Marc J. Mass

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.

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 PAHs. 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.

Human carcinogenesis by arsenic

Arsenic is one of the few human carcinogens for which there is not yet a reliable animal cancer model. As such, the classification of arsenic as a carcinogen is based upon data derived from human epidemiologic studies. Although the mechanisms of action of arsenic as a toxic agent have been known for many years, the inability to produce cancer with arsenic in laboratory animals has confounded the operational characterisation of arsenic as initiator, promoter, complete carcinogen, or cocarcinogen for humans. Arsenic is clearly a genotoxic agent that induces chromosomal aberrations, micronuclei and sister chromatid exchange in mammalian cells as well as neoplastically transforms Syrian hamster embryo cells; however, it is not a classical point mutagen. This paper reviews some of the scientifically based issues relating to arsenic and risk assessment.

K-ras mutations in lung carcinomas from nonsmoking women exposed to unvented coal smoke in China

Lung cancer mortality rate in nonsmoking women in Xuan Wei (XW) County is the highest in China. The XW lung cancer rate is associated with exposure to coal smoke, containing high concentrations of polycyclic aromatic hydrocarbons (PAHs), in unvented homes. Here we investigated codon 12 K-ras mutations in lung tumors or sputum samples from 102 XW lung cancer patients (41 nonsmoking women and 61 smoking men). In addition, we analyzed specimens from 50 lung cancer patients (14 nonsmoking women, 33 smoking men and three nonsmoking men), from Beijing and Henan (B&H), where natural gas is the main domestic fuel. K-ras mutations were found in nine women (21.9%) and 14 men (22.9%) from XW, with G to T transversions accounting for 66.7 and 85.7%, respectively. Among B&H patients, one woman (7.1%) and six men (16.7%) had K-ras mutations, with G to T transversions accounting for 66.7% of the mutations in the men. Therefore, the frequency and type of K-ras mutations in XW nonsmoking women are similar to those of K-ras mutations found in both XW and B&H smoking men. On the other hand, the mutation frequency in XW women is higher than, although not statistically significant from, that in the B&H nonsmoking women (P=0.28, two-sided Fishers Exact Test). These results suggest an association between exposure to coal smoke and the increased K-ras mutation frequency in XW nonsmoking female lung cancer patients. They also suggest that the mutagens and/or mechanisms of mutations in these nonsmoking women are similar to those responsible for K-ras mutations in cigarette smoking lung cancer patients, which are probably induced largely by chemicals such as PAHs.

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

ABSTRACT: 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 activation. 1 Moreover, an increase in the mRNA coding for the 14.3.3 protein, a known regulator of the MAP kinase pathway, 2 appears to be involved in methamphetamine neurotoxicity. 3 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), 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 C57B1/6J 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, 3–6hours post MPTP. These findings are suggestive of a role of the MAP kinase cascade in the early phase of injury‐induced glial activation.

A rat tracheal cell culture transformation system for assessment of environmental agents as carcinogens and promoters

Abstract A tracheal cell culture system which can be used for detection of hazardous environmental agents is described. The culture system makes use of primary tracheal cells that are isolated from rats by protease digestion of the tracheal epithelium. The epithelial cells are plated on a film of collagen or onto a layer of gamma-ray- or mitomicin-C-inactivated mouse 3T3 cells. One day after the rat tracheal cells are plated they are exposed to toxicants, and the effect on colony formation is tabulated after 1 week. By altering the culture conditions it has become possible to construct an assay for carcinogen-induced transformation of rat tracheal cells, which takes advantage of the observation that normal rat tracheal cells have a finite lifetime in culture. Rat tracheal cell transformants are visualized as discrete colonies of proliferating epithelial cells that arise and survive after the normal population has died out. Their number is proportional to concentration of carcinogen to which the cells were initially exposed. Agents that have been shown to induce tracheal cell transformation in culture include polycyclic hydrocarbons, metal salts, nitroso compounds, and cigarette smoke condensate. Tracheal cells in culture also respond to tumor promoters in a number of ways. The phorbol ester tumor promoters dramatically increas the colony-forming ability of primary rat tracheal cells in a concentration dependent fashion. This class of agents also induces permanent cell line formation in rat tracheal cells derived from explants exposed to phorbol esters. In two-stage exposure experiments in culture it has also been shown that phorbol esters enhance the number of cell lines initiated by carcinogen exposure in vitro . Because rat tracheal cell culture has evolved to the state where the above studies are readily performed, these limited results suggest that the inclusion of rat tracheal cells in environmental research may be useful.

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.

Interaction Analyses of Binary Mixtures of Carcinogenic PAHs Using Morphological Cell Transformation of C3H10T1/2CL8 Mouse Embryo Fibroblasts in Culture

Abstract Studies of defined mixtures of carcinogenic polycyclic aromatic hydrocarbons (PAH) have identified three major categories of interactions: antagonism; synergism; and additivity depending on the biological model, tissue, route of exposure, and specific PAH. To understand the bases of these interactions we studied binary mixtures of benzo[a]pyrene (B[a]P) and dibenz[a,h]anthracene (DBA) in transformable C3H10T1/2C18 (C3H10T1/2) mouse embryo fibroblast cells in culture. C3H10T1/2 cells treated with binary mixtures of B[a]P and DBA gave less than additive morphological cell transformation based on response additivity. These results were consistent with those reported in mice and rats on the antagonistic effects of B[a]P and DBA on tumorigenesis. 32P-Postlabeling DNA adduct studies revealed that DBA reduced B[a]P-DNA adduct levels by 47% with no effect on DBA-DNA adduct levels. This suggests that one mechanism for the inhibition of morphological cell transformation of binary mixtures of B[a]P and DBA is due to alterations in the metabolic activation of B[a]P.

Time-Integrated DNA Adduct Levels and Relative Tumorigenic Potencies of Six Polycyclic Aromatic Hydrocarbons in Strain A/J Mouse Lung

Abstract We have investigated the induction of DNA adducts and adenomas in the lungs of strain A/J mice following the i.p. administration of several polycyclic aromatic hydrocarbons (PAH): pyrene, dibenz[a,h]anthracene (DBA), benzo[a]pyrene (B[a]P), benzo[b]fluoranthene (B[b]F), 5-methylchrysene (5-MeC), 3-methylcholanthrene (3-MC), and cyclopenta[cd]pyrene (CPP). All of the PAH induced lung adenomas, with relative tumor potency rankings as a function of administered dose: DBA = 3-MC > 5-MeC > CPP > B[a]P > B[b]F. DNA adducts reached maximal levels between 3 and 7 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 out to 240 days for each PAH at each dose level. Tumorigenic potencies as a function of TIDAL values for 5-MeC, B[a]P, B[b]F, and CPP were all equal, while 3-MC was 2.6-fold more potent and DBA was 25.8-fold more potent.

Consistent oncogene methylation changes in epithelial cells chemically transformed in vitro.

We have examined the restriction digest patterns of CCGG sequences in Kiras, Ha-ras, and c-myc oncogenes in rat tracheal epithelial cells transformed in vitro by 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene/12-O-tetradecanoylphorbol-13-acetate (TPA), or TPA alone. Oncogenes c-myc and Ha-ras in transformed cell lines, compared to normal rat tracheal epithelial cells and untreated primary cultures, had altered Hpa II restriction patterns as demonstrated by hybridizing bands of different molecular weight, or loss of bands. Ki-ras was hypermethylated in all cell derivations, including normal cells. These molecular alterations have not previously been reported for epithelial cells transformed in vitro by polycyclic hydrocarbons and tumor promoters, and suggest common mechanisms of action for agents with diverse molecular targets.