Carcinogens induce genetic tandem duplications in Salmonella

Extensive studies have shown that chemical carcinogenesis involves an initiation-promotion pattern. A gene amplification model of carcinogenesis predicts that initiation involves induction of a genetic tandem duplication. We use a system developed by Anderson and Roth to select for tandem duplication of the histidine operon of Salmonella typhimurium by selection for resistance to 3-amino-1,2,4-triazole. Evidence reported here shows that, consistent with prediction, 10 carcinogens are all active in inducing tandem duplications. Two toxic noncarcinogens show little or no activity under the conditions used in inducing tandem duplication but azide, a mutagenic noncarcinogen, did show some activity. 9 types of evidence now support the gene amplification initiation-promotion model of carcinogenesis.     

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.     

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.     

Targeted disruption of the microsomal epoxide hydrolase gene. Microsomal epoxide hydrolase is required for the carcinogenic activity of 7,12-dimethylbenz[a]anthracene.

Microsomal epoxide hydrolase (mEH) is a conserved enzyme that is known to hydrolyze many drugs and carcinogens, and a few endogenous steroids and bile acids. mEH-null mice were produced and found to be fertile and have no phenotypic abnormalities thus indicating that mEH is not critical for reproduction and physiological homeostasis. mEH has also been implicated in participating in the metabolic activation of polycyclic aromatic hydrocarbon carcinogens. Embryonic fibroblast derived from the mEH-null mice were unable to produce the proximate carcinogenic metabolite of 7,12-dimethylbenz[a]anthracene (DMBA), a widely studied experimental prototype for the polycylic aromatic hydrocarbon class of chemical carcinogens. They were also resistant to DMBA-mediated toxicity. Using the two-stage initiation-promotion skin cancer bioassay, the mEH-null mice were found to be highly resistant to DMBA-induced carcinogenesis. In a complete carcinogenesis bioassay, the mEH mice were totally resistant to tumorigenesis. These data establish in an intact animal model that mEH is a key genetic determinant in DMBA carcinogenesis through its role in production of the ultimate carcinogenic metabolite of DMBA, the 3,4-diol-1,2-epoxide.     

On the mechanism of production of tandem genetic duplications in phage lambda

We have asked whether the mechanism by which tandem genetic duplications arise in the chromosome of phage lambda is inter- or intramolecular. Two parental phages carrying genetic markers at opposite ends of the phage chromosome have been grown in mixed infection, and progeny phages carrying newly-arising tandem duplications have been analysed to determine whether they carry the markers in parental or recombinant configuration. Ordinary genetic recombination of the markers has been prevented by mutations in the phage and host. Phages carrying tandem duplications are isolated by use of CsCl density gradients and an Escherichia coli strain that does not plate deletion phages. Of the duplication mutants isolated under these conditions, 13% carry the input markers in recombinant configuration. This suggests that tandem duplications can be produced via an intermolecular route which joins sequences originally present on different DNA molecules.     

Histochemical characteristics of spontaneous and chemically induced hepatocellular neoplasms in mice and the development of neoplasms with gamma-glutamyl transpeptidase activity during phenobarbital exposure

Summary The histochemical characteristics of spontaneous hepatocellular neoplasms in mice of both sexes were examined and compared with those of hepatocellular neoplasms induced in female mice by administration of polycyclic aromatic hydrocarbon carcinogens as initiators with or without subsequent phenobarbitone treatment. Controls treated with phenobarbitone alone were also included. Spontaneous neoplasms in the livers of mice rendered siderotic by subcutaneous iron injection were deficient in cellular accumulation of stainable iron. Glucose-6-phosphatase activity was deficient in the majority of spontaneous and induced neoplasms. ATPase activity was increased in about half of spontaneous and carcinogen-induced neoplasms but all induced neoplasms in mice treated with phenobarbitone showed deficient activity. -Glutamyltransferase activity was present in very few of the spontaneous neoplasms or in the neoplasms induced in the absence of phenobarbitone administration. However, all induced neoplasms in the mice receiving phenobarbitone showed some degree of -glutamyltransferase activity together with deficient glucose-6-phosphatase and ATPase activities. It is concluded that the histochemical characteristics of spontaneous or induced mouse hepatocellular neoplasms are variable and may be influenced by the inducing factors.     

Mechanism of action of food-associated polycyclic aromatic hydrocarbon carcinogens

The polycyclic aromatic hydrocarbon carcinogens are formed in the inefficient combustion of organic matter and contaminate foods through direct deposition from the atmosphere or during cooking or smoking of foods. These potent carcinogens and mutagens require metabolism to dihydrodiol epoxide metabolites in order to express their biological activities. In vitro studies show that these reactive metabolites can react with the bases in DNA with different specificities depending upon the hydrocarbon from which they are derived. Thus, the more potent carcinogens react more extensively with adenine residues in DNA than do the less potent carcinogens, with the result that mutation at A . T base pairs is enhanced for the more potent carcinogens. In the past few years, considerable clarification of the mechanism of metabolic activation have been achieved and the focus for the immediate future is expected to be on how the reactive metabolites actually bring about biological responses.     

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.     

Next-Generation Sequencing of Duplication CNVs Reveals that Most Are Tandem and Some Create Fusion Genes at Breakpoints

Interpreting the genomic and phenotypic consequences of copy-number variation (CNV) is essential to understanding the etiology of genetic disorders. Whereas deletion CNVs lead obviously to haploinsufficiency, duplications might cause disease through triplosensitivity, gene disruption, or gene fusion at breakpoints. The mutational spectrum of duplications has been studied at certain loci, and in some cases these copy-number gains are complex chromosome rearrangements involving triplications and/or inversions. However, the organization of clinically relevant duplications throughout the genome has yet to be investigated on a large scale. Here we fine-mapped 184 germline duplications (14.7 kb–25.3 Mb; median 532 kb) ascertained from individuals referred for diagnostic cytogenetics testing. We performed next-generation sequencing (NGS) and whole-genome sequencing (WGS) to sequence 130 breakpoints from 112 subjects with 119 CNVs and found that most (83%) were tandem duplications in direct orientation. The remainder were triplications embedded within duplications (8.4%), adjacent duplications (4.2%), insertional translocations (2.5%), or other complex rearrangements (1.7%). Moreover, we predicted six in-frame fusion genes at sequenced duplication breakpoints; four gene fusions were formed by tandem duplications, one by two interconnected duplications, and one by duplication inserted at another locus. These unique fusion genes could be related to clinical phenotypes and warrant further study. Although most duplications are positioned head-to-tail adjacent to the original locus, those that are inverted, triplicated, or inserted can disrupt or fuse genes in a manner that might not be predicted by conventional copy-number assays. Therefore, interpreting the genetic consequences of duplication CNVs requires breakpoint-level analysis.     

Generation of Tandem Direct Duplications by Reversed-Ends Transposition of Maize Ac Elements

Tandem direct duplications are a common feature of the genomes of eukaryotes ranging from yeast to human, where they comprise a significant fraction of copy number variations. The prevailing model for the formation of tandem direct duplications is non-allelic homologous recombination (NAHR). Here we report the isolation of a series of duplications and reciprocal deletions isolated de novo from a maize allele containing two Class II Ac/Ds transposons. The duplication/deletion structures suggest that they were generated by alternative transposition reactions involving the termini of two nearby transposable elements. The deletion/duplication breakpoint junctions contain 8 bp target site duplications characteristic of Ac/Ds transposition events, confirming their formation directly by an alternative transposition mechanism. Tandem direct duplications and reciprocal deletions were generated at a relatively high frequency (∼0.5 to 1%) in the materials examined here in which transposons are positioned nearby each other in appropriate orientation; frequencies would likely be much lower in other genotypes. To test whether this mechanism may have contributed to maize genome evolution, we analyzed sequences flanking Ac/Ds and other hAT family transposons and identified three small tandem direct duplications with the structural features predicted by the alternative transposition mechanism. Together these results show that some class II transposons are capable of directly inducing tandem sequence duplications, and that this activity has contributed to the evolution of the maize genome.     

Studies on pulmonary aryl hydrocarbon hydroxylase activity in inbred strains of mice.

Pulmonary and hepatic levels of aryl hydrocarbon hydroxylase (AHH) were studied in inbred strains of mice following intratracheal (i.t.) instillation of 3-methylcholanthrene (MCA). I.t. instillation of 188 mug MCA in sterile 0.2% gelatin in saline resulted in preferential induction of pulmonary AHH. After treatment with this dose of MCA, the pulmonary AHH levels of strains C57BL/6Cum, C57BL/6J, BALB/cMai, C3H/fMai, and C57L/J were observed to be induced within 24 h after treatment. Strains DBA/2Cum, AKR/J, SJL/J, DBA/2J and RF/J expressed no such increase. At a dose of 500 mug MCA, the pulmonary tissue of DBA/2 mice did express a 4-fold increase. This increase in AHH was determined to be quite different from the increase observed in C57BL/6 mice by: (1) specific activity of the enzymes, (2) genetic regulation, (3) susceptibility to inhibition by 7,8-benzoflavone, and (4) spectral properties of the associated cytochromes. It was of major importance that induction of pulmonary AHH was observed to be regulated by a single dominant gene in crosses involving the C57BL/6Cum and DBA/2Cum strains of mice. Results were discussed with the view in mind that these genetically regulated levels of AHH may play a role in susceptibility to cancers induced by polycyclic aromatic hydrocarbon carcinogens.     

Nebulin: A Study of Protein Repeat Evolution

Protein domain repeats are common in proteins that are central to the organization of a cell, in particular in eukaryotes. They are known to evolve through internal tandem duplications. However, the understanding of the underlying mechanisms is incomplete. To shed light on repeat expansion mechanisms, we have studied the evolution of the muscle protein Nebulin, a protein that contains a large number of actin-binding nebulin domains.Nebulin proteins have evolved from an invertebrate precursor containing two nebulin domains. Repeat regions have expanded through duplications of single domains, as well as duplications of a super repeat (SR) consisting of seven nebulins. We show that the SR has evolved independently into large regions in at least three instances: twice in the invertebrate Branchiostoma floridae and once in vertebrates.In-depth analysis reveals several recent tandem duplications in the Nebulin gene. The events involve both single-domain and multidomain SR units or several SR units. There are single events, but frequently the same unit is duplicated multiple times. For instance, an ancestor of human and chimpanzee underwent two tandem duplications. The duplication junction coincides with an Alu transposon, thus suggesting duplication through Alu-mediated homologous recombination.Duplications in the SR region consistently involve multiples of seven domains. However, the exact unit that is duplicated varies both between species and within species. Thus, multiple tandem duplications of the same motif did not create the large Nebulin protein.Finally, analysis of segmental duplications in the human genome reveals that duplications are more common in genes containing domain repeats than in those coding for nonrepeated proteins. In fact, segmental duplications are found three to six times more often in long repeated genes than expected by chance.     

Lowered induction of genetic tandem duplications in Salmonella by the pKM101 plasmid

Summary Selection for 3-amino-1,2,4-triazole (AT) resistance in certain strains of Salmonella typhimurium has been previously shown to select for genetic tandem duplications of the histidine operon. We show here that agents which induce tandem duplications are less effective in such induction in the presence of the pKM101 plasmid. The presence of the plasmid also produces an increase in AT-resistance due to mechanisms other than duplication, presumably because pKM101 produces high levels of error-prone repair. We suggest that high levels of error-prone repair may cause decreases in tandem duplication induction and propose that error-prone repair and tandem duplication may be alternative cellular responses to certain DNA lesions.     

Properties and Evolutionary Potential of Newly Induced Tandem Duplications in DROSOPHILA MELANOGASTER

Most of some 33 X-ray-induced duplications recovered as Suppressors of Minute loci proved to be direct tandem duplications. When heterozygous, most duplications were crossover suppressors, and duplications of short to moderate size did not reduce the fitness of their bearers. Crossover suppression by tandem duplication may be attributed to intrastrand foldbacks of the type regularly seen in somatic polytene chromosomes. As a consequence, linkage disequilibrium between duplicated elements and normal chromosomes should be more profound than has been supposed. Tandem duplications appear to be predisposed by reason of frequency of generation, crossover suppression and fitness effects to serve as the primary source of new genes.     

Unequal genetic exchange in Escherichia coli tandem duplications may represent a special pathway of homologous recombination

Heterozygous tandem duplications that appear in Escherichia coli conjugation matings segregate different types of haploid and diploid recombinants because of unequal crossing over between sister chromosomes. As shown previously, the frequency of segregants in the extended duplication D104 (approximately 150 kb or more than 3 min of the genetic map) heterozygous for E. coli deo-operon genes (deoA deoB::Tn5/deoC deoD) is not decreased in strains with defective RecBCD and RecF recombination pathways. Analysis of a shorter duplication of this type (approximately 46 kb) showed that the frequency of segregants in the strain recBC sbcBC recF was similar to that in a strain with undamaged system of recombination. Thus, genetic exchange between direct DNA repeats in tandem duplications may follow a special pathway of homologous recombination, which is independent of the recBC and recF genes.     

p53 mutational spectra and the role of methylated CpG sequences

The occurrence of tumor-specific mutational spectra in the p53 mutation database provides indirect evidence that implicates certain exogenous and possibly endogenous mutagenic events in human carcinogenesis. In some cases, the distribution of DNA damage along the p53 gene caused by environmental carcinogens can be correlated with the mutational spectra, i.e. hotspots and types of mutations of certain cancers, most notably for nonmelanoma skin cancers and lung cancers in smokers. This concept has been validated by experiments with sunlight and cigarette smoke components representing the polycyclic aromatic hydrocarbon class of carcinogens. A disproportionally high number of mutations in p53 (and other genes) are found at methylated CpG dinucleotides. These sequences are particularly prone to mutagenesis involving endogenous events as well as modification by exogenous carcinogens.     

Tandem duplications of the histidine operon observed following generalized transduction in Salmonella typhimurium

Unstable merodiploid transductants may be observed among the progeny of certain generalized transductional crosses between complementing mutations in the histidine operon of Salmonella typhimurium. In the presence of a functional recombination system, these transductants are unstable and they segregate His^? clones of both parental genotypes. The properties of these His⁺ transductants suggest that they contain tandem duplications of a region of DNA which includes the histidine operon, such that each copy of the duplication contains one of the two complementing mutations involved in the transduction. Transductional duplications have been observed from 14 pairs of his mutations, but only with complementing pairs of parental mutations. The length of duplicated material may be quite large: two duplications were found to include genetic markers ten minutes removed from the histidine operon on the Salmonella chromosomal map.These transductants appear to arise in a subpopulation of recipient cells which contain pre-existing tandem duplications of the histidine operon. As much as 0.01 to 0.1% of the cell population appears to be tandemly duplicated for a chromosomal region which includes the histidine operon.     

DNA adducts in humans as dosimeters of exposure to environmental, occupational, or dietary genotoxins.

The quantitation of adducts of genotoxins with DNA is probably one of the best indicators of genetic damage due to exposure to toxins or carcinogens. It is generally believed that such adducts can lead to mutations, which in turn can trigger the initiation of the carcinogenic process. DNA adducts have been quantitated in white blood cells and in various tissues of smokers, persons in certain high-exposure occupations, and persons consuming foods contaminated with certain carcinogens. The feasibility of this approach for biochemical epidemiologic studies has been demonstrated using methods such as 32P-postlabeling, enzyme-linked immunosorbent assay, and synchronous fluorescence spectrophotometry. Relatively large interindividual differences in DNA adducts have been observed in both exposed and nonexposed persons. As a result, there are only a few studies in which clear quantitative and qualitative differences between these two groups have been observed. In addition, it appears that in some studies the 32P-postlabeling method does not detect the presence of the polycyclic aromatic hydrocarbon DNA adducts that are detectable by immunoassays. More extensive studies in additional populations at risk should shed further light on the utility of DNA adduct analysis in biochemical monitoring, especially if further refinements in methodology would result in increased sensitivity and specificity.     

Induction of choline kinase by polycyclic aromatic hydrocarbons in rat liver. I. A comparison of choline kinases from normal and 3-methylcholanthrene-induced rat liver cytosol

Choline kinase in rat liver has been shown to be induced up to 2-fold by the administration of polycyclic aromatic hydrocarbon carcinogens such as 3-methylcholanthrene and 3,4-benzo[a]pyrene (Ishidate, K., Tsuruoka, M. and Nakazawa, Y., (1980) Biochem. Biophys. Res. Commun. 96, 946-952). In order to characterize the nature of choline kinase induction by these carcinogens, the 3-methylcholanthrene-induced form as well as the normal form of choline kinase were partially purified from rat liver cytosol through acid treatment, (NH4)2SO4 precipitation and DEAE-cellulose column chromatography with linear KCl-gradient elution, and the catalytic properties were compared between the two preparations. Both enzyme activities were purified about 17-fold with a yield of 50% through the purification steps and there appeared no detectable difference in the elution pattern from either DEAE-cellulose column or Sephadex G-200 gel filtration. On the other hand, some differences were observed in catalytic properties between the two enzyme preparations; (1) the induced form showed a higher apparent Km value for choline (0.19 mM) when compared to the normal form (0.11 mM) and (2) the addition of polyamines caused a considerable increase in the maximum reaction velocity for the normal form whereas no remarkable change for the induced form, when the activities were plotted as a function of choline concentration. The overall results suggest that the 3-methylcholanthrene-induced form of choline kinase in rat liver could be different from the normal form, or that there exist several isoenzymes of choline kinase in rat liver, and one or some of them are inducible by the administration of polycyclic aromatic hydrocarbons.     

Recombination between Chromosomal Is200 Elements Supports Frequent Duplication Formation in Salmonella Typhimurium

Spontaneous tandem chromosomal duplications are common in populations of Escherichia coli and Salmonella typhimurium. They range in frequency for a given locus from 10(-2) to 10(-4) and probably form by RecA-dependent unequal sister strand exchanges between repetitive sequences in direct order. Certain duplications have been observed previously to confer a growth advantage under specific selective conditions. Tandem chromosomal duplications are unstable and are lost at high frequencies, representing a readily reversible source of genomic variation. Six copies of a small mobile genetic element IS200 are evenly distributed around the chromosome of S. typhimurium strain LT2. A survey of 120 independent chromosomal duplications (20 for each of six loci) revealed that recombination between IS200 elements accounted for the majority of the duplications isolated for three of the loci tested. Duplications of the his operon were almost exclusively due to recombination between repeated IS200 elements. These data add further support to the idea that mobile genetic elements provide sequence repeats that play an important role in recombinational chromosome rearrangements, which may contribute to adaptation of bacteria to stressful conditions.