Induction of genotoxic damage is not correlated with the ability to methylate arsenite in vitro in the leukocytes of four mammalian species.

Arsenic is a natural drinking water contaminant that impacts the health of large populations of people throughout the world; however, the mode or mechanism by which arsenic induces cancer is unclear. In a series of in vitro studies, we exposed leukocytes from humans, mice, rats, and guinea pigs to a range of sodium arsenite concentrations to determine whether the lymphocytes from these species showed differential sensitivity to the induction of micronuclei (MN) assessed in cytochalasin B-induced binucleate cells. We also determined the capacity of the leukocytes to methylate arsenic by measuring the production of MMA [monomethylarsinic acid (MMA(V)) and monomethylarsonous acid (MMA(III))] and DMA [dimethylarsinic acid (DMA(V)) and dimethylarsonous acid (DMA(III))]. The results indicate that cells treated for 2 hr at the G(0) stage of the cell cycle with sodium arsenite showed only very small to negligible increases in MN after mitogenic stimulation. Treatment of actively cycling cells produced induction of MN with increasing arsenite concentration, with the human, rat, and mouse lymphocytes being much more sensitive to MN induction than those of the guinea pig. These data gave an excellent fit to a linear model. The leukocytes of all four species, including the guinea pig (a species previously thought not to methylate arsenic), were able to methylate arsenic, but there was no clear correlation between the ability to methylate arsenic and the induction of MN.

Lung tumor KRAS and TP53 mutations in nonsmokers reflect exposure to PAH-rich coal combustion emissions.

We determined the TP53 and codon 12 KRAS mutations in lung tumors from 24 nonsmokers whose tumors were associated with exposure to smoky coal. Among any tumors studied previously, these showed the highest percentage of mutations that (a) were G --> T transversions at either KRAS (86%) or TP53 (76%), (b) clustered at the G-rich codons 153-158 of TP53 (33%), and (c) had 100% of the guanines of the G --> T transversions on the nontranscribed strand. This mutation spectrum is consistent with an exposure to polycyclic aromatic hydrocarbons, which are the primary component of the smoky coal emissions. These results show that mutations in the TP53 and KRAS genes can reflect a specific environmental exposure.

Both hypomethylation and hypermethylation of DNA associated with arsenite exposure in cultures of human cells identified by methylation-sensitive arbitrarily-primed PCR.

In a previous study we reported that methylation within the promoter region of p53 was altered in human lung A549 cells exposed to arsenite over a 2-week period in culture. In the present study the methylation status of the 5' control region of the tumor suppressor gene, von Hippel Lindau syndrome (VHL), a gene known to be silenced transcriptionally by CpG methylation was assessed. No changes in DNA methylation in VHL in human kidney UOK cell lines exposed to arsenite were seen after 4 weeks in culture, assessed by simple HpaII digestion followed by PCR amplification. Using methylation-sensitive arbitrarily-primed PCR we identified eight differentially methylated regions of genomic DNA of approximately 300--500 bp from three UOK cell lines and from human lung A549 cells after arsenite exposure in culture. Six fragments were hypermethylated, and two were hypomethylated, relative to untreated controls. Sequence analysis revealed two DNA fragments contained repeat sequences of mammalian-apparent LTR retrotransposons, five contained promoter-like sequences, and 13 CpG islands were identified. Three fragments had 99-100% homology to regions on human chromosomes 6, 9, and 15 but these genes have not yet been identified. Our findings are consistent with a potential role for both hypermethylation and hypomethylation of DNA that coexist after exposure to arsenite. The results, in total, could support the existence of a state of DNA methylation imbalance that could conceivably disrupt appropriate gene expression in arsenite exposed cells.

Methylated trivalent arsenic species are genotoxic.

The reactivities of methyloxoarsine (MAs(III)) and iododimethylarsine (DMAs(III)), two methylated trivalent arsenicals, toward supercoiled phiX174 RFI DNA were assessed using a DNA nicking assay. The induction of DNA damage by these compounds in vitro in human peripheral lymphocytes was assessed using a single-cell gel (SCG, "comet") assay. Both methylated trivalent arsenicals were able to nick and/or completely degrade phiX174 DNA in vitro in 2 h incubations at 37 degrees C (pH 7.4) depending on concentration. MAs(III) was effective at nicking phiX174 DNA at 30 mM; however, at 150 microM DMAs(III), nicking could be observed. Exposure of phiX174 DNA to sodium arsenite (iAs(III); from 1 nM up to 300 mM), sodium arsenate (from 1 microM to 1 M), and the pentavalent arsenicals, monomethylarsonic acid (from 1 microM to 3 M) and dimethylarsinic acid (from 0.1 to 300 mM), did not nick or degrade phiX174 DNA under these conditions. In the SCG assay in human lymphocytes, methylated trivalent arsenicals were much more potent than any other arsenicals that were tested. On the basis of the slopes of the concentration-response curve for the tail moment in the SCG assay, MAs(III) and DMAs(III) were 77 and 386 times more potent than iAs(III), respectively. Because methylated trivalent arsenicals were the only arsenic compounds that were observed to damage naked DNA and required no exogenously added enzymatic or chemical activation systems, they are considered here to be direct-acting forms of arsenic that are genotoxic, though they are not, necessarily, the only genotoxic species of arsenic that could exist.

The enigma of arsenic carcinogenesis: role of metabolism.

Inorganic arsenic is considered a high-priority hazard, particularly because of its potential to be a human carcinogen. In exposed human populations, arsenic is associated with tumors of the lung, skin, bladder, and liver. While it is known to be a human carcinogen, carcinogenesis in laboratory animals by this metalloid has never been convincingly demonstrated. Therefore, no animal models exist for studying molecular mechanisms of arsenic carcinogenesis. The apparent human sensitivity, combined with our incomplete understanding about mechanisms of carcinogenic action, create important public health concerns and challenges in risk assessment, which could be met by understanding the role of metabolism in arsenic toxicity and carcinogenesis. This symposium summary covers three critical major areas involving arsenic metabolism: its biodiversity, the role of arsenic metabolism in molecular mechanisms of carcinogenesis, and the impact of arsenic metabolism on human risk assessment. In mammals, arsenic is metabolized to mono- and dimethylated species by methyltransferase enzymes in reactions that require S-adenosyl-methionine (SAM) as the methyl donating cofactor. A remarkable species diversity in arsenic methyltransferase activity may account for the wide variability in sensitivity of humans and animals to arsenic toxicity. Arsenic interferes with DNA methyltransferases, resulting in inactivation of tumor suppressor genes through DNA hypermethylation. Other studies suggest that arsenic-induced malignant transformation is linked to DNA hypomethylation subsequent to depletion of SAM, which results in aberrant gene activation, including oncogenes. Urinary profiles of arsenic metabolites may be a valuable tool for assessing human susceptibility to arsenic carcinogenesis. While controversial, the idea that unique arsenic metabolic properties may explain the apparent non-linear threshold response for arsenic carcinogenesis in humans. In order to address these outstanding issues, further efforts are required to identify an appropriate animal model to elucidate carcinogenic mechanisms of action, and to define dose-response relationships.

Lung tumorigenic interactions in strain A/J mice of five environmental polycyclic aromatic hydrocarbons.

The binary, ternary, quaternary, and quintary interactions of a five-component mixture of carcinogenic environmental polycyclic aromatic hydrocarbons (PAHs) using response surface analyses are described. Initially, lung tumor dose-response curves in strain A/J mice for each of the individual PAHs benzo[a]pyrene (B[a]P), benzo[b]fluoranthene (B[b]F), dibenz[a,h]anthracene (DBA), 5-methylchrysene (5MC), and cyclopenta[cd]pyrene (CPP) were obtained. From these data, doses were selected for the quintary mixture study based on toxicity, survival, range of response, and predicted tumor yields. The ratios of doses among PAHs were designed to simulate PAH ratios found in environmental air and combustion samples. Quintary mixtures of B[a]P, B[b]F, DBA, 5MC, and CPP were administered to male strain A/J mice in a 2(5) factorial 32-dose group dosing scheme (combinations of five PAHs each at either high or low doses) and lung adenomas were scored. Comparison of observed lung adenoma formation with that expected from additivity identified both greater than additive and less than additive interactions that were dose related i.e., greater than additive at lower doses and less than additive at higher doses. To identify specific interactions, a response surface analysis using response addition was applied to the tumor data. This response surface model contained five dose, ten binary, ten ternary, five quaternary, and one quintary parameter. This analysis produced statistically significant values of 16 parameters. The model and model parameters were evaluated by estimating the dose-response relationships for each of the five PAHs. The predicted dose-response curves for all five PAHs indicated a good estimation. The binary interaction functions were dominated for the most part by DBA and were inhibitory. The response surface model predicted, to a significant degree, the observed lung tumorigenic responses of the quintary mixtures. These data suggest that although interactions between PAHs do occur, they are limited in extent.

The MAP kinase cascade is activated prior to the induction of gliosis in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of dopaminergic neurotoxicity.

Injury to the central nervous system (CNS) provokes microglial activation and astrocytic hypertrophy at the site of damage. The signaling events that underlie these cellular responses remain unknown. Recent evidence has implicated tyrosine phosphorylation systems, in general, and the mitogen-activated protein kinase (MAP kinase) cascade, in particular, in the mediation of growth-associated events linked to neural degeneration, such as glial action. Moreover, an increase in the mRNA coding for the 14.3.3 protein, a known regulator of the MAP kinase pathway, appears to be involved in methamphetamine neurotoxicity. To examine the potential role of these protein kinase pathways in drug-induced damage to the CNS, we used the dopaminergic neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and to damage nerve terminals in the mouse neostriatum and elicit a glial reaction. The onset of reactive gliosis then was verified by Northern blot analysis of glial fibrillary acidic protein (GFAP) mRNA and qualified by enzyme-linked immunosorbent assay (ELISA) of GFAP (protein). A single administration of MPTP (12.5 mg/kg, subcutaneously (s.c.)) to the C57B1/66J mouse resulted in a 10-fold increase in GFAP mRNA by 1 day and a 4-fold increase in GFAP (protein) by 2 days. To determine the potential role of protein tyrosine phosphorylation and MAP kinase activation in these events, blots of striatal homogenates were probed with antibodies directed against phospho-tyr 204 and phospho-thr 202, residues corresponding to the active sites of p42/44 MAP kinase. After mice were sacrificed by focused microwave irradiation to preserve steady-state phosphorylation, proteins from striatal homogenates were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblots of these samples showed a number of phosphotyrosine-labeled bands, but there were no apparent differences between control and MPTP groups. In contrast, phospho-MAP kinase was elevated over 1.5 fold, 3-6 hours post MPTP. These findings are suggestive of a role of the MAP kinase cascade in the early phase of injury-induced glial activation.

Mechanistic relationships between DNA adducts, oncogene mutations, and lung tumorigenesis in strain A mice.

This paper describes a series of studies on the lung tumorigenic activities of polycyclic aromatic hydrocarbons (PAHs) in strain A/J mice, their ability to form PAH-DNA adducts in lung tissues, and their ability to mutate the Ki-ras oncogene in PAH-induced tumors. Seven PAHs were studied: cyclopenta[cd]pyrene (CPP), benzo[a]pyrene (B[a]P), benzo[b]fluoranthene (B[b]F), dibenz[a,h] anthracene (DBA), 5-methylchrysene (5MC), benz[j]aceanthrylene (B[j]A), and dibenzo[a,l]pyrene (DB[a,l]P). The dose-response data for the PAHs revealed 100-fold differences in tumor potency based on dose, with the order of activity DB[a,l]P, DBA > B[j]A > 5MC > CPP B[a]P > B[b]F. Large differences in tumor multiplicity were also observed between the PAHs. DNA adducts were measured by 32P-postlabeling techniques on DNA from lungs of mice treated with these PAH's. DB[a,l]P gave syn- and anti-fjord-region diol-epoxide adducts of dAdo and dGuo; DBA gave both bay-region diol-epoxide-dGuo and bisdihydrodiol-epoxide adducts; CPP gave cyclopenta-ring-dGuo adducts; B[j]A gave a mixture of cyclopenta-ring-dGuo and -dAdo adducts; 5MC gave anti-bay-region diol-epoxide-dGuo adducts; B[a]P gave bay-region diol-epoxide-dGuo adducts; and B[b]F gave 5-hydroxy-B[b]F-diol-epoxide-dGuo adducts. Ki-ras codon 12 and 61 mutation analysis of PAH induced tumors was performed using PCR and dideoxy sequencing methods. DB[a,l]P gave both codon 12 and codon 61 mutations. High proportions of codon 12 TGT mutations from B[a]P-, B[b]F- and 5MC-, induced tumors and CGT mutations from CPP- and B[j]A-induced tumors were observed. DBA produced no mutations in Ki-ras codons 12 or 61 by direct sequencing. The interrelationships between the tumorigenesis, DNA adduct, and oncogene mutation data are discussed.

Dibenzo[a,l]pyrene-induced DNA adduction, tumorigenicity, and Ki-ras oncogene mutations in strain A/J mouse lung.

Dibenzo[a,l]pyrene (DB[a,l]P), an environmental polycyclic aromatic hydrocarbon, is the most potent carcinogen ever tested in mouse skin and rat mammary gland. In this study, DB[a,l]P was examined for DNA adduction, tumorigenicity, and induction of Ki-ras oncogene mutations in tumor DNA in strain A/J mouse lung. Groups of mice received a single i.p. injection of 0.3, 1.5, 3.0, or 6.0 mg/kg DB[a,l]P in tricaprylin. Following treatment, DNA adducts were measured at times between 1 and 28 days, while tumors were counted at 250 days and analyzed for the occurrence of point mutations in codons 12 and 61 of the Ki-ras oncogene. DB[a,l]P in strain A/J mouse lung induced six major and four minor DNA adducts. Maximal levels of adduction occurred between 5 and 10 days after injection followed by a gradual decrease. DB[a,l]P-DNA adducts in lung tissue were derived from both anti- and syn-11,12-dihydroxy-13,14-epoxy- 11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE) and both deoxyadenosine (dAdo) and deoxyguanosine (dGuo) residues in DNA as revealed by cochromatography. The major adduct was identified as a product of the reaction of an anti-DB[a,l]PDE with dAdo in DNA. DB[a,l]P induced significant numbers of lung adenomas in a dose-dependent manner, with the highest dose (6.0 mg/kg) yielding 16.1 adenomas/mouse. In tricaprylin-treated control animals, there were 0.67 adenomas/mouse. Based on the administered dose, DB[a,l]P was more active than other environmental carcinogens including benzo[a]pyrene. As a function of time-integrated DNA adduct levels, DB[a,l]P induced lung adenomas with about the same potency as other PAHs, suggesting that the adducts formed by DB[a,l]P are similar in carcinogenic potency to other PAHs in the strain A/J mouse lung model. Analysis of the Ki-ras mutation spectrum in DB[a,l]P-induced lung tumors revealed the predominant mutations to be G-->T transversions in the first base of codon 12, A-->G transitions in the second base of codon 12, and A-->T transversions in the second or third base of codon 61, concordant with the DNA adduct profile.

Dichloroacetic acid reduces Ha-ras codon 61 mutations in liver tumors from female B6C3F1 mice.

Dichloroacetic acid (DCA), a disinfection by-product of chlorination found in drinking water, is a hepatocarcinogenic in the B6C3F1 mouse. Previous studies have shown that DCA does not significantly alter the incidence of Ha-ras codon 61 mutations in male mouse liver carcinomas from that observed in spontaneous tumors (approximately 50% have Ha-ras mutations) but it alters the proportions of mutations that occur in Ha-ras codon 61. Twenty-two tumors were produced in female B6C3F1 mice after treatment with 3.5 g DCA per liter of drinking water over a period of 104 weeks. To detect potential Ha-ras mutations in the liver tumor tissue of female B6C3F1 mice, genomic DNA was isolated from tumors that had been frozen. The polymerase chain reaction (PCR) and single-stranded conformational polymorphism (SSCP) was used to screen tumor DNA for mutations in Ha-ras exon 2. In DNA from liver tumors in female B6C3F1 mice induced by DCA-treatment we found only one mutation in exon 2 among the 22 tumors analyzed (4.5%). Direct-sequencing of exon 2 revealed a CAA to CTA transversion in Ha-ras codon 61. The result of this study indicates that tumor formation in DCA-treated female B6C3F1 mice is, therefore, not associated with a mutationally activated Ha-ras codon 61. This result differs from previous results obtained in male B6C3F1 mice.

CpG methylation inactivates the transcriptional activity of the promoter of the human p53 tumor suppressor gene.

Alterations of the methylation patterns of DNA are common in cancer cells and could conceivably comprise a subset of causal events in the carcinogenesis process. Although it has previously been shown that methylation of CpG islands in the 5'-control regions of tumor suppressor genes such as p16, Von Hippel-Lindau (VHL) syndrome gene, and the retinoblastoma (RB) gene can suppress expression and function of these gene products, the elements that control the expression of the p53 gene have not been examined in detail. In this study we examined the effect of CpG methylation in a region of the p53 promoter containing major transcription start sites. A region of the p53 promoter (from -199 to +142) containing 15 CpG dinucleotides was placed in a pCAT reporter plasmid and reporter activity was assessed in host CV-1 cells. We show for the first time that transcriptional activation of the p53 tumor suppressor gene, as assessed by a reporter plasmid construct, can be down-regulated by cytosine methylation in the basal promoter region. We believe these data suggest a role for methylation of CpG sequences in the regulation of transcription of p53. This implies that the tumor suppressor gene p53 could, therefore, contribute to carcinogenesis by inactivation via methylation of a key element in cell cycle control.

Arsenic alters cytosine methylation patterns of the promoter of the tumor suppressor gene p53 in human lung cells: a model for a mechanism of carcinogenesis.

Arsenic is a potent human carcinogen to which there is significant worldwide exposure through natural contamination of food and drinking water sources. Because arsenic is detoxified via methylation using a methyltransferase (MTase) and S-adenosylmethionine (SAM) as the methyl donor, we hypothesized that a mechanism of carcinogenesis of arsenic could involve alterations of MTase/SAM-dependent DNA methylation of a tumor suppressor gene. We found that exposure of human lung adenocarcinoma A549 cells to sodium arsenite (0.08-2 microM) or sodium arsenate (30-300 microM), but not dimethylarsenic acid (2-2000 microM), produced significant dose-responsive hypermethylation within a 341-base pair fragment of the promoter of p53. This was determined by quantitative PCR/HpaII restriction site analysis to analyze methylation status of two CCGG sites. In experiments with arsenite, DNA sequencing using bisulfite to visualize 5-methylcytosine (5-MeC) over the entire promoter region confirmed data obtained by restriction analysis. Limited data using SssI methylase also suggested that over-methylation of CpG sequences may exist over the entire genome in response to arsenite exposure. We propose that alteration of DNA methylation by arsenic offers a plausible, unified hypothesis for the carcinogenic mechanism of action of arsenic, and we present a model for arsenic carcinogenesis that utilizes perturbations of DNA methylation as the basis for the carcinogenic effects of arsenic.

Benzo[b]fluoranthene: tumorigenicity in strain A/J mouse lungs, DNA adducts and mutations in the Ki-ras oncogene.

The polycyclic aromatic hydrocarbon benzo[b]fluoranthene (B[b]F) is a pervasive constituent of environmental combustion products. We sought to examine the lung tumorigenic activity of B[b]F in strain A/J mice, to study the relationship between formation and decay of B[b]F-DNA adducts and to examine mutations in the Ki-ras proto-oncogene in DNA from B[b]F-induced tumors. Mice were given i.p. injections of 0, 10, 50, 100 or 200 mg/kg body wt and lung adenomas were scored after 8 months. B[b]F induced significant numbers of mouse lung adenomas in a dose-related fashion, with the highest dose (200 mg/kg) yielding 6.95 adenomas/ mouse, with 100% of the mice exhibiting an adenoma. In mice given tricaprylin, the vehicle control, there were 0.60 adenomas/mouse, with 55% of the mice exhibiting an adenoma. Based on dose, B[b]F was less active than benzo[a]pyrene. DNA adducts were analyzed qualitatively and quantitatively by 32P-post-labeling in lungs of strain A/J mice 1, 3, 5, 7, 14 and 21 days after i.p. injection. Maximal levels of adduction occurred 5 days after treatment with the 200 mg/kg dose group, producing 1230 amol B[b]F-DNA adducts/microgram DNA. The major B[b]F-DNA adduct was identified by co-chromatography as trans-9, 10-dihydroxy-anti-11, 12-epoxy-5-hydroxy-9, 10, 11, 12-tetra-hydro-B[b]F-deoxyguanosine. Approximately 86% of the tumors had a mutation in codon 12 of the Ki-ras oncogene, as determined by direct DNA sequencing of PCR-amplified exon 1 and single-stranded conformation polymorphism analysis. Analysis of the Ki-ras mutation spectrum in 25 of 29 B[b]F-induced tumors revealed the predominant mutation to be a G-->T transversion in the first or second base of codon 12, congruous with the DNA adduct data. Our data are consistent with previous reports in mouse skin implicating a phenolic diol epoxide as the proximate carcinogenic form of B[b]F that binds to guanine.

Mechanistic linkage between DNA adducts, mutations in oncogenes and tumorigenesis of carcinogenic environmental polycyclic aromatic hydrocarbons in strain A/J mice.

Five polycyclic aromatic hydrocarbons (PAHs), benzo[a]pyrene (B[a]P), benzo[b]fluoranthene (B[b]F), dibenz[a,h]anthracene (DBA), 5-methylchyrsene (5MC), and cyclopenta[cd]pyrene (CPP) were examined for their lung tumorigenic activities in strain A/J mice, their ability to form PAH-DNA adducts in lung tissues, and their ability to mutate the Ki-ras oncogene in PAH-induced tumors. PAHs dissolved in tricapyrlin were administered by single intraperitoneal injection to male strain A/J mice (20 mice/dose) at doses up to 200 mg/kg depending on the PAH. Animals were sacrificed 8 months later and the lungs removed, fixed, and surface adenomas enumerated. DBA produced maximal tumor multiplicity at the highest dose, 10 mg/kg, giving 32.2 lung adenomas per mouse. At 100 mg/kg, B[a]P, B[b]F, 5MC, and CPP gave 12.8, 5.3, 93.1, and 32.2 lung adenomas per mouse, respectively. The dose response data for each PAH was fit to y = 0.6 + bx1.6, where y is the observed mean lung adenomas per mouse at dose x (in mg/kg), 0.6 is the observed background of lung adenomas per mouse, and b is the fitted constant representing the potency of each PAH. Statistical analysis indicated that the fit of the data to the equation was extremely high with adjusted R2 values > 0.985 and small fit standard errors. Based on this equation, the relative potencies of B[b]F, DBA, 5MC, and CPP compared to B[a]P were PAH (relative activity): DBA (118); 5MC (8.8); CPP (2.9); B[a]P (1.0); B[b]F (0.43). DNA adducts were measured by 32P-postlabeling techniques on DNA from lungs of mice treated with these PAHs. Adducts identified by cochromatography with standards were: from B[a]P, 7R,8S,9S-trihydroxy-10R-(N2-2'-deoxyguanosyl)-7,8,9,10-tetrahydro- B[a]P, and two adducts resulting from the metabolic activation of 9-hydroxy-B[a]P and trans-7,8-dihydroxy-7,8-dihydro-B[a]P; from B[b]F, 5-hydroxy-B[b]F-9,10-diol-11,12-oxide-2'-deoxyguanosine; from DBA, three adducts from the metabolic activation of trans,trans-3,4,10,11-tetrahydroxy-3,4,10,11-tetrahydro-DBA and two anti-DBA-3,4-diol-1,2-oxide-N2-[2'-deoxyguanosine] adducts; from 5MC, 1R,2S,3S-trihydroxy-4-(N2-2'-deoxyguanosyl)-1,2,3,4-tetrahydro- 5MC; from CPP, four CPP-3,4-oxide-2'-deoxyguanosine adducts. Ki-ras codon 12 mutation analysis of PAH-induced tumors was performed using PCR and dideoxy sequencing methods. Mutations from lung tumors from tricaprylin-treated mice were GGT-->GAT, GGT-->CGT, and GGT-->GTT. DBA produced no mutations in Ki-ras codon 12 above spontaneous levels. High proportions (> or = 50%) of GGT-->TGT mutations from B[a]P, B[b]F and 5MC induced tumors and GGT-->CGT mutations from CPP tumors were observed and were statistically significant compared to mutations in tricaprylin control tumors. We conclude from the DNA adduct and Ki-ras mutation studies that bay region diol-epoxide-2'-deoxyguanosine PAH-DNA adducts are associated with the GGT-->TGT mutations, and cyclopenta-ring oxide-2'-deoxyguanosine adducts associated with the GGT-->CGT mutations.

Comparison of experimental and theoretical parameters of the Moolgavkar-Venzon-Knudson incidence function for the stages of initiation and promotion in rat hepatocarcinogenesis.

Mathematical descriptions of complex biological phenomena, such as cancer, require an experimental format that faithfully recapitulates the biological process. In addition, the biological process must dictate the parameters in the mathematical formula. Evidence from the epidemiology of several human cancers and from experimental carcinogenesis in several organ systems indicates that cancer is a multistage process. The initiation-promotion-progression format of experimental carcinogenesis mimics the development of cancer in humans and other animals. In rats, the altered hepatic focus model of hepatocarcinogenesis has been well characterized and, coupled with the method of quantitative stereology, permits accurate determination of the number and the volume fraction of such altered foci per liver. The placental isozyme of glutathione S-transferase (PGST) is reportedly the best single marker of preneoplasia in the rat liver. Recently, single hepatocytes expressing PGST have been proposed as putatively initiated cells. Quantitation of individual hepatic cells and altered hepatic foci expressing PGST in the livers of rats subjected to an initiation-promotion protocol permits determination of the congruence of the Moolgavkar-Venzon-Knudson (MVK) model with experimental data. The best fit of the MVK model for the preneoplastic stages of hepatocarcinogenesis assumes that all hepatocytes are susceptible and that single hepatocytes expressing PGST are the initiated cell population for the focal lesions that express PGST. Further refinement of the initiation-promotion-progression model to permit accurate quantitation of early malignant conversion should allow a more complete analysis of the congruence of the MVK model for human cancer risk determination. In addition, the MVK model may be extended to other model systems and to human cancers in which early preneoplasia can be quantitated. Furthermore, the use of a more biologically based risk-assessment protocol, such as the MVK model rather than the stochastic one-hit model presently used, would permit incorporation of the present knowledge on the pathogenesis of cancer. To apply experimental data to a mathematical model that reflects the biological processes underlying human cancer development will require integration of the cell kinetics and experimental data to a mathematical model that reflects the biological processes underlying human cancer development including the pharmacokinetic and pharmacodynamic properties of the treatment chemicals.

Adenomas induced by polycyclic aromatic hydrocarbons in strain A/J mouse lung correlate with time-integrated DNA adduct levels.

The induction of DNA adducts and adenomas in the lungs of strain A/J mice has been investigated following the single i.p. administration of each of the following polycyclic aromatic hydrocarbons (PAH): pyrene, dibenz[a,h]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, 5-methylchrysene, and cyclopenta[c,d]pyrene. DNA adducts were measured by 32P-postlabeling at times between 1 and 21 days following injection, while adenomas were counted at 240 days after treatment. Pyrene did not induce either DNA adducts or lung adenomas at any of the doses examined. Each of the remaining PAH induced both adenomas and DNA adducts in a dose-dependent manner, with dibenz[a,h]anthracene > 5-methylchrysene > cyclopenta[c,d]pyrene > benzo[a]pyrene > benzo[b]fluoranthene. DNA adducts reached maximal levels between 3 and 9 days after injection, followed by a gradual decrease. The time-integrated DNA adduct level (TIDAL) was calculated by numerically integrating the areas under the adduct persistence curves extrapolated to 240 days for each PAH at each dose level. This value represents the effective total molecular dose of PAH that was delivered to the lung DNA over the entire course of tumorigenesis. A strong correlation of lung adenoma induction with the TIDAL values was observed for each PAH. The slopes of the tumors versus TIDAL value relationships were essentially identical for 5-methylchrysene, cyclopenta[cd]pyrene, benzo[a]pyrene, and benzo[b]fluoranthene. The slope of this relationship for dibenz[a,h]anthracene was markedly greater. The essentially identical induction of adenomas as a function of TIDAL values for these PAH suggests that the formation and persistence of DNA adducts determines their carcinogenic potency.

Tumor multiplicity, DNA adducts and K-ras mutation pattern of 5-methylchrysene in strain A/J mouse lung.

This study was undertaken to evaluate the carcinogenic potential of 5-methylchrysene (5-MeC) in strain A/J mouse lung and to correlate the 5-MeC-DNA adduct profile in lung tissue with the mutation spectrum in the K-ras gene of lung tumors. Strain A/J mice received a single i.p. injection of 5-MeC at doses of 10, 50, 100 and 200 mg/kg and after 24, 48 and 72 h their lungs were collected for DNA adduct analysis. Eight months later, lungs from the remaining mice were harvested and the lung tumors counted and collected for subsequent mutational analysis of the K-ras gene. 5-MeC was found to be a potent lung carcinogen in strain A/J mice, inducing more than 100 tumors/mouse at a concentration of 200 mg/kg. Six 5-MeC-DNA adducts were observed; one adduct comigrated with the standard N2-deoxyguanosine adduct of 5-MeC-diol-epoxide I [1R,2S,3S-trihydroxy-4R-(N2-deoxy-guanosyl-3'-phosphate)- 1,2,3,4-tetrahydro-5-methyl-chrysene], derived from the bay-region diol-epoxide of 5-MeC. DNAs isolated from 5-MeC-induced lung tumors were evaluated for activating mutations in the K-ras gene by polymerase chain reaction-single strand conformation polymorphism and direct DNA sequencing analysis. Mutations were detected in 44 of 49 (90%) 5-MeC-induced tumors and the mutations were GGT-->TGT (50%), GGT-->GTT (23%) and GGT-->CGT (27%) in codon 12 of the gene. These results suggest that the N2-deoxyguanosine adduct of 5-MeC-diol-epoxide I may be one of the promutagenic adducts of 5-MeC in strain A/J mouse lung.

Biochemical events during initiation of rat hepatocarcinogenesis.

Carcinogenesis is a multistep, multistage process that begins with irreversible, but heritable damage to a single cell. The partial hepatectomy/diethylnitrosamine (DEN) model of rat hepatocarcinogenesis has been well characterized and many aspects of the stage of initiation are known. Recently, it has been suggested that hepatocytes expressing the placental isozyme of glutathione S-transferase (PGST) may be one population of initiated cells. Male Fischer rats were subjected to a 70% partial hepatectomy and at the peak of cell proliferation 24 h later were administered either the solvent trioctanoin, or 10 mg DEN/kg. The rats were administered 100 mg bromodeoxyuridine (BrdU)/kg 1 h prior to death at various times after DEN administration. Since initiation of the carcinogenesis process requires the division of cells containing DNA damage to induce mutations, we examined the concentration of alkylated adducts and the labeling index at various times after DEN administration. In addition, the time course of hepatic PGST expression was determined concurrent with the adduct concentration and labeling index. During the first day after DEN or solvent administration to a rat subjected to a 70% partial hepatectomy, a diurnal variation in labeling index was observed. A recovery to postsurgical labeling index levels was demonstrated for both the solvent- and DEN-treated groups by 7 days. The concentration of three promutagenic lesions was maximal at 6 h after DEN administration. The detectable level of the O6EG adduct was negligible by 24 h after DEN administration, while the two O-alkylpyrimidines, O2ET and O4ET, were retained for much longer periods. Single hepatocytes expressing PGST were observed by 2 days after DEN administration, while small foci of PGST-expressing hepatocytes could be reliably detected by 2 weeks. Two phases of PGST expression in single hepatocytes were observed. The first phase was maximal at day 3 and complete by day 6, while the second reached a plateau by day 8 and was maintained for the 28 days of the study. The presence of the three O-alkylation adducts during a time of enhanced cellular proliferation suggests that all three promutagenic adducts may contribute to the initiation that results in the partial hepatectomy/DEN model of rat hepatocarcinogenesis.

Ki-ras oncogene mutations in tumors and DNA adducts formed by benz[j]aceanthrylene and benzo[a]pyrene in the lungs of strain A/J mice.

Strain A/J mice received intraperitoneal injections of benz[j]aceanthrylene (B[j]A) or benzo[a]pyrene (B[a]P). At 24, 48, and 72 h, lung tissues were removed for analysis of B[a]P- or B[j]A-derived DNA adduct formation during the first 3 d of exposure. One group of mice exposed to these hydrocarbons was kept for 8 mo to determine lung tumor multiplicity, the occurrence of mutations in codons 12 and 61 of the Ki-ras gene in the tumors that arose, the relationship between Ki-ras oncogene mutations in tumors, and the presence and quantity of genomic DNA adducts. The major DNA adduct in the lungs of mice exposed to B[a]P was N2-(10 beta-[+B, 7 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl)-deoxyguanosine (BPDE-I-dGuo) arising from bay-region diolepoxide activation of B[a]P and was consistent with the occurrence of tumors with mutations GGT-->TGT (56%), GGT-->GTT (25%), and GGT-->GAT (19%) in codon 12, all involving mutations of a guanine. B[j]A, a demethylated analogue of 3-methylcholanthrene (3-MCA) with an unsaturated cyclopenta ring, produced 16-to 60-fold more tumors at equivalent doses than did B[a]P; the mutations in tumors were GGT-->TGT (4%), GGT-->GTT (30%), and GGT-->CGT (65%). Analysis of adduction patterns in DNA suggested that B[j]A was activated to form DNA-binding derivatives in A/J mouse lungs primarily at the cyclopenta ring even though B[j]A contains a bay region. As reported in the published literature, the mutation spectrum induced by 3-MCA in Ki-ras codon 12 of mouse cells is similar to that of B[a]P but not to that of its close relative B[j]A. In contrast to B[j]A, 3-MCA is activated mostly via a bay-region diol-epoxide since its cyclopenta ring is saturated and not easily epoxidates. Therefore, we propose that the GGT-->CGT mutations produced by B[j]A in Ki-ras codon 12 were mostly the result of cyclopenta-ring-derived adducts.