Jimmy W. Crott

Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells

Micronuclei (MN) and other nuclear anomalies such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) are biomarkers of genotoxic events and chromosomal instability. These genome damage events can be measured simultaneously in the cytokinesis-block micronucleus cytome (CBMNcyt) assay. The molecular mechanisms leading to these events have been investigated over the past two decades using molecular probes and genetically engineered cells. In this brief review, we summarise the wealth of knowledge currently available that best explains the formation of these important nuclear anomalies that are commonly seen in cancer and are indicative of genome damage events that could increase the risk of developmental and degenerative diseases. MN can originate during anaphase from lagging acentric chromosome or chromatid fragments caused by misrepair of DNA breaks or unrepaired DNA breaks. Malsegregation of whole chromosomes at anaphase may also lead to MN formation as a result of hypomethylation of repeat sequences in centromeric and pericentromeric DNA, defects in kinetochore proteins or assembly, dysfunctional spindle and defective anaphase checkpoint genes. NPB originate from dicentric chromosomes, which may occur due to misrepair of DNA breaks, telomere end fusions, and could also be observed when defective separation of sister chromatids at anaphase occurs due to failure of decatenation. NBUD represent the process of elimination of amplified DNA, DNA repair complexes and possibly excess chromosomes from aneuploid cells.

Diet-induced obesity elevates colonic TNF-α in mice and is accompanied by an activation of Wnt signaling: a mechanism for obesity-associated colorectal cancer

Inflammation associated with obesity may play a role in colorectal carcinogenesis, but the underlying mechanism remains unclear. This study investigated whether the Wnt pathway, an intracellular signaling cascade that plays a critical role in colorectal carcinogenesis, is activated by obesity-induced elevation of the inflammatory cytokine tumor necrosis factor-alpha (TNF-α). Animal studies were conducted on C57BL/6 mice, and obesity was induced by utilizing a high-fat diet (60% kcal). An inflammation-specific microarray was performed, and results were confirmed with real-time polymerase chain reaction. The array revealed that diet-induced obesity increased the expression of TNF-α in the colon by 72% (P=.004) and that of interleukin-18 by 41% (P=.023). The concentration of colonic TNF-α protein, determined by ex vivo culture assay, was nearly doubled in the obese animals (P=.002). The phosphorylation of glycogen synthase kinase 3 beta (GSK3β), an important intermediary inhibitor of Wnt signaling and a potential target of TNF-α, was quantitated by immunohistochemistry. The inactivated (phosphorylated) form of GSK3β was elevated in the colonic mucosa of obese mice (P<.02). Moreover, β-catenin, the key effector of canonical Wnt signaling, was elevated in the colons of obese mice (P<.05), as was the expression of a downstream target gene, c-myc (P<.05). These data demonstrate that diet-induced obesity produces an elevation in colonic TNF-α and instigates a number of alterations of key components within the Wnt signaling pathway that are protransformational in nature. Thus, these observations offer evidence for a biologically plausible avenue, the Wnt pathway, by which obesity increases the risk of colorectal cancer.

Status of Vitamins B-12 and B-6 but Not of Folate, Homocysteine, and the Methylenetetrahydrofolate Reductase C677T Polymorphism Are Associated with Impaired Cognition and Depression in Adults

The C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene differs in frequency in various ethnic groups that have differing prevalence of age-related cognitive impairments. We used a series of neuro-psychological tests to examine the association of the MTHFR C677T polymorphism with cognition and depression and also to assess whether genotype modifies the association of folate and homocysteine with these outcomes. This study analyzed pooled cross-sectional data from 2 ethnically diverse cohorts of community-living adults: the Boston Puerto Rican Health Study (n = 939) and the Nutrition, Aging, and Memory in Elders study (n = 1017). Individuals in both cohorts underwent anthropometric and laboratory measurements and dietary and health assessments using validated questionnaires between the years 2003 and 2007. Cognitive outcomes included measures of global cognition [Mini-Mental Status Exam (MMSE)], depression (Center for Epidemiological Studies Depression Scale), and 3 factor scores for the domains of attention, executive function, and memory that were derived from a detailed set of neuropsychological tests. Low plasma vitamin B-12 concentrations were associated with poorer MMSE scores and higher depression scores, and low vitamin B-6 concentrations were associated with lower MMSE and worse attention and executive function in the multivariate analysis. In contrast, MTHFR genotype, folate, and homocysteine were not associated with cognition or depression in either ethnicity-pooled or stratified analysis. The current study did not find evidence of an association between the MTHFR C677T TT genotype and impaired cognition or depression in a population with adequate folate status and a high prevalence of cognitive impairment and depression.

A Comparison of Carotenoids, Retinoids, and Tocopherols in the Serum and Buccal Mucosa of Chronic Cigarette Smokers versus Nonsmokers

Background: Cigarette smoking, a major risk factor for oropharyngeal cancer, is reported to alter oral levels of carotenoids and tocopherols. Such effects may be important because these nutrients, as well as retinoids, are putative chemoprotective agents. Objectives: To determine whether chronic smoking is associated with altered concentrations of these nutrients in serum and buccal mucosa; to distinguish whether such effects are ascribable to diet; and to determine whether oral concentrations of these nutrients correlate with a putative biomarker of oral cancer risk. Methods: Serum and buccal mucosal cells (BMC) were analyzed for these nutrients and for BMC micronuclei in smokers (n = 35) and nonsmokers (n = 21). Results: General linear regression with adjustments for dietary intake showed that smokers possess lower serum concentrations of β- and α-carotene, cryptoxanthin, lutein, and zeaxanthin (P ≤ 0.01) and a significantly higher serum γ-tocopherol (P = 0.03). In BMCs, smokers had significantly lower concentrations of β- and α-carotene, lycopene, and α-tocopherol (P < 0.05) but significantly higher γ-tocopherol (P < 0.01). Among nonsmokers, many serum carotenoid concentrations correlated with concentrations of the corresponding nutrient in BMCs whereas no such correlations existed among smokers. BMC micronuclei did not correlate with the oral concentration of any micronutrient. Conclusions: Chronic cigarette smokers have lower concentrations of many dietary antioxidants in serum and BMCs compared with nonsmokers, an effect which is not entirely ascribable to diet. Nevertheless, the lack of concordance between oral concentrations of these nutrients and genetic damage in the BMCs of smokers does not support a protective role for these nutrients in oral carcinogenesis. (Cancer Epidemiol Biomarkers Prev 2006:15(5):993–9)

Maternal one-carbon nutrient intake and cancer risk in offspring

Dietary intake of one-carbon nutrients, particularly folate, vitamin B(2) (riboflavin), vitamin B(6) , vitamin B(12) , and choline have been linked to the risk of cancers of the colon and breast in both human and animal studies. More recently, experimental and epidemiological data have emerged to suggest that maternal intake of these nutrients during gestation may also have an impact on the risk of cancer in offspring later in life. Given the plasticity of DNA methylation in the developing embryo and the established role of one-carbon metabolism in supporting biological methylation reactions, it is plausible that alterations in maternal one-carbon nutrient availability might induce subtle epigenetic changes in the developing embryo and fetus that persist into later life, altering the risk of tumorigenesis throughout the lifespan. This review summarizes the current literature on maternal one-carbon nutrient intake and offspring cancer risk, with an emphasis on cancers of the colon and breast, and discusses specific epigenetic modifications that may play a role in their pathogenesis.

Methylenetetrahydrofolate Reductase Variants Associated with Hypertension and Cardiovascular Disease Interact with Dietary Polyunsaturated Fatty Acids to Modulate Plasma Homocysteine in Puerto Rican Adults

Although methylenetetrahydrofolate reductase (MTHFR) genetic variants are associated with plasma homocysteine (Hcy) and cardiovascular disease (CVD), little is known whether dietary fatty acid intake modulates these associations. The goal was to examine the interaction of MTHFR variants with dietary fatty acids influencing plasma Hcy in 995 Boston Puerto Rican adults. We found that plasma Hcy concentration was negatively correlated with (n-3) PUFA intake (r = -0.117; P = 0.022), and the ratio of (n-3):(n-6) PUFA in the diet (r = -0.122; P = 0.009). Further, 2 functional MTHFR variants, 1298A>C and 677C>T, which are not in linkage disequilibrium in this population, were significantly associated with hypertension (OR = 1.72, P = 0.024, and OR = 1.60, P = 0.002, respectively). In addition, the 1298A>C variant was significantly associated with CVD (OR = 3.32; P = 0.030). Importantly, this variant exhibited significant interactions with intakes of total and (n-6) PUFA and the (n-3):(n-6) PUFA ratio of the diet. The plasma Hcy concentration of carriers of risk allele 1298C was greater than that of noncarriers only when participants had consumed a high-PUFA diet (>7.8% energy) but was not greater when they had low intake of PUFA (≤7.8% energy). In addition, participants with combined genotypes of both SNP (677 TT with 1298 AC or CC) who consumed high levels of (n-3) PUFA (>0.66% energy) had lower plasma Hcy compared with those who had the same genotype and consumed low levels of (n-3) PUFA (≤0.66% energy). Our study suggests that dietary PUFA intake modulates the effect of 2 MTHFR variants on plasma Hcy in Boston Puerto Rican adults.

Multiple B-vitamin inadequacy amplifies alterations induced by folate depletion in p53 expression and its downstream effector MDM2.

Folate is required for biological methylation and nucleotide synthesis, aberrations of which are thought to be the mechanisms that enhance colorectal carcinogenesis produced by folate inadequacy. These functions of folate also depend on the availability of other B‐vitamins that participate in “one‐carbon metabolism,” including B2, B6 and B12. Our study therefore investigated whether combined dietary restriction of these vitamins amplifies aberrations in the epigenetic and genetic integrity of the p53 gene that is induced by folate depletion alone. Ninety‐six mice were group pair‐fed diets with different combinations of B‐vitamin depletion over 10 weeks. DNA and RNA were extracted from epithelial cells isolated from the colon. Within the hypermutable region of p53 (exons 5–8), DNA strand breaks were induced within exons 6 and 8 by folate combined with B2, B6 and B12 restriction (p < 0.05); such effects were not significantly induced by mild folate depletion alone. Similarly, a minor degree of hypomethylation of exon 6 produced by isolated folate depletion was significantly amplified (p ≤ 0.05) by simultaneous depletion of all 4 B‐vitamins. Furthermore, the expression of p53 and MDM2 were significantly decreased (p ≤ 0.05) by the combined depletion state but not by folate depletion alone. These data indicate that inadequacies of other 1‐carbon vitamins may amplify aberrations of the p53 gene induced by folate depletion alone, implying that concurrent inadequacies in several of these vitamins may have added tumorigenic potential beyond that observed with isolated folate depletion.

MAT1A variants are associated with hypertension, stroke, and markers of DNA damage and are modulated by plasma vitamin B-6 and folate

BACKGROUND The S-adenosylmethionine synthetase type 1 (MAT1A) gene encodes a key enzyme in one-carbon nutrient metabolism. OBJECTIVE This study aimed to determine the association of MAT1A variants with homocysteine, DNA damage, and cardiovascular disease (CVD). DESIGN Eight variants of MAT1A were examined for associations with hypertension, stroke, CVD, homocysteine, and DNA damage in 1006 participants of the Boston Puerto Rican Health Study. Two variants were replicated in 1147 participants of the Nutrition, Aging, and Memory in Elders Study. RESULTS Two variants and haplotypes were strongly associated with hypertension and stroke, independent of methylenetetrahydrofolate reductase (MTHFR) variants. Homozygotes of the MAT1A d18777A (rs3851059) allele had a significantly greater likelihood of stroke (odds ratio: 4.30; 95% CI: 1.34, 12.19; P = 0.006), whereas 3U1510A (rs7087728) homozygotes had a lower likelihood of hypertension (odds ratio: 0.67; 95% CI: 0.48, 0.95; P = 0.022) and stroke (odds ratio: 0.35; 95% CI: 0.15, 0.82; P = 0.015). A similar trend of association was observed in a second elderly population. Furthermore, strong interactions between MAT1A genotypes and vitamin B-6 status were found. Carriers of the nonrisk allele 3U1510A had a lower 8-hydroxydeoxyguanosine concentration--a biomarker of oxidative DNA damage--when plasma vitamin B-6 was high, whereas homozygotes for the risk-allele 3U1510G had higher 8-hydroxydeoxyguanosine concentrations, regardless of vitamin B-6 status. CONCLUSIONS MAT1A variants were strongly associated with hypertension and stroke. Improving folate and vitamin B-6 status might decrease the CVD risk of only a subset of the population, depending on genotype. These findings suggest that impairments in methylation activity, independent of homocysteine, have an effect on CVD risk.

Maternal B vitamin supplementation from preconception through weaning suppresses intestinal tumorigenesis in Apc1638N mouse offspring

Objective Variations in the intake of folate are capable of modulating colorectal tumorigenesis; however, the outcome appears to be dependent on timing. This study sought to determine the effect of altering folate (and related B vitamin) availability during in-utero development and the suckling period on intestinal tumorigenesis. Design Female wildtype mice were fed diets either mildly deficient, replete or supplemented with vitamins B2, B6, B12 and folate for 4 weeks before mating to Apc1638N males. Females remained on their diet throughout pregnancy and until weaning. After weaning, all Apc1638N offspring were maintained on replete diets for 29 weeks. Results At 8 months of age tumour incidence was markedly lower among offspring of supplemented mothers (21%) compared with those of replete (59%) and deficient (55%) mothers (p=0.03). Furthermore, tumours in pups born to deficient dams were most likely to be invasive (p=0.03). The expression of Apc, Sfrp1, Wif1 and Wnt5a—all of which are negative regulatory elements of the Wnt signalling cascade—in the normal small intestinal mucosa of pups decreased with decreasing maternal B vitamin intake, and for Sfrp1 this was inversely related to promoter methylation. β-Catenin protein was elevated in offspring of deficient dams. Conclusions These changes indicate a de-repression of the Wnt pathway in pups of deficient dams and form a plausible mechanism by which maternal B vitamin intake modulates tumorigenesis in offspring. These data indicate that maternal B vitamin supplementation suppresses, while deficiency promotes, intestinal tumorigenesis in Apc1638N offspring.

MAT1A variants modulate the effect of dietary fatty acids on plasma homocysteine concentrations

BACKGROUND AND AIM Dietary n-3 polyunsaturated fatty acids (PUFAs) are associated with decreased plasma homocysteine (Hcy), an important biomarker for cardiovascular disease. The S-adenosylmethionine synthetase type-1 (MAT1A), an essential enzyme in the conversion of methionine to S-adenosylmethionine, plays a key role in homocysteine metabolism. This study investigated the interaction between dietary fatty acids and MAT1A genotypes on plasma Hcy concentrations among Boston Puerto Ricans. METHODS AND RESULTS Plasma Hcy and MAT1A genotypes were determined in 994 subjects of the Boston Puerto Rican Health Study. Dietary fatty acid intakes were assessed by interviews using a questionnaire adapted from the NCI/Block food frequency form. RESULT In the cross-sectional analysis, genetic variant MAT1A 3U1510 displayed a significant interaction with dietary n-3:n-6 PUFA ratio in determining plasma Hcy (p-value for interaction = 0.025). 3U1510G homozygotes had significantly lower plasma Hcy concentration than major allele homozygotes and heterozygotes (AA + AG) (p-value for trend = 0.019) when the n-3:n-6 ratio was >0.09. Two other MAT1A variants, d18777 and i15752, also showed significant interactions with different constituents of dietary fat influencing Hcy concentrations. Furthermore, haplotypes consisting of three variants displayed a strong interaction with n3:n6 ratio influencing Hcy concentrations. CONCLUSIONS Our results suggest that MAT1A genotypes appear to modulate effects of dietary fat on plasma Hcy.