Mitochondrial DNA copy number in colorectal cancer: between tissue comparisons, clinicopathological characteristics and survival.

Low mitochondrial DNA (mtDNA) copy number in tumors has been associated with worse prognosis in colorectal cancer (CRC). This study further deciphers the role of mtDNA copy number in CRC by comparing mtDNA copy number between healthy, adenoma and carcinoma tissue, by investigating its association according to several clinicopathological characteristics in CRC, and by relating it to CRC-specific survival in CRC patients. A hospital-based series of samples including cancer, adenoma and adjacent histologically normal tissue from primary CRC patients (n = 56) and recurrent CRC (n = 16) was studied as well as colon mucosa samples from healthy subjects (n = 76). Furthermore, mtDNA copy number was assessed in carcinomas of 693 CRC cases identified from the population-based Netherlands Cohort Study (NLCS). MtDNA copy number was significantly lower in carcinoma tissue (P = 0.011) and adjacent tissue (P < 0.001) compared to earlier resected adenoma tissue and in primary CRC tissue compared to recurrent CRC tissue (P = 0.011). Within both study populations, mtDNA copy number was significantly lower in mutated BRAF (P = 0.027 and P = 0.006) and in microsatellite unstable (MSI) tumors (P = 0.033 and P < 0.001) and higher in KRAS mutated tumors (P = 0.004). Furthermore, the association between mtDNA and survival seemed to follow an inverse U-shape with the highest HR observed in the second quintile of mtDNA copy number (HR = 1.70, 95% CI = 1.18, 2.44) compared to the first quintile. These results might reflect an association of mtDNA copy number with various malignant processes in cancer cells and warrants further research on tumor energy metabolism in CRC prognosis.

Bayesian Network Inference Enables Unbiased Phenotypic Anchoring of Transcriptomic Responses to Cigarette Smoke in Humans.

Microarray-based transcriptomic analysis has been demonstrated to hold the opportunity to study the effects of human exposure to, e.g., chemical carcinogens at the whole genome level, thus yielding broad-ranging molecular information on possible carcinogenic effects. Since genes do not operate individually but rather through concerted interactions, analyzing and visualizing networks of genes should provide important mechanistic information, especially upon connecting them to functional parameters, such as those derived from measurements of biomarkers for exposure and carcinogenic risk. Conventional methods such as hierarchical clustering and correlation analyses are frequently used to address these complex interactions but are limited as they do not provide directional causal dependence relationships. Therefore, our aim was to apply Bayesian network inference with the purpose of phenotypic anchoring of modified gene expressions. We investigated a use case on transcriptomic responses to cigarette smoking in humans, in association with plasma cotinine levels as biomarkers of exposure and aromatic DNA-adducts in blood cells as biomarkers of carcinogenic risk. Many of the genes that appear in the Bayesian networks surrounding plasma cotinine, and to a lesser extent around aromatic DNA-adducts, hold biologically relevant functions in inducing severe adverse effects of smoking. In conclusion, this study shows that Bayesian network inference enables unbiased phenotypic anchoring of transcriptomics responses. Furthermore, in all inferred Bayesian networks several dependencies are found which point to known but also to new relationships between the expression of specific genes, cigarette smoke exposure, DNA damaging-effects, and smoking-related diseases, in particular associated with apoptosis, DNA repair, and tumor suppression, as well as with autoimmunity.

Dietary methyl donors, methyl metabolizing enzymes, and epigenetic regulators: diet-gene interactions and promoter CpG island hypermethylation in colorectal cancer.

Dietary methyl donors might influence DNA methylation during carcinogenesis of colorectal cancer (CRC). Among 609 CRC cases and 1,663 subcohort members of the Netherlands Cohort Study on diet and cancer (n = 120,852), we estimated CRC risk according to methyl donor intake across genotypes of folate metabolizing enzymes and methyltransferases.Although diet-gene interactions were not statistically significant, methionine intake was inversely associated with CRC among subjects having both common rs2424913 and rs406193 DNMT3B C > T genotypes (highest versus lowest tertile: RR = 0.44; p (trend) = 0.05). Likewise, vitamin B2 was modestly inversely associated among individuals with the MTHFR c.665CC (rs1801133) genotype (RR = 0.66; p (trend) = 0.08), but with a significant reduced risk when ≤ 1 rare allele occurred in the combination of folate metabolizing enzymes MTHFR, MTRR and MTR (RR = 0.30; p (trend) = 0.005). Folate or vitamin B6 were neither inversely associated with CRC nor was methyl donor intake associated with the CpG island methylator phenotype (CIMP).Despite the absence of heterogeneity across genotypes, might an effect of methyl donors on CRC be more pronounced among individuals carrying common variants of folate metabolizing enzymes or DNA methyltransferases. Combining genotypes may assist to reveal diet associations with CRC, possibly because rare variants of related genes may collectively affect specific metabolic pathways or enzymatic functions.

Prevalence of at-risk genotypes for genotoxic effects decreases with age in a randomly selected population in Flanders: a cross sectional study.

BACKGROUND: We hypothesized that in Flanders (Belgium), the prevalence of at-risk genotypes for genotoxic effects decreases with age due to morbidity and mortality resulting from chronic diseases. Rather than polymorphisms in single genes, the interaction of multiple genetic polymorphisms in low penetrance genes involved in genotoxic effects might be of relevance. METHODS: Genotyping was performed on 399 randomly selected adults (aged 50-65) and on 442 randomly selected adolescents. Based on their involvement in processes relevant to genotoxicity, 28 low penetrance polymorphisms affecting the phenotype in 19 genes were selected (xenobiotic metabolism, oxidative stress defense and DNA repair, respectively 13, 6 and 9 polymorphisms). Polymorphisms which, based on available literature, could not clearly be categorized a priori as leading to an 'increased risk' or a 'protective effect' were excluded. RESULTS: The mean number of risk alleles for all investigated polymorphisms was found to be lower in the 'elderly' (17.0 ± 2.9) than the 'adolescent' (17.6 ± 3.1) subpopulation (P = 0.002). These results were not affected by gender nor smoking. The prevalence of a high (> 17 = median) number of risk alleles was less frequent in the 'elderly' (40.6%) than the 'adolescent' (51.4%) subpopulation (P = 0.002). In particular for phase II enzymes, the mean number of risk alleles was lower in the 'elderly' (4.3 ± 1.6 ) than the 'adolescent' age group (4.8 ± 1.9) P < 0.001 and the prevalence of a high (> 4 = median) number of risk alleles was less frequent in the 'elderly' (41.3%) than the adolescent subpopulation (56.3%, P < 0.001). The prevalence of a high (> 8 = median) number of risk alleles for DNA repair enzyme-coding genes was lower in the 'elderly' (37,3%) than the 'adolescent' subpopulation (45.6%, P = 0.017). CONCLUSIONS: These observations are consistent with the hypothesis that, in Flanders, the prevalence of at-risk alleles in genes involved in genotoxic effects decreases with age, suggesting that persons carrying a higher number of at risk alleles (especially in phase II xenobiotic-metabolizing or DNA repair genes) are at a higher risk of morbidity and mortality from chronic diseases. Our findings also suggest that, regarding risk of disease associated with low penetrance polymorphisms, multiple polymorphisms should be taken into account, rather than single ones.

Potential role of cytochrome P450-1B1 in the metabolic activation of 4-aminobiphenyl in humans.

Metabolites of the human carcinogen 4-aminobiphenyl (4-ABP) form hemoglobin (Hb) adducts, which represent a useful biomarker for exposure. However, not every individual responds to a similar degree to 4-ABP exposure, and variations in 4-ABP-Hb adduct formation might be explained by genetic polymorphisms in genes coding for enzymes involved in 4-ABP metabolism. 4-ABP-Hb adducts were measured in blood samples from 57 smoking and 10 non-smoking volunteers. An association was found between cigarette smoking and 4-ABP-Hb adduct levels in smokers (R(2) = 0.5, P < 0.001). Subsequently, subjects were genotyped for 12 polymorphisms in seven genes involved in biotransformation reactions. From this selection of polymorphisms, a significant impact was found for the CYP1B1 Leu(432)Val polymorphism (P = 0.021), which has been reported to lead to a decrease in enzyme activity. Indeed higher levels of 4-ABP-Hb adducts were observed in homo- and heterozygous carriers of the CYP1B1 (432)Leu as compared to the double CYP1B1 (432)Val genotype. A significant interaction between these CYP1B1 genotypes and the level of exposure was found (P = 0.003). Noteworthy, a saturation effect was observed for 4-ABP-Hb adduct formation at high smoking doses limited to carriers of the CYP1B1 (432)Leu allele. No effect of polymorphisms in other genes were found. This is the first study in humans suggesting a crucial role of the CYP1B1 enzyme in 4-ABP metabolism, indicating a protective effect of the CYP1B1 Leu(432)Val polymorphism against the formation of 4-ABP-Hb adduct levels, depending on the smoking dose.

Genomic analysis suggests higher susceptibility of children to air pollution.

Differences in biological responses to exposure to hazardous airborne substances between children and adults have been reported, suggesting children to be more susceptible. Aim of this study was to improve our understanding of differences in susceptibility in cancer risk associated with air pollution by comparing genome-wide gene expression profiles in peripheral blood of children and their parents. Gene expression analysis was performed in blood from children and parents living in two different regions in the Czech Republic with different levels of air pollution. Data were analyzed by two different approaches: one method first selected significantly differentially expressed genes and analyzed these gene lists for overrepresented biological processes, whereas the other applied the T-profiler tool to directly perform pathway analyses on the total gene set without preselection of significantly modulated gene expressions. In addition, gene expressions in both children and adults were investigated for associations with micronuclei frequencies. Both analysis approaches returned considerably more genes or gene groups and pathways that significantly differed between children from both regions than between parents. Very little overlap was observed between children and adults. The two most important biological processes or molecular functions significantly modulated in children, but not in adults, are nucleosome and immune response related. Our study suggests differences between children and adults in relation to air pollution exposure at the transcriptome level. The findings underline the necessity of implementing environmental health policy measures specifically for protecting children's health.

Multiplex genotyping as a biomarker for susceptibility to carcinogenic exposure in the FLEHS biomonitoring study.

Cancer has been suggested to result from interactions between genetic and environmental factors, and certain subgroups in the general population may be at increased risk because of their relatively higher susceptibility to environmental carcinogens. The current study, part of a large biomonitoring study conducted in Flanders from 2002 to 2006 (The Flanders Environment and Health Survey), aims to determine these susceptible subpopulations based on multiple genotypic differences between individuals. A random selection of 429 adolescents and 361 adults was genotyped for 36 polymorphisms in 23 genes selected because of their known role in carcinogen metabolism, DNA repair, and oxidative stress. In both age groups, relationships between endogenous exposure to organochloride substances (polychlorinated biphenyl, hexachlorobenzene, dichlorodiphenyl dichloroethane), metals (cadmium, lead), and urinary metabolites (1-hydroxypyrene, trans-trans muconic acid) versus genotoxic effects (Comet assay and micronuclei in lymphocytes, and urinary 8-hydroxydeoxyguanosine) were investigated. In addition, in the study among adults, the relationship of these exposures with several tumor markers (prostate-specific antigen, carcinoembryonic antigen, and p53) was tested. The impact of the genotype on established exposure-effect relationships was evaluated. Eight exposure-effect relationships were found, including three novel associations, with an impact of various genotypes, predominantly affecting biotransformation and oxidative stress response. This study shows that at least part of the interindividual differences in relationships between carcinogen exposure and genotoxic effect can be explained by genotypic differences, enabling the identification of more susceptible subgroups for environmental cancer risks. This may be of relevance for environmental health policy setting.

Transcriptome analysis in peripheral blood of humans exposed to environmental carcinogens: a promising new biomarker in environmental health studies.

BACKGROUND: Human carcinogenesis is known to be initiated and/or promoted by exposure to chemicals that occur in the environment. Molecular cancer epidemiology is used to identify human environmental cancer risks by applying a range of effect biomarkers, which tend to be nonspecific and do not generate insights into underlying modes of action. Toxicogenomic technologies may improve on this by providing the opportunity to identify molecular biomarkers consisting of altered gene expression profiles. OBJECTIVES: The aim of the present study was to monitor the expression of selected genes in a random sample of adults in Flanders selected from specific regions with (presumably) different environmental burdens. Furthermore, associations of gene expression with blood and urinary measures of biomarkers of exposure, early phenotypic effects, and tumor markers were investigated. RESULTS: Individual gene expression of cytochrome p450 1B1, activating transcription factor 4, mitogen-activated protein kinase 14, superoxide dismutase 2 (Mn), chemokine (C-X-C motif) lig-and 1 (melanoma growth stimulating activity, alpha), diacylglycerol O-acyltransferase homolog 2 (mouse), tigger transposable element derived 3, and PTEN-induced putative kinase1 were measured by means of quantitative polymerase chain reaction in peripheral blood cells of 398 individuals. After correction for the confounding effect of tobacco smoking, inhabitants of the Olen region showed the highest differences in gene expression levels compared with inhabitants from the Gent and fruit cultivation regions. Importantly, we observed multiple significant correlations of particular gene expressions with blood and urinary measures of various environmental carcinogens. CONCLUSIONS: Considering the observed significant differences between gene expression levels in inhabitants of various regions in Flanders and the associations of gene expression with blood or urinary measures of environmental carcinogens, we conclude that gene expression profiling appears promising as a tool for biological monitoring in relation to environmental exposures in humans.

Interactions between polycyclic aromatic hydrocarbons in binary mixtures: effects on gene expression and DNA adduct formation in precision-cut rat liver slices.

Although exposure to polycyclic aromatic hydrocarbons (PAHs) occurs mostly through mixtures, hazard and risk assessment are mostly based on the effects caused by individual compounds. The objective of the current study was to investigate whether interactions between PAHs occur, focusing on gene expression (as measured by cDNA microarrays) and DNA adduct formation. The effects of benzo[a]pyrene or dibenzo[a,h]anthracene (DB[a,h]A) alone and in binary mixtures with another PAH (DB[a,h]A, benzo[b]fluoranthene, fluoranthene or dibenzo[a,l]pyrene) were investigated using precision-cut rat liver slices. All compounds significantly modulated the expression of several genes, but overlap between genes affected by the mixture and by the individual compounds was relatively small. All mixtures showed an antagonistic response on total gene expression profiles. Moreover, at the level of individual genes, mostly antagonism was evident, with additivity and synergism observed for only a few genes. As far as DNA adduct formation is concerned, the binary mixtures generally caused antagonism. The effects in liver slices suggest a lower carcinogenic potency of PAH mixtures than estimated based on additivity of individual compounds.

Whole Exome Sequencing Is the Preferred Strategy to Identify the Genetic Defect in Patients With a Probable or Possible Mitochondrial Cause.

Mitochondrial disorders, characterized by clinical symptoms and/or OXPHOS deficiencies, are caused by pathogenic variants in mitochondrial genes. However, pathogenic variants in some of these genes can lead to clinical manifestations which overlap with other neuromuscular diseases, which can be caused by pathogenic variants in non-mitochondrial genes as well. Mitochondrial pathogenic variants can be found in the mitochondrial DNA (mtDNA) or in any of the 1,500 nuclear genes with a mitochondrial function. We have performed a two-step next-generation sequencing approach in a cohort of 117 patients, mostly children, in whom a mitochondrial disease-cause could likely or possibly explain the phenotype. A total of 86 patients had a mitochondrial disorder, according to established clinical and biochemical criteria. The other 31 patients had neuromuscular symptoms, where in a minority a mitochondrial genetic cause is present, but a non-mitochondrial genetic cause is more likely. All patients were screened for pathogenic variants in the mtDNA and, if excluded, analyzed by whole exome sequencing (WES). Variants were filtered for being pathogenic and compatible with an autosomal or X-linked recessive mode of inheritance in families with multiple affected siblings and/or consanguineous parents. Non-consanguineous families with a single patient were additionally screened for autosomal and X-linked dominant mutations in a predefined gene-set. We identified causative pathogenic variants in the mtDNA in 20% of the patient-cohort, and in nuclear genes in 49%, implying an overall yield of 68%. We identified pathogenic variants in mitochondrial and non-mitochondrial genes in both groups with, obviously, a higher number of mitochondrial genes affected in mitochondrial disease patients. Furthermore, we show that 31% of the disease-causing genes in the mitochondrial patient group were not included in the MitoCarta database, and therefore would have been missed with MitoCarta based gene-panels. We conclude that WES is preferable to panel-based approaches for both groups of patients, as the mitochondrial gene-list is not complete and mitochondrial symptoms can be secondary. Also, clinically and genetically heterogeneous disorders would require sequential use of multiple different gene panels. We conclude that WES is a comprehensive and unbiased approach to establish a genetic diagnosis in these patients, able to resolve multi-genic disease-causes.

Binary PAH mixtures cause additive or antagonistic effects on gene expression but synergistic effects on DNA adduct formation.

Polycyclic aromatic hydrocarbons (PAHs) cover a wide range of structurally related compounds which differ greatly in their carcinogenic potency. PAH exposure usually occurs through mixtures rather than individual compounds. Therefore, we assessed whether the effects of binary PAH mixtures on gene expression, DNA adduct formation, apoptosis and cell cycle are additive compared with the effects of the individual compounds in human hepatoma cells (HepG2). Equimolar and equitoxic mixtures of benzo[a]pyrene (B[a]P) with either dibenzo[a,l]pyrene (DB[a,l]P), dibenzo[a,h]anthracene (DB[a,h]A), benzo[b]fluoranthene (B[b]F), fluoranthene (FA) or 1-methylphenanthrene (1-MPA) were studied. DB[a,l]P, B[a]P, DB[a,h]A and B[b]F dose-dependently increased apoptosis and blocked cells cycle in S-phase. PAH mixtures showed an additive effect on apoptosis and on cell cycle blockage. DNA adduct formation in mixtures was higher than expected based on the individual compounds, indicating a synergistic effect of PAH mixtures. Equimolar mixtures of B[a]P and DB[a,l]P (0.1, 0.3 and 1.0 microM) were assessed for their effects on gene expression. Only at 1.0 microM, the mixture showed antagonism. All five compounds were also tested as a binary mixture with B[a]P in equitoxic concentrations. The combinations of B[a]P with B[b]F, DB[a,h]A or FA showed additivity, whereas B[a]P with DB[a,l]P or 1-MPA showed antagonism. Many individual genes showed additivity in mixtures, but some genes showed mostly antagonism or synergism. Our results show that the effects of binary mixtures of PAHs on gene expression are generally additive or slightly antagonistic, suggesting no effect or decreased carcinogenic potency, whereas the effects on DNA adduct formation show synergism, which rather indicates increased carcinogenic potency.

Flavone as PARP-1 inhibitor: its effect on lipopolysaccharide induced gene-expression.

The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) which was initially known for its role in the repair of oxidative stress-induced DNA damage, has also been reported to play a mediating role in the inflammatory response. Studies with PARP-1 knockout models have shown that PARP-1 is a co-activator of Nuclear Factor-kappa B (NF-kappaB), although this appears not to require its enzyme activity. In addition, drug-induced inhibition of the enzyme activity of PARP-1 was observed to reduce the production of pro-inflammatory mediators. In this study, the flavonoid compound flavone was demonstrated to significantly inhibit the enzyme activity of PARP-1. Further evaluation of flavone in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated human pulmonary epithelial and vascular endothelial cells revealed that both the decrease in NAD(+) levels, as well as the formation of PAR-polymers was dose-dependently attenuated by flavone. In addition, flavone was found to reduce the lipopolysaccharide (LPS)-induced interleukin (IL)-8 production in pulmonary epithelial cells, which was confirmed by transcription analysis. Furthermore, the transcription Inhibitor kappa B alpha (of IkappaBalpha) was significantly increased by flavone. The results of the present study indicate that the flavonoid flavone could be a potential candidate for application in treatment of chronic inflammatory diseases. PARP-1 inhibition could have beneficial effects in such diseases as Chronic Obstructive Pulmonary Disease (COPD) and diabetes, by preservation of cellular NAD(+) levels and attenuating inflammatory conditions.

The role of glutathione in the regulation of nucleotide excision repair during oxidative stress.

Nucleotide excision repair (NER) mainly repairs bulky DNA adducts and helix distorting lesions, but is additionally considered to be a back-up system for base excision repair to remove oxidative stress induced DNA damage. Therefore, it can be speculated that NER is up-regulated or primed by oxidative stress. Exposure of human pulmonary epithelial cells (A549) to non-toxic doses of 100muM H(2)O(2) indeed showed a 2 to 4.5-fold increase in expression of XPA, XPC, ERCC4, and ERCC5, whereas the expression of ERCC1 was 5-fold decreased. Phenotypical assessment of NER capacity (i.e. recognition and incision of benzo[a]pyrene-DNA adducts) showed a significant decrease to less than 50% after H(2)O(2) exposure, which paralleled the effects of H(2)O(2) on ERCC1 expression. To study the possible involvement of glutathione (GSH) in the regulation of NER, cells were pre-incubated with 0.5mM BSO, resulting in total GSH depletion and increased intracellular oxidative stress. In GSH-depleted cells, the down-regulation of ERCC1 expression by H(2)O(2) was completely abolished and the up-regulation of ERCC4 expression was potentiated from 2.5-fold to >10-fold. Similarly, the H(2)O(2)-induced decrease in NER capacity was absent in GSH-depleted cells. Overall, our data suggest that NER capacity as well as the expression of NER related genes can be modulated by oxidative stress. ERCC1 expression and NER capacity correlated strongly (R(2)=0.85, P<0.01) after oxidant exposure, indicating ERCC1 as a specific target for oxidative stress induced modification of NER.

The environmental carcinogen benzo[a]pyrene induces expression of monocyte-chemoattractant protein-1 in vascular tissue: a possible role in atherogenesis.

Exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs) has been implicated in the aetiology of atherosclerosis. Previously we showed that chronic exposure of ApoE-/- mice to the prototype PAH benzo[a]pyrene (B[a]P) causes enhanced progression of atherosclerosis, which was characterised by an increased inflammatory cell content in the atherosclerotic plaques. The aim of the present study was to evaluate the effect of B[a]P on vascular expression of monocyte-chemoattractant protein 1 (MCP-1), which is a crucial molecule promoting the recruitment of monocytes into atherosclerotic lesions. We hypothesised that B[a]P-induced expression of MCP-1 is mediated through aryl hydrocarbon receptor (AhR) activation. Initially we performed in vivo studies showing that acute treatment with B[a]P induces MCP-1 gene expression in aortic tissue of ApoE-/- mice. These observations could be confirmed by in vitro studies with human endothelial cells (RF24 cell line and primary HUVEC), showing a dose- and time-dependent increase in MCP-1 expression upon exposure to B[a]P. This was paralleled by an induction of cytochrome P450 1A1 and 1B1, indicating Ah receptor activation. No increased gene expression (MCP-1, CYP1A1 and 1B1) was found upon incubation with the structural isomer benzo[e]pyrene, which is a weak AhR agonist. Moreover, B[a]P-induced MCP-1 gene and protein expression was inhibited by co-treatment with the AhR antagonist alpha-naphthoflavone. In addition to its effect on basal gene expression, we showed that B[a]P significantly enhanced TNFalpha-induced expression of MCP-1. We were unable to block B[a]P-induced MCP-1 expression by antioxidant treatment. In contrast, we found that addition of N-acetylcysteine or vitamin C enhanced transcription of MCP-1 by B[a]P. In conclusion, our studies revealed potent vascular pro-inflammatory effects of B[a]P, as evidenced by AhR-mediated induction of MCP-1. These observations further contribute to the concept that induction of inflammation is a crucial process in PAH-enhanced atherogenesis.

Novel SLC25A32 mutation in a patient with a severe neuromuscular phenotype.

In a 51-year-old patient of consanguineous parents with a severe neuromuscular phenotype of early-onset ataxia, myoclonia, dysarthria, muscle weakness and exercise intolerance, exome sequencing revealed a novel homozygous variant (c.-264_31delinsCTCACAAATGCTCA) in the mitochondrial FAD-transporter gene SLC25A32. Flavin adenine dinucleotide (FAD) is an essential co-factor for many mitochondrial enzymes and impaired mitochondrial FAD-transport was supported by a reduced oxidative phosphorylation complex II activity in the patient's muscle, decreased ATP production in fibroblasts, and a deficiency of mitochondrial FAD-dependent enzymes. Clinically, the patient showed improvement upon riboflavin treatment, which is a precursor of FAD. Our results confirm the recently reported case of SLC25A32 as a cause of riboflavin-responsive disease. Our patient showed a more severe clinical phenotype compared with the reported patient, corresponding with the (most likely) complete absence of the SLC25A32-encoding MFT (Mitochondrial Folate Transporter) protein.

Interindividual variations in DNA adduct levels assessed by analysis of multiple genetic polymorphisms in smokers.

Genetic polymorphisms in genes involved in processes that affect DNA damage may explain part of the large interindividual variation in DNA adduct levels in smokers. We investigated the effect of 19 polymorphisms in 12 genes involved in carcinogen metabolism, DNA repair, and oxidant metabolism on DNA adduct levels (determined by (32)P post-labeling) in lymphocytes of 63 healthy Caucasian smokers. The total number of alleles that were categorized as putatively high-risk alleles seemed associated with bulky DNA adduct levels (P = 0.001). Subsequently, to investigate which polymorphisms may have the highest contribution to DNA adduct levels in these smokers, discriminant analysis was done. In the investigated set of polymorphisms, GSTM1*0 (P < 0.001), mEH*2 (P = 0.001), and GPX1*1 (P < 0.001) in combination with the level of exposure (P < 0.001) were found to be key effectors. DNA adduct levels in subjects with a relatively high number of risk alleles of these three genes were >2-fold higher than in individuals not having these risk alleles. Noteworthy, all three genes are involved in deactivation of reactive carcinogenic metabolites. This study shows that analysis of multiple genetic polymorphisms may predict the interindividual variation in DNA adduct levels upon exposure to cigarette smoke. It is concluded that discriminant analysis presents an important statistical tool for analyzing the effect of multiple genotypes on molecular biomarkers.

Polyhydramnios and cerebellar atrophy: a prenatal presentation of mitochondrial encephalomyopathy caused by mutations in the FBXL4 gene.

Severe recessive mitochondrial myopathy caused by FBXL4 gene mutations may present prenatally with polyhydramnios and cerebellar hypoplasia. Characteristic dysmorphic features are: high and arched eyebrows, triangular face, a slight upslant of palpebral fissures, and a prominent pointed chin. Metabolic investigations invariably show increased serum lactate and pyruvate levels.

Differential gene expression in human peripheral blood mononuclear cells induced by cigarette smoke and its constituents.

In current molecular epidemiology studies, a wide range of methods are used to monitor early biological effects after exposure to xenobiotic agents. Gene expression profiling is considered a promising tool that may provide more sensitive, mechanism-based biomarkers. As a first step toward obtaining information on the applicability of gene expression profiles as a biomarker for early biological effects of carcinogen exposure, we conducted in vitro studies on human peripheral blood mononuclear cells (PBMC). We used cigarette smoke condensate (CSC) and a selection of its genotoxic constituents as model agents, applying cDNA microarray technology to investigate modulated gene expression. In independent experiments using cells from several donors, quiescent PBMC were exposed for 18 h, followed by gene expression analyses on a microarray containing 600 toxicologically relevant genes. The search for candidate biomarker genes was binomial: first we looked for genes responding similarly to all agents; second, for agent-specific genes. Many genes were significantly deregulated by all compounds, but as the direction of deregulation frequently differed per agent, they are not useful as generic biomarkers. Cigarette smoke condensate modulated the expression of many more genes than any of its constituents, with the largest effect in SERPINB2. The affected genes are involved in immune or stress responses, but surprisingly no genes involved in DNA damage response were modulated, and only a few in DNA repair. In conclusion, several genes have been identified as potential biomarkers for population studies on early biological effects caused by cigarette smoke exposure, but no genes were identified that represent a generic biomarker.

Alcohol consumption, alcohol dehydrogenase 1C (ADH1C) genotype, and risk of colorectal cancer in the Netherlands Cohort Study on diet and cancer.

Within the Netherlands Cohort Study (1986), we examined associations between alcohol consumption, the alcohol dehydrogenase 1C (ADH1C) genotype, and risk of colorectal cancer (CRC). After a follow-up period of 7.3 years, 594 CRC cases with information on genotype and baseline alcohol intake were available for analyses. Adjusted incidence rate ratios (RRs) and 95% confidence intervals (CIs) were estimated using Cox proportional hazards models. In subjects who reported to have consumed equal amounts of total alcohol both 5 years before baseline and at baseline, drinkers of ≥30g of alcohol per day with the ADH1C*2/*2 genotype were associated-although not statistically significant-with an increased risk of CRC relative to abstainers with the ADH1C*1/*1 genotype (RR: 1.91, 95% CI: 0.68, 5.34). The risk estimate in this exposure group increased slightly when compared with light drinkers of ≥0.5-<5g/day with the ADH1C*1/*1 genotype (RR: 2.32, 95% CI: 0.80, 6.72). The interaction term however, was not statistically significant (P>.05). In subjects who reported to have consumed equal amounts of total alcohol both 5 years before baseline and at baseline, drinkers of ≥30g of alcohol per day were associated-although not statistically significant-with an increased risk of CRC relative to abstainers (RR: 1.38, 95% CI: 0.80, 2.38). This risk estimate for high-level drinkers became stronger when compared with light drinkers (RR: 1.74, 95% CI: 1.01, 2.99). As main effect of genotype, we observed that the ADH1C*2/*2 genotype was associated with a 42% increase in risk of CRC when compared with the ADH1C*1/*1 genotype. In conclusion, both genotype and alcohol consumption were associated with an increased risk of CRC. Owing to limited statistical power, we found no apparent evidence for the ADH1C genotype as effect modifier of the relationship between alcohol intake and CRC. Nevertheless, the interaction deserves further investigation in larger genetic epidemiologic studies.

Global gene expression analysis reveals differences in cellular responses to hydroxyl- and superoxide anion radical-induced oxidative stress in caco-2 cells.

Reactive oxygen species-induced oxidative stress in the colon is involved in inflammatory bowel diseases and suggested to be associated with colorectal cancer risk. However, our insight in molecular responses to different oxygen radicals is still fragmentary. Therefore, we studied global gene expression by an extensive time series (0.08, 0.25, 0.5, 1, 2, 4, 8, 16, or 24 h) analyses in human colon cancer (caco-2) cells after exposure to H(2)O(2) or the superoxide anion donor menadione. Differences in gene expression were investigated by hybridization on two-color microarrays against nonexposed time-matched control cells. Next to gene expression, correlations with related phenotypic markers (8-oxodG levels and cell cycle arrest) were investigated. Gene expression analysis resulted in 1404 differentially expressed genes upon H(2)O(2) challenge and 979 genes after menadione treatment. Further analysis of gene expression data revealed how these oxidant responses can be discriminated. Time-dependent coregulated genes immediately showed a pulse-like response to H(2)O(2), while the menadione-induced expression is not restored over 24 h. Pathway analyses demonstrated that H(2)O(2) immediately influences pathways involved in the immune function, while menadione constantly regulated cell cycle-related pathways Altogether, this study offers a novel and detailed insight in the similarities and differences of the time-dependent oxidative stress responses induced by the oxidants H(2)O(2) and menadione and show that these can be discriminated regarding their modulation of particular colon carcinogenesis-related mechanisms.