Distribution of radioactivity and anthracycline-fluorescence in tissues of mice one hour after [14C]-labeled AD 32 administration

Summary Levels of radioactivity and total anthracycline fluorescence in tissues of A/JAX mice were compared 1 h after IV administration of unlabeled or [¹⁴C]-labeled AD 32 (50 mg/kg). Highest levels of both fluorescence and radioactivity were found in the small intestine (including contents) and liver, a result consistent with the known hepatobiliary excretion of AD 32 and metabolites. Significant accumulations of radioactivity and fluorescence were found in kidney, spleen, large intestine (including contents), lung, and heart. Lesser levels were found in muscle and fat. Little radioactivity and fluorescence were found in brain. Liquid chromatographic analysis of extracts of small intestine and liver homogenates showed N-trifluoroacetyladriamycin (AD 41) as the major fluorescent species, and also revealed N-trifluoroacetyladriamycinol (AD 92) and occasional low levels of AD 32. In addition, there was a major peak of nonfluorescent radioactive material and two fluorescent nonradioactive signals (unknowns 1 and 2), indicative of cleavage of the radiolabel from the chromophore.The abbreviations used are AD 32 N-trifluoroacetyladriamycin-14-valerate - AD 41 N-trifluoroacetyladriamycin - AD 92 N-trifluoroacetyladriamycinol - ADR adriamycin - AD 48 adriamycin-14-valerate - AD 60 13-dihydro-N-trifluoroacetyladriamycin-14-valerate - AMNOL adriamycinol - HPLC high-performance liquid chromatography - TLC thin layer chromatography - SDS sodium dodecylsulfate     

Metabolism, excretion and pharmacokinetics of a single dose of [14C]-raltitrexed in cancer patients

Raltitrexed (Tomudex) is a specific inhibitor of thymidylate synthase and has recently been licensed in Europe for use in the treatment of advanced colorectal carcinoma. This study evaluated the metabolism, excretion and pharmacokinetics after a single dose of 3.0 mg/m² [¹⁴C]-raltitrexed in patients with advanced solid malignancies not amenable to curative therapy. From April 1994 to July 1995, nine patients (six men and three women) were recruited into the study. Pharmacokinetics analysis was performed during the first cycle of treatment in all patients and, in two patients, limited sampling was done prior to and during the second cycle of treatment. The mean observed peak plasma concentration (C_max) was 700.6 ng/ml and the median time (t _max) to reach maximal raltitrexed concentrations was 15 min after the initiation of the infusion. After reaching C_max the drug declined in a triexponential manner with a terminal half-life of 257 h. The AUC_0–∞ as predicted by the pharmacokinetic model was 2341.7 ng h ml⁻¹. Clearance was 41.3 ml/min, of which renal clearance accounted for 50–60%. Urinary collection for the measurement of radiolabeled drug revealed that renal excretion extrapolated to infinity accounted for 40% of the total radioactive dose. Faecal excretion accounted for only 3% of the dose when samples were collected to day 5 in the first six patients. Collection was extended to 10 days in the last three patients and faecal elimination accounted for 14% in these patients. Raltitrexed measurements prior to subsequent doses suggest that there was no accumulation of the drug with repeated administration. Low levels of radioactivity measured in the red cell pellets on days 15, 22 and 29 are likely to represent drug retained by newly forming red cells at the time of dosing. Examination of the urine revealed that the drug was excreted unchanged. The toxicities seen were in line with those encountered in previous studies. Grade 3 and 4 thrombocytopenia occurred in three patients and grade 3 neutropenia occurred in two patients. Received: 29 July 1997 / Accepted: 23 October 1997     

Radioactive species in rat urines and tissues after [14C] AD 32 administration

Summary [14-¹⁴C]N-Trifluoroacetyldoxorubicin-14-valerate ([¹⁴C]AD 32) was synthesized and administered IV to male rats at 9.09 mg/kg. Urinary radioactivity excreted in the 0–24 h interval was only 2.3% of the dose, N-trifluoroacetyldoxorubicin (AD 41), 13-dihydro-N-trifluoroacetyldoxorubicin (AD 92), and doxorubicin being the major urinary metabolites identified. Doxorubicin and AD 32 were the main radioactive species extracted from tissues at 24 h after treatment. The amount of doxorubicin present in the analysed tissue samples is in agreement with the relatively low toxicity of AD 32 compared with doxorubicin.     

Formation and persistence of DNA adducts in different target tissues of rats after multiple administration of benzo[a]pyrene

Exposure of humans to poly cyclic aromatic hydrocarbons is anongoing concern because of the carcinogenicity of these substances.DNA adducts are being increasingly used as indicators of carcinogenexposure. While considerable experimental evidence exists tosupport their use there are aspects that require further attention,especially after repeated exposure, which has led to this seriesof experiments. Male Sprague-Dawley rats were dosed with 10mg/kg benzo[a]pyrene (B[a]P) i.p., 3 times/week for 2 weeks.At 1, 3, 7, 14, 28 and 56 days after the last treatment liver,lung, spleen and peripheral blood mononuclear cells (PBMNs)were sampled. The DNA adduct levels, as measured by the ³²P-postlabellingtechnique, were significantlyincreased in all tissues, withlung having the highest levels. At day 14 total DNA adductsin lung, spleen and PBMNs were still >50% of the level atday 1. The removal of total DNA adducts was found to be fastestin liver > spleen < PBMNs > lung. A consistent correlationof total adducts between the lung and PBMNs was observed. Amajor adduct, designated adduct 1, was detected in all tissues,while adduct 4 was only found in liver and lung. Adduct 5 wasdetected only in lung, where it constituted     

Formation and removal of B[a]P diol epoxide -- DNA adducts in human fibroblasts

Using xeroderma pigmentosum fibroblasts, deficient in excisionrepair, as controls to measure the initial rate of (±)7ß,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (B[a]PDE)— DNA adduct removal in normal human fibroblasts, it wasfound that the maximum amount of carcinogen DNA adducts occurred1 h after the addition of B[a]PDE, and that during the firsthour 12% of the DNA — carcinogen adducts had already beenremoved. Thus the formation and removal of DNA — carcinogenadducts occurred simultaneously within the first hour afterB[a]PDE addition to confluent fibroblasts. Examination of excisionrepair over an extended period showed that during a further6 h, DNA adducts were removed at a rate four times slower thanthat observed during the first hour. Since the maximum levelof B[a]PDE — DNA adducts was observed 1 h after the additionof B[a]PDE to the cells in culture, this suggested that therate of breakdown of B[a]PDE was much slower than that observedin vitro. Further experiments indeed indicated that the rateof hydrolysis of B[a]PDE within the cell was significantly decreased.Thus, the stability of B[a]PDE inside the cell is governed byvery different parameters than those observed in vitro.     

Mucosa-specific DNA adducts in human small intestine: a comparison with the colon

The presence of several covalent DNA adducts in the human colonwas demonstrated by ³²p-postlabeling in a previous study. Wedemonstrated that DNA of all the colonlc mucosa tested wereselectively adducted by a single genotoxic agent and this modificationwas completely absent in the DNA of muscular layers. In thisstudy, the DNA adducts of the small intestine are compared withthose of the colon to understand the role of mucosa-specificDNA adduct (MSA) in intestinal carcinogenesis. The mucosal DNAof the small Intestine from 19 adults undergoing surgery dueto gastric carcinoma (seven cases), pancreatic carcinoma (fourcases), colon carcinoma (four cases), small intestinal tumor(two cases), intestinal trauma (one case) and ectopic pancreas(one case) were analyzed. The mucosa adjacent muscular layerDNA of the corresponding samples was examined as a control.Several common DNA adducts were observed in both mucosal andmuscular layers of all the adults. Besides these common backgroundDNA adducts, two MSAs (Si1, Si2) were detected in most of theadults as in colon cases. Si1 was present in all adults examined(19/19 cases) at a level of 0.04–0.22 adducts/10⁸ nucleotides(average 0.09) and Si2 was found in 13/19 patients at a levelof 0.03–0.08 adducts/10 nucleotides (average 0.05). Si2was the same adduct detected in the adult colonic mucosa. However,they were absent in the adjacent muscular layer as well as inthe neonatal intestine tested as a control. The total levelof the mucosa-specific DNA adducts of the small Intestine was     

DNA polymerase action on benzo[a]pyrene-DNA adducts.

A 16mer oligonucleotide containing a single guanine residue at nucleotide 13 from the 3' end was treated with the (+)-enantiomer of the 7,8-dihydrodiol 9,10-epoxide of benzo[a]pyrene (B[a]P). Oligonucleotides containing either an adduct in which the epoxide ring was opened trans or cis by the amino group of the guanine residue were separated by chromatography and identified by 32P postlabeling and circular dichroism spectroscopy. In the presence of nucleotide triphosphates and DNA polymerase (either Sequenase, version 2.0 or human polymerase alpha), it was found that the B[a]P adducts inhibited extension of an 11mer primer opposite the nucleotide 3' to the adduct in the template. Under various conditions, this inhibition was greater for the cis adduct than for the trans adduct. After a 10 min incubation with Sequenase, primer extension was reduced to approximately 20% of that seen with unmodified oligonucleotide by the trans adduct and was almost completely inhibited by the cis adduct. When a 12mer primer was used to examine nucleotide incorporation directly across from the guanine or adducted guanine residues, it was clear that deoxycytidylic acid was preferentially incorporated in all cases but that the incorporation was severely inhibited by both the cis and trans adducts. These findings suggest that a cis adduct is a more effective block to replication than a trans adduct, and that these adducts may not be very efficient mutagenic lesions.     

Liver DNA adducts in methyl-deficient rats administered a single dose of aflatoxin B1.

Using an 8 week Solt-Farber protocol with selection pressure (2-acetylaminofluorene/partial hepatectomy) applied during weeks 6 and 7, we have observed that a single oral administration of aflatoxin B1 (AFB1) to Fischer 344 rats on day 1 of the study, followed by a 3 week feeding regimen of either a methyl-deficient (CMD) or a basal (CMS) diet, results in a relative increase in hepatic preneoplastic lesions in CMD diet fed rats. It has previously been shown that a multiple dosing regimen with AFB1, started after 3 weeks of CMD diet, enhances tumor incidence. In the present study, the role of metabolic activation in the induction of preneoplastic lesions, and liver DNA adduct levels after the first dose of AFB1 in the tumorigenesis model have been investigated. AFB1-DNA adducts were determined at 2-168 h following a single non-necrogenic (100 micrograms/kg body wt) or necrogenic (600 micrograms/kg body wt) dose of AFB1 on day 1 or day 21 of a 3 week treatment with a complete basal or CMD diet. In all rats irrespective of dose, dietary treatment or time of AFB1 dosing, the patterns of adduct formation and repair did not change. In rats receiving AFB1 on day 1, total DNA adduct levels between the diet or dose groups were not significantly different, and quantitatively did not correlate with the observed increase in preneoplastic lesions, suggesting a contribution by additional factors in the initiation of these lesions. Administration of AFB1 on day 21, however, resulted in significantly reduced levels of total adducts at both dose levels in CMD diet fed rats compared to controls. Serum biochemistry data suggest that a prolonged exposure to CMD diet may cause pathological and/or biochemical alterations in hepatocytes with a resultant decrease in metabolic activation of AFB1, thus making it difficult to evaluate whether DNA damage is directly related to tumorigenesis.     

Metabolism of [14C]benzo[a]pyrene in vivo in the rat

[¹⁴C]Benzo[a]pyrene ([¹⁴C]B[a]P) injected intraperitoneally into rats appeared rapidly in liver, lung and kidney, and remained detectable in these tissues for at least 7 days. A large proportion (7–13%) of the ¹⁴C became covalently bound to tissue macromolecules, probably primarily proteins. Subcellular organelles of the liver were all found to bind the carcinogen, the microsomes most rapidly and the light mitochondrial fraction taking up ¹⁴C later. Nuclear bound ¹⁴C was detected in both liver and lung.Purification of the cytosolic ¹⁴C from liver revealed specific binding to the same cytosolic proteins purified from the in vitro reaction of [¹⁴C]B[a]P.     

Binding of benzo[a]pyrene metabolites in the rat intestinal lumen by magnetic polyethyleneimine microcapsules following an intragastric dose of [14C]benzo[a]pyrene

Semi-permeable magnetic polyethyleneimine (PEI) microcapsuleshave been developed to trap carcinogens and their metabolitesin vivo and their time-dependent binding of a model carcinogen,[¹⁴C]benzo[a]pyrene ([¹⁴C]BaP), is studied within the intestinallumen. Overall, 0.5% of an intragastrk BaP dose was bound bythese microcapsules recovered from faeces with specific bindingof metabolites (nmol/10⁶ recovered microcapsules) being similarin the 0–24-h and 24–48-h periods, but 10-foldlower in the 48–72-h period. Successive extractions ofmicrocapsules with ammoniacal methanol, 2.5 N HCI, methanoland dimethylsulfoxide released 60% of bound radiolabel andthe unextracted radiolabel was presumed to have been bound covalently.By contrast, > 90% of bound radiolabel was extractable fromthe faeces of the treated animals and from microcapsules treatedin vitro with [¹⁴C]7,8-dihydroxy-9,10-epoxytetrahydrobenzo[a]-pyrene(BaPDE), indicating that the in vivo microcapsule- bound metaboliteswere not derived either from adsorbed faecal material or from[¹⁴C]BaPDE formed in situ. A time-dependent appearance of BaP3,6-dione was found. Also the qualitative and quantitative patternsof metabolites trapped by microcapsules, as assayed by h.p.l.c,were consistent only with a unique set of BaP metabolites beingbound within the intestinal lumen. Hence these carcinogen-bindingmicrocapsules can be used to investigate the in situ formationof carcinogen metabolites within the intestinal tract.     

Fluorescence detection of benzo[a]pyrene--DNA adducts in human lung.

Improved techniques are described for the specific identification of benzo[a]pyrene-diolepoxide (BPDE)--DNA adducts in human tissues. Immunoaffinity chromatography, synchronous fluorescence spectroscopy and second-derivative synchronous fluorescence spectroscopy have previously been used to detect BPDE-DNA adducts in human placenta. Here we report how these methods, together with HPLC and the generation of complete fluorescence excitation--emission matrices, have been used to identify unequivocally BPDE-DNA adducts in samples of human lung. BPDE nucleotide adducts were isolated with immunoaffinity chromatography columns bearing antibodies raised against the (+/-)anti-7,8-diol-9,10-epoxide-deoxyguanosine adduct of benzo[a]pyrene. These adducts were hydrolyzed to tetrahydrotetrols and the hydrolysis products subjected to HPLC. The major product isolated by HPLC, benzo[a]-pyrene-7,10/8,9-tetrahydrotetrol, was determined by fluorescence spectroscopy. Using this method, levels of BPDE-DNA adducts in the range of 1-40 in 10(8) nucleotides were measured in 6 out of 25 samples, with a lower detection limit of one adduct in 10(8) nucleotides. The data may also indicate that adduct levels show regional variation in different parts of the same lung.     

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.     

DNA adducts and induction of sister chromatid exchanges in the rat following benzo[b]fluoranthene administration.

Benzo[b]fluoranthene (B[b]F) was administered (100 mg/kg by i.p. injection) to male Sprague--Dawley rats. Lungs, livers and peripheral blood lymphocytes (PBLs) were harvested 1, 3, 5, 7, 14, 28 and 56 days after treatment. Several DNA adducts were observed in each tissue, with maximal levels occurring at approximately 7 days after treatment. Lung DNA exhibited consistently higher adduct levels than liver or PBL DNA. At 56 days after B[b]F administration, the adducts in liver and PBL DNA were present at < 10 amol/microgram DNA, while in lung there were 100 amoles/microgram DNA. No significant differences were observed between tissues in the types of adducts produced. Co-chromatography with synthetic standards showed that only a minor adduct produced in vivo is derived from trans-9,10-dihydro-9,10-dihydroxybenzo[b]fluoranthene-11,12-oxide. Sister chromatid exchanges (SCEs) from whole blood cultures were significantly increased relative to concurrent controls between 1 and 14 days after B[b]F administration, with maximum levels at 14 days. By 28 days after treatment, SCEs had essentially returned to control levels. SCE induction did not correlate with the amount of B[b]F--DNA adducts remaining in the PBLs at harvest time.     

Absorption, metabolism and excretion of [14C]pomalidomide in humans following oral administration

Purpose

To investigate the pharmacokinetics and disposition of [¹⁴C]pomalidomide following a single oral dose to healthy male subjects.

Methods

Eight subjects were administered a single 2 mg oral suspension of [¹⁴C]pomalidomide. Blood (plasma), urine and feces were collected. Mass balance of radioactivity and the pharmacokinetics of radioactivity, pomalidomide and metabolites were determined. Metabolite profiling and characterization was performed. The enzymes involved in pomalidomide metabolism and the potential pharmacological activity of metabolites were evaluated in vitro.

Results

Mean recovery was 88 %, with 73 and 15 % of the radioactive dose excreted in urine and feces, respectively, indicating good oral absorption. Mean C _max, AUC_0?∞ and t _max values for pomalidomide in plasma were 13 ng/mL, 189 ng*h/mL and 3.0 h. Radioactivity and pomalidomide were rapidly cleared from circulation, with terminal half-lives of 8.9 and 11.2 h. Pomalidomide accounted for 70 % of the circulating radioactivity, and no circulating metabolite was present at >10 % of parent compound. Pomalidomide was extensively metabolized prior to excretion, with excreted metabolites being similar to those observed in circulation. Clearance pathways included cytochrome P450-mediated hydroxylation with subsequent glucuronidation (43 % of the dose), glutarimide ring hydrolysis (25 %) and excretion of unchanged drug (10 %). 5-Hydroxy pomalidomide, the notable oxidative metabolite, was formed primarily via CYP1A2 and CYP3A4. The hydroxy metabolites and hydrolysis products were at least 26-fold less pharmacologically active than pomalidomide in vitro.

Conclusions

Following oral administration, pomalidomide was well absorbed, with parent compound being the predominant circulating component. Pomalidomide was extensively metabolized prior to excretion, and metabolites were eliminated primarily in urine.     

Quinone-induced DNA single strand breaks in a human colon carcinoma cell line

It has been demonstrated that synthetic quinones, such as menadione, cause DNA damage in different cell systems, possibly being mediated by free radicals generated during redox cycling. It has been suggested that the damage caused could be related to tumor induction in different sites. To our knowledge it has not yet been demonstrated that the natural quinones, vitamin K1 and K2, exert the same activity. Using a colon carcinoma cell line, HT-29, we examined the extent of DNA damage induced by menadione, vitamin K1 and K2. Menadione caused significant DNA damage at low concentrations (25-200 microM) with a linear correlation of r = 0.95. In the presence of dicoumarol, a DT-diaphorase inhibitor, the damage was detected at concentrations five times lower indicating that free radicals generated during the redox cycling play a key role. Neither vitamin K1, incorporated in micelles, nor K2 caused detectable single strand breaks with respect to the controls either in the presence or in absence of dicoumarol. Our results demonstrate that, despite their redox cycling properties, the natural forms of vitamin K do not cause DNA damage in HT-29 cells as menadione does in the experimental conditions used.      

Non-random distribution of dibenzo[a, e]fluoranthene-induced DNA adducts in DNA loops in mouse fibroblast nuclei

The three-dimensional distribution of nuclear DNA damage inducedby dibenzo[a, e]fluoranthene (DBF), a potent carcinogen formouse fibroblasts, has been examined. The intact supercoilednuclear DNA obtained from nucleoids of mouse fibroblasts incubatedwith DBF was fractionated into loop DNA attached to the matrix(10%) and bulk loop DNA (90%). Preferential binding of DBF tothe DNA of the extremities of loops, which are rich in regulatorysequences, was observed in all experiments. An increase of thepreferential DBF binding was seen when fibroblasts were incubatedwith both DBF and novobiocin or hydroxyurea. The excess damageseen in loop DNA attached to the cage may be due to the functionalstate of chromatin proximate to the matrix and to the kineticsof diffusion to the interior of the nucleus of hydrophobic DBFmetabolites accumulated in lipid-rich nuclear membrane.     

Disposition and metabolism of [14-14C] 4-demethoxydaunorubicin HCl (idarubicin) and [14-14C]daunorubicin HCl in the rat

Summary The disposition of [14-¹⁴C]4-demethoxydaunorubicin HCl ([14-¹⁴C]idarubicin HCl, [¹⁴C]IDR) and of [14-¹⁴C]daunorubicin HCl ([¹⁴C]DNR) was studied in male Sprague Dawley rats. [¹⁴C]IDR was administered either IV at 0.25 mg/kg body weight or PO at 1 mg/kg body weight, whereas [¹⁴C]DNR was dosed IV at 1 mg/kg body weight. The main elimination route for both compounds was the bile, fecal excretion representing 0.75–0.8 times the total dose at 72 h. Radioactivity due to [¹⁴C]IDR-derived species is released by the tissues at a slower rate than activity derived from [¹⁴C]DNR. After IV treatment comparable plasma levels are obtained, but tissue radioactivity is markedly lower with [¹⁴C]IDR, in keeping with the lower dosage. The ratio of plasma to tissue radioactivity is even higher in animals treated PO with [¹⁴C]IDR, because of the more extensive metabolism after this route of administration. The 13-dihydro derivatives of both [¹⁴C]IDR and [¹⁴C]DNR are the main metabolites in tissues, but in the case of the former, products of phase II reactions become more important at later times in liver and kidney and in excreta.     

Binding of [14C]azaserine to DNA and protein in the rat and hamster

The binding of [¹⁴C]azaserine or its metabolites to DNA and protein in the organs of rats and hamsters was determined at various times after treatment with [¹⁴C]azaserine. The specific activity of ¹⁴C labelling of DNA and protein was determined. Rat liver DNA and protein were most extensively labelled at 90 min post-injection, but by 24 h the specific activity decreased to the levels found in pancreas and kidney. Thymus contained negligible amounts of radioactivity at all time-points. DNA and protein from hamster pancreas contained more label than did DNA and protein from rat pancreas. The results suggest that factors other than DNA binding play a role in determining the species and organ specificity of azaserine.     

Presence of benzo[a]pyrene diol epoxide adducts in target DNA leads to an increase in UV-induced DNA single strand breaks and supF gene mutations

Exposure to DNA damaging agents and mutagens often occurs as combinations of agents, or as complex mixtures of chemicals. We found that plasmid DNA adducted with benzo[a]pyrene diol epoxide (BPDE) was more susceptible to UV-induced single strand breaks than was control DNA. To determine whether the increase in DNA damage also applied to mutagenic lesions, the supF gene forward mutation assay was used to compare mutations induced by BPDE alone, UVB, UVC, BPDE followed by UVB and BPDE followed by UVC. It was found that the mutation frequency for BPDE + UVB (1167 in 10(4) transformants) was higher than BPDE alone (12 in 10(4) transformants) or UVB alone (446 in 10(4) transformants), and the mutation frequency for BPDE + UVC (197 in 10(4) transformants) was higher than BPDE alone or UVC alone (26 in 10(4) transformants). For BPDE + UVB and BPDE + UVC there was a significant increase in plasmids with multiple mutations. Whilst these indicate error prone repair due to the single strand breaks, the different mutation frequencies in plasmids treated to give similar levels of strand breaks suggest other mechanisms for the mutations in plasmids with single mutation events. The spectrum of non-multiple mutations in the two combined treatments included both UV signature mutations (GC-->AT as the most common mutation) and BPDE signature mutations (GC-->TA and GC-->CG as the most common mutations). However, the increase in absolute mutation frequency of BPDE signature mutations between BPDE treatment and BPDE + UV treatment was greater than the increase in absolute mutation frequency of UV signature mutations, even though the level of BPDE adducts was identical in each case. These results suggest two possibilities: (i) the BPDE adducts are photoactivated to a more mutagenic lesion, or (ii) the presence of UV lesions lead to the BPDE adducts becoming more mutagenic.     

DNA adducts of the ubiquitous enviromnental contaminant cyclopenta[cd]pyrene

The bioactivation of cydopenta[cd]pyrene (CPP) was investigatedto determine the major DNA adduct-forming metabolite(s) of thiswidespread environmental contaminant and suspect carcinogen.DNA adducts were analyzed by ³²P-postlabeling. Four major andat least seven minor adducts formed when CPP was incubated withcalf thymus DNA in the presence of rat liver microsomal systems.P450 subfamilies IA and IIB both activated CPP as microsomesfrom either phenobarbital- or ß-naphthoflavone-treatedrats produced quantitatively similar and qualitatively identicaladducts. When the epoxide hydrolase inhibitors, 1,1,1-trichloropropene-2,3-oxideor cydohexene oxide were added to the incubatlons, binding increased2.5- to 4-fold, suggesting epoxidation as a mechanism of adductformation in vitro. Sprague-Dawley rats were killed 1,3,7,18,45and 80 days postdosing i.p. with 50 mg/kg CPP. In all tissuesanalyzed, four major and several minor qualitatively Identicaladducts were produced. Binding was highest and most persistentIn lung followed by heart, white blood cells (WBCs) and liver.CPP adducts were detectable at doses from 1 µg/kg to 50mg/kg. Rat lung DNA adducts were cochromatographed with standardizeddeoxyguanosine and deoxyadenosine adducts produced by reactionof CPP-3,4-epoxide in vitro. All rat lung adducts comigratedwith the deoxyguanosine adducts but one was clearly deoxyadenosinederived. Mouse skin DNA adducts from NIH Swiss mice and mouselung DNA adducts from B6C3F1 mice were also analyzed. All adductsfrom either mouse tissue comigrated with rat lung DNA adducts,suggesting CPP-3,4-epoxide was also the major DNA adduct formingspecies in the mouse. CPP-3,4-epoxlde has been suggested tobe the key mediator of the biological activities of CPP. Evidencepresented here strongly suggests CPP-3,4-epoxlde as the majoradduct-forming species of CPP as catalyzed in vitro by rat liverpreparations known to mediate the mutagenic activation of CPP,in the rat in vivo, and in mouse skin and lung, two tissueswith known sensitivity to CPP tumorigenicity.