Hematotoxicity of lead, cadmium, mercury and arsenic low dosed mixtures relevant to environmental exposure: the Adverse Outcome Pathway framewor

Abstract

Lead (Pb), cadmium (Cd), mercury (Hg) and arsenic (As) are toxic metals/metalloids placed at the top of the list of the most significant environmental pollutants from the public aspect of health. The literature showed that exposure to individual metals, as well as their mixtures, could influence the development of numerous health disorders, including changes in hematological parameters, which mostly result in anemia. In the current study, in silico and in vivo analysis was performed to determine hematotoxicity of low dosed toxic metals mixture (Pb, Cd, Hg and As) relevant for exposure from life environment. AOP framework was applied to elucidate the mechanisms of hematotoxicity through: in silico approach – Comparative Toxicogenomics Database (CTD), GeneMANIA Gene Ontology tools, and in vivo approach – orally exposed male Wistar rats divided into control group and group treated with a mixture (MIX) of aqueous solutions of lead-acetate (1 mg/kg b.w.), cadmium-chloride (3 mg/kg b.w.), mercury(II)-chloride (0,06 mg/kg b.w.), arsenic(III)-oxide (0,6 mg/kg b.w.). Rats were euthanized after 28 days and hematological parameters were measured. CTD analysis showed connected genes for MIX and the development of: hematological diseases – FAS and ALAD; anemia – ACHE, GSR, TNF; thrombocytosis – JAK2, CALR, MPL, THPO. Further analysis singled out changes in protein and mRNA expression, as well as protein activity of the selected genes as 3 clusters of molecular initiating events (MIEs). These molecular changes, along with the main interactions among the related genes in each cluster (GeneMANIA) (physical interactions), led to the further macromolecular alterations. Cluster-specific subsequent key events (KEs) were obtained by Gene Ontology tools: hematological diseases – The FAS protein signaling pathways, apoptosis and heme synthesis; anemia – oxidative stress, glutathione metabolism, ROS induced apoptosis; thrombocytosis – activation of JAK and THPO, binding of THPO to receptors and their activation. Identified molecular pathways (KEs) could converge to the development of Disruption of Hematological Homeostasis as the final organ-specific adverse outcome (AO). In vivo approach showed statistically significant changes compared to the control in values of RCB, Hb, Hct, MCV, MCH, MCHC, strengthening the weight of evidence for anemia development, while an increase in platelets suggested the thrombocytosis development. This AOP framework provides an insight into the better mechanistic understanding of environmental metal mixture mode of action and its relation to hematotoxicity. Also, it suggested KEs useful for future biomarkers identification and enabled grouping the metals with similar toxicity (451-03-68/2020–14/200161).