Fred M. Farin

Vitamin D Related Genes, CYP24A1 and CYP27B1, and Colon Cancer Risk

Genetic association studies investigating the role of vitamin D in colon cancer have primarily focused on the vitamin D receptor (VDR), with limited data available for other genes in the vitamin D pathway, including vitamin D activating enzyme 1-α hydroxylase (CYP27B1) and vitamin D deactivating enzyme 24-α hydroxylase (CYP24A1). We evaluated whether 12 tagging single nucleotide polymorphisms (SNP) in CYP24A1, identified by resequencing the gene in 32 Caucasian samples, and 1 SNP in CYP27B1 were associated with colon cancer risk. In addition, we evaluated whether these two genes modify associations between colon cancer on the one hand and total vitamin D intake and UV-weighted sun exposure on the other, as well as other variants in VDR. Unconditional logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (95% CI) for the association between polymorphisms and haplotypes in CYP27B1 and CYP24A1 in a multicenter population-based case-control study of 1,600 cases and 1,949 controls. The CYP24A1 polymorphism IVS4-66T > G showed a statistically significant association with risk of colon cancer overall, particularly for proximal colon cancer. When stratified by anatomic site, we also found statistically significant associations for three CYP24A1 polymorphisms with risk of distal colon cancer (IVS4 + 1653C > T: OR for CT/TT versus CC, 0.81; 95% CI, 0.68-0.96; IVS9 + 198T > C: OR for CC versus TT, 1.33; 95% CI, 1.03-1.73; and within whites only: +4125bp 3′ of STPC > G: OR for GG versus CC, 1.44; 95% CI, 1-2.05). In addition, a possible interaction between CYP27B1 and UV-weighted sun exposure with proximal colon cancer was observed. As this is the first study to evaluate these genes in relation to colon cancer, additional studies are needed to confirm these results. (Cancer Epidemiol Biomarkers Prev 2009;18(9):2540–8)

Genetic polymorphisms as biomarkers of sensitivity to inhaled sulfur dioxide in subjects with asthma.

BACKGROUND Individuals with asthma are sensitive to inhaled sulfur dioxide (SO2); decrements in pulmonary function occur after exposure to low concentrations even for a short duration of time. There is a great amount of interindividual variation in response to SO2. OBJECTIVE It was our objective to determine whether one of the following polymorphism locations linked with asthma is associated with the bronchial hyperresponsiveness to SO2 observed in some asthmatic patients: the beta2-adrenergic receptor, interleukin-4 (IL-4) receptor alpha subunit, Clara cell secretory protein (CC16), TNF-alpha gene promoter, and first intron of the lymphotoxin alpha (LT-alpha) gene. METHODS Subjects were volunteers with physician-diagnosed asthma requiring regular asthma medication. Spirometry was performed before and after a 10-minute exposure to 0.5 ppm SO2. Subjects were classified as SO2 responders if forced expiratory volume in 1 second (FEV1) decreased > or = 12%. DNA obtained from buccal cell samples was analyzed for genetic polymorphisms. RESULTS Of the 62 subjects (21 male and 41 female), 13 had a 12% or greater decrement in FEV1 after SO2 exposure (range + 19% to -49%). Response to SO2 was associated with the wild-type allele of the TNF-alpha promoter polymorphism (12 of 12 SO2 responders versus 28 of 46 nonresponders; P < .05) but with no other polymorphisms. Medication category and atopic status showed no association with SO2 sensitivity. CONCLUSIONS The wild-type allele of the TNF-alpha promoter polymorphism may be associated with mechanisms of asthmatic sensitivity to inhaled SO2.

A randomized cross-over study of inhalation of diesel exhaust, hematological indices, and endothelial markers in humans

BackgroundExposure to traffic-related air pollution (TRAP) is considered a trigger for acute cardiovascular events. Diesel Exhaust (DE) is a major contributor to TRAP in the world. We evaluated the effect of DE inhalation on circulating blood cell populations, hematological indices, and systemic inflammatory cytokines in humans using a specialized facility.MethodsIn a randomized double-blind crossover study balanced to order, 17 metabolic syndrome (MetS) and 15 healthy subjects inhaled filtered air (FA) or DE exposure in two-hour sessions on different days with a minimum 2-week washout period. We collected blood pre-exposure, 7, and 22 hours after exposure initiation and measured the complete blood count and differential. We performed multiplex cytokine assay to measure the changes in the systemic inflammatory cytokines, and endothelial adhesion molecules (n=15). A paired analysis compared the effect of DE and FA exposures for the change from pre-exposure to the subsequent time points.ResultsA significant increase in the hematocrit was noted 7 hrs after DE [1.4% (95% CI: 0.9 to 1.9%)] compared to FA exposure [0.5% (95% CI: -0.09 to 1.0%); p=0.008. The hemoglobin levels increased non-significantly at 7 hrs post DE [0.3 gm/dL (95% CI: 0.2 to 0.5 gm/dL)] versus FA exposure [0.2 gm/dL (95% CI: 0 to 0.3 gm/dL)]; p=0.06. Furthermore, the platelet count increased 22 hrs after DE exposure in healthy, but not in MetS subjects [DE: 16.6 (95% CI: 10.2 to 23) thousand platelets/mL versus [FA: 3.4 (95% CI: -9.5 to 16.3) thousand platelets/mL)]; p=0.04. No DE effect was observed for WBC, neutrophils, lymphocytes or erythrocytes. Using the multiplex assay, small borderline significant increases in matrix metalloproteinase-9, interleukins (IL)-1beta, 6 and 10 occurred 7 hrs post exposure initiation, whereas E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule -1, and myeloperoxidase 22 hrs post exposure.ConclusionsOur results suggest that short-term DE exposure results in hemoconcentration and thrombocytosis, which are important determinants of acute cardiovascular events. Multiplex assay showed a non-significant increase in IL-1β and IL-6 immediately post exposure followed by myeloperoxidase and endothelial activation molecules. Further specific assays in a larger population will improve our understanding of the systemic inflammatory mechanisms following acute exposure to TRAP.Clinical trials registration numberStudy was conducted between 2004 to 2006, prior to expectation for registration.

Genetic Susceptibility to Prostate Cancer: Prostate-specific Antigen and its Interaction with the Androgen Receptor (United States)

Objective To determine whether directly observed prostate-specific antigen (PSA) promoter diploid haplotype, either alone or in conjunction with androgen receptor (AR) genotype, is associated with prostate cancer risk.Methods We conducted a case–control study nested within the US population-based Cardiovascular Health Study cohort. Incident prostate cancers were identified by linkage to cancer registry records for the years 1989–2000. We genotyped 193 cases and 391 controls for the PSA −252 G/A and −158 G/A SNPs and the AR CAG microsatellite, and developed methods to directly determine proximal PSA promoter haplotypes. Exact logistic regression was used to estimate odds ratios and significance levels.Results No significant associations were observed between PSA diplotype and prostate cancer overall. Short (<20) AR CAG repeat lengths were associated with modest increases in the risk of prostate cancer (OR, 1.46; 95% CI, 0.97–2.19; p = 0.071) that were significant for advanced disease (OR, 1.82; 95% CI, 1.02–3.26; p = 0.044). Men who possessed two copies of the PSA*2 (−252G/−158G) haplotype and short AR CAG repeat lengths had a 4-fold (95% CI, 1.05–20.75; exact p = 0.040) increased risk of prostate cancer, and a 7-fold (95% CI, 1.25–39.78; exact p = 0.026) increased risk of advanced disease.Conclusions We found evidence that the PSA*2*2 diplotype in combination with short AR CAG alleles increases a man’s risk of developing prostate cancer. These findings support an etiologic role in prostate cancer of genetic interactions between polymorphisms that increase AR transactivation strength and those that alter the regulatory regions of target genes such as PSA that are responsive to androgen stimulation.

Muscarinic receptors prevent oxidative stress-mediated apoptosis induced by domoic acid in mouse cerebellar granule cells

In mouse cerebellar granule neurons (CGNs) low concentrations of domoic acid (DomA) induce apoptotic cell death, which is mediated by oxidative stress; apoptosis is more pronounced in CGNs from Gclm (−/−) mice, which lack the modifier subunit of glutamate cysteine ligase (GCL) and have very low GSH levels. By activating M3 muscarinic receptors, the cholinergic agonist carbachol inhibits DomA‐induced apoptosis, and the anti‐apoptotic action of carbachol is more pronounced in CGNs from Gclm (+/+) mice. Carbachol does not prevent DomA‐induced increase in reactive oxygen species, suggesting that its anti‐apoptotic effect is downstream of reactive oxygen species production. Carbachol inhibits DomA‐induced activation of Jun N‐terminal (JNK) and p38 kinases, increased translocation to mitochondria of the pro‐apoptotic protein Bax, and activation of caspase‐3. Carbachol activates extracellular signal‐regulated kinases 1/2 (ERK1/2) MAPK and phospahtidylinositol‐3 kinase (PI3K) in CGNs from both genotypes. However, while the protective effect of carbachol is mediated by ERK1/2 MAPK in CGNs from both mouse genotypes, inhibitors of PI3K are only effective at antagonizing the action of carbachol in CGNs from Gclm (+/+) mice. In CGNs from both Gclm (+/+) and (−/−) mice, carbachol induces a MAPK‐dependent increase in the level of the anti‐apoptotic protein Bcl‐2. In contrast, carbachol causes a PI3K‐dependent increase in GCL activity and of GSH levels only in CGNs from Gclm (+/+) mice. Such increase in GCL is not because of a transcriptionally‐mediated increase in glutamate cysteine ligase catalytic subunit or glutamate cysteine ligase modifier subunit, but rather to an increase in the formation of the GCL holoenzyme. The results indicate that multiple pathways may contribute to the protective action of carbachol toward DomA‐induced apoptosis. Compromised GCLM expression, which is also found in a common genetic polymorphism in humans, leads to lower GSH levels, which can exacerbate the neurotoxicity of DomA, and decreases the anti‐apoptotic effectiveness of muscarinic agonists.

Infant sex-specific placental cadmium and DNA methylation associations

BACKGROUND Recent evidence suggests that maternal cadmium (Cd) burden and fetal growth associations may vary by fetal sex. However, mechanisms contributing to these differences are unknown. OBJECTIVES Among 24 maternal-infant pairs, we investigated infant sex-specific associations between placental Cd and placental genome-wide DNA methylation. METHODS We used ANOVA models to examine sex-stratified associations of placental Cd (dichotomized into high/low Cd using sex-specific Cd median cutoffs) with DNA methylation at each cytosine-phosphate-guanine site or region. Statistical significance was defined using a false discovery rate cutoff (<0.10). RESULTS Medians of placental Cd among females and males were 5 and 2 ng/g, respectively. Among females, three sites (near ADP-ribosylation factor-like 9 (ARL9), siah E3 ubiquitin protein ligase family member 3 (SIAH3), and heparin sulfate (glucosamine) 3-O-sulfotransferase 4 (HS3ST4) and one region on chromosome 7 (including carnitine O-octanoyltransferase (CROT) and TP5S target 1 (TP53TG1)) were hypomethylated in high Cd placentas. Among males, high placental Cd was associated with methylation of three sites, two (hypomethylated) near MDS1 and EVI1 complex locus (MECOM) and one (hypermethylated) near spalt-like transcription factor 1 (SALL1), and two regions (both hypomethylated, one on chromosome 3 including MECOM and another on chromosome 8 including rho guanine nucleotide exchange factor (GEF) 10 (ARHGEF10). Differentially methylated sites were at or close to transcription start sites of genes involved in cell damage response (SIAH3, HS3ST4, TP53TG1) in females and cell differentiation, angiogenesis and organ development (MECOM, SALL1) in males. CONCLUSIONS Our preliminary study supports infant sex-specific placental Cd-DNA methylation associations, possibly accounting for previously reported differences in Cd-fetal growth associations across fetal sex. Larger studies are needed to replicate and extend these findings. Such investigations may further our understanding of epigenetic mechanisms underlying maternal Cd burden with suboptimal fetal growth associations.

Paraoxonase 1 promoter and coding region polymorphisms in Parkinson’s disease

Parkinson’s disease (PD) is thought to be caused by a combination of genetic and environmental factors. Epidemiological studies have found associations of PD with pesticide exposure, or suspected pathways of pesticide exposure, such as rural residence and well water consumption. Many organophosphorous insecticides (for example, chlorpyrifos and diazinon) are bioactivated to potent cholinesterase inhibitors by the cytochromes P450, and the resulting toxic oxon forms are hydrolysed by paraoxonase (PON1). Genetic variants of detoxifying enzymes or pesticide metabolising enzymes, such as paraoxonase, may confer a predisposition to PD and thus are considered candidate genes for association studies. Multiple polymorphisms have been identified in the PON1 gene. The coding region contains two common polymorphisms, at amino acid codons 55 and 192. The glutamine (Q) to arginine (R) substitution at codon 192 causes substrate dependent differences in the kinetics of hydrolysis: compared with the PON1 192Q isoform, PON1 192R has higher activity towards paraoxon and chlorpyrifos oxon, but lower activity towards diazoxon, soman, and sarin. The leucine (L) to methionine (M) substitution at codon 55 does not affect the catalytic efficiency of substrate hydrolysis by the enzyme, but the PON1 55M allele is correlated with decreased mRNA and protein levels, because of linkage disequilibrium with a single nucleotide polymorphism (SNP) at position -108 of the promoter region of the gene. Five SNPs have been identified …

Functional variants of the lipoprotein lipase gene and the risk of preeclampsia among non-Hispanic Caucasian women

Hypertriglyceridemia is an important pathophysiologic feature of preeclampsia, a common vascular disorder of pregnancy. Three well‐documented functional variants (N291S, S447X, and D9N) of the lipoprotein lipase gene were related to hypertriglyceridemia. Results from the only two studies concerning the relationship between these variants and preeclampsia risk have been inconsistent. We investigated this relationship in a case–control study including 144 preeclamptic and 290 normotensive pregnant women (all non‐Hispanic Caucasians). We estimated odds ratios (OR) and 95% confidence intervals (CI) adjusted for maternal age, pre‐pregnancy body mass index, and parity. After adjusting for covariates, women with the 291 N/S or S/S genotype had significantly increased risk of preeclampsia (OR 6.9, 95% CI 1.9–25.4) compared with women with the common 291N/N genotype. The 447 S/X or X/X genotype was not significantly associated with preeclampsia risk. The frequency of the 9N variant allele was 1.8% in controls, while this allele was not observed among cases. Haplotype 9D/291S/447S was strongly associated with higher risk of preeclampsia as compared with the most common haplotype 9D/291N/447S (adjusted OR 6.6, 95% CI 1.7–25.0). Results from our study support the thesis that abnormal lipid metabolism is important in the pathogenesis of preeclampsia.

A Combination Therapy of Nicotinamide and Progesterone Improves Functional Recovery following Traumatic Brain Injury.

Neuroprotection, recovery of function, and gene expression were evaluated in an animal model of traumatic brain injury (TBI) after a combination treatment of nicotinamide (NAM) and progesterone (Prog). Animals received a cortical contusion injury over the sensorimotor cortex, and were treated with either Vehicle, NAM, Prog, or a NAM/Prog combination for 72 h and compared with a craniotomy only (Sham) group. Animals were assessed in a battery of behavioral, sensory, and both fine and gross motor tasks, and given histological assessments at 24 h post-injury to determine lesion cavity size, degenerating neurons, and reactive astrocytes. Microarray-based transcriptional profiling was used to determine treatment-specific changes on gene expression. Our results confirm the beneficial effects of treatment with either NAM or Prog, demonstrating significant improvements in recovery of function and a reduction in lesion cavitation, degenerating neurons, and reactive astrocytes 24 h post-injury. The combination treatment of NAM and Prog led to a significant improvement in both neuroprotection at 24 h post-injury and recovery of function in sensorimotor related tasks when compared with individual treatments. The NAM/Prog-treated group was the only treatment group to show a significant reduction of cortical loss 24 h post-injury. The combination appears to affect inflammatory and immune processes, reducing expression of a significant number of genes in both pathways. Further preclinical trials using NAM and Prog as a combination treatment should be conducted to identify the window of opportunity, determine the optimal duration of treatment, and evaluate the combination in other pre-clinical models of TBI.

The effect of nicotinamide on gene expression in a traumatic brain injury model

Microarray-based transcriptional profiling was used to determine the effect of nicotinamide on gene expression in an experimental traumatic brain injury (TBI) model. Ingenuity Pathway Analysis (IPA) was used to evaluate the effect on relevant functional categories and canonical pathways. At 24 h, 72 h, and 7 days, respectively, 70, 58, and 76%, of the differentially expressed genes were up-regulated in the vehicle treated compared to the sham animals. At 24 h post-TBI, there were 150 differentially expressed genes in the nicotinamide treated animals compared to vehicle; the majority (82%) down-regulated. IPA analysis identified a significant effect of nicotinamide on the functional categories of cellular movement, cell-to-cell-signaling, antigen presentation and cellular compromise, function, and maintenance and cell death. The canonical pathways identified were signaling pathways primarily involved with the inflammatory process. At 72 h post-cortical contusion injury, there were 119 differentially expressed genes in the nicotinamide treated animals compared to vehicle; the majority (90%) was up-regulated. IPA analysis identified a significant effect of nicotinamide on cell signaling pathways involving neurotransmitters, neuropeptides, growth factors, and ion channels with little to no effect on inflammatory pathways. At 7 days post-TBI, there were only five differentially expressed genes with nicotinamide treatment compared to vehicle. Overall, the effect of nicotinamide on counteracting the effect of TBI resulted in significantly decreased number of genes differentially expressed by TBI. In conclusion, the mechanism of the effect of nicotinamide on secondary injury pathways involves effects on inflammatory response, signaling pathways, and cell death.