Urinary Metabolomics Revealed Environmental Arsenic Or Cadmium Exposure Related To Metabolic Alterations In General Chinese Pregnant Women

Heavy metal, such as arsenic, cadmium, chromium and lead, is considered as a very important group of environmental pollutant which in larger quantity may be toxic and dangerous to human being. It has been proved by epidemiology and animal studies that type 2 diabetes mellitus, hypertension and chronic kidney disease are closely related to arsenic exposure. In addition, arsenic exposure during pregnancy may increase the risk of maternal gestational diabetes mellitus and gestational hypertension. A number of epidemiological studies have showed significant associations between prenatal arsenic exposure and adverse infant outcome, such as fetal loss, infant death, low birth weight and birth defect. However, most of the research works relied on the study of individuals exposed to high-levels of arsenic. There is no report about pregnant women exposed to environmental low-level arsenic. As we know, pregnancy is a period of particular vulnerability to environmental pollution exposure. Thus, it is a worthy question to explore the risk of relatively low-level arsenic exposure for pregnant women. This study had investigated the urinary metabolic changes of pregnant women exposed to environmental low-dose arsenic and identified biomarkers from metabolomics analysis.Cadmium, as a typical heavy metal, has become a major environmental threat to human health. Human can exposed to cadmium mainly by ingestion and breath. A large number of studies show that a high intake of cadmium during pregnancy may not only lead to gestational hypertension but also increase the risk of fetal loss, intrauterine growth retardation and low birth weight. Since the pregnant women are known to be more susceptible to some chemical exposures than ordinary people, the understanding regarding the health effects of low-level cadmium exposure on pregnant women is critical and remains unclear. In the present study, we applied metabolomics technology to investigate the urinary metabolic changes of pregnant women exposed to environmental low-level cadmium, and to identify metabolites that could serve as biomarkers to assist in assessment of cadmium toxicity during pregnancy. This study had also investigated the urinary metabolic alterations of pregnant women co-exposed to arsenic and cadmium.Part I:Environmental arsenic exposure induces urinary metabolite profile alterations in general Chinese pregnant womenObjective:To investigate the urinary metabolic changes of pregnant women exposed to environmental low-level arsenic, and to identify biomarkers from metabolomics analysis.Methods:Urine samples of 246 pregnant women were collected in the first trimester of pregnancy and were divided into three groups based on the urinary arsenic concentrations which were determined using inductively coupled plasma mass spectrometry (ICP-MS). Changes in the metabolite profile were measured using ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF MS). The partial least squares discriminant analysis (PLS-DA) was used to analyze the multivariate metabolomics datasets and select the possible arsenic-related metabolic biomarkers. The identified biomarkers were subjected to receiver operator characteristic (ROC) analysis to evaluate diagnostic ability. The hierarchical cluster analysis (HCA) algorithm was performed to visualize and characterize the differences between two groups in the abundances of potential biomarkers. The biological significance of the biomarkers was investigated by using data-bases such as HMDB, METLIN and KEGG.Results:The urinary metabolic profiles were principally different between groups exposed to different doses of arsenic. Arsenic-related metabolic biomarkers were investigated by comparing the samples of the first and third tertiles of arsenic exposure classifications using a PLS-DA model. Nine urine potential biomarkers were identified, including glutathione,18-carboxy-dinor-LTE4,20-COOH-LTE4, thiocysteine, cystathionine ketimin, 1-(beta-D-ribofuranosyl)-1,4-dihydronicotinamide, LysoPC (14:0), p-cresol glucuronid and vanillactic acid, and their combination pattern showed a moderate ability to discriminate the first and third tertiles of arsenic exposure with an area under the ROC curve (ROC-AUC) value of 0.80. It was found that the glutathione,18-carboxy-dinor-LTE4,20-COOH-LTE4, thiocysteine, cystathionine ketimin and 1-(beta-D-ribofuranosyl)-1,4-dihydronicotinamide might be associated with the oxidative stress induced by arsenic exposure; the LysoPC (14:0), p-cresol glucuronid and vanillactic acid might be related to the multiple organ damage induced by arsenic exposurey.Conclusion:In our study, the mean concentration of urinary arsenic of pregnant women was within the limits (total urinary arsenic≦100μg/L) laid down by the Agency for Toxic Substances and Disease Registry (ATSDR). However, the results of metabolomics analysis suggested that arsenic exposure, even at such low levels, has led to the metabolite alteration in pregnant women, and the changes might be associated with adverse health outcomes. To the best of our knowledge, this is the first report on metabolic changes in pregnant women for arsenic exposure. The findings may be valuable for the arsenic risk assessment for pregnant women.PartⅡ:Environmental cadmium exposure induces urinary metabolite profile alterations in general Chinese pregnant womenObjective:To investigate the urinary metabolic changes of pregnant women exposed to environmental low-level cadmium, and to identify biomarkers from metabolomics analysis.Methods:Urine samples of 246 pregnant women were collected in the first trimester of pregnancy and were divided into three groups based on the tertile distribution of cadmium concentrations in the urine which were determined using ICP-MS. Changes in the metabolite profile were measured using UPLC/Q-TOF MS. The PLS-DA was used to analyze the multivariate metabolomics datasets and select the possible cadmium-related metabolic biomarkers. The identified biomarkers were subjected to ROC analysis to evaluate diagnostic ability. The HCA algorithm was performed to visualize and characterize the differences between two groups in the abundances of potential biomarkers. The biological significance of the biomarkers was investigated by using data-bases such as HMDB, METLIN and KEGG.Results:The urinary metabolic profiles were principally different between groups exposed to different doses of cadmium. Cadmium-related metabolic biomarkers were investigated by comparing the samples of the first and third tertiles of cadmium exposure classifications using a PLS-DA model. Eight urine potential biomarkers were identified, including L-cystine, L-tyrosine, dityrosine, cysteineglutathione disulfide,16DHEA-S, prolylhydroxyproline, histamine and uric acid. The levels of L-cystine, L-tyrosine, dityrosine and cysteineglutathione disulfide were significantly higher in high cadmium exposure group than in low cadmium exposure group, which might be associated with the oxidative stress induced by cadmium exposure. The levels of uric acid and histamine were significantly lower in high cadmium exposure group than in low cadmium exposure group, which might be related to the nephrotoxic effect induced by cadmium exposure. The increased levels of 16DHEA-S and prolylhydroxyproline in high cadmium exposure group might be associated with the multiple organ damage induced by cadmium exposurey. In addition, the combination pattern of the eight biomarkers showed a high ability to discriminate the first and third tertiles of cadmium exposure with a ROC-AUC value of 0.91.Conclusion:In this study, the urine cadmium concentration of any pregnant woman was less than the health-based exposure limit of urinary cadmium proposed by the World Health Organization (WHO) (urinary cadmium at 5μg/g of creatinine). However, environmental cadmium exposure might cause metabolite changes in pregnant women even at low levels, and the changes might be associated with adverse health outcomes. The overall findings indicates that metabolomics is useful for revealing metabolic changes associated with low-dose cadmium exposure during pregnancy and providing a better understanding of the risk on pregnant women exposed to cadmium.Part III:Co-exposure of arsenic and cadmium induces urinary metabolite profile alterations in general Chinese pregnant womenObjective:To investigate the urinary metabolic changes of pregnant women co-exposed to arsenic and cadmium, and to identify biomarkers from metabolomics analysis.Methods:Urine samples of 246 women in early pregnancy were subjected to 2 X 2 factorial experimental design and divided into four groups (including the control group, the arsenic exposure group, the cadmium exposure group and the co-exposure group) based on the arsenic and cadmium concentrations in the urine which were determined using ICP-MS. Changes in the metabolite profile were measured using UPLC/Q-TOF MS. The PLS-DA was used to analyze the multivariate metabolomics datasets and select the possible co-exposure related urinary metabolites. The effect of arsenic and cadmium exposure on pregnant women was investigated by analyzing the biological significance and the metabolic pathway of co-exposure related urinary metabolites. In addition, the two-way analysis of variance (two-way ANOVA) was used to analyze the mode of the combined effects of arsenic and cadmium.Results:The urinary metabolic profiles were principally different between co-exposure group and other three groups. Co-exposure related urinary metabolites were investigated by comparing the samples of the control group ana the co-exposure group using a PLS-DA moaei. Eight co-exposure related urinary metabolites were identified:1-(beta-D-ribofuranosyl)-1,4-dihydronicotinamide, p-cresol glucuronide, L-cystine and uric acid were the potential biomarkers of arsenic or cadmium exposure that we have found; while 20-oxo-LTE4, N-acetyl-LTE4, gamma-CEHC and xanthosine were new-found urinary metabolites. This indicated that the effect of co-exposure of arsenic and cadmium might not only retain the effect of arsenic/cadmium exposure alone, but also create some new effects. In addition, the result of the two-way ANOVA revealed that the mode of the combined effects of arsenic and cadmium was additive.Conclusion:Based on the results of this study, exposure alone or combined exposure to arsenic and cadmium resulted indifferent effects on urinary metabolic profiles of pregnant women. Metabolomics is a promising new technology for the combined effects study of heavy metals.