Formation and persistence of safrole-DNA adducts over a 10 000-fold dose range in mouse liver

The spice constituent safrole (l-allyl-3, 4-methylenedioxy-benzene)and related allylbenzenes form DNA adducts and are rodent carcinogens.This study examined both dose and time dependence of hepaticsafrole-DNA adduct formation over a 10 000-fold dose range upto 30 days after single administration. Female CD-I mice weretreated with safrole i.p. at 0.001, 0.01, 0.1, 1.0, and 10.0mg/mouse in 0.2 ml tricaprylin or with vehicle alone. LiverDNA was analyzed at 0.5, 1, 2, 3, 7, 15 and 30 days via thedinucleotide/mono-phosphate version of the ³²P-postlabelingassay. An 10-fold increase in total safrole adduct levels witheach successive 10-fold increase in dose was observed, givingrelative adduct labeling (RAL) values of 10^-9-10^-5. Each doseelicited identical kinetics of adduct formation, showing peaklevels at 2 days and only slight decreases thereafter. The timecourse of adduct persistence was independent of the dose (0.01–10 mg/mouse). An in vitro experiment established thatthe assay responded in strictly linear fashion to adduct concentrationover a 10 000-fold range, and thus was suitable for in vivodosimetry. DNA synthesis, as measured by [³H]thymidine incorporation,was enhanced only for the 10.0 mg dose at 2, 3 and 7 days. Theseresults indicate a linear response of safrole-DNA adduct formationand persistence in mouse liver following administration of minute(0.001 mg/mouse) to high (10.0 mg/mouse) doses of the carcinogen.     

32P-post-labelling analysis of DNA adducts formed in the livers of animals treated with safrole, estragole and other naturally-occurring alkenylbenzenes. I. Adult female CD-1 mice

The binding of a series of alkenylbenzenes to liver DNA of adultfemale CD-1 mice, isolated 24 h after i.p. administration ofnon-radioactive test compound (2 or 10 mg/mouse), was investigatedby a modified ³²P-post-labelling assay. The known hepatocarcinogens,safrole, estragole and methyleugenol, exhibited the strongestbinding to mouse-liver DNA (1 adduct in 10 000–15 000DNA nucleotides or 200–300 pmol adduct/mg DNA after administrationof a 10 mg dose), while several related compounds, which havenot been shown thus far to be carcinogenic in rodent bioassays,bound to mouse-liver DNA at 3–200x lower levels. The lattercompounds included allylbenzene, anethole, myristicin, parsleyapiol, dill apiol and elemicin. Eugenol did not bind. Low bindingto mouse-liver DNA was also observed for the weak hepatocarcinogen,isosafrole. Two main ³²P-labelled adducts, which appeared tobe guanine derivatives, were detected for each of the bindingchemicals on thin-layer chromatograms. The loss of safrole adductsfrom liver DNA was biphasic: a rapid loss during the first week(t_frac12;{small tilde}3 days) was followed by a much slowerdecline up to 20 weeks after treatment (t_frac12;{small tilde}2.5months). Adducts formed by reaction of 1'-acetoxysafrole, amodel ultimate carcinogen, with mouseliver DNA in vitro werechromatographically identical to safrole-DNA adducts formedin vivo. Pretreatment with pentachlorophenol, a known inhibitorof sulphotransferases, inhibited the binding of safrole to mouse-liverDNA, providing further evidence that the metabolic activationof the allylbenzenes proceeds by the formation of 1'-hydroxyderivatives as proximate carcinogens and 1'-sulphoöxy derivativesas ultimate carcinogens.     

Possible mechanisms for PhIP-DNA adduct formation in the mammary gland of female Sprague-Dawley rats

2-Amino-l-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP), themost abundant heterocyclic amine in fried beef, is a mammarygland carcinogen in rats. Using the ³²PP-postlabeling method,PhIP-DNA adduct levels were measured in mammary epithelial cellsisolated from female Sprague-Dawely rats given 10 daily dosesof PhIP (75 mg/ kg, p.o.) according to a protocol previouslyshown to induce mammary gland cancer. At 24 h, 48 h, 1 weekand 5 weeks after the last dose of PhIP, PhIP-DNA adduct levels[relative adduct labeling (RAL) x 10⁷, mean ± SD] were10.2 ± 0.7, 7.9 ± 2.7, 2.2 ± 0.6 and 0.9± 0.03 respectively. When isolated rat mammary epithelialcells (from untreated rats) were incubated in vitro with N-hydroxy-PhIP(45 µM, 1 h, 37°C), PhIP-DNA adducts were detectedin cell DNA (RAL =     

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.     

DNA adduct levels of 2-amino-l-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) in tissues of cynomolgus monkeys after single or multiple dosing

DNA adducts of 2-amino-l-methyl-6-phenylimidazo[4,5-b]-pyridine(PhIP), a heterocyciic amine derived from cooked meat, weremeasured by the ³²P-postlabeling method in tissues of cynomolgusmonkeys given PhIP. Monkeys received either a single dose ofPhIP (20 mg/kg orally) or nine daily doses of PhIP (20 mg/kgorally, days 1–5 and 8–l1) and tissue samples wereobtained 24 h after the last dose. Over 28 different tissueswere examined for PhIP—DNA adducts. Adducts were detectedin all tissues examined except the fat and bone marrow. Aftera single dose, adduct levels (mean value/10⁷ nucleotides, n= 2 monkeys) were highest in the liver (2.1), followed by thelung (1.7), gall bladder (1.7) and pancreas (0.9). Low adductlevels were detected in the brain and aorta (0.06 and 0.02 respectively).Following multiple doses of PhIP, adduct levels (mean value/10⁷nucleotides ± SE, n = 3 monkeys) were highest in theheart (5.7 ± 2.0) followed by the liver (3.8 ±0.8), submandibular gland (2.7 ± 1.8) and pancreas (2.2± 0.5). Comparison of the adduct levels after a singledose with those found after multiple doses indicates that accumulationof PhlP—DNA adducts occurred in certain tissues. Adductlevels in liver, pancreas, kidney, small intestine and colonincreased about 1.5- to 2.4-fold. PhlP—DNA adduct levelsin submandibular gland and brain increased 4- to 5-fold. Adductlevels in heart increased 10-fold and levels in the aorta increased31-fold. Adducts in white blood cell DNA increased with dailydosing for 9 days. No apparent changes in adduct levels wereseen in the lung, stomach, bladder, muscle and spleen. The widedistribution of PhlP-DNA adducts and their presence in whiteblood cells suggests that there is transport of reactive metabolitesfrom the liver to extrahepatic tissues. The relatively highadduct levels in the gall bladder in comparison with the liversuggests biliary excretion and possible reabsorption of reactivemetabolites. The presence of DNA adducts in tissues implicatesPhIP as a potential carcinogen in non-human primates. The possibilitythat PhlP-DNA adducts in tissues such as the heart and aortamay have toxicological consequences is discussed.     

Comparative study of the formation and repair of O6-methylguanine in humans and rodents treated with dacarbazine

The mutagenic, carcinogenic and cytotoxic activity of dacarbazine,a drug employed in cancer chemotherapy, may be related to theinduction in DNA of O⁶-methyl-guanine (O⁶-meG), a quantitativelyminor but biologically important lesion. In the present studythe kinetics of O⁶-meG formation and repair in blood leukocyteDNA were examined in 20 Hodgkin's lymphoma patients treatedi.v. with 180 ± 13 (mean ± SD) mg/m² dacarbazineand compared with those observed in various tissues of rodentstreated with different doses of the drug. In Hodgkin's lymphomapatients adduct levels reached a value of 0.27± 0.14fmol/µg DNA 2 h after dacarbazine administration, whilethe rate of subsequent loss suggested an adduct half-life of     

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 (     

Carcinogenicity, DNA adduct formation and K-ras activation by 7H-dibenzo[c, g]carbazole in strain A/J mouse lung

N-Heterocyclic polynuclear aromatic hydrocarbons (NHA) are environmentalpollutants formed during the combustion of organic materials.7H-Dibenzo[c,g]carbazole (DBC) is a potent carcinogen in lung,liver and skin. We undertook these studies to determine whethertissue specificity for DBC lung carcinogenicity inthe strainA/J mouse is mirrored by formation of DBC-DNA adducts in lungtissue and whether these adducts are consistent with mutationpatterns in the K-ras gene. Strain A/J mice were given a singlei.p. injectionof DBC at doses of 0, 5, 10, 20 or 40 mg/kg andlevels of DNA adducts in the lung were monitored by ³²P-postlabelingon days 1,3,5,7,14 and 21. The remaining animals were sacrificed8 months after DBC treatment and lung tumor multiplicity andK-ras mutation patterns in the tumors were determined. The lungtumor response to DBC was dose related, with an average of 4.7± 1.2 tumors/mouse at 5 mg/kg and 48.1 ± 5.5 tumors/mouseat 40 mg/kg. As many as seven DBC-DNA adducts were observedin the lung. DNA binding levels in the lung were highest at40 mg/kg, with maximum binding at 5-7 days. At lower dose levelsthe maximum binding to DNA decreased and shifted to earliertime points. The DBC-DNA adduct in the lung with the highestlevel of binding at all dose levels was DBC-DNA adduct 3. Themajority of DBC-induced mutations in the K-ras gene in the lungwere A     

SHORT COMMUNICATION: Detection of DNA adducts in the white blood cells of B6C3F1 mice treated with benzene

We have employed the P₁-enhanced ³²P-postlabeling procedureto detect the formation DNA of adducts in the white blood cells(WBC) of B6C3F1 mice treated by i.p. injection with benzene.Treatment twice a day with 440 mg/kg benzene for 1–7 daysresulted in the formation of one major (adduct 1) and one minor(adduct 2) DNA adduct in the WBCs of mice. The same DNA adductpattern was also found in the bone marrow (BM) of benzene treatedmice. The relative adduct levels were dependent upon both benzenedose from 100–440 mg/kg and treatment time from 1 to 7days. The relative adduct levels ranged between 0.11 and 1.33adducts in 10⁷ nucleotides for WBCs and 0.16–1.21 adductsin 107 nucleotides for BM. Following treatment with benzene,the levels of DNA adducts formed in WBCs were significantlycorrelated with the levels of DNA adducts formed in BM (r² =0.97, P <0.001). Our results suggest that measurement ofDNA adducts in WBCs may be an indicator of DNA adduct formationin BM following BZ exposure.     

DNA adduct formation in B6C3F1 mice and Fischer-344 rats exposed to 1, 2, 3-trichloropropane

1, 2, 3-Trichloropropane (TCP) is a multispecies, multisitecarcinogen which has been found to be an environmental contaminantIn this study, we have characterized and measured DNA adductsformed in vivo following exposure to TCP. [¹⁴C]TCP was administeredto male B6C3F1 mice and Fischer-344 rats by gavage at dosesused in the NTP carcinogenesis bioassay. Both target and nontargetorgans were examined for the formation of DNA adducts. Adductswere hydrolyzed from DNA by neutral thermal or mild acid hydrolysis,isolated by HPLC, and detected and quanti-tated by measurementof radioactivity. The HPLC elution profile of radioactivitysuggested that one major DNA adduct was formed. To characterizethis adduct, larger yields were induced in rats by intraperitonealadministration of TCP (300 mg/kg). The DNA adduct was isolatedby HPLC based on coelution with the radiolabeled adduct, andcompared to previously identified adducts. The isolated adductcoeluted with S-[1-(hydroxymethyl)-2-(N⁷-guanyl)-ethyljglutathione,an adduct derived from the structurally related carcinogen 1,2-dibromo-3-chloropropane (DBCP). Analysis by electrospray massspectrometry suggested that the TCP-induced adduct and the DBCP-derivedadduct were identical. The ¹⁴C-labeled DNA adduct was distributedwidely among the organs examined. Adduct levels varied dependingon species, organ, and dose. In rat organs, adduct concentrationsfor the low dose ranged from 0.8 to 6.6 µmol per mol guanineand from 7.1 to 47.6 µmol per mol guanine for the highdose. In the mouse, adduct yields ranged from 0.32 to 28.1 µmolper mol guanine for the low dose and from 12.2 to 208.1 µmolper mol guanine for the high dose. The relationship betweenDNA adduct formation and organ-specific tumorigenesis was unclear.Although relatively high concentrations of DNA adducts weredetected in target organs, several nontarget sites also containedhigh adduct levels. Our data suggest that factors in additionto adduct formation may be important in TCP-induced carcinogenesis.     

Microsomal and peroxidase activation of 4-hydroxy-tamoxifen to form DNA adducts: comparison with DNA adducts formed in Sprague-Dawley rats treated with tamoxifen

Using rat liver microsomal preparations and peroxidase enzymes,we have investigated the formation of DNA adducts by the antiestrogencompound tamoxifen (TAM) and its metabolite 4-hydroxy-tamoxifen(4-OH-TAM). When reduced nicotinamide-adenine dinucleotide phosphate(NADPH) was used as a cofactor in microsomal activation of either4-OH-TAM or TAM, one DNA adduct and relative DNA adduct levelsof 4.6 and 3.1x10^–8, respectively were detected by 32P-postlabeling.The DNA adduct produced by microsomal activation of 4-OH-TAMand TAM was the same. With cumene hydroperoxide (CuOOH) as thecofactor for the microsomal activation of either 4-OH-TAM orTAM, three to six DNA adducts were produced; the relative adductlevels were 8.0 and 20.6x10^–8, respectively. Comparisonof the DNA adduct patterns produced by 4-OH-TAM and TAM showedthat they were distinct However one of the DNA adducts (a) producedby microsomal activation of 4-OH-TAM using CuOOH was the sameas adduct a produced by microsomal activation of 4-OH-TAM withNADPH. Activation of 4-OH-TAM with horseradish peroxidase resultedin the formation of a single DNA adduct and a relative adductlevel of 20.7x10^–8. Rechromatography analysis of thisDNA adduct showed that it was identical to that produced bymicrosomal activation of 4-OH-TAM with NADPH and one of theadducts produced using CuOOH as the cofactor. Ten DNA adductsand a relative adduct level of 15.3x10^–8 were detectedin the liver of female Sprague-Dawley rats treated daily with20 mg/kg of TAM for 7 days. The DNA adduct pattern in the liverof the treated animals was similar to that produced by microsomalactivation of TAM using CuOOH as the co-factor. The principalDNA adduct (no. 6) formed in the livers of rats treated withTAM was the same as the principal DNA adduct formed followingmicrosomal activation of TAM using CuOOH as a cofactor. TheDNA adduct formed following microsomal activation of eitherTAM or 4-OH-TAM using NADPH was also present as one of the adducts(1^*) formed in vivo following TAM treatment These studies demonstratethat 4-OH-TAM can be activated to form DNA adducts and thatit contributes to the formation of DNA adducts in the liverof rats treated with TAM.     

DNA adduct formation in the bone marrow of B6C3F1 mice treated with benzene

We used P₁-enhanced ³²P-postlabeling to investigate DNA adductformation in the bone marrow of B6C3F1 mice treated intraperitoneallywith benzene (BZ). No adducts were detected in the bone marrowof controls or mice treated with various doses of BZ once aday. After twice-daily treatment with BZ, 440 mg/kg, for 1 to7 days, one major and two minor DNA adducts were detected. Therelative adduct levels ranged from 0.06–1.46x10^–7.In vitro treatment of bone marrow from B6C3F1 mice with variousdoses of hydroquinone (HQ) for 24 h also produced three DNAadducts. These adducts were the same as those formed after invivo treatment of bone marrow with BZ. Co-chromatography experimentsindicated that the principal DNA adduct detected in the bonemarrow of B6C3F1 mice was the same as that detected in HL-60cells treated with HQ. This finding suggests that HQ may bethe principal metabolite of BZ leading to DNA adduct formationin vivo. DNA adduct 2 corresponds to the DNA adduct formed inHL-60 cells treated with 1,2,4-benzenetriol. DNA adduct 3 remainsunidentified. After a 7-day treatment with BZ, 440 mg/kg twicea day, the number of cells per femur decreased from 1.6x10⁷to 0.85xlO⁷, indicating myelotoxicity. In contrast, administrationof BZ once a day produced only a small decrease in bone marrowcellularity. These studies demonstrate that metabolic activationof BZ leads to the formation of DNA adducts in the bone marrow.Further investigation is required to determine the role of DNAadducts and other forms of DNA damage in the myelotoxic effectsof exposure to BZ.     

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.     

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.     

Seasonal variation of DNA adduct pattern in human lymphocytes analyzed by 32P-HPLC

³²P-HPLC is a recently published method to generate DNA adductprofiles after exposure to a complex of genotoxic substances.The low detection limit enables characterization of individualDNA adducts in the general population. The ³²P-HPLC method wasapplied to human lymphocytes and granulocytes from Silesia,apolluted industrial region in the south of Poland. Human sampleswere collected at the end of winter and summer to investigatethe seasonal influence on DNA adduct formation. In lymphocytesa strong seasonal variation was seen in total DNA adducts, withwinter values exceeding the summer values by 7.33±3.56times. Granulocytes did not show any seasonal variation. Inwinter-collected lymphocytes the DNA adduct levels were 21.4±16.6/10⁸normal nucleotides (NN) while the summer values were 2.96±2.46±10⁸NN.Granulocytes had 8.06± 7.76 and 9.59±6.19 DNAadducts/10⁸ NN during winter and summer respectively. The lymphocyteDNA adduct profile consisted of at least 16 individual or clustersof DNA adducts. All 16 had a clear winter influence, with awinter: summer ratio of 1.6–15.3, indicating exposureto a complex mixture of genotoxic substances. The DNA adductsanalyzed in human lymphocytes had retention times similar toDNA adducts generated by poycyclic aromatic hydrocarbons. Thesuggested candidates for DNA adducts displayed a similar seasonalvariation in airborne particles to that found in DNA adductsin lymphocytes of humans living in the area. This is the firstapplication of the ³²P-HPLC method to analysis of DNA adductsin human tissues.     

32P-Postlabeling analysis of liver DNA adducts in rats chronically fed a choline-devoid diet

Liver DNA, obtained at various time intervals from rats chronicallyfed a choline-devoid diet, was analysed for the presence ofaromatic or alkyl adducts by the ³²P-postlabeling assay. Alkyladducts were not detected. Aromatic DNA adduct lesions wererevealed, but only at levels (1 adduct per 0.5–3x10⁹ nucleotides)which are at the limits of the extremely high sensitivity ofthe method used, levels which remained constant throughout theperiod of feeding. Thus, contamination of the total environmentof the animals with chemical carcinogens does not appear tobe responsible for the genesis of the hepatocellular carcinomasthat develop in rats chronically fed a choline-devoid diet.The diet, therefore, either acts as a complete carcinogen, orpromotes the evolution to cancer of endogenous, ‘spontaneously’initiated liver cells.     

Cyclopenta[cd]pyrene-induced tumorigenicity, Ki-ras codon 12 mutations and DNA adducts in strain A/J mouse lung

Cyclopenta[cd]pyrene (CPP) is a ubiquitous cyclopenta-fusedpolycyclic aromatic hydrocarbon. CPP is highly genotoxic inbacterial and mammalian systems inducing gene mutations, sisterchromatid exchanges and morphological transformation. CPP isa mouse skin carcinogen, a mouse skin tumor initiator and inducespulmonary tumors in newborn mice. We have examined the tumorigenicactivity of CPP in strain A/J mice, have determined the formationand persistence of CPP-induced DNA adducts in lung tissue, andanalyzed the mutational spectrum in the Ki-ras oncogene fromCPP induced tumors. CPP dissolved in tricaprylin was administeredby i.p. injection to male A/J mice (20 mice/dose) at 0,10,50,100and 200 mg/kg. Animals were killed 8 months later and the lungsremoved, fixed, and surface adenomas enumerated. CPP provedto be highly tumorigenic in A/J mice in terms of inducing lungadenomas. The observed tumor multiplicities (lung adenomas/mouse)were: 97.7 ± 28.7 at 200 mg/kg, 32.8 ± 15.4 at100 mg/kg, 4.63 ± 2.11 at 50 mg/kg and 0.58 ±0.82 at 10 mg/kg. Tricaprylin-treated controls produced 0.60± 0.58 lung adenomas/mouse. Groups of mice treated underthe same dosing conditions as those in the tumor studies werekilled 1, 3, 7, 14 and 21 days after treatment. The lungs wereremoved, and the DNA was subjected to DNA adduct analysis bythe 32P-postlabeling method. Total CPP-DNA adducts in mouselung peaked at day 3 with 5870 amol CPP adducts/µg DNAafter a single dose of 200 mg/kg. DNA adduct levels decreasedto 1800 amol CPP adducts/µg DNA at day 21. QualitativeDNA adduct analysis revealed four major adducts and one minoradduct. Co-chromatography of the lung DNA from CPP-treated micewith calf thymus DNA treated with CPP-3,4-oxide indicated thatall DNA adducts were oxide derived and comparison with CPP-3,4-oxlde-treatedpolydeoxyguanylic acid suggests that almost all of these adductsare CPP-3,4-oxide-2'-deoxyguanosine adducts. Ki-ras codon 12mutation analysis of the DNA from twnors taken from the 100and 200 mg/kg CPP dose groups demonstrated the following patterns:GGT     

Macromolecular binding of [3H]6-nitrobenzo[a]pyrene in male rats

Generally labelled [³H)6-nitrobenzo[a]pyrene (6NBP) when administeredto male rats by i.p. injection was widely distributed throughoutthe body. After a single injection of 4.2 mg/kg the highestconcentration was found in the liver (5% at 2 h) followed bythe spleen > kidney > lung. Maximal covalent binding tomacromolecules occurred between 8 and 24 h depending upon theorgan; the order of binding/mg macromolecule was RNA > DNA> protein for all organs studied. In the liver maximal bindingwas found 8 h after 6NBP administration at which time therewere 45±7 pmol 6NBP/mg bound to DNA and 114±40pmol/mg to RNA following a dose of 4.2 mg/kg. These levels fallto 14±4 and 12±6 pmol/mg, respectively, after168 h, the longest time point so far studied. At a lower doseof 1.4 mg/kg the values for DNA at 8 h were 14±2 pmol/mgand at 168 h 3±2 pmol/mg while for RNA the correspondingvalues were 29±8 and 15±3 pmol/mg. After gastricintubation at a dose of 4.2 mg/kg there was no difference inliver macromolecular binding at 24 h between conventional andgerm-free animals. Enzymic digestion of liver DNA from [³H)6NBPinjected animals and reversed-phase h.p.l.c of the digests showedtwo peaks of radioactivity. The percentage of total radioactivityin each peak varied from preparation to preparation suggestingthat one peak may be a degradation product of the other althoughexperimental variability cannot be ruled out. The implicationsof these findings in relation to the potential carcinogenicityof 6NBP are discussed.     

Tamoxifen: evidence by 32P-postlabeling and use of metabolic inhibitors for two distinct pathways leading to mouse hepatic DNA adduct formation and identification of 4-hydroxytamoxifen as a proximate metabolite

Exposure to pentachlorophenol (PCP) strongly intensifies theformation of mouse hepatic DNA adducts elicited by oral administrationof tamoxifen (TAM), as previously shown by ³²P-postlabeling.To explain this effect, PCP was proposed to interfere with thedetoxication by sulfate conjugation of an as yet unidentifiedhydroxylated proximate TAM metabolite. A comparison of the presentand earlier results shows that the hepatic TAM adduct patternin female ICR mice depended on the route of administration ofTAM (120 µmol/kg), with oral administration primarilyeliciting formation of more polar adducts (termed group I adducts),while after i.p. administration less polar adducts (group II)predominated over group I adducts by a factor of 17.5. All theseadducts were also formed in female Sprague–Dawley ratsafter i.p. dosing with TAM, but total adduct levels were 3.5-to 5-fold higher than in mice. After four daily i.p. treatments,TAM adducts accumulated in mouse liver DNA in a non-linear fashion.Adduct levels were 30–50 times lower in mouse kidney andlung than in liver. The phenolic metabolite 4-hydroxy TAM (120µimol/kg) exclusively led to formation of polar (groupI) hepatic adducts, and this process was stimulated 8-fold bycoadministration of PCP (75 µimol/kg). Co-administrationof PCP with the parent compound led to an 11-fold enhancementof group I adduct formation; simultaneously, levels of groupII adducts were suppressed 6-fold. Another inhibitor of sulfateconjugation, 2,6-dichloro-4-nitrophenol, unlike PCP, had noeffect on group I adducts, but it reduced group II adduct formation2.2-fold. The PCP metabolite 2,3,5,6-tetrachlorohydroquinone(75 µimol/kg) did not significantly affect any major TAMadduct, suggesting that PCP itself was the active compound.Similar to group II TAM adducts, the formation of hepatic safrole–DNAadducts was inhibited in female ICR mice by both sulfotransferaseinhibitors, consistent with the proposal that metabolic     

Protection of conjugated linoleic acids against 2-amino-3-methylimidazo[4,5-f]quinoline-induced colon carcinogenesis in the F344 rat: a study of inhibitory mechanisms

Grilled ground beef contains a number of heterocyclic aminecarcinogens, such as 2-amino-3-methylimidazo[4,5-/Iquinoline(IQ), as well as anticarcinogenic conjugated linoleic acids(CLA). In the present study, CLA was administered to male F344rats by gavage on alternating days in weeks 1–4, whileIQ was given by gavage every other day in weeks 3 and 4 (100mg/kg body wt). Rats were killed 6 h after the final carcinogendose in order to quantify IQ-DNA adducts or after week 16 inorder to score colonic aberrant crypt foci (ACF). In the ACFstudy, CLA had no effect on the size of the foci, but inhibitedsignificantly (P <0.05) the number of ACF/colon, from 4.3± 2.4 in controls to 1.1 ± 1.3 in CLA-treatedrats (mean ± SD,n = 10). Rats given CLA also had significantlylower IQ—DNA adducts in the colon as determined by 32P-postlabelinganalysis; relative adduct labeling levels (RAL x 10⁷) for themajor adduct were 9.13 ± 2.6 in controls versus 5.42± 1.8 in CLA-treated animals (P < 0.05). Mechanismstudies indicated that CLA and other fatty acids interact withcertain heterocyclic amines in a manner consistent with substrate—ligandbinding. However, no such interaction occurred with IQ, andCLA failed to inhibit significantly the mutagenicity ofN-hydroxy-IQin the Salmonella assay, Liver microsomes from CLA-treated ratsexhibited lower activities for dealkylation of 7-ethoxyresorufinand methoxyresorufin and activated IQ to DNA binding speciesless effectively than microsomes from control animals. Directaddition of CLA to the in vitro incubation inhibited IQ-DNAbinding and was associated with increased recovery of unmetabolizedparent compound. In the Salmonella assay, CLA inhibited themuta-genic activity of IQ in the presence of S9 or ram seminalvesicle microsomes. Collectively, these results support a mechanisminvolving inhibition of carcinogen activation by CLA, as opposedto direct interaction with the procarcin-ogen, scavenging ofelectrophiles or selective induction of phase I detoxificationpathways.