Fecapentaenes and their precursors throughout the bowel--results of an autopsy study

The fecapentaenes are potent mutagens found in the stool of some humans and pigs. These compounds are produced by Bacteroides species in the gut from an uncharacterized family of precursor compounds, and have been postulated to pose a risk of human colorectal cancer. To better understand fecapentaene production in vivo, and to determine if excreted levels measured in epidemiologic studies are representative of the entire colon, fecapentaenes were assayed from multiple sites in the bowel in an autopsy study of 16 humans and 2 pigs. An indirect measurement of fecapentaene precursors was also made. Colonic concentrations of fecapentaenes and precursors varied widely between individuals, but were consistent for each individual throughout the colon. In addition, the measurements of rectal contents, assumed to approximate values in excreted stool, were equivalent to measurements from the colon.     

Relation of changes in amount and type of dietary fat to fecapentaenes in premenopausal women

Correlation studies suggest that fecal mutagenicity is increased in groups eating high-fat diets, the same groups who are often found to have high colorectal cancer incidence and mortality. The fecapentaenes are the best characterized class of fecal mutagens, but the relationship of dietary fat intake to the excretion of these potent genotoxins is unknown. We studied the effect of changes in amount and type of dietary fat on fecapentaene levels in 31 premenopausal women 20-40 years of age who participated in a controlled feeding study. After a pre-diet free-living period lasting 1 menstrual cycle, women were placed on a high-fat (40% energy from fat) diet for 4 menstrual cycles and then switched to a low-fat (20% energy from fat) diet for an additional 4 menstrual cycles. One-half the subjects were maintained throughout the study at a ratio of polyunsaturated-to-saturated fatty acids (P/S ratio) of 1.0, the other half at 0.3; body weight was constant. All meals during the controlled diet periods were prepared at the Human Study Facility of the Beltsville Human Nutrition Research Center. Fecapentaene and fecapentaene precursor levels were measured in acetone extracts from 3-day pooled stool samples collected during the study. No differences in fecapentaene or precursor levels were observed between the high- and low-fat diets at either P/S ratio. Fecapentaene and precursor levels were higher while on controlled diets than during the pre-diet free-living period, and levels declined again in the post-diet free-living period. We conclude that dietary fat has no significant effect on fecapentaene or precursor levels in acetone extracts of stool in premenopausal women. The effect of other dietary or non-dietary factors on fecapentaenes remains unknown.     

Chromatographic methods for the determination of toxicants in faeces

Modern chromatographic techniques and their application in the determination of toxic compounds in faeces are reviewed. Faecal analysis may be of importance in toxicokinetic studies of xenobiotics in order to determine factors such as metabolism, body burden and major routes of elimination. Compounds of interest include various food constituents, drugs and occupational or environmental factors. Further, various mutagenic or carcinogenic compounds which are excreted by faeces have been indicated to represent risk factors for colorectal cancer. In this context, the chromatographic determination of the endogenously generated fecapentaenes and bile acids, both postulated etiological factors in colorectal carcinogenesis, is reviewed. For fecapentaene determination, several high-performance liquid chromatographic (HPLC) methods are available; however, the applicability of some of these methods is limited owing to insufficient separation of various isomeric forms or discrimination between fecapentaenes and their precursors. For the determination of bile acids in faeces, many chromatographic procedures have been reported, and the characteristics of the most relevant methods are compared and discussed. It is concluded that separation by gas chromatography (GC) in combination with mass spectrometry provides the highest selectivity and sensitivity. A relatively rapid alternative analysis for the determination of total and aqueous faecal bile acids is proposed. Further, methods for the determination of polycyclic aromatic hydrocarbons (PAHs) are reviewed. Although the use of radiolabelled PAHs in animal studies has many advantages, it cannot be applied for human biological monitoring and HPLC and GC provide sensitive alternatives. An HPLC method for the determination of non-metabolized PAHs in faeces is described.     

Fecapentaene concentration and mutagenicity in 718 North American stool samples

The fecapentaenes (FP) are the predominant fecal mutagens identified to date, but they have not been shown to be carcinogenic. Epidemiologists looking for other fecal mutagens that may be related to colorectal cancer must disentangle from their investigations the pervasive mutagenic effect of the fecapentaenes. As a first step to studying the epidemiology of fecal mutagenicity independent of fecapentaenes, we compared FP measurements and Salmonella mutagenicity assay results for 718 acetone-extracted stool samples collected from a variety of subjects in the Washington DC metropolitan areas. In this large group, 50% of mutagenic samples contained elevated fecapentaenes. Specifically, three-quarters of the samples mutagenic in TA100 contained high FP levels. In contrast, mutagenicity in TA98 was not generally explainable by fecapentaenes, suggesting that non-fecapentaene TA98 mutagenicity should be one focus of future efforts to uncover colorectal carcinogens of etiologic importance.     

Metabolism of dietary genotoxins by the human colonic microflora; the fecapentaenes and heterocyclic amines

The microflora of the human colon is a complex ecosystem of anaerobic bacteria which have the capability of enzymatically transforming a variety of dietary (or biliary) compounds to genotoxic metabolites. In the past, most investigators studying the interplay between diet and colonic flora and its role in the etiology of cancers focused on the reductive and glycosidic potential of the bacterial enzymes--many of which reverse the oxidative and conjugative reactions performed by the liver. Recent work in our laboratory has focused on the metabolism of two relatively new classes of genotoxins, the fecapentaenes and the heterocyclic amines (pyrolysis carcinogens). The fecapentaenes (conjugated ether lipids) are produced in the colon by Bacteroides spp. from polyunsaturated ether phospholipids (plasmalogens) whose natural origin and function are unknown. The fecapentaenes are potent direct-acting genotoxins that are detected in the feces of most individuals on normal western diets. The heterocyclic amines, which originate from fried or broiled proteinaceous foods, normally require activation by the liver before being potent mutagens or carcinogens. However, the "IQ" subclass (e.g. IQ and MeIQ) can be activated in the colon by Eubacterium and Clostridium species to a 7-hydroxy form which is directly mutagenic in Salmonella. Although there is no direct evidence that the fecapentaenes or the 7-hydroxy "IQ" compounds influence risk for colon cancer, the potency and prevalence of these bacterial metabolites is cause for concern.     

Commensal bacteria, redox stress, and colorectal cancer: mechanisms and models

The potential role for commensal bacteria in colorectal carcinogenesis is explored in this review. Most colorectal cancers (CRCs) occur sporadically and arise from the gradual accumulation of mutations in genes regulating cell growth and DNA repair. Genetic mutations followed by clonal selection result in the transformation of normal cells into malignant derivatives. Numerous toxicological effects of colonic bacteria have been reported. However, those recognized as damaging epithelial cell DNA are most easily reconciled with the currently understood genetic basis for sporadic CRC. Thus, we focus on mechanisms by which particular commensal bacteria may convert dietary procarcinogens into DNA damaging agents (e.g., ethanol and heterocyclic amines) or directly generate carcinogens (e.g., fecapentaenes). Although these and other metabolic activities have yet to be linked directly to sporadic CRC, several lines of investigation are reviewed to highlight difficulties and progress in the area. Particular focus is given to commensal bacteria that alter the epithelial redox environment, such as production of oxygen radicals by Enterococcus faecalis or production of hydrogen sulfide by sulfate-reducing bacteria (SRB). Super-oxide-producing E. faecalis has conclusively been shown to cause colonic epithelial cell DNA damage. Though SRB-derived hydrogen sulfide (H(2)S) has not been reported thus far to induce DNA damage or function as a carcinogen, recent data demonstrate that this reductant activates molecular pathways implicated in CRC. These observations combined with evidence that SRB carriage may be genetically encoded evoke a working model that incorporates multifactorial gene-environment interactions that appear to underlie the development of sporadic CRC.     

Evaluation of fecal mutagenicity and colorectal cancer risk

Colorectal cancer is one of the most common internal malignancies in Western society. The cause of this disease appears to be multifactorial and involves genetic as well as environmental aspects. The human colon is continuously exposed to a complex mixture of compounds, which is either of direct dietary origin or the result of digestive, microbial and excretory processes. In order to establish the mutagenic burden of the colorectal mucosa, analysis of specific compounds in feces is usually preferred. Alternatively, the mutagenic potency of fecal extracts has been determined, but the interpretation of these more integrative measurements is hampered by methodological shortcomings. In this review, we focus on exposure of the large bowel to five different classes of fecal mutagens that have previously been related to colorectal cancer risk. These include heterocyclic aromatic amines (HCA) and polycyclic aromatic hydrocarbons (PAH), two exogenous factors that are predominantly ingested as pyrolysis products present in food and (partially) excreted in the feces. Additionally, we discuss N-nitroso-compounds, fecapentaenes and bile acids, all fecal constituents (mainly) of endogenous origin. The mutagenic and carcinogenic potency of the above mentioned compounds as well as their presence in feces, proposed mode of action and potential role in the initiation and promotion of human colorectal cancer are discussed. The combined results from in vitro and in vivo research unequivocally demonstrate that these classes of compounds comprise potent mutagens that induce many different forms of genetic damage and that particularly bile acids and fecapentaenes may also affect the carcinogenic process by epigenetic mechanisms. Large inter-individual differences in levels of exposures have been reported, including those in a range where considerable genetic damage can be expected based on evidence from animal studies. Particularly, however, exposure profiles of PAH and N-nitroso compounds (NOC) have to be more accurately established to come to a risk evaluation. Moreover, lack of human studies and inconsistency between epidemiological data make it impossible to describe colorectal cancer risk as a result of specific exposures in quantitative terms, or even to indicate the relative importance of the mutagens discussed. Particularly, the polymorphisms of genes involved in the metabolism of heterocyclic amines are important determinants of carcinogenic risk. However, the present knowledge of gene-environment interactions with regard to colorectal cancer risk is rather limited. We expect that the introduction of DNA chip technology in colorectal cancer epidemiology will offer new opportunities to identify combinations of exposures and genetic polymorphisms that relate to increased cancer risk. This knowledge will enable us to improve epidemiological study design and statistical power in future research.     

Testing the FMR1 promoter for mosaicism in DNA methylation among CpG sites, strands, and cells in FMR1-expressing males with fragile X syndrome

Variability among individuals in the severity of fragile X syndrome (FXS) is influenced by epigenetic methylation mosaicism, which may also be common in other complex disorders. The epigenetic signal of dense promoter DNA methylation is usually associated with gene silencing, as was initially reported for FMR1 alleles in individuals with FXS. A paradox arose when significant levels of FMR1 mRNA were reported for some males with FXS who had been reported to have predominately methylated alleles. We have used hairpin-bisufite PCR, validated with molecular batch-stamps and barcodes, to collect and assess double-stranded DNA methylation patterns from these previously studied males. These patterns enable us to distinguish among three possible forms of methylation mosaicism, any one of which could explain FMR1 expression in these males. Our data indicate that cryptic inter-cell mosaicism in DNA methylation can account for the presence of FMR1 mRNA in some individuals with FXS.     

A review and appraisal of the DNA damage theory of ageing

Given the central role of DNA in life, and how ageing can be seen as the gradual and irreversible breakdown of living systems, the idea that damage to the DNA is the crucial cause of ageing remains a powerful one. DNA damage and mutations of different types clearly accumulate with age in mammalian tissues. Human progeroid syndromes resulting in what appears to be accelerated ageing have been linked to defects in DNA repair or processing, suggesting that elevated levels of DNA damage can accelerate physiological decline and the development of age-related diseases not limited to cancer. Higher DNA damage may trigger cellular signalling pathways, such as apoptosis, that result in a faster depletion of stem cells, which in turn contributes to accelerated ageing. Genetic manipulations of DNA repair pathways in mice further strengthen this view and also indicate that disruption of specific pathways, such as nucleotide excision repair and non-homologous end joining, is more strongly associated with premature ageing phenotypes. Delaying ageing in mice by decreasing levels of DNA damage, however, has not been achieved yet, perhaps due to the complexity inherent to DNA repair and DNA damage response pathways. Another open question is whether DNA repair optimization is involved in the evolution of species longevity, and we suggest that the way cells from different organisms respond to DNA damage may be crucial in species differences in ageing. Taken together, the data suggest a major role of DNA damage in the modulation of longevity, possibly through effects on cell dysfunction and loss, although understanding how to modify DNA damage repair and response systems to delay ageing remains a crucial challenge.     

Fecapentaene causes sister-chromatid exchanges in human lymphocytes

Fecapentaenes are potent mutagenic compounds found in human feces that are considered as potential colon carcinogens. It is demonstrated that a synthetic racemic all-trans fecapentaene-12 (fec-12) causes a strong dose-dependent increase in the frequency of sister-chromatid exchanges (SCE) in human lymphocytes exposed at different stages of the cell cycle. The SCE-inducing capacity is consistent with published results on the DNA-damaging activity of fec-12 such as formation of DNA single-strand breaks and interstrand cross-links.     

Circadian clock,carcinogenesis, chronochemotherapy connections

The circadian clock controls the expression of nearly 50% of protein coding genes in mice and most likely in humans as well. Therefore, disruption of the circadian clock is presumed to have serious pathological effects including cancer. However, epidemiological studies on individuals with circadian disruption because of night shift or rotating shift work have produced contradictory data not conducive to scientific consensus as to whether circadian disruption increases the incidence of breast, ovarian, prostate, or colorectal cancers. Similarly, genetically engineered mice with clock disruption do not exhibit spontaneous or radiation-induced cancers at higher incidence than wild-type controls. Because many cellular functions including the cell cycle and cell division are, at least in part, controlled by the molecular clock components (CLOCK, BMAL1, CRYs, PERs), it has also been expected that appropriate timing of chemotherapy may increase the efficacy of chemotherapeutic drugs and ameliorate their side effect. However, empirical attempts at chronochemotherapy have not produced beneficial outcomes. Using mice without and with human tumor xenografts, sites of DNA damage and repair following treatment with the anticancer drug cisplatin have been mapped genome-wide at single nucleotide resolution and as a function of circadian time. The data indicate that mechanism-based studies such as these may provide information necessary for devising rational chronochemotherapy regimens.     

Deletion of the neuron-specific protein delta-catenin leads to severe cognitive and synaptic dysfunction

Delta-catenin (delta-catenin) is a neuron-specific catenin, which has been implicated in adhesion and dendritic branching. Moreover, deletions of delta-catenin correlate with the severity of mental retardation in Cri-du-Chat syndrome (CDCS), which may account for 1% of all mentally retarded individuals. Interestingly, delta-catenin was first identified through its interaction with Presenilin-1 (PS1), the molecule most frequently mutated in familial Alzheimer's Disease (FAD). We investigated whether deletion of delta-catenin would be sufficient to cause cognitive dysfunction by generating mice with a targeted mutation of the delta-catenin gene (delta-cat(-/-)). We observed that delta-cat(-/-) animals are viable and have severe impairments in cognitive function. Furthermore, mutant mice display a range of abnormalities in hippocampal short-term and long-term synaptic plasticity. Also, N-cadherin and PSD-95, two proteins that interact with delta-catenin, are significantly reduced in mutant mice. These deficits are severe but specific because delta-cat(-/-) mice display a variety of normal behaviors, exhibit normal baseline synaptic transmission, and have normal levels of the synaptic adherens proteins E-cadherin and beta-catenin. These data reveal a critical role for delta-catenin in brain function and may have important implications for understanding mental retardation syndromes such as Cri-du-Chat and neurodegenerative disorders, such as Alzheimer's disease, that are characterized by cognitive decline.     

Do mtDNA deletions drive premature aging in mtDNA mutator mice?

Deletions in mitochondrial DNA (mtDNA) have long been suspected to be involved in mammalian aging, but their role remains controversial. Recent research has demonstrated that relatively higher levels of mtDNA deletions correlate with premature aging in mtDNA mutator mice, which led to the conclusion that premature aging in these mice is driven by mtDNA deletions. However, it is reported here that the absolute level of deletions in mutator mice is quite low, especially when compared with the level of point mutations in these mice. It is thus argued that the available data are insufficient to conclude that mtDNA mutations drive premature aging in mtDNA mutator mice. It remains possible that clonal expansion of mtDNA deletions may result in sufficiently high levels to play a role in age-related dysfunction in some cells, but assessing this possibility will require studies of the distribution of these deletions among different cell types and in individual cells.     

Lack of Evidence for mtDNA as a Biomarker of Innate Immune Activation in HIV Infection

Many human immunodeficiency virus (HIV) infected individuals suffer from persistent immune activation. Chronic inflammation and immune dysregulation have been associated with an increased risk of age-related diseases even among patients on highly active antiretroviral therapy. The factors leading to immune activation are complex, but have been hypothesized to include persistent viral replication with cellular death as well as microbial translocation across the gastrointestinal tract. Both processes may trigger innate immune responses since many native molecules released from dying cells are similar in structure to pathogen associated molecular patterns. These damage associated molecular patterns include mitochondrial DNA and formylated peptides. We hypothesized that circulating mitochondrial nucleic acid could serve as a biomarker for HIV-associated cell death and drive innate immune activation in infected individuals. We developed a quantitative polymerase chain reaction assay for plasma mitochondrial DNA and validated it on normal blood donors. We then measured mitochondrial DNA levels in acute and chronic HIV infection. While the assay proved to be accurate with a robust dynamic range, we did not find a significant association between HIV disease status and circulating mitochondrial DNA. We did, however, observe a negative correlation between age and plasma mitochondrial DNA levels in individuals with well-controlled HIV.     

Epithelial proliferation and differentiation in the mammary gland do not correlate with cFABP gene expression during early pregnancy

Cardiac fatty acid binding protein (cFABP) is abundantly expressed in the nondividing, functionally differentiated mammary ephithelium. It is very closely related, if not identical to, a previously described protein termed mammary derived growth inhibitor (MDGI). In vitro studies suggest that low concentrations of diffusible cFABP/MDGI may play a hormone-like role in limiting proliferative activity and promoting functional differentiation of this tissue, but no in vivo data to support this idea have been published. To test this hypothesis, we compared the levels of cFABP mRNA with both the epithelial DNA labelling index and levels of β-casein mRNA in wild-type mice. We also investigated the effect of a precocious experimental increase of cFABP levels in the mammary gland of transgenic mice on the labelling index and β-casein mRNA levels. This was accomplished by expressing a bovine cFABP cDNA under the control of the ovine β-lactoglobulin (BLG) gene promoter. We found that although both the DNA labelling index, β-casein mRNA levels, and cFABP mRNA levels in wild-type mice are developmentally regulated, they do not correlate with each other during early pregnancy in individual mice. Moreover, a three- to fourfold increase of total cFABP mRNA in two transgenic lines did not affect the DNA labelling index or the levels of β-casein mRNA, an established marker of differentiation of the mammary epithelium, at this developmental stage. These data suggest that epithelial DNA synthesis, β-casein gene expression, and expression of the cFABP gene are regulated independently in the proliferatively active mammary gland and that the rapidly dividing mammary epithelial cells are not susceptible to the action of cFABP during early pregnancy. © 1995 Wiley-Liss, Inc.     

Intergenerational genomic DNA methylation patterns in mouse hybrid strains

Background

DNA methylation is a contributing factor to both rare and common human diseases, and plays a major role in development and gene silencing. While the variation of DNA methylation among individuals has been partially characterized, the degree to which methylation patterns are preserved across generations is still poorly understood. To determine the extent of methylation differences between two generations of mice we examined DNA methylation patterns in the livers of eight parental and F1 mice from C57BL/6J and DBA/2J mouse strains using bisulfite sequencing.

Results

We find a large proportion of reproducible methylation differences between C57BL/6J and DBA/2J chromosomes in CpGs, which are highly heritable between parent and F1 mice. We also find sex differences in methylation levels in 396 genes, and 11% of these are differentially expressed between females and males. Using a recently developed approach to identify allelically methylated regions independently of genotypic differences, we identify 112 novel putative imprinted genes and microRNAs, and validate imprinting at the RNA level in 10 of these genes.

Conclusions

The majority of DNA methylation differences among individuals are associated with genetic differences, and a much smaller proportion of these epigenetic differences are due to sex, imprinting or stochastic intergenerational effects. Epigenetic differences can be a determining factor in heritable traits and should be considered in association studies for molecular and clinical traits, as we observed that methylation differences in the mouse model are highly heritable and can have functional consequences on molecular traits such as gene expression.     

Genetic tagging: contemporary molecular ecology

Population genetic analyses have been highly successful in deciphering inter- and intra-specific evolutionary relationships, levels of gene flow, genetic divergence and effective population sizes. Parameters estimated by traditional population genetic analyses are evolutionary averages and thus not necessarily relevant for contemporary ecological or conservation issues. Molecular data can, however, also provide insight into contemporary patterns of divergence, population size and gene flow when a sufficient number of variable loci are analysed to focus subsequent data analyses on individuals rather than populations. Genetic tagging of individuals is an example of such individual-based approaches and recent studies have shown it to be a viable alternative to traditional tagging methods. Owing to the ubiquitous presence of hyper-variable DNA sequences in eukaryote genomes it is in principle possible to tag any eukaryote species and the required DNA can be obtained indirectly from substrates such as faeces, sloughed skin and hair. The purpose of this paper is to present the concept of genetic tagging and to further advocate the extension of individual-based genetic analyses beyond the identification of individuals to other kinds of relationships, such as parent-offspring relations, which more fully exploit the genetic nature of the data.     

Quantitative analysis of human tissue-specific differences in methylation

Tissue-specific differentially methylated regions (tDMRs) have been identified and implicated for their indispensable involvement in mammalian development and tissue differentiation. In this report, a quantitative DNA methylation analysis was performed for 13 human orthologous regions of recently confirmed mouse tDMRs by using Sequenom Mass Array, by which bisulfite-treated fragments are quantitatively detected using time of flight mass spectroscopy analysis. Eight regions were shown as tDMRs in various tissues from three independent individuals. Testis DNA samples from eight individuals were also analyzed for methylation. Interestingly, there is evidence that the DNA methylation level is divergent among individuals. DNA methylation levels of five testis-specific DMRs were significantly inversely correlated with the number of spermatocytes. However, a positive correlation was seen at tDMRs located near the TRIM38 and CASZ1 genes. Our results indicate that tDMRs are conserved between mouse and human and may have an important role in regulating tissue function, differentiation, and aging.     

Formation of 7-methylguanine by dichlorvos in bacteria and mice

The methylating capability of dichlorvos, as assayed by the formation of labelled 7-methylguanine from dichlorvos radioactively labelled in the methyl groups, has been studied in an Escherichia coli test system and in mice. Labelled 7-methylguanine is present in both DNA and RNA isolated from E. coli exposed to dichlorvos. Mice exposed to dichlorvos by inhalation as well as by i.p. injection excrete in the urine labelled 7-methylguanine which most likely originates from the non-enzymatic methylation of guanine moieties. The methylating capability of dichlorvos is less, by a factor of 10 to 100, than that of strongly genotoxic methylating compounds.