Inhibition of 2-Amino-3, 8-dimethylimidazo[4,5-f]quinoxaline-mediated DNA-adduct Formation by Chlorophyllin in Drosophila

The effect of chlorophyllin on 2-ammo-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx)-mediated DNA-adduct formation in Drosophila was studied. Third-instar larvae of Drosophila were fed MeIQx at 1 mg/6.5 g-feed/bottle, with or without chlorophyllin (100–300 ing). After a 6 h feeding exposure to MeIQx, the larvae were divided into 2 groups. The first group was examined for covalent DNA adducts by ³²P-postlabeIing assay. The second group was assayed for DNA damage by allowing the larvae to develop to adults and measuring the male/female ratio (males, DNA repair-deficient; females, DNA repair-proficient). The ³²P-postlabeling results indicated a significant decrease in DNA adduct levels in larvae treated with MeIQx and 300 mg chlorophyllin (1.7±0.7 adducts/10⁷ nucleotides) as compared with MeIQx-treated larvae (6.5±2.1 adducts/10⁷ nucleotides). The results on male/female sex ratios also indicated a chlorophyllin-indnced decrease in DNA damage by exposure to MeIQx. The suppressive effect of chlorophyllin on the genotoxic actions of a polycyclic mutagen, MeIQx, may be a result of complex formation between chlorophyllin and the 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.     

Analysis of 7-methylbenz[a]anthracene-DNA adducts formed in SENCAR mouse epidermis by 32P-postlabeling

The present study has analysed the DNA adducts formed in SENCAR mouse epidermis following topical application of 7-methylbenz[a]anthracene (7- MBA). Mice were treated with 400 nmol of 7-MBA, which represents an initiating dose of this hydrocarbon for SENCAR mice. DNA adducts were analysed 24 h after topical application of the hydrocarbon by 32P- postlabeling coupled with either HPLC analysis or an improved TLC procedure giving better resolution of DNA adducts through the use of a D6 solvent [isopropanol:4N NH4OH (1:1)] following D5. Twenty-four hours after topical application of 400 nmol 7-MBA, the level of total covalent binding was 0.37 +/- 0.07 pmol/mg DNA as determined by 32P- postlabeling. This level of binding correlated well with the relative tumor initiating activity of this hydrocarbon compared to 7,12- dimethylbenz[a]anthracene (6.4 +/- 0.01 pmol/mg DNA) and dibenz[a,j]anthracene (0.03 +/- 0.01 pmol/mg DNA). Analysis of the 32P- labeled 3',5'-diphosphodeoxyribonucleosides by HPLC and TLC revealed the presence of deoxyguanosine (dGuo) and deoxyadenosine (dAdo) adducts formed from both the anti- and syn-bay-region diol-epoxides of 7-MBA (anti- and syn-7-MBADEs). The major DNA adduct derived from 7-MBA in mouse epidermis was tentatively identified as (+) anti-7-MBADE-trans-N2- dGuo. In addition, a minor dGuo adduct derived from the bay-region syn- diol-epoxide of 7-MBA was detected as well as a minor dAdo adduct from this diol-epoxide. Another minor dAdo adduct was also detectably present which arose from either the anti- or syn-diol epoxide. Furthermore, several unidentified DNA adducts were present in both HPLC and TLC chromatograms of DNA samples from 7-MBA-treated mice. These results are discussed in terms of the role of specific 7-MBA-DNA adducts in tumor initiation by this hydrocarbon.      

Intra- and interindividual variability in systemic exposure in humans to 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl- 6-phenylimidazo[4,5-b]pyridine, carcinogens present in cooked beef.

During the cooking of beef, the genotoxic heterocyclic aromatic amines 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are formed. Little is known about the fate of these compounds in humans or the factors affecting it. We have developed assays based on capillary column gas chromatography-negative ion mass spectrometry capable of the simultaneous measurement of MeIQx, DiMeIQx, and PhIP in cooked meat and in human urine using stable isotope labeled analogues. Ten normal, healthy male volunteers were invited to consume a standard cooked meat meal (400-450 g lean beef, cooked as patties on a griddle hotplate) on four separate occasions over a period of 14 months. Following consumption of the test meals, urine was collected from 0 to 8 h, during which time all free amines were excreted and analyzed for MeIQx, DiMeIQx, and PhIP. Subjects ingested 240 +/- 9 (SEM) g cooked meat, which contained 2.2 +/- 0.2 ng MeIQx/g meat, 0.7 +/- 0.1 ng DiMeIQx/g meat, and 16.4 +/- 2.1 ng PhIP/g meat. The variability in relative systemic bioavailability was assessed from the percentage of ingested amine excreted unchanged in the urine. Subjects excreted 2.1 +/- 1.1% of MeIQx and 1.1 +/- 0.5% of PhIP ingested as unchanged amine in the urine. Levels of DiMeIQx in urine, if present, were below the sensitivity of our assay (20 pg/ml) and could not be detected in any of the samples analyzed. Irrespective of dose, urinary excretion of unchanged MeIQx or PhIP (expressed as a percentage of the ingested dose) remained constant for each individual subject. The intraindividual coefficients of variation for MeIQx (28.4%) and PhIP (23.7%) were low and the pooled interday (intrasubject) coefficients of variation for both compounds were only 19 and 3.4%, respectively. In contrast, inter-subject (intraday) variation was greater, with pooled coefficients of variation of 145% for MeIQx and 71% for PhIP. Based on these studies, it should be possible to use the percentage excretion of MeIQx and PhIP to assess the relative bioavailability of these compounds in humans.     

Benzo[a]pyrene-globin adducts detected by synchronous fluorescence spectrophotometry: method development and relation to lung DNA adducts in mice.

A simple synchronous fluorescence spectrophotometry (SFS) to detect benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-globin adducts is described. SFS for BPDE-DNA, which measures detached benzo[a]pyrene (B[a]P)-tetrols after acid hydrolysis of DNA, was applied for BPDE-globin adducts in B[a]P-treated C57BL/6 (B6) mice. Unlike DNA samples, globin is not measurable as such after acid hydrolysis because proteins give a background in SFS. Furthermore, proteinase incubation before acid hydrolysis of globin gave too much background even after purification to be useful in this assay. Of several purification procedures tried after acid hydrolysis (protein precipitation, elution through Sep-Pak C18, filtration, ether extraction of tetrols), the lowest background fluorescence was obtained with ether extractions of B[a] moieties. Ether phases were evaporated to dryness and the remainder dissolved in distilled water (1 ml), which was measured by SFS. Compared to DNA, somewhat milder hydrolysis conditions were optimal for globin samples (0.05 M HCl, 1.5 h, + 90 degrees C). Globin samples from B[a]P-treated mice gave a peak at the same wavelength (345 nm excitation) as the hydrolysis products of BPDE-DNA adducts, indicating B[a]P-tetrols and triols in the sample. Less than half of B[a]P measured in globin was from covalently bound BPDE. In mice injected i.p. with 1-160 mg/kg of B[a]P there was a dose-dependent increase in the amount of BPDE adducts in globin and a positive correlation with lung and liver DNA. Globin adducts were a more sensitive indicator of B[a]P exposure than DNA adducts because more globin can be used for the assay. Although both covalently and non-covalently bound BPDE in globin are detected by SFS, this method is the simplest described so far, reproducible and theoretically sensitive enough for human biomonitoring.     

Formation of DNA adducts from N-acetoxy-2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene in rat hemopoietic tissues in vivo

Administration of [ring-3H]-N-acetoxy-2-acetylaminofluorene (10 mg/kg i.v.) to male F344 rats resulted in substantial binding of [ring-3H]-N-acetoxy-2-acetylaminofluorene to DNA isolated from bone marrow [20.3 +/- 1.7 (SD) pmol/mg DNA] and spleen (23.6 +/- 5.8 pmol/mg DNA) compared to liver (39.4 +/- 2.1 pmol/mg DNA) and kidney (27.1 +/- 1.0 pmol/mg DNA) 2 h after dosing. High-performance liquid chromatography analyses of trifluoroacetic acid hydrolyzed DNA from bone marrow and spleen revealed the presence of N-(guanin-8-yl)-2-aminofluorene as the major adduct comprising more than 80% of total adducts, while N-(guanin-8-yl)-2-acetylaminofluorene and ring opened derivatives of N-(guanin-8-yl)-2-aminofluorene were only minor adducts. Dose dependent binding of [ring-3H]-N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to DNA and formation of individual adducts in spleen and bone marrow was observed at a dose range of 1.0-10.0 mg/kg. There was a 3- and 6-fold more DNA adduct formation in bone marrow and spleen, respectively, following treatment with [ring-3H]-N-acetoxy-2-acetylaminofluorene compared to N-OH-AAF. However, the pattern of DNA adducts formed was similar. Pretreatment of rats with the cytotoxic agent 5-fluorouracil (150 mg/kg i.p.), which causes transient depletion of hemopoietic cells, on days -10, -7, -4, -2, and -1 prior to the administration of [ring-3H]-N-OH-AAF (10 mg/kg) on day 0 resulted in different levels of N-OH-AAF binding to spleen and bone marrow DNA without altering the pattern of DNA adducts compared to that in control animals. These data suggest a possible existence of a target cell population for N-OH-AAF and perhaps other aromatic amines and amides in both bone marrow and spleen of F344 rat.     

Genotoxic effects of cyclopenta-fused polycyclic aromatic hydrocarbons in different types of isolated rat lung cells

The genotoxic effects of the environmental contaminants benz[j]aceanthrylene (B[j]A), benz[l]aceanthrylene (B[l]A) and benzo[a]pyrene (B[a]P), and the metabolism of radiolabelled B[j]A, were studied using rat lung microsomes and various types of isolated rat lung cells from control and Aroclor 1254 (PCB) treated animals. All three compounds (10 or 20 microg/plate) resulted in low, but detectable, levels of His+ revertants in the Salmonella assay when plated with control lung microsomes. The two cyclopenta polycyclic aromatic hydrocarbons (CP-PAH) B[j]A and B[l]A, gave increased levels of revertants when plated with microsomes from PCB-treated animals. Clara cells, type 2 cells and alveolar macrophages isolated from control rats were exposed to B[j]A, B[l]A or B[a]P (30 microg/ml, 1 h), but neither of the cell types showed any DNA damage when measured by alkaline filter elution. However, both B[j]A and B[l]A (30 microg/ml, 2 h) caused DNA adducts in all three cell types, measured by the 32P-post- labelling technique, whereas no B[a]P adducts were detected (30 microg/ml, 2 h). The total DNA adduct levels in Clara cells, type 2 cells and macrophages exposed to B[j]A were 0.085 +/- 0.033, 0.053 +/- 0.001 and 0.170 +/- 0.030 fmol/microg DNA, respectively, whereas the total levels in cells exposed to B[l]A were 0.140 +/- 0.070, 0.140 +/- 0.030 and 0.220 +/- 0.080 fmol/microg DNA, respectively. Cells exposed to B[j]A revealed only one adduct which corresponds with the B[j]A-1,2- oxide DNA adduct. Judged from high performance liquid chromatography (HPLC) analysis using radiolabelled B[j]A (30 microg/ml, 30 min), the major metabolite formed in control microsomes was B[j]A-1,2-diol. Thus, oxidation at the cyclopenta ring appears to be the most important activation pathway for B[j]A with control rat lung cells. Exposure of lung cells to CP-PAH (30 microg/ml, 2 h) isolated from PCB pretreated rats resulted in slightly increased DNA adduct levels in Clara cells and macrophages when compared to cells isolated from control rats. Furthermore, the adduct pattern had shifted, and no apparent B[j]A-1,2- oxide adduct could be detected on the thin layer chromatography (TLC) plate. In contrast, the major metabolite formed with microsomes from PCB-treated animals was still the B[j]A-1,2-diol.      

N-hydroxy-MeIQx is the major microsomal oxidation product of the dietary carcinogen MeIQx with human liver.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant of the heterocyclic aromatic amines formed during the cooking of meat, is genotoxic and carcinogenic in rodents. MeIQx requires metabolic activation by P450 before it can exert these effects. Whilst there is indirect evidence that the mutagenic product is N-hydroxy-MeIQx (N-OHMeIQx), we have now identified this unequivocally following incubation of the amine with human hepatic microsomal fraction. A mixture of unlabelled MeIQx, [13C,15N2]MeIQx and [14C]MeIQx was used as substrate and the products analysed by HPLC-thermospray mass spectrometry. Characteristic doublet ions, 3 mass units apart, were found at m/z 214/217 ([M+H]+) from the parent compound, MeIQx and at 230/233 ([M+H]+) from N-OHMeIQx. The presence of a doublet ion at m/z 214/217 with the doublet at 230/233 [M+H+] provided additional evidence that this was N-OHMeIQx, as facile loss of 'O' is characteristic of N-hydroxylamines. Further evidence for the identity of the major metabolite, which accounted for approximately 90% of all microsomal metabolism, was obtained by comparing the mutagenicity of the HPLC eluate using Salmonella typhimurium YG1024, which is particularly sensitive to N-hydroxylamines, and TA98/1,8-DNP6 which is resistant to most N-hydroxylamines. Ninety-five per cent of direct-acting mutagenicity present in the reaction mixture was associated with a single peak, which co-eluted with N-OHMeIQx, as indicated by mass spectrometry. In the presence of a metabolic activation system, only one additional mutagenic peak, corresponding to unchanged MeIQx, could be detected. MeIQx (5 microM) was N-hydroxylated at a rate of 77 +/- 11 pmol/mg/min (mean +/- SEM, n = 4) by human liver microsomes. The specific inhibitor of human CYP1A2, furafylline (5 microM) inhibited the N-hydroxylation of MeIQx by > 90%. These data show that N-OHMeIQx is both the major oxidation product and the major genotoxic product of MeIQx generated by microsomal fractions of human liver and that the reaction is catalysed almost exclusively by CYP1A2.     

Formation and Removal of DNA Adducts in the Liver of Rats Chronically Fed the Food-borne Carcinogen, 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline

Effects of chronic administration of 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx) at 0.4, 8 and 400 ppm in the diet on DNA adduct formation and removal in the rat liver were examined by the ³²P-postlabeling method. The 0.4 and 8 ppm doses for 40 weeks resulted in time-dependent increases in MeIQx-DNA adduct levels until 16 and 8 weeks, respectively, with constant values being maintained thereafter. In the case of a carcinogenic dose (400 ppm) of MeIQx, the adduct levels reached a maximum at week 12, and then gradually decreased. Alteration of metabolism of MeIQx during liver carcinogenesis might be related to this decrease in DNA adduct levels. When MeIQx administration was stopped at week 20, 60–90% of the MeIQx-DNA adducts formed with the three doses (0.4, 8 and 400 ppm) of MeIQx were removed in a biphasic manner after return to a basal diet, with initial rapid removal followed by a slow change. No difference in the pattern of MeIQx-DNA adducts was detected on thin layer chromatography at any dose at any time point. Thus, it is suggested that there may be at least two types of damaged DNA, susceptible and resistant to removal of MeIQx-DNA adducts, after chronic administration of MeIQx.     

Identification and in vivo formation of 32P-postlabeled rat mammary DMBA-DNA adducts

The in vivo formation of 32P-postlabeled mammary 7,12-dimethylbenz[a]anthracene (DMBA)-DNA adducts was evaluated for female Sprague-Dawley rats following administration of DMBA (i.g.) at 1, 3, 5, 10 and 20 mg/rat. Adduct formation was also measured as a function of time following DMBA intubation. At least eight adducts were formed in vivo in mammary epithelial cells. The identities of four of these nucleoside bisphosphate adduct spots were determined by cross-referencing with previously characterized 3H-labeled nucleoside DMBA adducts. These identified adducts constitute four of the five major adducts formed in vivo. Two adducts were identified as the anti-dihydrodiolepoxide of DMBA reacted with deoxyguanosine (dGuo). Two other major adducts were derived from the syn-dihydrodiolepoxide and bound to dGuo and to deoxyadenosine (dAdo). Total DMBA-DNA binding increased at all DMBA doses investigated (r = 0.94). Total binding values were (mean +/- SEM) 39.3 +/- 6.1, 158.0 +/- 16.9, 194.7 +/- 9.9, 326.9 +/- 21.5 and 443.2 +/- 20.8 nmol DMBA/mol DNA for rats administered DMBA at 1, 3, 5, 10 and 20 mg/rat respectively. The anti-dGuo adduct predominated at all doses and times evaluated, contributing to approximately 52% of total binding. The occurrence of anti-derived adducts was greater than that of syn-derived adducts. Binding of DMBA to dGuo substantially exceeded binding to dAdo. The 32P-postlabeling procedure represents a sensitive technique for detecting specific DMBA-DNA adducts formed in vivo in the rat mammary gland.     

Mutagenic activation of IQ, PhIP, and MeIQx by hepatic microsomes from rat, monkey and man: low mutagenic activation of MeIQx in cynomolgus monkeys in vitro reflects low DNA adduct levels in vivo

Cooked meat, poultry and fish contain a number of mutagenicand carcinogenic heterocyclic amines, including 2-amino-3-methylimidazo[4,5-f]quinoline(IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).In the present study we examined the capacity of hepatic microsomesfrom Fischer 344 rats, cynomolgus monkeys and humans to metabolicallyactivate IQ, MeIQx and PhIP in vitro using the Ames Salmonellamutagenicity assay. The mutagenic activation of IQ was similaramong the three species; however, there were significant differencesamong the species in the activation of PhIP and MeIQx. Livermicrosomes from humans showed the greatest capacity to activatePhIP and MeIQx, followed by rats, and then monkeys. The largestdifferences between the species were observed when MeIQx wasused as the mutagen. MeIQx–DNA adducts formed in vivowere then compared among rats and monkeys given MeIQx by gavage(20 mg/kg/day, 10 doses). ³²P-Postlabeling analysis, carriedout under intensification conditions, was used to examine MeIQx–DNAadducts in the liver, kidney, heart, colon and white blood cells.MeIQx–DNA adducts were highest in all tissues examinedfrom male rats, followed by female rats, and much lower in monkeys.In the liver, the total MeIQx–DNA adduct levels of monkeyswere {small tilde}19 and {small tilde}10 times lower than inmale and female rats respectively. In extrahepatic tissues,the differences in MeIQx–DNA adduct levels between monkeysand rats were even greater. The results suggest that the lowlevel of MeIQx–DNA adducts found in vivo in cynomolgusmonkeys reflects a low capacity to activate MeIQx via the hepaticcytochrome P450 monooxygenase system.     

Heterozygous p53-deficient mice are not susceptible to 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) carcinogenicity.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a very potent mutagen which induces tumors in the liver, lung and hematopoietic system of CDF1 mice and the liver, Zymbal gland and skin in F344 rats. The recent development of transgenic knockout mice allows their introduction for sensitive screening of environmental carcinogens due to the rapid development of tumors. P53 gene deficient mice (p53-/-) were found to spontaneously develop malignant lymphoma and hemangiosarcoma, whereas heterozygotes (p53+/-) mice display a high incidence of tumors of the urinary bladder when treated with N-butyl-N-(4-hydroxybutyl)nitrosamine. In the present study, to determine whether p53 gene knockout mice can be utilized in a short-term assay model for the screening of heterocyclic amines (HCAs), the effects of MeIQx, as a representative compound, at low doses were examined. Male and female p53+/- mice and wild type littermates (p53+/+) were continuously given diets containing 0, 0.1, 1, 10 and 100 ppm MeIQx for 1 year. No significant difference in tumor induction was observed other than an increase in liver adenomas in males receiving 10 ppm MeIQx treatment. The results indicate that p53+/- mice have no practical advantages for use in short-term carcinogenicity tests of HCAs.     

DNA Adducts Formed by 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline in Rat Liver: Dose-Response on Chronic Administration

The effect of administration of 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx) at various doses on DNA adduct formation in male rats was examined by ³²P-postlabeling analysis. Administration of MeIQx in the diet at 0.4 ppm, 4 ppm, 40 ppm and 400 ppm for one week resulted in the formations of 0.04, 0.28, 3.34 and 39.0 adducts per 10⁷ nucleotides in rat liver cells. Continuous administration of 400 ppm of MeIQx in the diet for 61 weeks to rats induced hepatocellular carcinomas in all rats. The carcinogenicity of MeIQx at doses of 40 ppm or less is not known yet, but the above results show a linear relationship between the level of MeIQx administered and the adduct level. In rats treated with low doses of 0.4, 4 and 40 ppm of MeIQx, adduct levels increased linearly with time of treatment, the levels in week 12 being two to three times those in week 1. In contrast, on treatment with 400 ppm of MeIQx, the adduct level in the liver increased until week 4, when it was 110 adducts per 10⁷ nucleotides, and then remained constant for the next 8 weeks. Induction of the multidrug-resistance gene was suggested to be involved in development of this plateau level.     

Disposition and metabolism of the food mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in rats

The disposition and metabolism of a common food mutagen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), was studied in rats. Five rats of both sexes were given a single oral dose of 14C-labeled MeIQx (3-4 mg/kg body wt). The male rats excreted 36% of the radioactivity and 15% of the mutagenic activity of the dose given in the urine collected during the first 24 h. In the females the corresponding urine contained 41% of the radioactivity and 12% of the mutagenicity. During the next 48 h only 1-3% of the radioactive dose was excreted in urine. The remaining dose was excreted in the feces except of less than 1% that was retained by the tissues after 72 h. The liver and kidney retained more radioactivity than other organs. In a separate study the metabolites of bile, urine and feces of both sexes were investigated. After a single oral dose of 20 mg 14C-labeled MeIQx/kg body wt, three major non-mutagenic metabolites were identified. These were 2-amino-4(or 5)-(beta-D-glucuronopyranosyloxy)-3,8-dimethylimidazo[4,5-f] quinoxaline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxalin-4(or 5)-yl sulfate and N-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl) sulfamate. Another two metabolites present in bile, urine and feces were 2-(beta-D-glucuronopyranosylamino)-3,8-dimethylimidazo[4,5-f ] quinoxaline and 2-amino-8-hydroxymethyl-3-methylimidazo[4,5-f]quinoxalin-4 (or 5)yl sulfate. All metabolites were essentially non-mutagenic. Most of the mutagenicity still present in bile, urine and feces could be explained by unchanged MeIQx. Unchanged MeIQx was the most abundant form excreted in urine.     

Molecular dosimetry of DNA adducts and sister chromatid exchanges in human lymphocytes treated with benzo[a]pyrene

We examined the relationship between benzo[a]pyrene-DNA adducts and sister chromatid exchanges (SCEs) in human lymphocytes. Cultures of isolated phytohemagglutinin (PHA)-stimulated lymphocytes from two normal donors were treated with 0.01-5.0 microM B[a]P from 24 to 72 h of culture. Using the highly sensitive 32P-postlabeling assay, we identified seven B[a]P-DNA adducts, one of which accounted for greater than 90% of the total DNA modifications. This adduct comigrated on polyethylenimine plates with the adduct produced by (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydro-benzo[a]pyrene. B[a]P-DNA adduct levels ranged from 0.02 to 8 adducts/10(7) nucleotides. SCE frequencies measured in parallel cultures ranged from 8 to 46 SCEs/cell. At the same B[a]P concentrations, B[a]P-induced SCE frequencies and B[a]P-DNA adduct levels were higher in lymphocytes from donor 1 than in lymphocytes from donor 2. There was a linear correlation between the number of B[a]P-DNA adducts and the number of SCEs induced; slopes of the linear regressions of induced SCEs on B[a]P-DNA adducts were similar for both donors. Our data suggest that SCE induction by B[a]P in human lymphocytes results from covalent DNA modification.     

The acridonecarboxamide GF120918 potently reverses P-glycoprotein-mediated resistance in human sarcoma MES-Dx5 cells

The doxorubicin-selected, P-glycoprotein (P-gp)-expressing human sarcoma cell line MES-Dx5 showed the following levels of resistance relative to the non-P-gp-expressing parental MES-SA cells in a 72 h exposure to cytotoxic drugs: etoposide twofold, doxorubicin ninefold, vinblastine tenfold, taxotere 19-fold and taxol 94-fold. GF120918 potently reversed resistance completely for all drugs. The EC50s of GF120918 to reverse resistance of MES-Dx5 cells were: etoposide 7+/-2 nM, vinblastine 19+/-3 nM, doxorubicin 21+/-6 nM, taxotere 57+/-14 nM and taxol 91+/-23 nM. MES-Dx5 cells exhibited an accumulation deficit relative to the parental MES-SA cells of 35% for [3H]-vinblastine, 20% for [3H]-taxol and [14C]-doxorubicin. The EC50 of GF120918, to reverse the accumulation deficit in MES-Dx5 cells, ranged from 37 to 64 nM for all three radiolabelled cytotoxics. [3H]-vinblastine bound saturably to membranes from MES-Dx5 cells with a KD of 7.8+/-1.4 nM and a Bmax of 5.2+/-1.6 pmol mg(-1) protein. Binding of [3H]-vinblastine to P-gp in MES-Dx5 membranes was inhibited by GF120918 (K = 5+/-1 nM), verapamil (Ki = 660+/-350 nM) and doxorubicin (Ki = 6940+/-2100 nM). Taxol, an allosteric inhibitor of [3H]-vinblastine binding to P-gp, could only displace 40% of [3H]-vinblastine (Ki = 400+/-140 nM). The novel acridonecarboxamide derivative GF120918 potently overcomes P-gp-mediated multidrug resistance in the human sarcoma cell line MES-Dx5. Detailed analysis revealed that five times higher GF120918 concentrations were needed to reverse drug resistance to taxol in the cytotoxicity assay compared to doxorubicin, vinblastine and etoposide. An explanation for this phenomenon had not been found.     

Correlation between DNA or protein adducts and benzo[a]pyrene diol epoxide I-triglyceride adduct detected in vitro and in vivo

In this study, we demonstrated the in vitro and in vivo formation of carcinogen-lipid adduct and its correlation with DNA or protein adducts. The lipids from serum or hepatocyte membranes of Sprague-Dawley rats, human serum and standard major lipids were in vitro reacted with benzo[a]pyrene (B[a]P) and B[a]P metabolites. 7, 8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE-I), an ultimate carcinogenic form of B[a]P, was covalently bound to triglyceride (TG). BPDE-I-TG adducts isolated by thin-layer chromatography (TLC) were further detected by high-performance liquid chromatography. TGs, including triolein, tripalmitin and tristearin, showed positive reactions with BPDE-I. However, cholesterol, phospholipids (phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidyl-inositol and sphingomyelin) and non-esterified fatty acids (palmitic acid, oleic acid, linoleic acid and stearic acid) did not react with BPDE-I. In addition, other B[a]P metabolites (B[a]P-phenols and -diols) did not react with TG. TG appeared to be the most reactive lipid yet studied with respect to its ability to form an adduct with BPDE-I. There was a clear-cut dose-related formation of [1,3-(3)H]BPDE-I-lipid adducts in vitro between TG and [1,3-(3)H]BPDE-I. In an animal study, BPDE-I-TG was also formed in the serum of rats orally treated with B[a]P (25 mg/rat). Also, obvious correlations between [(3)H]B[a]P related-biomolecule adducts (DNA or protein) or lipid damage and the BPDE-I-TG adducts were obtained in various tissues of mice i.p. treated with [(3)H]B[a]P. These data suggest that TG can form an adduct with BPDE-I, as do other macromolecules (DNA, RNA and protein). Therefore, a carcinogen-lipid adduct would be a useful biomarker for chemical carcinogenesis research and cancer risk assessment.     

Formation and persistence of DNA adducts in the liver of brown bullheads exposed to benzo[a]pyrene

The formation and persistence of benzo[a]pyrene (BP)-DNA adducts in the liver of brown bullheads (Ictalurus nebulosus) treated with the hydrocarbon (20 mg/kg body wt, i.p.) was investigated using the 32P-postlabeling assay. The highest level of covalent binding of BP to liver DNA (188 fmol BP adducts/mg DNA) was observed 25-30 days following treatment. After 70 days, the adduct level in liver DNA had declined to approximately 26% of the maximum adduct level. One major BP-DNA adduct and several minor ones were detected in the liver. The major adduct co-chromatographed with anti-BP-7,8-diol-9,10-epoxide-deoxyguanosine (anti-BPDE-dGuo) adduct. The data suggest that brown bullheads metabolically activate BP by the same mechanism as the mammalian systems susceptible to carcinogenic effects of the hydrocarbon.     

Excretion of the mercapturic acid S-[2-(N7-guanyl)ethyl]-N-acetylcysteine in urine following administration of ethylene dibromide to rats

Administration of the carcinogen ethylene dibromide (EDB) to rats resulted in the urinary excretion of S-[2-(N7-guanyl)ethyl]-N-acetylcysteine, which is derived from the nucleic acid adduct S-[2-(N7-guanyl)ethyl]glutathione. This mercapturic acid was isolated from urine by reversed-phase and propylamino high-performance liquid chromatography and was quantitated by measurement of fluorescence intensity. The urinary mercapturic acid was identified as S-[2-(N7-guanyl)ethyl]-N-acetylcysteine on the basis of cochromatography and UV, fluorescence, 1H nuclear magnetic resonance, and fast atom bombardment mass spectra, all of which were identical with the authentic synthesized material. The excretion of mercapturic acid into urine of rats given injections of various doses of EDB occurred in a dose-dependent, linear manner over the range of 0.5-37 mg EDB/kg body weight. A good correlation was found between the excretion of mercapturic acid and the (in vivo) formation of DNA adducts in liver and kidney DNA. The higher level of urinary mercapturic acid compared to the level of hepatic DNA adduct indicates that extra-hepatic DNA adducts and RNA adducts may contribute to the mercapturic acid production. The measurement of the mercapturic acid may provide a means of noninvasive estimation of DNA adducts derived from EDB exposure.