fold (100µM) higher 8-OH-dGuo levels than cells treated with methyleneblue not exposed to light or untreated cells exposed to light.There was no significant change in 8-OH-dGuo levels in culturestreated with 1–5 µg/ml BaP for 24 h in the absenceof light. However, both the human and hamster cell culturestreated with BaP and then exposed to fluorescent light for 4h contained 3-fold (1 µg/ml) and 8- to 10-fold (5 µg/ml)higher 8-OH-dGuo levels than those not exposed to light or nottreated with BaP. These results indicate that BaP treatmentdoes not cause 8-OH-dGuo formation in DNA of cells maintainedin darkness. Exposure of BaP-treated cells to fluorescent lightcauses formation of significant amounts of oxidative DNA damageas measured by 8-OH-dGuo formation. These findings suggest thatoxidative damage of DNA could be involved in tumor inductionby BaP in tissues, such as skin, in which exposure to BaP canoccur In the presence of light.     

Establishment of a spontaneously transformed human fetal lung cell line and its biological characteristics

A spontaneously transformed human fetal lung cell line (HFLT) was derived from a human fetal lung diploid fibroblast cell line (2BS) by continuous culture. The biological characteristics of this cell line were studied and compared with those of 2BS cells. Accompanying the morphological alteration, the growth rate of the transformed cell was accelerated and the maximum cell density was increased. The anchorage-independent growth was shown by its ability to form colonies in soft agar. The collagen synthesis phenotype of 2BS cells was changed. In addition, the increased chromosome number and the nodule formation after heterotransplantation were pathognomonic of malignant transformation.     

Mammary carcinogenicity in female CD rats of a diol epoxide metabolite of fluoranthene, a commonly occurring environmental pollutant

We examined the mammary carcinogenicity in CD rats of anti-2,3-dihydroxy-1,10b-epoxy-10b,1,2,3-tetrahydrofluoranthene (FDE), a genotoxic metabolite ofthe environmental pollutant fluoranthene. FDE (2 µmolor 10 µmol) in 0.1 ml dimethyl sulfoxide (DMSO) was injectedbeneath each of the three left thoracic nipples of groups of20 rats each, with 0.1 ml DMSO alone being injected under theright nipples. On the next day, the procedure was repeated forthe three inguinal nipples on each side. anti-3, 4-Dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]-phenanthrene(BcPDE, 2 µmol per nipple) was used as a positive controland DMSO alone as a negative control. Tumor development wasassessed weekly by palpation and the experiment was terminatedafter 41 weeks. Eighty five percent of the rats in each of theFDE treated groups developed histologically confirmed mammarytumors, compared to 11% in the DMSO treated annuals (P <0.01). Most tumors were on the left side. The lower dose ofFDE induced a significant number of adenomas while the higherdose induced significant incidences of both adenomas and adenocarcinomascompared to controls. BcPDE was a powerful mammary carcinogen,confirming our previous observation. The results of this studydemonstrate the carcinogenicity of FDE to the CD rat mammarygland. Since FDE is a potentially transportable human metaboliteof fluoranthene, its possible role as an etiologic factor inbreast cancer deserves further study.     

Recombinagenic activity of the phorbol ester 12-O-Metradecanoylphorbol-13-acetate in human lymphoblastoid cells

The potent mouse skin tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate(TPA) was examined for its mutagenic and recombinagenic activityat the heterozygous thymidine kinase (tk +/–) locus andthe hemizygous hypoxanthine phosphoribosyltransferase (hprt+/0) locus in the TK6 human lymphoblastoid cell line. TPA atconcentrations of 0.01–1.0 µg/ml induced a low frequencyof tk mutants showing the slow growth phenotype in a dose-dependentmanner, but few normal growth tk mutants or hprt mutants. Concentrationsof 1.0–10 µg/ml TPA induced all three types of mutants.The molecular structure of tk mutants arising spontaneouslyor induced by 1.0 and 10 µg/ml TPA was investigated bySouthern hybridization with a human tk cDNA probe: 86% of allmutants arising after incubation with 10 µg/ml TPA lostthe entire active tk allele, resulting in loss of heterozygosity(LOH), while 71% of spontaneously arising mutants showed LOH.Densitometric analysis indicated that the majority of LOH mutantsinduced by TPA were homozygous at the tk locus (retained twocopies of the mutant allele), consistent with the occurrenceof inter-chromosomal homologous recombination. These resultssupport the hypothesis that tumor promoters such as TPA mayincrease the rate of chromosomal mitotic recombination and hencefacilitate the segregation of recessive mutations. TPA may thusinduce a type of genetic instability during the process of tumorpromotion that involves enhanced recombinagenic activity.     

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.     

Transformation of human breast epithelial cells by chemical carcinogens

The present study was carried out to determine whether humanbreast epithelial cells (HBEC) are transformed by chemicalsthat have been proven to be carcinogenic in other model systems.A spontaneously immortalized human breast epithelial cell line,MCF-10F, was treated with dimethylbenz[a]anthracene (DMBA),benzo[a]pyrene (B[a]P), N-methyl-N-nitrosoguanidine (MNNG) orN-methyl-N-nitrosourea (NMU) for 24 h. MCF-7 and T24 malignantcell lines were used as positive controls. All the carcinogensinduced alterations in both cell morphology and pattern of growth,increased growth rate and anchorage-independent growth in agar—methocel,which became evident by the 8th to 10th passages post-treatment,at     

Aflatoxin B1-induced immortalization of cultured skin fibroblasts from a patient with Li-Fraumeni syndrome

To examine the mechanisms of immortalization in human cells,normal human diploid fibroblasts (WHE-7) and skin fibroblastsfrom a patient with Li-Fraumeni syndrome (MDAH 087) and a mutantp53 allele were treated with aflatoxin B₁ (AFB₁). Exogenousmetabolic activation of AFB₁ with rat liver post-mitochondrialsupernatant (PMS) was used and the optimal treatment conditionsneeded were determined by the inducibility of unscheduled DNAsynthesis. The same degree of cytotoxicity was observed withMDAH 087 cells and normal WHE-7 cells treated with AFB1 at 0.1,0.3 or 1 (µg/ml for 2h with a 2% PMS mixture. All WHE-7cell cultures (AFB₁-treated and controls) failed to escape fromsenescence, whereas three out of nine AFB₁-treated culturesof MDAH 087 cells escaped senescence. MDAH 087 cells treatedwith 0.1 µg/ml of AFB1 two or three times initially decreasedin growth {small tilde}40 days [10 population doublings (PD)]after the first treatment. However, the cells recovered withfaster growth rates after {small tilde}100 additional days andgrew continuously. Both cultures were immortal, defined as continuousgrowth for over 300 PD. Cells treated once with 03 µg/mlof AFB₁ also escaped senescence, although they had about a 230day time lag before restoration of cell growth. The three AFB₁-treated cell lines exhibited altered morphologies, chromosomeaberrations (numerical and structural aberrations) and lossof the wild-type p53 allele. Although immortal, the cells werenon-tumorigenic in nude mice. Spontaneous immortalization ofuntreated MDAH 087 was not observed in this study. The resultsindicate that AFB₁ treatment of cells from a Li-Fraumeni patient,but not cells from normal individuals, can induce immortalization.This model may be useful for studying mechanisms of chemicallyinduced immortalization.     

Human hepatocyte-mediated mutagenesis and DNA repair activity

Combined cultures of human hepatocytes and human fibroblastsconstitute a system composed entirely of normal human cellsthat can be used to investigate the mutagenicity of chemicalsrequiring metabolic activation. Addition of diethylnilrosamine(DEN) to this system resulted in mutations at the hypoxanthine-guaninephosphoribosyltransferase locus of the human fibroblasts. Inseparate experiments with cultures of hepatocytes alone, DENinduced unscheduled DNA synthesis (UDS) in the human hepatocytes.A comparative analysis of UDS and hepatocyte-mediated mutagensisindicates a great degree of similarity between the human andpreviously studied rat hepatocytes in their response to DENin vitro.     

Allyl isothiocyanate is selectively toxic to transformed cells of the human colorectal tumour line HT29

Allyl isothiocyanate, a constituent of mustard and certain vegetablesfound in the human diet, was tested for cytotoxic and cytostaticeffects in HT29 human colon carcinoma cells in vitro. For anexposure time of 24 h, allyl isothiocyanate exhibited a D_q of0.32 µg/ml and a D₀ of 0.74 µg/ml. Following detransformationof the cells by treatment with sodium butyrate or dimethylformamidethe cells became more resistant to the cytotoxic effects ofallyl isothiocyanate, the D_q increasing to 0.74 µg/mland the D₀ to 0.96 µg/ml (with butyrate) or 0.84 µg/ml(with dimethylformamide). At the D_q value for detransformedcells the survival of the control cells was reduced to 56%.Allyl isothiocyanate was also found to be less cytostatic tothe mass growth of detransformed populations in that daily dosesof 1.6 µg/ml over a week reduced the final number of detransformedcells relative to untreated cultures by <25% whilst growthof the transformed cultures was reduced by >60%. Given thisincreased sensitivity of the cells to allyl isothiocyanate whenin the transformed state, it is hypothesized that, when consumedin the human diet, this compound may protect against the developmentof colorectal cancer by selectively inhibiting the growth oftransformed cell clones within the gastrointestinal mucosa.     

The measurement of cisplatin-DNA adduct levels in testicular cancer patients

Seventeen patients with ‘poor prognosis’ non-seminomatoustesticular cancer were monitored for formation of intrastrandbidentate N⁷-d(ApG)- and N⁷-d(GpG)- diammineplatinum adductsin peripheral blood cell DNA during the course of cisplatin-basedchemotheraphy. Adduct values from blood cell DNA samples werecompared with disease response data from the same individuals.Patients who received a dose of 40 mg/m² cisplatin for 5 daysgenerally formed more adducts than patients receiving 20 mg/m²for 5 days, and adduct levels ranged from 0 to approximately300 amol/µg DNA. Among the individuals who achieved acomplete response, the median adduct level was 170 amol/µgDNA and the mean was 162. Among the individuals who achieveda partial response, the median adduct level was 78 amol/ µgDNA and the mean was 83. Comparison of adduct levels betweenresponse groups using the Mann-Whitney test gave a two-sidedP value of 0.072 (one-sided P value 0.036). Of 11 patients forminghigh levels of adduct (>140 amol/µg DNA), 10 achieveda complete response; this compares with two complete respondersin the group of six patients forming low levels (<100 amol/µg DNA) of adduct (P = 0.055, two-sided Fisher exact test).We conclude that cisplatin-DNA adduct formation in peripheralblood cell DNA correlates with the occurrence of complete responsein patients with poor prognosis testicular cancer.     

MOLECULAR EPIDEMIOLOGY AND CANCER PREVENTION: Prepubertal genistein exposure suppresses mammary cancer and enhances gland differentiation in rats

Genistein, a component of soy, was administered to pre-pubertalfemale Sprague-Dawley CD rats and investigated for chemopreventionagainst mammary cancer. Genistein, at 500 µg/g body wtor an equivalent volume of the vehicle, dimethylsulfoxide (DMSO),was injected (s.c) on days 16, 18 and 20 post-partum. At day50 post-partum all animals were exposed to 80 µg dimethylbenz[a]anthracene(DMBA) per g body wt. Animals treated prepubertally with genisteinas compared to DMSO had reduced incidence and significantlyfewer adenocarcinomas per animal. Mammary whole mount analysisshowed that prepubertal genistein treatment resulted in mammaryglands of 50-day-old rats developing fewer terminal end budsand more lobules II. Cell proliferation studies with bromodeoxyuridine(BrdU) showed that terminal end buds from mammary glands of50-day-old females treated prepubertally with genistein hadsignificantly fewer cells in S-phase of the cell cycle. Serumgenistein concentrations in 21- and 50-day-old females followingprepubertal genistein treatment were 4.2 ± 0.6 µMand 102 ± 30 nM, respectively. Animals treated prepubertallywith genistein as compared to vehicle spent more time in theestras phase of the estrus cycle, although all animals did cycle.In 50-day-old females, circulating estradiol-17ß andprogesterone concentrations were not significantly altered bythe prepubertal genistein treatment Oocyte/follicle counts andnumbers of atretic follicles and corpora lutea were not significantlydifferent between the genistein- and vehicle-treated animals.We conclude that genistein treatment during the prepubertalperiod can suppress the development of chemically-induced mammarycancer without significant toxicity to the endocrine/reproductivesystem.     

Inhibition of aflatoxin B1 mutagenesis in Salmonella typhimurium and DNA damage in cultured rat and human tracheobronchial tissues by ellagic acid

Ellagic acid (EA), a plant phenol found in various fruits andnuts, was examined for its ability to inhibit aflatoxin B₁ (AFB₁)mutagenesis in strain TA 100 of Salmonella typhimurium. In thepresence of rat liver S-9 microsomal preparation, EA (1.5 µg/plate)inhibited the number of mutations induced by AFB, (0.5 µg/plate)by 50%. EA at a dose of 1000 µg/plate inhibited the mutationfrequency by >90%. EA was also tested for its ability toinhibit the DNA binding and adduct formation of AFB₁ in culturedexplants of rat trachea and human tracheobronchus. Explantswere incubated in medium containing EA at concentrations of10, 50 and 100 µM for 16 h foUowed by the addition of1 µM [₃H]AFB₁ and EA for 24 h. DNA was isolated by phenolextraction and hydroxylapatite chromatography. EA caused a dose-dependentinhibition in the covalent binding of AFB₁ to the DNA of boththe rat trachea (9—57% inhibition) and human tracheobronchus(24—79% inhibition). After acid hydrolysis of the isolatedDNA, the AFB₁—DNA adducts were separated by h.p.l.c. Intissues from both species, the major AFB₁—DNA adductswere AFB₁-N⁷-Gua [8,9-dihydro-8-(N⁷-guanyl)-9-hydroxyAFB₁] andAFB₁-N⁷-FaPyr (major) [8,9-dihydro-8- (2, 6-diamino-4-oxo-3,4-dihydro-pyrimid-5-ylformamido)-9-hydroxyAFB₁], and the formation of these adductswas reduced by 28—76% in the presence of EA. These dataindicate that EA has the potential to act as a naturally occurringinhibitor of AFB₁-related respiratory damage in rats and inhumans.     

Assessment of chemically-induced DNA repair in rat tracheal epithelial cells

An assay for measuring chemically-induced DNA repair in primarycultures of rat tracheal epithelial (TE) cells has been developedand characterized. Chemical exposure may be either in vitroor in vivo. Epithelial cells were removed from the trachea byprotease digestion, allowed to attach to collagen-coated glassslides, and incubated with [³H]-thymidine. DNA repair was assessedas unscheduled DNA synthesis by quantitative autoradiography.The direct acting genotoxicants methyl methanesulfonate (100µM) and N-methyl-N'-nitro-N-nitrosoguanidine (10 µM)yielded a positive response in vitro. 1, 6-Dinitropyrene (DNP)(0.05 µM) and dimethylnitrosamine (DMN) (1 mM) were alsopositive in vitro demonstrating that TE cells have the capacityto metabolically activate these compounds. 2-Acetylaminofluorene(AAF), aflatoxin B₁ (AFB₁), and benzo[a]pyrene (BP) were allnegative in vitro, suggesting organ specific patterns of metabolicactivation. DMN, which has been shown to induce DNA repair inTE cells following exposure by inhalation, was negative whenadministered by gavage. 1, 6-DNP, BP and AAF did not induceDNA repair or alter the fraction of cells in S-phase when administeredby gavage. Formaldehyde did not induce DNA repair or increasethe fraction of cells in S-phase in TE cells following eitherin vivo exposure by inhalation (0.47, 2, 5.9 or 14.8 p.p.m.for 1, 3 or 5 days) or exposure of the cultured cells in vitro(100 µM). This assay provides the means to assess thegenotoxic potential of environmental chemicals in the epithelialcells of the respiratory system.