Mammalian cell transformation and cell-mediated mutagenesis by carcinogenic polycyclic hydrocarbons.

The introduction of a polycyclic hydrocarbon such as benzo(alpha)pyrene (BP) into normal golden hamster embryo cell cultures results, in addition to cytotoxicity, in malignant cell transformation. Studies on the effect of different doses of BP on the normal cells showed that the frequency of transformed colonies was directly related to the dose of the carcinogen. Analysis of this dose-response curve suggests a one-event ("one-hit") response for transformation by this carcinogen. The one-event response for transformation by carcinogenic polycyclic hydrocarbons and the fact that these carcinogens bind to DNA in susceptible cells suggests that transformation can involve a single alteration in the genetic constitution of the treated cells. Carcinogens may, therefore, produce somatic mutations, some of which may involve the genes that control malignancy. Recently, considerable progress has been made in developing models for the study of chemical mutagenesis in mammalian cells. Using resistance to 8-azaguanine as a marker, positive correlations between mutagenicity and transformation were obtained with chemically reactive carcinogens such as N-acetoxy-N-2-fluorenyl-acetamide, N-methyl-N'-nitro-N-nitrosoguanidine and K-region epoxides of polycyclic hydrocarbons. However, no such correlations were obtained with the carcinogenic polycyclic hydrocarbons themselves, since the cell lines used in chemical mutagenesis do not metabolize these carcinogens. In order to obtain better correlations, we have developed a cell-mediated mutagenic assay with carcinogenic hydrocarbons in which Chinese hamster cells, which are susceptible for mutagenesis, were co-cultivated with lethally irradiated rodent cells that can metabolize these compounds. Using this cell mediated assay, we obtained mutagenesis with the carcinogenic hydrocarbons 7,12-dimethylbenz(alpha)anthracene (DMBA), BP, 3-methylcholanthrene and 7-methylbenz(alpha)anthracene; the most potent carcinogen, DMBA, gave the highest frequency of mutations. The polycyclic hydrocarbons, pyrene and benz(alpha)anthracene, which are not carcinogenic were also not mutagenic. We have therefore demonstrated a relationship between the carcinogenecity of polycyclic hydrocarbons and their mutagenicity in mammalian cells, without having to isolate their reative metabolic intermediates. It should be possible to use in this system human cells from different organs and individuals to screen for environmental chemicals hazardous to humans which have to be metabolically activated.     

Identifying human cells capable of metabolizing various classes of carcinogens

Human cells that appear capable of metabolizing various classes of carcinogens have been identified using one of two methods: metabolism of tritiated benzo(a)pyrene to aqueous-acetone soluble forms or inhibition of cellular DNA synthesis. Each of the assay systems was optimized and the results on 15 human epithelial cell lines were compared. One or more cell lines were found to activate each of four classes of carcinogens examined: polycyclic hydrocarbons, aromatic amines, heterocyclic hydrocarbons, and nitrosamines. Cells that appeared capable of metabolizing polycyclic hydrocarbons or aromatic amines by these methods were also found to produce metabolites which were cytotoxic to cocultivated human xeroderma pigmentosum fibroblasts after a 48-hr exposure to the carcinogen.     

Roles of UDP-glucuronosyltransferases in chemical carcinogenesis

UDP-glucuronosyltransferases (UGT) play a major role in the elimination of nucleophilic metabolites of carcinogens, such as phenols and quinols of polycyclic aromatic hydrocarbons. In this way they prevent their further oxidation to electrophiles, which may react with DNA, RNA, and protein. They also inactivate carcinogenic, N-oxidized metabolites of aromatic amines. Furthermore, glucuronides may be stable transport forms of proximate carcinogens excreted via the biliary or urinary tract, thereby liberating the ultimate carcinogen at the target of carcinogenicity. Isozymes of the UGT enzyme superfamily that control the glucuronidation of metabolites of aromatic hydrocarbons and of N-oxidized aromatic amines have been identified in rats and humans. Phenol UGT appears to be coinduced with other drug-metabolizing enzymes via the Ah or dioxin receptor. This isozyme probably controls various proximate carcinogens and contributes to the persistently altered enzyme pattern, leading to the "toxin-resistance phenotype" at cancer prestages. Knowledge about UGTs in different species, their regulation, and their tissue distribution will improve the risk assessment of carcinogens.     

Mutagenicity of Epoxides of Polycyclic Hydrocarbons correlates with Carcinogenicity of Parent Hydrocarbons

MANY chemical carcinogens are mutagenic¹ and some non-mutagenic carcinogens are metabolized to mutagenic derivatives^2,3. Recent work^4–6 has confirmed that epoxides are intermediates in the metabolism of the aromatic double bonds of carcinogenic polycyclic hydrocarbons to hydroxylated derivatives, as Boyland suggested⁷. In addition to chemical reactions with nucleic acids and histone⁸, epoxides derived from polycyclic hydrocarbons bind more extensively to the nucleic acids of cells in culture than the parent hydrocarbons⁹. Hydrocarbon epoxides are also more active in inducing malignant transformation in vitro of hamster embryo and mouse prostate cells¹⁰ although, in whole animals, they were less potent carcinogens than the hydrocarbons themselves^11–13. As potential mutagens, polycyclic hydrocarbon epoxides are therefore of particular interest, mainly because of the support positive results would give to the somatic mutation theory of carcinogenesis. In the work described here we have tested K-region epoxides of hydrocarbons for their ability to cause host range mutations of T₂h⁺ bacteriophage, specifically because there is no possibility, in this test system, of the epoxides being further metabolized. The epoxides tested were phenanthrene 9,10-oxide (Ph-E), benz(a)anthracene 5,6-oxide (BA-E), dibenz(a,h)anthracene 5,6-oxide (DBA-E), 7-methylbenz(a)anthracene 5,6-oxide (7-MeBA-E), 3-methylcholanthrene 11,12-oxide (MCA-E) and chrysene 5,6-oxide (Ch-E). Ethylene oxide and propylene oxide were used as examples of aliphatic epoxides which do not increase the frequency of host range mutants of T₂ bacteriophage and ethyl methanesulphonate (EMS) was used as a known mutagen¹⁴.     

Effect of some polycyclic aromatic hydrocarbons on protein synthesis in vitro

1. The effect of the strongly carcinogenic polycyclic aromatic hydrocarbons benzo[a]pyrene, 3-methylcholanthrene and dibenz[a,h]anthracene and of the non-carcinogenic anthracene, pyrene and phenanthrene on protein synthesis was studied in vitro with subcellular systems from rat liver. 2. Both types of hydrocarbons affect amino acid activation and inhibit transfer of labelled amino acids from transfer RNA to ribosomes. 3. Only the carcinogenic compounds stimulate the incorporation of labelled algal-protein hydrolysate and of some individual amino acids into transfer RNA. The most active dose was 10mmug. under the conditions used. This effect is abolished by preincubation of pH5 enzymes with the carcinogens before the addition of radioactive amino acids. 4. The carcinogens stimulate the incorporation of some amino acids into ribosomal protein whereas the non-carcinogenic compounds have no such effects. 5. Polynucleotide-dependent stimulation of protein synthesis is greatly enhanced in the presence of the carcinogenic hydrocarbons when either free amino acids or transfer RNA charged with labelled amino acids are used. The non-carcinogenic compounds induce a partial inhibition of this process. 6. It is concluded, in agreement with other authors, that carcinogens may increase the number of active incorporation sites on both transfer and ribosomal RNA. Possible mechanisms of such an effect are discussed.     

Modulation of rat guanylate cyclase activity in vitro by chemical carcinogens.

The nucleotide cyclic GMP has been reported to be involved in cell proliferation and malignant transformation. Nitroso chemical carcinogens activate the enzyme guanylate cyclase (EC 4.6.1.2) which catalyzes the production of cyclic GMP. The present investigation demonstrates that compounds from other major classes of carcinogens including (1) alpha-halo ethers (chloromethyl methyl ether); (2) aromatic amines (benzidine and B-naphthylamine); (3) polycyclic hydrocarbons (1,2-benzanthracene and acridine); (4) azo dyes (p-dimethylaminoazobenzene), and (5) aflatoxins (B1, B2, G1, G2) produced a striking and significant inhibition of guanylate cyclase over a general concentration range of 0.5-13 mmol/1 in a variety of tissues. Some of the nitrosamides which increase guanylate cyclase activity, increase DNA synthesis whereas carcinogens which decrease guanylate cyclase activity inhibit DNA or RNA synthesis suggesting a relationship between cyclic GMP, DNA synthesis, and chemical carcinogenesis.     

Roles of UDP-Glucuronosyltransferases in Chemical Carcinogenesi

Abstract

UDP-glucuronosyltransferases (UGT) play a major role in the elimination of nucleophilic metabolites of carcinogens, such as phenols and quinols of polycyclic aromatic hydrocarbons. In this way they prevent their further oxidation to electrophiles, which may react with DNA, RNA, and protein. They also inactivate carcinogenic, N-oxidized metabolites of aromatic amines. Furthermore, glucuronides may be stable transport forms of proximate carcinogens excreted via the CCor urinary tract, thereby liberating the ultimate carcinogen at the target of carcinogenicity. Isozymes of the UGT enzyme super family that control the glucuronidation of metabolites of aromatic hyharbons and of N-oxidized aromatic amines have been identified in rats and humans. Phenol UGT appears to be conduced with other drug-metabolizing enzymes via the Ah or dioxin receptor. This isozyme probably controls various proximate carcinogens and contributes to the persistently altered enzyme pattern, leading to the “toxin-resistance phenotype” at cancer prestages. Knowledge about UGTs in different species, their regulation, and their tissue distribution will improve the risk assessment of carcinogens.     

Potential identification of chemical carcinogens in a viral transformation system

Chemical carcinogens from several diverse chemical classes i.e.; aromatic amines, polycyclic hydrocarbons, nitrosamines, hormonal derivatives, metals and direct alkylating agents cause a 6.2–60.5-fold increase in the frequency of murine sarcoma virus (MSV)-induced transformation in a normal rat kidney (NRK) cell system. Exogenous metabolic activation with a rat liver S-9 homogenate is required for expression of this activity by procarcinogens. Non-carcinogenic analogs of these compounds fail to cause significant increases in the transformation frequency either with or without prior metabolic activation. Iododeoxyuridine, a mutagen also does not cause enhancement of transformation. This system may serve as the basis for a rapid and quantifiable means of identifying chemical carcinogens while introducing a new model for the understanding of the interactions between oncorna-viruses and chemical carcinogens.     

Ah receptor in primate liver: binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and carcinogenic aromatic hydrocarbons

Ah receptor in hepatic cytosols from adult cynomolgus monkeys (Macaca fasicularis) was identified and quantitated by its binding of the highly toxic chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the carcinogens 3-methylcholanthrene, benzo[a]pyrene, and dibenz[a,h]anthracene. The concentration of Ah receptor in cynomolgus hepatic cytosols (approximately 10 fmol/mg cytosol protein) was about one-quarter of that typically detected in rodent hepatic cytosols. Receptor concentrations were equal in male and female cynomolgus. [3H]TCDD bound to cytosolic receptor with high affinity (Kd approximately 3 nM). In rodents, Ah receptor is known to play a central role in toxicity caused by halogenated aromatic compounds and in carcinogenesis caused by polycyclic aromatic hydrocarbons. Existence of Ah receptor in monkeys indicates that the receptor also may mediate such responses in primates.     

Induction of sister-chromatid exchanges by procarcinogens in metabolically competent Chinese hamster epithelial liver cells

An epithelial cell strain has been established from the livers of male Chinese hamsters (CHEL cells). These cells, which proliferate in culture and retain their metabolic enzymatic activities during several subcultures, were used in a sister-chromatid exchange assay to evaluate the effectiveness of polycyclic aromatic hydrocarbons (PAHs), aflatoxin B1 (AFB1) and cyclophosphamide (CP). The results obtained demonstrate that CHEL cells are metabolically competent to activate different classes of procarcinogens into biologically active metabolites. Moreover, they showed a selective capacity to discriminate chemical carcinogens from noncarcinogens. Thus, the CHEL cell system appears to be a promising alternative to the short-term tests that include cell-free rodent liver homogenate to evaluate new promutagens and/or procarcinogens.     

An alternatively spliced cytochrome P4501A1 in human brain fails to bioactivate polycyclic aromatic hydrocarbons to DNA-reactive metabolites

CYP1A1, a cytochrome P450 enzyme, metabolizes polycyclic aromatic hydrocarbons to genotoxic metabolite(s) that bind to DNA and initiate carcinogenesis. RT-PCR amplification of the complete open reading frame of CYP1A1 generated an amplicon of 1593 bp having deletion of 87 bp of exon-6 that translated into functional P450 enzyme. Unlike wild type CYP1A1, exon 6 del CYP1A1 did not metabolize polycyclic aromatic hydrocarbons such as, benzo(a)pyrene to genotoxic, ultimate carcinogens that form DNA adducts. Exon 6 del CYP1A1 metabolized ethoxyresorufin (the classical substrate for CYP1A1) less efficiently compared with wild type CYP1A1 while pentoxy and benzyloxyresorufin (classical substrates for CYP2B) were dealkylated more efficiently. In silico docking showed alteration of the substrate access channel in exon 6 del CYP1A1 such that benzo(a)pyrene does not bind in any orientation that would permit the formation of carcinogenic metabolites. Genotyping revealed that the splice variant was not generated due to differences in genomic DNA sequence and the variant was present only in brain but not in liver, kidney, lung, or heart from the same individual. We provide evidence that unique P450 enzymes, generated by alternate splicing in a histiospecific manner can modify genotoxic potential of carcinogens such as benzo(a)pyrene by altering their biotransformation pathway.     

The Escherichia coli K-12 SOS chromotest agar spot test for simple, rapid detection of genotoxic agents

The Escherichia coli K-12 SOS chromotest is a colorimetric (beta-galactosidase induction) system for detecting genotoxic chemicals as agents which induce filamentation in response to DNA damage. The chromotest was modified from a liquid suspension assay to a simple, convenient agar spot test, which was performed in the manner of a related colorimetric prophage induction assay (BIA). Chromotest agar dishes yielded optimal results after 16-18 h incubation, presumably because of the agar growth characteristics of tester strain PQ37. Of 44 tested chemicals, nitro aromatics, cytotoxic/antitumor agents, polycyclic hydrocarbons and aflatoxins showed good activity. Alkylating agents such as MNNG and MMS were active only at high concentrations. Compounds active in both the chromotest and BIA were active at 10-100-fold lower concentrations in the chromotest. The chromotest appeared to be less effective than the Salmonella Ames mutagenicity test in the detection of diverse classes of chemical carcinogens. The chromotest may be a useful alternative to the BIA in the study of particular classes of genotoxic compounds.     

Quantum chemical studies of polycyclic aromatic hydrocarbons and their metabolites: correlations to carcinogenicity.

In the context of the bay region hypothesis for polycyclic aromatic hydrocarbon (PAH) carcinogenesis, molecular properties were calculated for seventeen polycyclic aromatic hydrocarbons related to (1) intrinsic substrate reactivities towards activating and detoxifying metabolism and (2) the stabilities of the putative carbocation ultimate carcinogens. All-valence electron methods were used, avoiding the inherent difficulties found in the pi-electron methods. The calculated substrate reactivities were found to predict major metabolites successfully, supporting the validity of their use in attempted correlations with observed carcinogenic potencies. Positive correlations were found between observed carcinogenic potencies and (1) the reactivities of the parent polycyclic aromatic hydrocarbons towards the initial distal bay region epoxidation and (2) the stabilities of the diol epoxide carbocations. The reactivities of the distal bay region diol epoxides, were high for both carcinogenic and non-carcinogenic compounds, implying that the second epoxidation does not determine relative carcinogenic activity. Support for a possible alternative hypothesis, that polycyclic aromatic hydrocarbons are activated by one electron oxidation, was also found.     

Bioactivation of xenobiotics by prostaglandin H synthase

Prostaglandin H synthase (PHS) catalyzes the oxidation of arachidonic acid to prostaglandin H2 in reactions which utilize two activities, a cyclooxygenase and a peroxidase. These enzymatic activities generate enzyme- and substrate-derived free radical intermediates which can oxidize xenobiotics to biologically reactive intermediates. As a consequence, in the presence of arachidonic acid or a peroxide source, PHS can bioactivate many chemical carcinogens to their ultimate mutagenic and carcinogenic forms. In general, PHS-dependent bioactivation is most important in extrahepatic tissues with low monooxygenase activity such as the urinary bladder, renal medulla, skin and lung. Mutagenicity assays are useful in the detection of compounds which are converted to genotoxic metabolites during PHS oxidation. In addition, the oxidation of xenobiotics by PHS often form metabolites or adducts to cellular macromolecules which are specific for peroxidase- or peroxyl radical-dependent reactions. These specific metabolites and/or adducts have served as biological markers of xenobiotic bioactivation by PHS in certain tissues. Evidence is presented which supports a role for PHS in the bioactivation of several polycyclic aromatic hydrocarbons and aromatic amines, two classes of carcinogens which induce extrahepatic neoplasia. It should be emphasized that the toxicities induced by PHS-dependent bioactivation of xenobiotics are not limited to carcinogenicity. Examples are given which demonstrate a role for PHS in pulmonary toxicity, teratogenicity, nephrotoxicity and myelotoxicity.     

Induction of genetic tandem duplications in Salmonella by polycyclic aromatic hydrocarbon and amine carcinogens

6 polycyclic aromatic hydrocarbon or similar amine carcinogens were tested as inducers of genetic tandem duplications in a rough strain of Salmonella typhimurium. When metabolically activated by rat-liver microsomes, all 6 were active in inducing genetic tandem duplications, yielding from over 3 times to almost 14 times as many tandem duplicants per viable bacterium as did concurrent uninduced control cultures. These results extend the number and chemical diversity of carcinogens shown to induce genetic duplications in bacterial tester systems. We suggest that polycyclic hydrocarbon carcinogens may act in carcinogenesis by inducing genetic duplications or other genetic rearrangements. Duplication induction may be a useful genetic endpoint for screening potential carcinogens.     

Inverse correlation between species life span and capacity of cultured fibroblasts to metabolize polycyclic hydrocarbon carcinogens.

Many investigators have hypothesized that the aging process may result from an accumulation of DNA damage, and, if valid, this necessitates a means by which this accumulation can be related to the potential life span of an organism. Using an assay for cell-mediated mutagenesis, we have tested multiple diploid fibroblast strains from six mammalian species of widely differing life spans, and found a very good inverse correlation between species life span and ability to activate 7,12-dimethylbenz(a)anthracene (DMBA) to mutagenic forms. We have also found a very good inverse correlation between species life span and ability to activate DMBA to forms capable of covalent binding to DNA. Since the polycyclic hydrocarbon carcinogens such as DMBA and benzo(a)pyrene (BP) are chemically non-reactive in their native forms and must be metabolically activated by mixed-function oxidases to their biologically active forms, these data indicate that the capacity of fibroblasts to activate polycyclic hydrocarbon carcinogens to DNA-damaging forms is a species property related to potential life span. To determine the role of carcinogen metabolism in this phenomenon the capacity of diploid fibroblasts from eight mammalian species to convert BP and DMBA to water-soluble metabolites was then determined. This rate of conversion varies widely among different species and shows a very good inverse correlation with species life span. As a whole, these findings suggest that the ability of cultured cells to metabolize the polycyclic hydrocarbon carcinogens is related to species life span, and may be important in the occurrence of spontaneous cancer.     

Effect of various chemicals on the aldehyde dehydrogenase activity of the rat liver cytosol

The cytosolic activity of aldehyde dehydrogenase (ALDH) was studied in the rat liver, after acute administration of various carcinogenic and chemically related compounds. Male Wistar rats were treated with 27 different chemicals, including polycyclic aromatic hydrocarbons, aromatic amines, nitrosamines, azo dyes, as well as with some known direct-acting carcinogens. The cytosolic ALDH activity of the liver was determined either with propionaldehyde and NAD (P/NAD), or with benzaldehyde and NADP (B/NADP). The activity of ALDH remained unaffected after treatment with 1-naphthylamine, nitrosamines and also with the direct-acting chemical carcinogens tested. On the contrary, polycyclic aromatic hydrocarbons, polychlorinated biphenyls (Arochlor 1254) and 2-naphthylamine produced a remarkable increase of ALDH. In general, the response to the effectors was disproportionate between the two types of enzyme activity, being much in favour for the B/NADP activity. This fact resulted to an inversion of the ratio B/NADP vs. P/NAD, which under constitutive conditions is lower than 1. In this respect, the most potent compounds were found to be polychlorinated biphenyls, 3-methylcholanthrene, benzo(a)pyrene and 1,2,5,6-dibenzoanthracene. Our results suggest that the B/NADP activity of the soluble ALDH is greatly induced after treatment with compounds possessing aromatic ring(s) in their molecule. It is not known, if this response of the hepatocytes is related with the process of chemical carcinogenesis.     

Electrofluorescence study of polycyclic hydrocarbon diol-epoxide binding to DNA

By the use of a novel electrofluorescence method, estimates have been made of the geometry of binding to DNA of racemic mixtures of the anti-diol-epoxide derivatives of three polycyclic hydrocarbon carcinogens. These anti-configurations bind in a manner consistent with the planar diol-epoxide ring's being inclined at approximately 50 degrees to the DNA axis. This is true for the derivatives of benzo(a)pyrene, benz(a)anthracene and 3-methylcholanthrene. This binding is thus different from the regular intercalative interaction associated with the native hydrocarbons. As the (+ anti)-diol-epoxides are thought to be the initiatory compounds for carcinogenesis, the common binding characteristics for the three hydrocarbons may be significant in understanding the molecular interactions precursive to cancer.