Showing papers in "Chemical Research in Toxicology in 2002"

Metabolism and kinetics of bisphenol a in humans at low doses following oral administration.

Abstract: Bisphenol A is a widely used industrial chemical with many potential sources of human exposure. Bisphenol A is a weak estrogen and has been implicated as an "endocrine disruptor". This term is used for a variety of chemicals encountered in the environment which have estrogenic activity. It has been postulated that human exposure to these chemicals may elicit unwanted estrogenic effects in humans such as reduced fertility, altered development and cancer. Up to now the body burden of bisphenol A in humans is unknown. Therefore, we investigated the metabolism and toxicokinetics of bisphenol A in humans exposed to low doses since systemic bioavailability has a major influence on possible estrogenic effects in vivo. Human subjects (three males and three females, and four males for detailed description of blood kinetics) were administered d(16)-bisphenol A (5 mg). Blood and urine samples were taken in intervals (up to 96 h), metabolites formed were identified by GC/MS and LC-MS/MS and quantified by GC/MS-NCI and LC-MS/MS. d(16)-Bisphenol A glucuronide was the only metabolite of d(16)-bisphenol A detected in urine and blood samples, and concentrations of free d(16)-bisphenol A were below the limit of detection both in urine (6 nM) and blood samples (10 nM). d(16)-Bisphenol A glucuronide was cleared from human blood and excreted with urine with terminal half-lives of less than 6 h; the applied doses were completely recovered in urine as d(16)-bisphenol A glucuronide. Maximum blood levels of d(16)-bisphenol A glucuronide (approximately 800 nM) were measured 80 min after oral administration of d(16)-bisphenol A (5 mg). The obtained data indicate major species differences in the disposition of bisphenol A. Enterohepatic circulation of bisphenol A glucuronide in rats results in a slow rate of excretion, whereas bisphenol A is rapidly conjugated and excreted by humans due to the absence of enterohepatic circulation. The efficient glucuronidation of bisphenol A and the rapid excretion of the formed glucuronide result in a low body burden of the estrogenic bisphenol A in humans following oral absorption of low doses.

Protective role of Kupffer cells in acetaminophen-induced hepatic injury in mice.

Abstract: Hepatic injury induced by various toxic agents, including acetaminophen (APAP), has been attributed, in part, to the production of proinflammatory cytokines and other mediators by resident Kupffer cells within the liver. However, recent evidence from our laboratory has demonstrated that hepato-protective factors, such as interleukin (IL)-10 and cyclooxygenase-derived mediators, are also upregulated in response to hepatic damage to help protect against exacerbated injury, and Kupffer cells have been suggested to be a source of these modulatory factors. In other models, Kupffer cells also serve important regulatory functions in pathophysiological states of the liver. Therefore, we reevaluated the role of Kupffer cells in a murine model of APAP-induced liver injury using liposome-entrapped clodronate (liposome/clodronate) as an effective Kupffer cell-depleting agent. We show that in contrast to pretreatment of mice with a widely used macrophage inhibitor, gadolinium chloride, which did not deplete Kupffer cells but moderately protected against APAP-induced hepatotoxicity as reported previously, the intravenous injection of liposome/clodronate caused nearly complete elimination of Kupffer cells and significantly increased susceptibility to APAP-induced liver injury as compared with mice pretreated with empty liposomes. This increased susceptibility was apparently unrelated to the metabolism of APAP since liposome/clodronate pretreatment did not alter APAP-protein adduct levels. Instead, Kupffer cell depletion by liposome/clodronate led to significant decreases in the levels of hepatic mRNA expression of several hepato-regulatory cytokines and mediators, including IL-6, IL-10, IL-18 binding protein and complement 1q, suggesting that Kupffer cells are a significant source for production of these mediators in this model. Our findings indicate that, in addition to their protoxicant activities, Kupffer cells can also have an important protective function in the liver through the production of a variety of modulatory factors which may counteract inflammatory responses and/or stimulate liver regeneration.

Oxidation of proximal protein sulfhydryls by phenanthraquinone, a component of diesel exhaust particles.

Abstract: Diesel exhaust particles (DEP) contain quinones that are capable of catalyzing the generation of reactive oxygen species in biological systems, resulting in induction of oxidative stress. In the present study, we explored sulfhydryl oxidation by phenanthraquinone, a component of DEP, using thiol compounds and protein preparations. Phenanthraquinone reacted readily with dithiol compounds such as dithiothreitol (DTT), 2,3-dimercapto-1-propanol (BAL), and 2,3-dimercapto-1-propanesulfonic acid (DMPS), resulting in modification of the thiol groups, whereas minimal reactivities of this quinone with monothiol compounds such as GSH, 2-mercaptoethanol, and N-acetyl-L-cysteine were seen. The modification of DTT dithiol caused by phenanthraquinone proceeded under anaerobic conditions but was accelerated by molecular oxygen. Phenanthraquinone was also capable of modifying thiol groups in pulmonary microsomes from rats and total membrane preparation isolated from bovine aortic endothelial cells (BAEC), but not bovine serum albumin (BSA), which has a Cys34 as a reactive monothiol group. A comparison of the thiol alkylating agent N-ethylmaleimide (NEM) with that of phenanthraquinone indicates that the two mechanisms of thiol modification are distinct. Studies revealed that thiyl radical intermediates and reactive oxygen species were generated during interaction of phenanthraquinone with DTT. From these findings, it is suggested that phenanthraquinone-mediated destruction of protein sulfhydryls appears to involve the oxidation of presumably proximal thiols and the reduction of molecular oxygen.

Covalent modification of amino acid nucleophiles by the lipid peroxidation products 4-hydroxy-2-nonenal and 4-oxo-2-nonenal.

Abstract: Lipid peroxidation yields the aldehydes 4-hydroxynonenal (4HNE) and 4-oxononenal (4ONE). Protein adduction by 4HNE is thought to be involved in the pathogenesis of several diseases. Currently, the reactivity of 4ONE toward proteins is unknown. The purpose of this study was to identify amino acids that react with 4HNE and 4ONE, characterize the chemical structure of the adduct, and determine the preference for amino acid modification. Model peptides containing one or more nucleophilic residues (i.e., Arg, Cys, His, Met, and Lys) were reacted with 4HNE and 4ONE and analyzed using matrix-assisted laser desorption/ionization mass spectrometry. Post-source decay analysis was used to confirm peptide modification. The bimolecular rate constant for adduction of amino acids and peptides by 4HNE and 4ONE was measured. Results of this work indicate that Cys, His, and Lys are modified by 4HNE and 4ONE. In addition, Arg was adducted by 4ONE. The predominant adduct resulting from modification of peptides by 4HNE or 4ON...

Combined functional genomic maps of the C-elegans DNA damage response

Abstract: Many human cancers originate from defects in the DNA damage response (DDR). Although much is known about this process, it is likely that additional DDR genes remain to be discovered. To identify such genes, we used a strategy that combines protein-protein interaction mapping and large-scale phenotypic analysis inCaenorhabditis elegans. Together, these approaches identified 12 worm DDR orthologs and 11 novel DDR genes. One of these is the putative ortholog of hBCL3, a gene frequently altered in chronic lymphocytic leukemia. Thus, the combination of functional genomic mapping approaches in model organisms may facilitate the identification and characterization of genes involved in cancer and, perhaps, other human diseases.

Analytical reproducibility in (1)H NMR-based metabonomic urinalysis.

Abstract: Metabonomic analysis of biofluids and tissues utilizing high-resolution NMR spectroscopy and chemometric techniques has proven valuable in characterizing the biochemical response to toxicity for many xenobiotics. To assess the analytical reproducibility of metabonomic protocols, sample preparation and NMR data acquisition were performed at two sites (one using a 500 MHz and the other using a 600 MHz system) using two identical (split) sets of urine samples from an 8-day acute study of hydrazine toxicity in the rat. Despite the difference in spectrometer operating frequency, both datasets were extremely similar when analyzed using principal components analysis (PCA) and gave near-identical descriptions of the metabolic responses to hydrazine treatment. The main consistent difference between the datasets was related to the efficiency of water resonance suppression in the spectra. In a 4-PC model of both datasets combined, describing all systematic dose- and time-related variation (88% of the total variation), differences between the two datasets accounted for only 3% of the total modeled variance compared to ca. 15% for normal physiological (pre-dose) variation. Furthermore, 95% correlation (r(2)) between sites, with an analytical error comparable to normal physiological variation in concentration (4-8%). The excellent analytical reproducibility and robustness of metabonomic techniques demonstrated here are highly competitive compared to the best proteomic analyses and are in significant contrast to genomic microarray platforms, both of which are complementary techniques for predictive and mechanistic toxicology. These results have implications for the quantitative interpretation of metabonomic data, and the establishment of quality control criteria for both regulatory agencies and for integrating data obtained at different sites.

Identification and characterization of methylated and ring-fission metabolites of tea catechins formed in humans, mice, and rats.

Abstract: (-)-Epigallocatechin gallate (EGCG), the most abundant tea catechin, has been proposed to be beneficial to human health based on its strong antioxidative and other biological activities in vitro. Inadequate knowledge regarding the bioavailability and biotransformation of EGCG in humans, however, has limited our understanding of its possible beneficial health effects. In this study, 4',4' '-di-O-methyl-EGCG (4',4' '-DiMeEGCG) was detected in human plasma and urine by LC/MS/MS following green tea ingestion. Both 4',4' '-DiMeEGCG and EGCG reached peak plasma values (20.5 +/- 7.7 and 145.4 +/- 31.6 nM, respectively, in 4 subjects) at 2 h after the dose. The half-lives of 4',4' '-DiMeEGCG and EGCG were 4.1 +/- 0.8 and 2.7 +/- 0.9 h, respectively. The cumulative urinary excretion of 4',4' '-DiMeEGCG during a 24 h period was 140.3 +/- 48.6 microg, about 5-fold higher than that of EGCG, but the excreted 4',4' '-DiMeEGCG and EGCG in urine only accounted for about 0.1% of ingested EGCG. (-)-5-(3',4',5'-Trihydroxyphenyl)-gamma-valerolactone (M4) and (-)-5-(3',4'-dihydroxyphenyl)-gamma-valerolactone (M6), along with another possible ring-fission metabolite, (-)-5-(3',5'-dihydroxyphenyl)-gamma-valerolactone (M6'), were detected in human urine after green tea ingestion. The cumulative excretion of M4, M6', and M6 during a 24 h period ranged from 75 microg to 1.2 mg, 0.6 to 6 mg, and 0.6 to 10 mg, respectively. The combined excretion of all three ring-fission metabolites accounted for 1.5-16% of ingested catechins. M4, M6', and M6 were all observed after the ingestion of pure EGCG or EGC by human subjects, whereas only M6 was produced after EC ingestion. These metabolites as well as monomethylated EGCG were detected in mice and rats after tea or EGCG administration, and the tissue levels reflected the rather low bioavailability of EGCG in rats. The presently characterized methylated EGCG metabolites and ring-fission products exist in substantial quantities and may contribute to the biological activities of tea.

DNA damage induced by methylated trivalent arsenicals is mediated by reactive oxygen species.

Abstract: Arsenic is a human carcinogen; however, the mechanisms of arsenic's induction of carcinogenic effects have not been identified clearly. We have shown previously that monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)) are genotoxic and can damage supercoiled phiX174 DNA and the DNA in peripheral human lymphocytes in culture. These trivalent arsenicals are biomethylated forms of inorganic arsenic and have been detected in the urine of subjects exposed to arsenite and arsenate. We show here by molecular, chemical, and physical methods that reactive oxygen species (ROS) are intermediates in the DNA-damaging activities of MMA(III) and DMA(III). Using the phiX174 DNA nicking assay we found that the ROS inhibitors Tiron, melatonin, and the vitamin E analogue Trolox inhibited the DNA-nicking activities of both MMA(III) and DMA(III) at low micromolar concentrations. The spin trap agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) also was effective at preventing the DNA nicking induced by MMA(III) and DMA(III). ESR spectroscopy studies using DMPO identified a radical as a ROS intermediate in the DNA incubations with DMA(III). This radical adduct was assigned to the DMPO-hydroxyl free radical adduct on the basis of comparison of the observed hyperfine splitting constants and line widths with those reported in the literature. The formation of the DMPO-hydroxyl free radical adduct was dependent on time and the presence of DMA(III) and was completely inhibited by Tiron and Trolox and partially inhibited by DMSO. Using electrospray mass spectrometry, micromolar concentrations of DMA(V) were detected in the DNA incubation mixtures with DMA(III). These data are consistent with the conclusions that the DNA-damaging activity of DMA(III) is an indirect genotoxic effect mediated by ROS-formed concomitantly with the oxidation of DMA(III) to DMA(V).

Regioselectivity of phase II metabolism of luteolin and quercetin by UDP-glucuronosyl transferases.

Abstract: The regioselectivity of phase II conjugation of flavonoids is expected to be of importance for their biological activity. In the present study, the regioselectivity of phase II biotransformation of the model flavonoids luteolin and quercetin by UDP-glucuronosyltransferases was investigated. Identification of the metabolites formed in microsomal incubations with luteolin or quercetin was done using HPLC, LC-MS, and (1)H NMR. The results obtained demonstrate the major sites for glucuronidation to be the 7-, 3-, 3'-, or 4'-hydroxyl moiety. Using these unequivocal identifications, the regioselectivity of the glucuronidation of luteolin and quercetin by microsomal samples from different origin, i.e., rat and human intestine and liver, as well as by various individual human UDP-glucuronosyltransferase isoenzymes was characterized. The results obtained reveal that regioselectivity is dependent on the model flavonoid of interest, glucuronidation of luteolin and quercetin not following the same pattern, depending on the isoenzyme of UDP-glucuronosyltransferases (UGT) involved. Human UGT1A1, UGT1A8, and UGT1A9 were shown to be especially active in conjugation of both flavonoids, whereas UGT1A4 and UGT1A10 and the isoenzymes from the UGTB family, UGT2B7 and UGT2B15, were less efficient. Due to the different regioselectivity and activity displayed by the various UDP-glucuronosyltransferases, regioselectivity and rate of flavonoid conjugation varies with species and organ. Qualitative comparison of the regioselectivities of glucuronidation obtained with human intestine and liver microsomes to those obtained with human UGT isoenzymes indicates that, in human liver, especially UGT1A9 and, in intestine, UGT1A1 and UGT1A8 are involved in glucuronidation of quercetin and luteolin. Taking into account the fact that the anti-oxidant action as well as the pro-oxidant toxicity of these catechol-type flavonoids is especially related to their 3',4'-dihydroxyl moiety, it is of interest to note that the human intestine UGT's appear to be especially effective in conjugating this 3',4' catechol unit. This would imply that upon glucuronidation along the transport across the intestinal border, the flavonoids loose a significant part of these biological activities.

Retrospective detection of exposure to organophosphorus anti-cholinesterases: Mass spectrometric analysis of phosphylated human butyrylcholinesterase

Abstract: In this paper a novel and general procedure is presented for detection of organophosphate-inhibited human butyrylcholinesterase (HuBuChE), which is based on electrospray tandem mass spectrometric analysis of phosphylated nonapeptides obtained after pepsin digestion of the enzyme. The utility of this method is exemplified by the positive analysis of serum samples from Japanese victims of the terrorist attack with sarin in the Tokyo subway in 1995.

Surface modification of quartz inhibits toxicity, particle uptake, and oxidative DNA damage in human lung epithelial cells

Abstract: Quartz (crystalline silica) is not consistently carcinogenic across different industries where similar quartz exposure occurs. In addition, there are reports that surface modification of quartz affects its cytotoxicity, inflammogenicity, and fibrogenicity. Taken together, these data suggest that the carcinogenicity of quartz is also related to particle surface characteristics, and so we determined the genotoxic effects of DQ12 quartz particles versus DQ12 whose surface was modified by treating with either aluminum lactate or polyvinylpyridine-N-oxide (PVNO). The different particle preparations were characterized for hydroxyl-radical generation using electron spin resonance (ESR). DNA damage was determined by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG) and the alkaline comet-assay using A549 human lung epithelial cells. Cytotoxicity was measured using the LDH- and MTT-assays, and particle uptake by the A549 cells was quantified by light microscopy, using digital light imaging evaluation of 800 nm sections. The ability of quartz to generate hydroxyl-radicals in the presence of hydrogen peroxide was markedly reduced upon surface modification with aluminum lactate or PVNO. DNA strand breakage and 8-OHdG formation, as produced by quartz at nontoxic concentrations, could be completely prevented by both coating materials. Particle uptake into A549 cells appeared to be significantly inhibited by the PVNO-coating, and to a lesser extent by the aluminum-lactate coating. Our data demonstrate that respirable quartz particles induce oxidative DNA damage in human lung epithelial cells and indicates that surface properties of the quartz as well as particle uptake by these target cells are important in the cytotoxic and the genotoxic effects of quartz in vitro.

Hypochlorite-Induced Damage to DNA, RNA, and Polynucleotides: Formation of Chloramines and Nitrogen-Centered Radicals

Abstract: Stimulated monocytes and neutrophils generate hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is a key bactericidal agent, but can also damage host tissue. As there is a strong link between chronic inflammation and some cancers, we have investigated HOCl damage to DNA, RNA, and polynucleotides. Reaction of HOCl with these materials is shown to yield multiple semistable chloramines (RNHCl/RR'NCl), which are the major initial products, and account for 50-95% of the added HOCl. These chloramines decay by thermal and metal-ion catalyzed processes, to give nucleoside-derived, nitrogen-centered, radicals. The latter have been characterized by EPR spin trapping. The propensity for radical formation with polynucleotides is cytidine > adenosine = guanosine > uridine = thymidine. The rates of decay, and yield of radicals formed, are dependent on the nature of the nucleobase on which they are formed, with chloramines formed from ring heterocyclic amine groups being less stable than those formed on exocyclic amines (RNH2 groups). Evidence is presented for chlorine transfer from the former, kinetically favored, sites to the more thermodynamically favored exocyclic amines. EPR experiments have also provided evidence for the rapid addition of pyrimidine-derived nitrogen-centered radicals to other nucleobases to give dimers and the oxidation of DNA by radicals derived from preformed nucleoside chloramines. Direct reaction of HOCl with plasmid DNA gives rise to single- and double-strand breaks via chloramine-mediated reactions. Preformed nucleoside chloramines also induce plasmid cleavage, though this only occurs to a significant extent with unstable thymidine- and uridine-derived chloramines, where radical formation is rapid. Overall the data rationalize the preferential formation of chlorinated 2'-deoxycytidine and 2'-deoxyadenosine in DNA and suggest that DNA damage induced by HOCl, and preformed chloramines, occurs at sequence-specific sites.

Cytochrome P450 3A4-mediated bioactivation of raloxifene: irreversible enzyme inhibition and thiol adduct formation.

Abstract: Raloxifene is a selective estrogen receptor modulator which is effective in the treatment of osteoporosis in postmenopausal women. We report herein that cytochrome P450 (P450)3A4 is inhibited by raloxifene in human liver microsomal incubations. The nature of the inhibition was irreversible and was NADPH- and preincubation time-dependent, with K(I) and k(inact) values estimated at 9.9 microM and 0.16 min(-1), respectively. The observed loss of P450 3A4 activity was attenuated partially by glutathione (GSH), implying the involvement of a reactive metabolite(s) in the inactivation process. Subsequently, GSH adducts of raloxifene were identified in incubations with human liver microsomes; substitution with GSH occurred at the 5- or 7-position of the benzothiophene moiety or at the 3'-position of the phenol ring, with the 7-glutathionyl derivative being most abundant based on LC/MS and NMR analyses. These adducts are postulated to derive from addition of GSH to raloxifene arene oxides followed by dehydration and aromatization. Alternatively, raloxifene may be oxidized to an extended quinone intermediate, which then is trapped by GSH conjugation. The bioactivation of raloxifene most likely is catalyzed by P450 3A4, since the formation of GSH adducts was almost abolished when liver microsomes were pretreated with ketoconazole or with an inhibitory anti-P450 3A4 IgG. The GSH adducts also were detected in incubations of raloxifene with rat or human hepatocytes, while the corresponding N-acetylcysteine adducts were identified in the bile and urine from rats treated orally with the drug at 5 mg/kg. Taken together, these data indicate that P450 3A4-mediated bioactivation of raloxifene in vitro is accompanied by loss of enzyme activity. The significance of these findings with respect to the clinical use of raloxifene remains to be determined.

Pesticide Roundup provokes cell division dysfunction at the level of CDK1/cyclin B activation.

Abstract: To assess human health risk from environmental chemicals, we have studied the effect on cell cycle regulation of the widely used glyphosate-containing pesticide Roundup. As a model system we have used sea urchin embryonic first divisions following fertilization, which are appropriate for the study of universal cell cycle regulation without interference with transcription. We show that 0.8% Roundup (containing 8 mM glyphosate) induces a delay in the kinetic of the first cell cleavage of sea urchin embryos. The delay is dependent on the concentration of Roundup. The delay in the cell cycle could be induced using increasing glyphosate concentrations (1-10 mM) in the presence of a subthreshold concentration of Roundup 0.2%, while glyphosate alone was ineffective, thus indicating synergy between glyphosate and Roundup formulation products. The effect of Roundup was not lethal and involved a delay in entry into M-phase of the cell cycle, as judged cytologically. Since CDK1/cyclin B regulates universally the M-phase of the cell cycle, we analyzed CDK1/cyclin B activation during the first division of early development. Roundup delayed the activation of CDK1/cyclin B in vivo. Roundup inhibited also the global protein synthetic rate without preventing the accumulation of cyclin B. In summary, Roundup affects cell cycle regulation by delaying activation of the CDK1/cyclin B complex, by synergic effect of glyphosate and formulation products. Considering the universality among species of the CDK1/cyclin B regulator, our results question the safety of glyphosate and Roundup on human health.

Possible role of dimethylarsinous acid in dimethylarsinic acid-induced urothelial toxicity and regeneration in the rat.

Abstract: Dimethylarsinic acid (DMA(V)) is carcinogenic to the rat urinary bladder when administered at high doses in the diet or drinking water. At a dietary dose of 100 ppm (microg/g), it produces cytotoxicity within 6 h and increased proliferation (hyperplasia) by 7 days of administration. We hypothesize that formation of the reactive organic intermediate dimethylarsinous acid (DMA(III)) is involved in the induction of the cytotoxicity. To evaluate the possibility that DMA(V) administration produces urothelial toxicity and regeneration by the formation of trivalent arsenicals, 2,3-dimercaptopropane-1-sulfonic acid (DMPS, 5600 ppm), a chelator of trivalent arsenicals, was co-administered with DMA(V) (100 ppm) for 2 weeks to groups of female Fischer F344 rats. Based on light and scanning electron microscopy, and bromodeoxyuridine labeling index, DMA(V) produced cytotoxicity and regenerative hyperplasia of the urothelium which was inhibited by co-administration with DMPS. The major forms of arsenic in the 24-h urine of rats administered DMA(V) were high concentrations of DMA(V) (66.4 +/- 2.7 microM) itself and the pentavalent organic arsenical trimethylarsine oxide (TMAO) (73.2 +/- 9.5 microM). Co-administration with DMPS led to an increase in DMA(V) (507 +/- 31 microM) with a decrease in TMAO (2.8 +/- 0.4 microM) excretion. The formation of TMAO from DMA(V) mechanistically suggests formation of the intermediate trivalent metabolite, DMA(III). In a second experiment evaluating fresh void urines collected on study days 1, 71, and 175, we detected DMA(III) in the urine of DMA(V) and DMA(V) plus DMPS-treated rats at approximately micromolar concentrations. Using rat (MYP3) and human (1T1) urothelial cells, cytotoxicity for trivalent arsenicals, sodium arsenite, monomethylarsonous acid (MMA(III)), and DMA(III) was demonstrated at 0.4-4.8 microM concentrations, whereas MMA(V), DMA(V), and TMAO were cytotoxic at millimolar concentrations. The presence of DMA(III) at micromolar concentrations in the urine of rats fed 100 ppm DMA(V) suggests that DMA(III) produced in vivo may be involved in the toxic effects in the rat urinary bladder after dietary administration of DMA(V).

Catechol and hydroquinone have different redox properties responsible for their differential DNA-damaging ability.

Abstract: We examined the redox properties of the “carcinogenic” catechol and the “noncarcinogenic” hydroquinone in relation to different DNA damaging activities and carcinogenicity using 32P-labeled DNA fragments obtained from the human genes. In the presence of endogenous NADH and Cu2+, catechol induces stronger DNA damage than hydroquinone, although the magnitudes of their DNA damaging activities were reversed in the absence of NADH. In both cases, DNA damage resulted from base modification at guanine and thymine residues in addition to strand breakage induced by Cu+ and H2O2, generated during the oxidation of catechol and hydroquinone into 1,2-benzoquinone and 1,4-benzoquinone, respectively. EPR and 1H NMR studies indicated that 1,2-benzoquinone is converted directly into catechol through a nonenzymatic two-electron reduction by NADH whereas 1,4-benzoquinone is reduced into hydroquinone through a semiquinone radical intermediate through two cycles of one-electron reduction. The reduction of 1,2-benzoquinone by ...

Arsenate reductase II. Purine nucleoside phosphorylase in the presence of dihydrolipoic acid is a route for reduction of arsenate to arsenite in mammalian systems.

Abstract: An arsenate reductase has been partially purified from human liver using ion exchange, molecular exclusion, hydroxyapatite chromatography, preparative isoelectric focusing, and electrophoresis. When SDS−β-mercaptoethanol−PAGE was performed on the most purified fraction, two bands were obtained. One of these bands was a 34 kDa protein. Each band was excised from the gel and sequenced by LC-MS/MS, and sequest analyses were performed against the OWL database SWISS−PROT with PIR. Mass spectra analysis matched the 34 kDa protein of interest with human purine nucleoside phosphorylase (PNP). The peptide fragments equal to 40.1% of the total protein were 100% identical to the corresponding regions of the human purine nucleoside phosphorylase. Reduction of arsenate in the purine nucleoside arsenolysis reaction required both PNP and dihydrolipoic acid (DHLP). The PNP rate of reduction of arsenate with the reducing agents GSH or ascorbic acid was negligible compared to that with the naturally occurring dithiol DHLP ...

Reactive oxygen species generated by PAH o-quinones cause change-in-function mutations in p53.

Abstract: Polycyclic aromatic hydrocarbons (PAHs) in tobacco smoke may cause human lung cancer via metabolic activation to ultimate carcinogens. p53 is one of the most commonly mutated tumor suppressor genes in this disease. An analysis of the p53 mutational database shows that G to T transversions are a signature mutation of lung cancer. Aldo-keto reductases (AKRs) activate PAH trans-dihydrodiol proximate carcinogens to yield their corresponding reactive and redox-active o-quinones, e.g., benzo[a]pyrene-7,8-dione (BP-7,8-dione). We employed a yeast reporter system to determine whether PAH o-quinones or the ROS they generate cause change-in-function mutations in p53. N-Methyl-N-nitroso-N'-nitro-guanidine, a standard alkylating mutagen was used as a positive control. MNNG caused a dose-dependent increase in mutant yeast colonies and at the highest concentrations 8-14% of the yeast colonies were mutated and were characterized by G:C to A:T transitions in the p53 DNA binding domain. Treatment of p53 cDNA with micromolar concentrations of (+/-)-anti-7,8-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene, (anti-BPDE, an ultimate carcinogen) or sub-micromolar concentrations of BP-7,8-dione in the presence of redox-cycling conditions (NADPH and CuCl(2)) also caused p53 mutations in a dose-dependent manner. We found that no mutants were observed with PAH o-quinones or NADPH alone. p53 mutagenesis by BP-7,8-dione was attenuated by ROS scavengers and completely abrogated by a combination of superoxide dismutase and catalase, indicating that both superoxide anion and hydroxyl radicals were the responsible mutagens. The bulk of the mutations detected were single-point mutations and were not random in occurrence. Over 46% of BP-7,8-dione-induced mutations were G:C to T:A transversions, consistent with the formation of 8-oxo-dGuo or its secondary oxidation products. In addition, 25% of these mutations were at hotspots in p53 which are known to be mutated in lung cancer. Together these data suggest that PAH o-quinones generate an endogenous mutagen (ROS) which leads to p53 inactivation. These observations provide an alternative route to G to T transversions that dominate in p53 in lung cancer.

Acetaldehyde in mainstream tobacco smoke: formation and occurrence in smoke and bioavailability in the smoker.

Abstract: A review is presented of the scientific literature on the effects of sugars (mono- and disaccharides), when used as tobacco additives, on the formation of acetaldehyde in mainstream (MS) smoke and the potential bioavailablity of MS smoke acetaldehyde derived from sugars to the smoker. The experimental data supports the following conclusions. Sugars, e.g., D-glucose, D-fructose, and sucrose, do not produce greater yields of acetaldehyde in MS smoke than are produced from tobacco itself on a weight-for-weight basis. A variety of studies suggests that natural tobacco polysaccharides, including cellulose, are the primary precursors of acetaldehyde in MS smoke. In a number of different studies using commercial cigarette brands, MS smoke yields of acetaldehyde correlate (r > 0.9) with both MS smoke "tar" and carbon monoxide. MS smoke acetaldehyde yields are affected more by cigarette design characteristics that influence total smoke production, such as filter ventilation, filtration, and paper porosity, than by reducing sugars. MS smoke acetaldehyde deposits primarily in the upper respiratory tract, including the mouth, of the smoker. Acetaldehyde is rapidly metabolized by aldehyde dehydrogenase in the blood and elsewhere in the body, including at the blood-brain barrier. Tobacco sugar-derived MS smoke acetaldehyde from commercial cigarettes is unlikely to result in direct central nervous system effects on the smoker.

Glutathione conjugation and DNA adduct formation of dibenzo[a,l]pyrene and benzo[a]pyrene diol epoxides in V79 cells stably expressing different human glutathione transferases.

Abstract: Mammalian V79 cells stably expressing human glutathione transferase (GST) A1-1, M1-1, and P1-1 (the allelic variant with Val105 and Ala114) have been constructed and characterized. The cells have been used to study the capacity of individual GST isoenzymes in conjunction with GSH to detoxify diol epoxides from dibenzo[a,l]pyrene (DBPDE), the most carcinogenic polycyclic aromatic hydrocarbon (PAH) identified so far, and diol epoxides from benzo[a]pyrene (BPDE). The relationship between GSH-conjugation and DNA adduct-formation has been investigated as well as factors governing the accessibility of lipophilic diol epoxide substrates for the soluble GSTs in the cells. Relative to control cells, those expressing GSTA1-1 showed the highest rate (about 50-fold increase) to perform GSH-conjugation of (-)-anti-DBPDE (R-absolute configuration at the benzylic oxirane carbon in the fjord-region) followed by GSTM1-1 (25-fold increase) and GSTP1-1 (10-fold increase). GSTA1-1 was found to be strongly inhibited when expressed in cells (10% of fully functional protein). Taking this factor into account, the rates of conjugation found in the cells fairly well reflected the order of catalytic efficiencies (k(cat)/K(m)) obtained with the pure enzymes. Increased GSH conjugation of (-)-anti-DBPDE was associated with a reduction in DNA adduct formation. GSTA1-1 inhibited the formation of adducts more than 6-fold and GSTM1-1 and GSTP1-1 about 2-fold. With (+)-anti-BPDE, GSTP1-1-expressing cells demonstrated a substantially higher rate of GSH-conjugate formation than cells with GSTA1-1 and GSTM1-1 cells (33- and 10-fold increase, respectively). Relative to control cells, GSTM1-1 was found to inhibit DNA adduct formation of (+)-anti-BPDE most effectively followed by GSTP1-1 and GSTA1-1 (12-, 4-, and 3-fold, respectively). Values of k(cat)/K(m) and estimated oil/water partition coefficients of DBPDE and BPDE were used to calculate the concentration of free diol epoxides in solution and expected rates of GSH conjugate formation in cells, and these theoretical results were compared with the observed ones. With the highly reactive (+)-anti-BPDE, 1-2% of the expected activity was observed, whereas the corresponding values for the less reactive (-)-anti-DBPDE were up to 13%. The most obvious explanations for the low observed rate with (+)-anti-BPDE are rapid and competing reactions such as hydrolysis and/or more unspecific chemical and physical reactions with cellular constituents (proteins, lipids, nucleic acids, etc.). In addition, the difference between the theoretical and observed rates may also reflect participation of factors such as macromolecular crowding and reduced rates of diffusion, factors expected to further restrict the accessibility of GST and the diol epoxides in the intact cell.

First Principles Calculations of the Tautomers and pKa Values of 8-Oxoguanine: Implications for Mutagenicity and Repair

Abstract: 8-Oxoguanine is a mutagenic oxidative damage product of guanine that has been the subject of many experimental studies. Despite numerous references to this damaged base, its precise configuration or population of configurations in equilibrium are unknown, as it can be drawn in over 100 potential neutral and ionized tautomeric forms. The structural uncertainty surrounding 8-oxoguanine complicates mechanistic studies of its mutagenicity and capacity to be recognized for repair. Experimental measurements on the tautomeric equilibria and pK_a values of 8-oxoguanine are complicated by its insolubility in water. Therefore, we used first principles quantum mechanics (density functional theory, B3LYP, in combination with the Poisson−Boltzmann continuum-solvation model) to investigate the relative stabilities and site-specific pK_a values of various neutral and ionized tautomers of 8-oxoguanine. We show that the major tautomer of neutral 8-oxoguanine in aqueous solution is the 6,8-diketo form 2, and that 8-oxoguanine has increased acidity at N1 relative to guanine. Our calculations on 2‘-deoxyguanosine-3‘,5‘-bisphosphate and its 8-oxo analogue support the accepted conclusion that repulsion between the O8 of 8-oxoguanine and O5‘ of the backbone sugar promote 8-oxoguanine:adenine pairings in the syn:anti conformation. Further, we show that the N7 proton of 8-oxoguanine is difficult to remove either through tautomerization or ionization, consistent with its involvement as an important landmark in distinguishing guanine from 8-oxoguanine. The possibility of additional structural landmarks that distinguish 8-oxoguanine from guanine, and a possible mechanism for glycosylase removal of 8-oxoguanine are discussed.

Characterization of nucleoside adducts of cis-2-butene-1,4-dial, a reactive metabolite of furan.

Abstract: Furan is a hepatic toxicant and carcinogen in rodents. Its microsomal metabolite, cis-2-butene-1,4-dial, is mutagenic in the Ames assay. Consistent with this observation, cis-2-butene-1,4-dial reacts with 2‘-deoxycytidine, 2‘-deoxyguanosine, and 2‘-deoxyadenosine to form diastereomeric adducts. HPLC analysis indicated that the rate of reaction with deoxyribonucleosides was dependent on pH. At pH 6.5, the relative reactivity was 2‘-deoxycytidine > 2‘-deoxyguanosine > 2‘-deoxyadenosine whereas it was 2‘-deoxyguanosine > 2‘-deoxycytidine > 2‘-deoxyadenosine at pH 8.0. Thymidine did not react with cis-2-butene-1,4-dial. The primary 2‘-deoxyguanosine and 2‘-deoxyadenosine reaction products were unstable and decomposed to secondary products. NMR and mass spectral analysis indicated that the initial 2‘-deoxyadenosine and 2‘-deoxyguanosine reaction products were hemiacetal forms of 3-(2‘-deoxy-β-d-erthyropentafuranosyl)-3,5,6,7-tetrahydro-6-hydroxy-7-(ethane-2‘ ‘-al)-9H-imidazo[1,2-α]purine-9-one (structure 2) an...

Cross-Linked Thymine-Purine Base Tandem Lesions: Synthesis, Characterization, and Measurement in γ-Irradiated Isolated DNA

Abstract: 5-(Phenylthiomethyl)-2‘-deoxyuridine has been recently shown to be a specific photolabile precursor of 5-(2‘-deoxyuridilyl)methyl radical that is involved in the formation of tandem base lesion with vicinal guanine in oxygen-free aqueous solution. The thionucleoside was incorporated by either liquid or solid-phase phosphoramidite synthesis into dinucleoside monophosphates with a 2‘-deoxyadenosine residue as the vicinal nucleoside located either at the 3‘ or 5‘-extremity. UV-C irradiation of the modified dinucleoside monophosphate under anaerobic conditions gives rise to cross-linked thymineCH2-C8adenine tandem base lesions which were isolated and characterized by 1H NMR and mass spectrometry analyses. The formation of the latter tandem lesions involved an intramolecular addition of the 5-(2‘-deoxyuridilyl)methyl radical to the C8 of the adenine moiety. A sensitive and specific assay aimed at monitoring the formation of the four thymineCH2-C8purine adducts, namely d(T∧G), d(G∧T), d(T∧A), d(A∧T), within DNA...

Identification of tobacco-derived compounds in human pancreatic juice

Abstract: Cancer of the pancreas is the fourth leading cause of cancer mortality in the USA with an estimated 28 900 deaths in 2001. Several factors have been implicated in the etiology of this disease. However, at present, only cigarette smoking has been positively associated with pancreatic cancer. It is our working hypothesis that tobacco-derived compounds can be delivered to the pancreas where, upon metabolic activation, they can initiate carcinogenesis. Our current investigation was conducted to determine whether cotinine and tobacco-specific nitrosamines (TSNA) are present in human pancreatic juice. Smoking status was assessed by the determination of levels of urinary cotinine and was further supported by quantifying nicotine in hair. The TSNA were extracted from the pancreatic juice of 18 smokers and 9 nonsmokers by supercritical carbon dioxide that contained 10% methanol. The extracts were analyzed for TSNA, namely, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), by gas chromatography with mass spectrometric detection using a selected ion monitoring technique (GC-SIM-MS). Twenty-three extracts of human pancreatic juice were also analyzed for the presence of the NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by GC-SIM-MS and by gas chromatography interfaced wit a thermal energy analyzer (GC-TEA; TEA, a nitrosamine-specific detector). Cotinine was detected in all analyzed samples of pancreatic juice from smokers (129 +/- 150 ng/mL juice; mean +/- standard deviation) and was present in only two of the nine samples of pancreatic juice from nonsmokers. Its levels in these two samples were 7 and 9 ng/mL juice. NNK was detected in 15 of 18 samples (83%) from smokers at levels from 1.37 to 604 ng/mL pancreatic juice. In nine samples of pancreatic juice from nonsmokers, NNK ranged from not detected (in three samples) to 96.8 ng/mL juice. In pancreatic juice from smokers the mean level of NNK (88.7 +/- 161 ng/mL juice) was significantly higher (p < 0.04) than in that from nonsmokers (12.4 +/- 31.7 ng/mL juice). In addition to NNK, NNN was found in two samples of pancreatic juice of smokers at levels of 68.1 and 242 ng/mL juice; NNN was not detected in any other sample. NNAL was present in 8 of 14 pancreatic juice samples (57%) from smokers and in three of nine samples (33%) from nonsmokers. This research presents preliminary data that supports the hypothesis that pancreatic tissue is exposed to TSNA and that they may be important contributors to pancreatic carcinogenesis in humans.

Cu(II)-Catalyzed Oxidation of β-Amyloid Peptide Targets His13 and His14 over His6: Detection of 2-Oxo-histidine by HPLC-MS/MS

Abstract: The interaction of β-amyloid peptide (βAP) with Cu(II) leads to the formation of reactive oxygen species, neurotoxicity, and the chemical modification of the peptide. To study product formation and the potential selectivity of oxidation, we have exposed specific βAP congeners, βAP1−16, βAP1−28, and βAP1−40, to ascorbate/Cu(II)-induced metal-catalyzed oxidation and electrospray ionization-time-of-flight (ESI-TOF) MS/MS analysis. Incubation of 30 μM βAP with 15−150 μM Cu(II) and (physiologically relevant) 720 μM ascorbate in 20 mM phosphate buffer, pH 7.4, leads to significant oxidation of the peptides within remarkably short reaction times of as low as 6 min. Initial oxidation targets are His13 and His14, which are converted to 2-oxo-His, whereas the other two metal-binding residues, His6 and Tyr10, remain intact. Longer oxidation times then also target His6. Even in βAP1−40 the oxidation of His13 and His14 precedes the oxidation of Met35. Especially, the insensitivity of Tyr10 is noteworthy and may be exp...

Mitogen-Activated Protein Kinases Contribute to Reactive Oxygen Species-Induced Cell Death in Renal Proximal Tubule Epithelial Cells

Abstract: Extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNK/SAPK), and p38 mitogen-activated protein kinase (MAPK) were all rapidly activated in a ROS-dependent manner during 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ)-mediated oxidative stress and oncotic cell death in renal proximal tubule epithelial cells (LLC-PK1). TGHQ-induced phosphorylation of ERK1/2 and JNK MAPKs required epidermal growth factor receptor (EGFR) activation, whereas p38 MAPK activation was EGFR independent. In contrast to their established roles in cell survival, TGHQ-activated ERK1/2 and p38 MAPK (but not JNK) appear to contribute to cell death, since inhibition of ERK1/2 or p38 MAPKs with PD098059 or SB202190 respectively, attenuated TGHQ-mediated cell death. TGHQ increased AP-1 and NFkappaB DNA-binding activity, but whereas pharmacological inhibition of ERK1/2 or p38 MAPKs attenuated AP-1 DNA binding activity, it potentiated TGHQ-mediated NFkappaB activation. Consistent with a role for NFkappaB activation in the cytoprotective response to ROS in renal epithelial cells, an anti-NFkappaB peptide SN50 suppressed the protective effects of ERK inhibition (PD098059 treatment). The data provide evidence that the activation of MAPKs by ROS in renal epithelial cells plays an important role in oncotic cell death, and NF-kB is involved in the cytoprotective effects of PD098059.

Oxidized phospholipids derived from ozone-treated lung surfactant extract reduce macrophage and epithelial cell viability.

Abstract: Ozone is known to be a highly toxic gas present in the urban air which exerts its effect on pulmonary tissue through its facile chemical reactions with target molecules in the airway. One of the first barriers encountered by ozone is epithelial lining fluid which contains pulmonary surfactant rich in glycerophosphocholine lipids. The reaction of ozone with calf lung surfactant extract was found to result in the production of 1-palmitoyl-2-(9'-oxo-nonanoyl)-glycerophosphocholine (16:0a/9-al-GPCho) as an expected product of the ozonolysis of abundant unsaturated phospholipids containing unsaturated fatty acyl groups with a double bond at carbon-9. This oxidized phospholipid was identified as a biologically active product in that it reduced elicited macrophage viability by necrosis with an ED(50) of 6 microM. Further studies of the biological activity of 16:0a/9-al-GPCho revealed that concentrations from 100 to 200 nM initiated apoptosis in pulmonary epithelial-like A549 cells as assessed by TUNEL staining, nuclear size, and caspase-3 activation with loss of viability indicated by reduction of mitochondrial dehydrogenase activity. The release of IL-8, a neutrophil chemokine, from A549 cells was also stimulated by 50-100 nM 16:0a/9-al-GPCho. Exposure of calf lung surfactant to low levels of ozone (62.5, 125, and 250 ppb) for various time periods from 2 to 48 h in a feedback-regulated ozone exposure chamber resulted in a dose- and time-dependent increase in the formation of 16:0a/9-al-GPCho as measured by a specific and sensitive LC/MS/MS assay. The quantity of this biologically active chain-shortened glycerophosphocholine lipid generated even at 125 ppb ozone for 2-4 h (50-100 nM) was consistent with this product mediating the toxic effects of ozone on cells in contact with surfactant.

Role of cytochrome P450 1a1 and 1b1 in the metabolic activation of 7,12-dimethylbenz[a]anthracene and the effects of naturally occurring furanocoumarins on skin tumor initiation.

Abstract: The current study was designed to determine the mechanistic basis for differences in the effects of naturally occurring furanocoumarins on skin tumor initiation by 7,12-dimethylbenz[a]anthracene (DMBA). Female SENCAR mice were pretreated topically with bergamottin, imperatorin, or isopimpinellin (100-3200 nmol), 7,8-benzoflavone (7,8-BF, 5-40 nmol, a known inhibitor of DMBA skin carcinogenesis in mice), or acetone (vehicle control) 5 min prior to topical treatment with DMBA (10 nmol). Imperatorin, isopimpinellin, and 7,8-BF, but not bergamottin, significantly blocked total DMBA-DNA adduct formation. HPLC analysis of DNA adducts revealed that bergamottin preferentially inhibited formation of anti-DMBA diol-epoxide (DMBADE) derived DNA adducts, imperatorin, and isopimpinellin inhibited both anti- and syn- derived adducts, whereas 7,8-BF showed some selectivity for reduction of syn-DMBADE-DNA adducts. Mouse embryo fibroblast C3H/10T1/2 (10T1/2) cells, and mouse hepatoma-derived 1c1c7 (Hepa-1) cells, which preferentially express P450 1b1 and P450 1a1, respectively, were co-incubated with 2 microM bergamottin, imperatorin, isopimpinellin, and 7,8-BF, and with DMBA (2 microM). Hepa-1 cells (P450 1a1) formed mainly anti-DMBADE-DNA adducts. In contrast, 10T1/2 cells (P450 1b1) formed mainly syn-DMBADE-DNA adducts. Bergamottin inhibited DMBA metabolism to DMBA-3,4-diol and blocked DNA adduct formation in Hepa-1 cells, but had little effect in 10T1/2 cells. In contrast, 7,8-BF completely blocked DMBA metabolism and DNA adduct formation in 10T1/2 cells, but had little effect in Hepa-1 cells. Imperatorin and isopimpinellin inhibited DMBA bioactivation in both cell lines. These results indicate that bergamottin is a more selective inhibitor of P450 1a1 and overall a less effective inhibitor of the metabolic activation of DMBA in mouse epidermis. In contrast, imperatorin, isopimpinellin, and especially 7,8-BF, which block metabolic activation of DMBA in mouse epidermis, appear more selective for P450 1b1. On the basis of our studies using 10T1/2 cells and Hepa-1 cells, it appears that P450 1a1 is primarily responsible for converting DMBA-3,4-diol to anti-DMBADE, whereas P450 1b1 is primarily responsible for converting DMBA-3,4-diol to syn-DMBADE. These data demonstrate the role of P450 1a1 and 1b1 in the metabolic activation of DMBA in mouse epidermis and provide a mechanistic explanation for the differential effects of naturally occurring furanocoumarins (and 7,8-BF) on polycyclic aromatic hydrocarbon skin carcinogenesis.

Cytochrome P450 1B1 Determines Susceptibility to Dibenzo(a,l)pyrene-Induced Tumor Formation

Abstract: Metabolic activation, DNA binding, and tumorigenicity of the carcinogenic polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P) catalyzed by murine cytochrome P450 (P450) enzymes were investigated. DNA binding of DB[a,l]P in human mammary carcinoma MCF-7 and human P450-expressing Chinese hamster V79 cell lines was previously shown to occur preferentially with metabolically generated fjord region DB[a,l]P-11,12-dihydrodiol 13,14-epoxides (DB[a,l]PDE). To elucidate different capabilities of murine P450 1A1 and 1B1 for metabolic activation of DB[a,l]P, V79 cell cultures stably expressing P450s 1A1 or 1B1 from mice were exposed to 10 or 100 nM DB[a,l]P. Both cell lines transformed DB[a,l]P to DNA binding intermediates. As with V79 cells expressing the corresponding human P450 enzyme [Luch et al. (1998) Chem. Res. Toxicol. 11, 686-695], murine P450 1B1-catalyzed metabolism and DNA binding proceeded exclusively through generation of fjord region DB[a,l]PDE. In addition, only DB[a,l]PDE-derived DNA adducts were found in V79 cells expressing P450 1A1 from mice. This is in contrast to our recent findings with V79 cells expressing P450 1A1 from humans or rats which catalyzed the formation of both highly polar DNA adducts as well as nonpolar DB[a,l]PDE-DNA adducts. To establish the role of P450 1B1 in DB[a,l]P-induced tumor formation in vivo, we treated P450 1B1-null and wild-type mice intragastrically and monitored survival rates and appearance of neoplasias in various organs. All wild-type mice (n = 17) used in this study developed at least one tumor at one site (tumor rate of 100%). In contrast, 5 of 13 P450 1B1-null mice were observed to be free from any tumor (tumor rate of 62%). The organ sites of tumor formation and the dignity of tumors were different between wild-type and P450 1B1-null mice. Wild-type mice were diagnosed with both benign and malignant tumors of the ovaries, lymphoid tissues, as well as with skin and endometrial hyperplasias, whereas P450 1B1-null mice developed only lung adenomas and endometrial hyperplasias. DNA binding studies using embryonic fibroblasts isolated from these animals provided further evidence that P450 1B1-catalyzed formation of fjord region DB[a,l]PDE-DNA adducts is the critical step in DB[a,l]P-mediated carcinogenesis in mice, and probably also in man.