Induction of oxygen-dependent lethal damage by monochromatic UVB (313 nm) radiation: strand breakage, repair and cell death

The action of 313 nm radiation in cellular inactivation (biologicalmeasurements) and induction and repair of DNA strand breaks(physical measurements) were studied in a repair proficientstrain and three repair deficient strains (polA, recA, uvrA)of Escherichia coli K-12. Although the induction of breaks waslinear in purified T₄ DNA (6.3 x 10^–4 breaks/2.5 x 10⁹daltons/Jm^–2) and the polA strain (4 x 10^–4 breaks/2.5x 10⁹ daltons/Jm^–2), simultaneous induction and repairof breaks were observed in the uvrA, recA and repair proficientstrains at doses <5 x 10⁴ Jm^–2. The final rates ofinduction in these strains were 1 x 10^–4, 7.5 x 10^–5and 7.5 x 10^–5 breaks/2.5 x 10⁹ daltons/Jm^–2, respectively.A highly efficient polA-dependent repair occurring at 0°Cin minimal buffer and a second slower type of repair occurringat 31°C in the polA strain were detected. Oxygen dependenceof cellular inactivation was observed for the polA and repairproficient strains irradiated at 313 nm thus providing biologicalevidence for an oxygen-dependent lesion involved in lethalityin the short wavelength range of the solar u.v. The lower hypoxicbreak induction rates of the pol4 (1.6 x 10^–4 breaks/2.5x 10⁹ daltons/Jm^–2) and the repair proficient (3.6 x 10^–5breaks/2.5 x 10⁹ daltons/Jm^–2) strains, indicate oxygen-enhancedDNA breakage by 313 nm radiation.     

Effect of indomethacin on N-(4-(5-nitro-2-firyl)-2-thiazolyl)formamide-induced urinary tract carcinogenesis

The effects of indomethacin on the urinary bladder and renalpelvis in rats treated with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide(FANFT) were studied. Two hundred female Sprague-Dawley ratswere divided into four groups. Group 1 received control dietwithout added chemicals. Group 2 was treated with indomethacin(1 mg/kg per day) in the drinking water throughout the experiment.Groups 3 and 4 received 0.2% FANFT in the diet for seven weeksfollowed by control diet. In addition to FANFT, Group 4 receivedindomethacin, 1 mg/kg per day, for the entire experiment. Therats were sacrificed after 92 weeks. There were no urothelialtumors in the control group, one renal pelvic tumor in the indomethacingroup, 4 tumors in the FANFT group and 10 urothelial tumorsin the FANFT ⁺ indomethacin group. The difference between Groups3 and 4 was statistically significant (P < 0.05). Moderateand severe hyperplasia of the renal pelvic and papillary epitheliumwas found in 15 of 48 rats in Group 2 (indomethacin only) ascompared with 6 of 49 control rats (P < 0.05). Moderate andsevere hyperplasia was equally frequent in Groups 3 and 4 (14and 17 animals in each group, respectively). Twenty-four ratsin Group 2 had mammary tumors as compared to 12 animals in Group1 (P < 0.01). Five of the tumors in Group 2 were adenocarcinomas.There was no difference between the number of mammary tumorsin Groups 3 and 4 (36 and 32 animals in each group, respectively).The results suggest that indomethacin enhances FANFT-inducedurinary tract carcinogenesis. Indomethacin also seems to exertsome tumorigenic activity in the mammary gland.     

32P-Post-labeling detection of DNA adducts in mononuclear cells of workers occupationally exposed to styrene

³²-Post-labeling was used to analyze for the presence of DNAadducts in 47 workers exposed to styrene in a boat manufacturingfacility. Individual airborne exposures measured several timesover the course of 1 year ranged from 1to 235 mg/m³ with a meanvalue of 65.6 mg/m³. Two adducts were detected in the DNA ofmononuclear cells of these workers. The following levels ofadducts were detected: adduct 1, range 0.6–102x10^–8(mean 15.8x10^–8); adduct 2, range 0.1–70.9x10^–8(mean 14.2x10^–8). Significant linear relationships werefound between styrene exposure and both DNA adducts (adduct2, r = 0.330, P = 0.012; adduct 1, r = 0.244, P = 0.049). Co-chromatographyexperiments identified DNA adduct 1 in the exposed samplesasN²-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine-3', 5'-bisphosphate.DNA adduct 2 remains unidentified. No significant linear relationshipswere observed between the level of DNA adducts and sister chromatidexchanges, possibly because of the poor precision of the ³²-post-labelingassay (the estimated coefficients of variation for adducts 1and 2 were 2.54 and 1.96, respectively). These results demonstratethat occupational exposure to styrene results in the formationof DNA adducts in human mononuclear cells.     

Mutagenicity of butadiene and its epoxide metabolites: II. Mutational spectra of butadiene, 1,2-epoxybutene and diepoxybutane at the hprt locus in splenic T cells from exposed B6C3F1 mice

The mutagenic potential and mutational spectra of butadiene(BD), 1,2-epoxybutene (EB), and diepoxybutane (DEB) were determinedin splenic T cells from exposed B6C3F1 mice. Mice exposed byinhalation to 625 p.p.m. BD for 2 weeks displayed an averagehprt^– mutation frequency of 6.2 x10^–6 compared to1.2x10^–6 in controls. Mice were also given three dailyi.p. doses of 60, 80 and 100 mg EB/kg or 7, 14 and 21 mg DEB/kg.Average hprt^– frequencies of 5.4x10^–6, 4.lx10^–6and 8.6x10^–6 were seen in the EB groups, respectively,while average frequencies of 4.6x10^–6, 9.4x10^–6and 13x10^–6 were seen in the DEB groups. DNA sequencingrevealed that approximately half of the mutations induced invivo by BD, EB and DEB were frameshift mutations. A +1 frameshift‘hotspot’ in six consecutive guanine bases in exon3 was observed with all three compounds. The remaining mutationsproduced by BD, EB and DEB were transition and transversionmutations at both AT and GC base pairs. Base pair substitutionsinduced by BD were biased in favor of mutation at AT base pairs.The mutational spectra produced by BD, EB and DEB were verysimilar to that observed previously with ethylene oxide, suggestingthat these epoxide agents may be working through a similar mutagenicmechanism.     

A comparative study of the biochemical properties of human and mouse recombinant O6-methylguanine-DNA methyltransferases

The O⁶-methylguanine-DNA methyltransferase (MGMT) repairs mutagenicand carcinogenic O⁶-alkylguanine in DNA by accepting stoichiometricallythe alkyl group from the base. Although the mouse MGMT is largerthan the human protein because of an additional tetrapeptidesequence, these proteins are 70% homologous. Recombinant MGMTsof the human, the mouse and a mouse mutant with the tetrapeptidedeleted were purified to homogeneity from Escherichia coli.The N-terminal amino acid sequences of these proteins are identicalto those predicted from the nucleotide sequences, and theirmolecular masses deter mined by SDS-PAGE agreed with the predictedvalues. However, the observed isoelectric points of 9.3, 9.2and 9.3, for the human, mouse and mutant mouse proteins respectivelywere significantly different from the values, 8.09, 7.47 and7.49 calculated from the amino acid composition. The extinctioncoefficients E_1%^{280 nm} of human, mouse and mutant mouse proteinwere calculated from amino acid composition to be 18.2, 11.1and 11.3 respectively. These values agree fairly well with calculatedvalues. Human and wild-type mouse MGMTs react with the alkylatedbase in a synthetic DNA substrate poly(dC, dG, m⁶dG) with comparablesecond-order rate constants of 2.2x10⁸ and 3.7x10⁸ 1/M/min at37°C respectively and were inactivated by O⁶-benzylguanineat similar rates. The initial reaction rate (K_in) and rate ofinactivation (k_inact) constants for reaction with the base werecalculated to be 1.8x10^–4 M and 1.4x10^–3/s for thehuman protein, 2.3x10^–4 M and 1.1x10^–3/s for thewild-type mouse protein, and 2.1x10^–4 and 1.4x10^–3/sfor the mutant mouse protein respectively. The MGMTs were inactivatedto the extent of 55—65% after heating at 50°C in 20mMTris-HCI, pH 8.0, 1 mM EDTA, 1 mM DTT and 10% glycerol. However,in the presence of DNA (200 µg/ml), only 25—35%of the protein was inactivated. Both DNA and RNA inhibited allthree enzymes in a concentration-dependent fashion, althoughDNA was a better inhibitor than RNA. High salt (0.2 M NaCl)inhibited human MGMT by 80%, while the wild-type and the mutantmouse MGMTs were inhibited by 55%. The human protein had higheraffinity for binding to duplex DNAs than the mouse proteins.     

Relationship between hprt mutant frequency, aromatic DNA adducts and genotypes for GSTM1 and NAT2 in bus maintenance workers

We have studied the mutant frequency in the human gene for hypoxanthine-guaninephosphoribayl transferase (hprt) using the T-cell cloning assay,the aromatic DNA adduct level using the ³²P-postlabelling assay,and related the levels of these biomarkers to the genotypesfor glutathione transferase (GSTµ) and N-acetyltransferase(NAT2) in non-smoking bus maintenance workers exposed to dieselexhaust. No difference in mutant frequency was observed betweenthe 47 exposed (8.6x10^–6, age range 27–45) and the22 control individuals (8.4x10^–6, age range 23–61),while the difference in adduct level (3.2 versus 23x10^–8)was highly significant (P = 0.0009). Both mutant frequency andadduct level were highest in the 16 most heavily exposed workers.Overall, a significant increase of mutant frequency was obsenedwith adduct level (P = 0.008) as well as with age (P < 0.0001).The age dependence was higher in the GSTM1-negative slow acetylators(3.l%year) as compared to the three other genotype combinations(2.4-2.5%/year). There was no signiEicant difference in mutantfrequency or in adduct level between the GSTM1- negative (49.3%of the population) and positive individuals, or between theslow (60.9% of the population) and rapid acetylators. Amongthe slow acetylators, however, a significantly higher adductlevel (P = 0.03) was obtained for the GSTM1-negative individualsas compared to the GSTM1-positive individuals. These resultssuggest a posible role of both GSTµ and NAT2 for individualsusceptibility to carcinogen exposure.     

Induction of sister chromatid exchanges in cultured adult rat hepatocytes by directly and indirectly acting mutagens/carcinogens

Primary cultures of adult rat hepatocytes were tested for theirsuitability to assess sister chromatid exchange (SCE)-inducingDNA damage produced by both directly and indirectly acting mutagens/carcinogens.Compared to other genotoxicity assay systems which utilize themetabolizing activity of liver microsomes, this system is atleast 1–2 orders of magnitude more sensitive. The approximatedrug concentrations leading to a doubling of control SCE levelswere 2.5x10^–4 M for cyclophosphamide, 4.5x10^–5 Mfor dimethylnitrosamine, 2.5x10^–6 M for N-methyl-N-nitro-N-nitrosoguanidine,2x10^–10 M for aflatoxin B₁ (AFB₁) and 30 mJ for u.v. Themost potent inducer of SCE proved to be AFB₁, leading to a significantlyelevated level of exchanges at a concentration of 10^–12M. The increased background SCE levels observed (0.75 SCE/chromosome)appears to reflect the sensitivity of hepatocytes to SCE-inducingDNA damage resulting from the dietary intake of mutagenic/carcinogeniccompounds. In view of the high sensitivity and versatility ofthis genotoxicity assay system, it will be of use for the detectionof the low levels of mutagenic/carcinogenic compounds foundin the environment.     

Characterization and properties of the DNA adducts formed from N-methyl-4-aminoazobenzene in rats during a carcinogenic treatment regimen

Chronic oral administration of the carcinogenic aminoazo dyeN-methyl-4-aminoazobenzene (MAB) to rats is known to resultin the induction of liver tumors. In order to assess the roleof carcinogen-DNA adduct formation in MAB hepatocarcinogenesis,male rats were fed 0.06% [3'-³H]MAB in the diet for 1, 3 or5 weeks. Groups were sacrificed at 0, 24 and 72 h after dosing,and DNA was isolated from the liver and from two non-targettissues, the kidney and spleen. Upon enzymatic hydrolysis ofthe DNA, [³H]aminoazo dye-nucleoside adduct levels in thesetissues were determined by h.p.l.c. Rats concurrently administeredunlabeled MAB for 5 weeks and continued on a control diet for9 months developed hepatocellular carcinomas (16/30 animals).No tumors were observed in 21 rats given only control diets.After chronic administration of [³H]MAB, three major MAB-DNAadducts were found in vivo: N-(deoxyguanosin-8-yl)-MAB (C8-dG-MAB),3-(deoxyguanosin-N²-yl)-MAB (N²-dG-MAB) and 3-(deoxyadenosin-N⁶-yl)-MAB(N⁶-dA-MAB). In addition, several minor products were identifiedas: (i) an (8,9)-purine ring-opened derivative of C8-dG-MABthat may represent an intermediate in DNA repair; (ii) N-guanosin-8-yl-MABwhich is present due to trace RNA contamination; (iii) cis isomersof C8-dG-MAB and N-guanosin-8-yl-MAB, formed by photo-illuminationduring analyses; and (iv) N-(guanin-8-yl)-MAB, a deribosylatedproduct resulting from thermal depurination of C8-dG-MAB. Inaddition, N-(deoxyguanosin-8-yl)-4-aminoazobenzene (C8-dG-AB),a major adduct previously detected in mouse liver after a singledose of 4-aminoazobenzene, was found in rat liver but appearedto be present in significant amounts only after chronic treatmentwith MAB. This product co-chromatographed with N⁶-dA-MAB butcould be removed by selective decomposition in 0.1 N NaOH. Forall tissues examined N²-dG-MAB and C8-dG-MAB were the majoradducts observed with each accounting for 40-50% of the totalcarcinogen bound to DNA in rats that were sacrificed immediatelyafter MAB feeding for 1, 3 or 5 weeks. The levels of total MAB-DNAadducts in the liver were 2–10 times greater than in thekidney or spleen and appeared to increase 2- to 3-fold overthe dosing period. However, by 24–72 h after cessationof MAB treatment, hepatic C8-dG-MAB showed a rapid decline tolevels similar to that found in non-target tissues. The minoradducts, N⁶-dA-MAB and C8-dG-AB, exhibited similar behaviorand never accounted for > 5–10% of the total DNA binding.In contrast, hepatic N²-dG-MAB was a persistent lesion throughoutthe treatment regimen; at 72 h after dosing, it accounted for60–90% of the hepatic DNA adducts and was the only adductwhose levels correlated with target tissue specificity aftera complete hepatocarcinogenic dose of MAB.     

The intensity of vanadium(V)-induced cytotoxicity and morphological transformation in BALB/3T3 cells is dependent on glutathione-mediated bioreduction to vanadium(IV)

Cytotoxicity and morphological transformation has been studiedin BALB/3T3 CI A31–1–1 mouse embryo cells for ammoniumvanadate [vanadium(V)] and vanadyl sulphate[vanadium(IV)] aloneor in combination with diethylmaleate(DEM), a cellular glutathione(GSH)-depleting agent.Cells exposed for 24 h to 10^–5 Mvanadium(V) alone or in combination with 3x10^–6M DEM showedthe characteristic hyperfine EPR signal of vanadium(IV), whichwas more obvious in the case of exposure to vanadium(V) alone.Thissuggests that the amount of vanadium(V) reduced to vanadium(IV)decreased in GSH-depleted cells. While vanadium(IV) at concentrationsof 3x10^–6M and 10^–5 M was not transforming in thecells, vanadium(V) showed neoplastic transforming activity (P<0.025 and P< 0.001 for the two doses, respectively) in comparisonto controls(vanadium unexposed cells). Cytotoxicity and morphologicaltransformation in cells exposed to vanadium(V) in combinationwith 3x^–6M DEM were significantly more intensive (P <0.005 and P < 0.01 for the two doses of vanadate tested)compared to the corresponding values observed in cells exposedto vanadium(V) alone.This suggests that the final transformingactivity response is dependent on the intracellular GSH-mediatedmechanism of reduction of vanadium(V) to vanadium (IV): (i)the extent to which vanadium(V) should be bioreduced to lesstoxic vanadium(IV) via intracellular GSH is a key point in determiningthe intensity of the observed neoplastic action; (ii) the carcinogenicpotential of vanadium(V) should be strictly dependent on itsintracellular persistence which could lead to changes in normalmetabolic patterns of vanadium(V) in the oxidized form due tolack of GSH-mediated reduction.     

DNA adduct formation and assessment of aberrant crypt foci in vivo in the rat colon mucosa after treatment with N-methyl-N-nitrosourea

N-Nitroso-compound DNA adduct formation in vivo and occurrenceof aberrant crypt foci (ACF) were studied in the rat colon mucosaafter a single, local treatment with a carcinogen, N-methyl-N-nitrosourea(MNU), using a simple surgical approach. A segment of F344 ratcolon was ligated to make a pouch and injected with MNU solution.For the study of DNA adduct formation, the solution contained50 µCi of [³H]MNU. The results demonstrated that similarranges of carcinogen dose, i.e. 0.15x10^–2 –1.5x10^–2MMNU, could induce both DNA adduct formation and appearanceof ACF in the rat colon with both parameters showing a nearbylinear dose dependence. HPLC analysis revealed the DNA adductsto include both 7-methylguanine (7-mGua) and O⁶-methylguanine(O⁶-mGua) with the 7-mGua/O⁶-mGua ratio being 8.2–11.3:1in the system used. Assessment of ACF development from 4 to16 weeks after MNU treatment at a dose of 7.5x10^–2 M showedthe numbers to increase up to the 8th week, followed by a decreaseat weeks 12 and 16, when 40% of the ACF counted at the peaktime point were still present. The percentage of large ACF (     

Clinical and Pathological Analyses of Patients with a Family History of Colorectal Cancer

Patients who were registered by the Japanese Society for Cancerof the Colon and Rectum between 1978 and 1983 were examinedclinically and pathologically, in terms of colorectal cancerwith familial accumulation. The incidence of patients with afamily history of colorectal cancer (FH⁺ group)—patientswith adenomatosis coli were excluded—was 6.5% in 15,369colorectal cancer patients. The incidence of patients with afamily history of malignant tumors other than colorectal cancerwas 27.7%. Comparison of the FH⁺ group with the FH^– group(patients without a family history of colorectal cancer) revealedthe incidence of colonic cancer to be significantly higher thanthat of rectal cancer in the FH⁺ group (P<0.01). The patientswith colonic cancer in the FH⁺ group were significantly youngerthan those in the FH^– group (P<0.01), but there wasno age-dependent difference between patients with rectal cancerin the two groups. There was no difference in sex ratio andthere was little difference in the subsite of the primary lesionin the colon between the FH⁺ and FH^– groups. The incidenceof multiple primary colorectal cancer was significantly higherin patients with colonic cancer in the FH⁺ group than in theFH^– group (P<0.01). The incidence of multiple primarycancer in sites other than the colon and rectum was significantlyhigher in the FH⁺ group (P<0.01), but no significant differencewas found in the site of lesions. The prognosis of patientsin the FH⁺ group was significantly better than that of thosein the FH^– group; however, there were no differences inbackground factors such as findings of the primary lesion, statusof metastasis, clinical stage and rate of curative resectionbetween the groups.     

Malignant transformation of mouse BALB/c3T3 cells induced by NaNO2

The addition of sodium nitrite (NaNO₂; 5–20 mM) for 72h to mouse BALB/c3T3 cells resulted in the induction of transformedfoci (type III foci) in a dose-dependent manner. The cells isolatedfrom the NaNO₂-induced transformed foci produced progressivelygrowing tumors when inoculated into nude mice subcutaneouslyat an inoculum size of 1 x 10⁶ cells per site. In contrast,the original untreated cells did not take even at an inoculumsize of 1 x 10⁶ cells per site. The possibility that NaNO₂ mightreact with cellular or medium components to make carcinogenicN-nitrosamines and that these might induce cell transformationwas examined and almost excluded. Thus, nitrite itself seemsto have a cell transforming activity. Recent evidence suggeststhat NO₂^– is produced by the activated macrophage of mammals.We also detected NO₂^– production in culture media in themouse macrophage-like cell Line J774–A1 after lipopolysaccharide(LPS) treatment, and also in the human promyeloleukemia cellline HL60 after differentiation into macrophage-like cells by12-O-tetradecanoyl phorbol-13-acetate and further activationby LPS.     

Production of benzoquinone adducts with hemoglobin and bone-marrow proteins following administration of [13C6]benzene to rats

Adduction of hemoglobin (Hb) and bone-marrow proteins with 1,2-and 1,4-benzoquinone (1,2-BQ and 1,4-BQ) and 4,4'-diphenoquinonewas examined following oral administration of [¹³C₆)benzeneto F344 rats. Linear production of [¹³C₆]1,4-BQ adducts wasobserved with both Hb and bone-marrow proteins over the entirerange of dosages of 0–400 mg/kg. The slopes of the regressionswere 3.4 x 10^–4 (r² = 0.997) and 1.6 x 10^–3 (r²= 0.926) nmol/g protein/mg/kg respectively, for Hb and bone-marrowproteins. Production of [¹³C₆)1,2-BQ adducts of Hb and bone-marrowproteins also increased with benzene dosage. Although the shapesof the relationships between 1,2-BQ adducts and dosage werenonlinear, the levels were     

Spontaneous and 2-nitropropane induced levels of 8-hydroxy-2'-deoxyguanosine in liver DNA of rats fed iron-deficient or manganese- and copper-deficient diets

Rats (Wistar, female, 4 weeks old) were fed iron-deficient (Fe^–;2.2 µg Fe/g) or manganese- and copper-deficient (Mn.Cu^–;0.3 µg Mn/g, 0.4 ug Cu/g) diets for 8 weeks to determinethe oxidative damage of DNA by element deficiency. After feedingof the diets, 2-nitropropane (2-NP, 80 mg/kg body weight) wasadministered i. p. as an inducer of 8-hydroxy-2'-deoxyguanosine(8-OH-dG) to the element-deficient rats. The hemoglobin concentrationof rats in the Fe- group showed an induction of severe anemia(8.4 g/100 ml whole blood). In the Mn.Cu^– group, Mn-super-oxidedismutase (SOD) activities of plasma and Cu.Zn-SOD activitieswere significantly lower than that of the normal diet group.However, total SOD activities of plasma were not depressed severelyin contrast to that of the liver in the Mn.Cu^– group.Background (spontaneous) levels of 8-OH-dG in normal diet groupwere 0.96 + 0.37/105 deoxyguanosine (dG), however, significantlyhigher levels were detected in the Fe^– group (1.56±0.19,P < 0.01). Conversely, a lower (but not significant) levelof 8-OH-dG than the normal diet group were detected in the Mn.Cu^–group (0.78 ±0.08). Six hours after 2-NP treatment,8-OH-dG levels in liver DNA were significantly induced to 1.44+ 0.24 in the normal diet fed group 1.89±0.22 in theFe^– and 1.08±0.12 in the Mn.Cu^– groups. Comparedto the normal diet group, these induced levels of 8-OH-dG inthe Fe-group were significantly higher (P < 0.05), and thatin Mn. Cu^– group were significantly lower (P < 0.05).The high levels of 8-OH-dG in severe iron deficiency might bethe results of (i) an increase of hydroxyl radical generationby accumulated copper in hepatocytes; or (ii) a depression ofenzymatic activity for removing 8-hydroxy-2' -deoxyguanosinein DNA, which is dependent on divalent cations. On the otherhand, the low level of 8-OH-dG in manganese and copper deficiencymight be the result of a decrease of lipid peroxidation whichhas been suggested to be an intermediator from active oxygenspecies to hydroxyl radical.     

Inhibition of gap junctional intercellular communication by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat hepatocytes

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent rodenthepatic tumor promoter. Unlike observations with the majorityof tumor promoting chemicals studied to date, most investigationshave failed to demonstrate down- regulation of gap junctionalintercellular communication (GJIC) in cultured cells by TCDD.The present study examined the effect of TCDD on GJIC in rathepatocytes in primary culture. At non-cytolethal doses TCDDinhibited GJIC In a time- (1, 4, 24 and 48 h) and concentration(1x10^–8–1x10^–14M)-dependent manner. This inhibitionoccurred within 4 h of treatment at doses of 1x10^–81x10–^–12MTCDD and persisted for up to 48 h, despite removal of TCDD.Treatment of rat hepatocytes with TCDD resulted in a decreasein hepatocyte connexin 32 mRNA, but had no apparent effect onconnexin 26 mRNA. Co-incubation of rat hepatocytes with TCDDand     

Biomarkers of styrene exposure in lamination workers: levels of 06-guanine DNA adducts, DNA strand breaks and mutant frequencies in the hypoxanthine guanine phosphoribosyltransferase gene in T-lymphocytes

Occupational exposure to styrene was studied in nine workersof a hand lamination plant in Bohemia. Personal dosimeters wereused to monitor the styrene workplace exposure, and the levelsof styrene in blood and mandelic acid in urine were measured.Blood samples were taken at four occasions during a 7 monthperiod to determine styrene-specific 0⁶-guanine DNA adductsin lymphocytes and granulocytes, DNA strand breaks and hypoxanthineguanine phosphoribosyltransferase (HPRT) mutant frequency inT-lymphocytes. Seven administrative employees in the same factory(factory controls) and eight persons in a research laboratory(laboratory controls) were used as referents. DNA adduct levelsdetermined by the ³²P-postlabelling method in lymphocytes oflamina-tors were remarkably constant and significantly higher(P < 0.0001) than in factory controls at all four samplingtimes. HPRT mutant frequencies (MF) measured by the T-cell cloningassay were higher in the laminators (17.5 x10^–6, groupmean) than in the factory controls (15.7x10^–6, group mean)at three of the four sampling times, but the differences werenot statistically significant. However, a statistically significant(P = 0.021) difference between MF in the laminators (18.0 x10^–6,group mean) and laboratory controls (11.8 xl0^–6, groupmean) was observed at sampling time 4 (the only sampling timewhen this latter group was studied). This result indicates thatstyrene exposure may induce gene mutation in T-cells in vivo.DNA strand breaks were studied by the ‘Comet assay’at the fourth sampling time. The laminators were found to havesignificantly higher levels of DNA strand breaks than the factorycontrols (P = 0.032 for tail length, TL; P = 0.007 for percentageof DNA in tail, T%; and P = 0.020 for tail moment, TM). A statisticallysignificant correlation was also found between the levels oflymphocyte DNA adducts and all three DNA strand break parameters(TL P = 0.046; T% P = 0.026 and TM P = 0.034). On the contrary,no significant correlations were found between DNA adduct levelsand the HPRT mutant frequencies or between the mutant frequenciesand DNA strand breaks. Taken together, these results add furthersupport to the genotoxic and possibly mutagenic effects of styreneexposure in vivo. However, no simple quantitative relationshipseems to exist between the levels of styrene-induced DNA damageand frequency of HPRT mutation in T-lymphocytes.     

Genotoxicity of the food mutagen 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) and analogs

The food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)is an analogue of quinoline, a hepatocarcinogen. 2-Aminofluorene,benzldine and 3,2'-dimethyl-4-aminobiphenyl (DMAB) are potentinducers of unscheduled DNA repair in primary culture rat liverhepatocytes, as was IQ (151 grains/ nucleus at 1 x 10^–6M). Quinoline, on the other hand, is only weakly positive inthis assay (15 grains/nucleus at 1 x 10^–3 M). IQ, quinolineand DMAB were applied topically to shaved skin of Sencar micewith promotion by 12-O-tetradecanoylphorbol 13-acetate (TPA)for 20 weeks, when 14 of 20 mice in the quinoline group had25 tumors, but only one of 30 animals in the IQ group and fiveof 30 in the DMAB group were tumor-bearing. Analogs of IQ synthesizedby substitution at the 2- or 3-position with amino or methylgroups were assayed with the Ames Salmonella typhimurium testerstrains TA98 and TA100. Mutagenicity for TA98 is reduced inthe absence of the 3-methyl group and is completely abolishedwith removal of the 2-amino moiety. None of these analogs arestrong mutagens for TA100. Exocyclic N-oxidation is a likelyobligatory step in the activation of IQ to a mutagen.     

Dose-response studies of MeIQx in rat liver and liver DNA at low doses

2-Amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx) is aheterocyclic amine mutagen found in cooked meats and is carcinogenicin mice and rats at high doses (mg/kg body wt). Humans, however,are exposed to low amounts (p.p.b.) in the diet, and the effectscaused by exposure to human equivalent doses of MeIQx have beendifficult to determine accurately. We report on the effect ofMeIQx exposure on liver bioavailability, hepatic DNA bindingand MeIQx persistence in both liver tissue and liver DNA afteracute (24 h), and subchronic (7 day and 42 day) exposures inmale Sprague-Dawley rats. Male Sprague-Dawley rats were administered[2-¹⁴C] MeIQx either by gavage or in the diet for 1, 7 or 42days (1x10^-6mg/kg day up to 3.4x10^-2 mg/kg day dose) and the[2-¹⁴C]MeIQx was measured by accelerator mass spectrometry (AMS).Assessment of the kinetics of hepatic MeIQx DNA adduct formationover 42 days (1.1x10^-4 mg [2-¹⁴C]MeIQx kg daily dose) showsthat steady-state [2-¹⁴C]MeIQx tissue concentrations of 138± 15 pg/g liver and DNA adduct levels of 113 ±10 ag adduct/µg DNA were reached at 14–28 days and28 days respectively. The relationship between administereddose and either hepatic MeIQx DNA adduct levels or MeIQx tissuelevels are linear for the 24 h, 7 day and 42 day exposures.Furthermore, MeIQx adducts persist for at least 14 days afterexposure ceases. These data suggest that bloavailability andDNA adduction by MeIQx increase linearly with increasing dosefor both acute and subchronic exposures. These data also showthat MeIQx DNA adducts are useful in predicting daily exposureand support a linear extrapolation in the risk assessment ofMeIQx. However, the quantitative relationship between DNA adductsand tumor formation will also depend on the specific tissueand the subsequent steps needed for tumor progression.     

Mammary carcinoma induced by oral administration of ethyl methanesulphonate. Determination of some of the parameters affecting tumor induction

The carcinogenic activity of ethyl methanesulphonate (EMS),an alkylating agent and a potent mutagen, was examined in WistarKing A and Sprague Dawley rats following oral administration.In the Wistar King A rats, mammary carcinomas were detectedin all of the young female rats by the 32nd week after initiationof the experiment. To determine the optimal experimental conditionsfor the rapid and invariable induction of mammary carcinomas,the relationship among age, sex, strain of the rats and concentrationand duration of EMS administration, and tumor production wasinvestigated. The tumor incidence was higher in the youngerfemale rats and the Wistar rats were more susceptible than Sprague-Dawleyrats. Mammary carcinomas were also induced in the younger malerats. In addition, concommitant production of renal tumors occurredby the 40th week in young rats administered 10^–2 M or2 x 10^–2 M of EMS solution, while renal and uterine tumorsdeveloped eoncommitantly in the older female rats given 3 x10^–3 EMS by the 56th week. Histologically, the prevailingfeatures of tumors were infiltrating ductal adenocarcinoma inthe mammary glands, mesenchymal tumor in the kidney, and leiomyosarcomain the uterus. Methyl methanesulphonate, an analogue of EMS,did not induce tumors in any organs.     

Adenylate cyclase activity in crude liver membranes during chemical hepatocarcinogenesis in portacaval shunted rats

Hepatocarcinogenesis was initiated in rats with diethylnitrosamine(DEN) followed by a selection with 2-acetylaminofluorene (2-AAF).Portacaval shunt was then performed in order to promote tumordevelopment. Control rats were not submitted to the initiation-selectionprotocol and were sham-operated. In control rats, adenylatecyclase activity from crude liver membranes was stimulated 7-to 8-fold by maximal doses of glucagon (10^–6 M) or guanyl-5'-yl-imidophosphate[Gpp(NH)p] (10^–3 M), and 17-fold by a maximal (10^–5M) dose of forskolin. Guanosine-5'-O-(2-thiodiphosphate) inhibitedthe response to forskolin (–38%) and to low doses of glucagon(–50%). The initiation-selection protocol increased theactivity in basal conditions and in response to various stimuli.The portacaval shunt did not modify the activity of the enzymewith respect to basal activity or the response to glucagon.It significantly decreased the response to Gpp(NH)p (–45%)and to forskolin (–27%). The initiation-selection protocolincreased the basal activity of the enzyme (+150%) and its responseto Gpp(NH)p (+300%). When tumors developed, the activity ofthe cyclase further increased (+200%) and an inhibitory effectof GTP on the hormone-stimulated enzyme appeared (–40%).From these results, it is concluded that the promotion of hepatocarcinogenesisby portacaval shunt is coupled with modifications in the activityof adenylate cyclase in response to glucagon and guanylnucleotides.