Dietary Intake Estimates and Urinary Cadmium Levels in Danish Postmenopausal Women

<div><p>Background</p><p>Cadmium is a known carcinogen that can disrupt endocrine signalling. Cigarette smoking and food are the most common routes of non-occupational exposure to cadmium. Cadmium accumulates in the kidney and can be measured in urine, making urine cadmium (U-Cd) a biomarker of long-term exposure. However dietary-cadmium (D-Cd) intake estimates are often used as surrogate indicator of cadmium exposure in non-smoking subjects. It is therefore important to investigate the concordance between D-Cd estimates obtained with Food Frequency Questionnaires and U-Cd.</p><p>Methods</p><p>U-Cd levels were compared with estimated dietary-cadmium (D-Cd) intake in 1764 post-menopausal women from the Danish Diet, Cancer and Health cohort. For each participant, a food frequency questionnaire, and measures of cadmium content in standard recipes were used to judge the daily intake of cadmium, normalized by daily caloric intake. Cadmium was measured by ICP-MS in spot urine sampled at baseline and normalized by urinary creatinine. Information on diet, socio-demographics and smoking were self-reported at baseline.</p><p>Results</p><p>Linear regressions between U-Cd and D-Cd alone revealed minimal but significant positive correlation in never smokers (R² = 0.0076, <i>β</i> = 1.5% increase per 1 ng Cd kcal^-1, p = 0.0085, <i>n</i> = 782), and negative correlation in current smokers (R² = 0.0184, <i>β</i> = 7.1% decrease per 1 ng Cd kcal^-1 change, p = 0.0006, <i>n</i> = 584). In the full study population, most of the variability in U-Cd was explained by smoking status (R² = 0.2450, <i>n</i> = 1764). A forward selection model revealed that the strongest predictors of U-Cd were age in never smokers (Δ R² = 0.04), smoking duration in former smokers (Δ R² = 0.06) and pack-years in current smokers (Δ R² = 0.07). Food items that contributed to U-Cd were leafy vegetables and soy-based products, but explained very little of the variance in U-Cd.</p><p>Conclusions</p><p>Dietary-Cd intake estimated from food frequency questionnaires correlates only minimally with U-Cd biomarker, and its use as a Cd exposure indicator may be of limited utility in epidemiologic studies.</p></div

Forward model selection to predict creatinine-adj. U-Cd^a (μg Cd g creatinine^-1).

<p>^aU-Cd is natural-log transformed</p><p>^b Percent change for creatinine-adj. U-Cd for a one unit increase of each independent variable was calculated as (e^β-1)*100</p><p>^cR² adjusted by degrees of freedom.</p><p>Parameter estimates are listed for the factors selected by the procedure for inclusion in each model (p value cut-off = 0.2).</p

Associations between D-Cd (ng kcal^-1) and creatinine-adj. U-Cd^d (μg Cd g creatinine^-1).

<p>p <0.05 were highlighted in bold</p><p>^aR² adjusted by degrees of freedom</p><p>^bPercent change for creatinine-adj. U-Cd for a one ng kcal^-1 increase of D-Cd was calculated as (e^β-1)*100 n100</p><p>^cEach model was adjusted for age, smoking status (in the all population only), BMI, pack-years (in current smokers only) and iron intake</p><p>^dU-Cd is natural-log transformed.</p><p>Associations between D-Cd (ng kcal^-1) and creatinine-adj. U-Cd<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138784#t003fn005" target="_blank">^d</a> (μg Cd g creatinine^-1).</p

Perturbation of microRNAs in Rat Heart during Chronic Doxorubicin Treatment

<div><p>Anti-cancer therapy based on anthracyclines (DNA intercalating Topoisomerase II inhibitors) is limited by adverse effects of these compounds on the cardiovascular system, ultimately causing heart failure. Despite extensive investigations into the effects of doxorubicin on the cardiovascular system, the molecular mechanisms of toxicity remain largely unknown. MicroRNAs are endogenously transcribed non-coding 22 nucleotide long RNAs that regulate gene expression by decreasing mRNA stability and translation and play key roles in cardiac physiology and pathologies. Increasing doses of doxorubicin, but not etoposide (a Topoisomerase II inhibitor devoid of cardiovascular toxicity), specifically induced the up-regulation of miR-208b, miR-216b, miR-215, miR-34c and miR-367 in rat hearts. Furthermore, the lowest dosing regime (1 mg/kg/week for 2 weeks) led to a detectable increase of miR-216b in the absence of histopathological findings or alteration of classical cardiac stress biomarkers. <em>In silico</em> microRNA target predictions suggested that a number of doxorubicin-responsive microRNAs may regulate mRNAs involved in cardiac tissue remodeling. In particular miR-34c was able to mediate the DOX-induced changes of Sipa1 mRNA (a mitogen-induced Rap/Ran GTPase activating protein) at the post-transcriptional level and in a seed sequence dependent manner. Our results show that integrated heart tissue microRNA and mRNA profiling can provide valuable early genomic biomarkers of drug-induced cardiac injury as well as novel mechanistic insight into the underlying molecular pathways.</p> </div

Chronic DOX treatment (3 mg/kg/week) alters levels of 25 microRNAs from week 2 onwards.

<p>Variation of cardiac microRNA levels versus vehicle are reported for animals treated with DOX 3 mg/kg/week for 2 and 4 weeks. Values were calculated via the relative quantification (ΔΔCt) method by using the mammalian U6 snRNA as a normalizer. MicroRNAs showing same trend at 2 and 4 weeks are italicized. # indicates microRNAs selected for further analysis. Significant P values (<0.05) are in bold. FC  =  fold change.</p

Study design and representative micrograph showing DOX-related vacuolation in the myocardium.

<p>(A) Six adult male rats were injected with the indicated doses of vehicle, doxorubicin (DOX), dexrazoxane (DZR), etoposide (EPS) or a combination of DOX and DZR for 2, 4 or 6 weeks. Cardiac tissue was excised and deep frozen for gene expression and microRNA profiling experiments. A representative micrograph of a toluidine blue stained myocardial section of a control (B) and of a DOX treated animal (C). Black arrows indicate sarcoplasmic micro- and macro- vacuolation of cardiomyocytes.</p

Ambra1 expression was induced by DOX treatment <i>in vivo</i> and miR-34c could control its endogenous expression levels in H9c2 cardiac myoblasts.

<p>(A) Fold change of Ambra1 probe set in rat heart tissue treated with DOX. Fold change and statistical significance were assessed <i>vs.</i> each vehicle group. n = 4 to 6 (except #, n = 2) (B) Endogenous levels of Ambra1 were measured after miR-34c over-expression (miR-34c mimic) or inhibition (miR-34c HI) in absence of presence of DOX 0.1. Fold change value were normalized <i>vs.</i> the respective negative transfection controls in the untreated condition (n = 3). *P<0.05, **P<0.01, ***P<0.005. FC  =  fold change.</p

DOX 3 mg/kg/week altered levels of genomic cardiomyopathy indicators (Ankrd/Carp, Nppb, Myh7 and Myh6).

<p>Expression fold change relative to vehicle were represented for DOX 3 mg/kg/week at 2, 4 and 6 weeks time point (n = 6) for (A) Ankrd/Carp, (B) Nppb, (C) Myh7 and (D) Myh6. For each time point and each probe set, vehicle values were averaged and normalized to 1. The same correction was applied to the DOX treated values. Affymetrix probe-set number is indicated in brackets. Error bars represent standard deviation. T-test was performed for vehicle- vs. DOX-treated at each time point. *P<0.05, **P<0.01, ***P<0.005, NS = Non-Significant. (No t-test for #, as n = 2).</p

Relative quantification of DOX-responder microRNAs in rat heart across all groups.

<p>Relative quantification of (A) miR-208b, (B) miR-215, (C) miR-216b, (D) miR-367 and (E) miR-34c in DOX, DOX + DZR, EPS groups, normalized versus vehicle treated animals. Expression levels were measured by single assay qPCR (n = 3, except #, n = 2). DOX: Doxorubicin, DZR: dexrazoxane, EPS: etoposide; numbers indicate the weekly dose of each compound in mg/kg/week. Empty spaces represent non-sampled animals. The vehicle treated is the first column of each time-point. The animals used in this experiment were distinct from the ones represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040395#pone-0040395-t001" target="_blank">Table 1</a>. Error bars represent SD. T-test results are indicated by asterisks for significant DOX-treated groups vs. their own vehicle-treated, unless otherwise specified by horizontal range bars; *P<0.05, **P<0.01, ***P<0.005, NS = Non-Significant).</p

DOX-induced upregulation of miR-216 and miR-367 expression precedes the detection of overt histopathological lesions in cardiac tissue.

<p>Blue bars show cumulative vacuolation grade. X axis shows the DOX regimen in mg/kg/week received by the animals at 2 weeks. Y axes report cumulative histopathological scores and microRNA fold change vs. untreated cardiac tissues (normalized at value 1). Path grading  =  cumulative vacuolation score. FC  =  fold change. SEM  =  standard error on the mean.</p