RESUMEN
Citalopram, a selective serotonin reuptake inhibitor (SSRI), is often detected in aquatic ecosystems. In this investigation, developing zebrafish were continuously exposed to one nominal concentration of either 0, 10, or 1000 µg/L citalopram for 7 days. Ribonucleic acids were then extracted from zebrafish for RNA-sequencing using the NovoSeq 6000 (Illumina). Clean reads were obtained following the removal of both the adapter and poly-N sequences. Alignment and differential gene expression analysis was conducted using programs HISAT2 and StringTie assembler. Data were converted to FPKM to quantify differentially expressed transcripts. Significant clinical subnetworks enriched following citalopram exposure included sympathetic nerve activity, blood pressure, vascular tone, and arterial pressure. Regulated transcripts were related to diseases such as mechanical hyperalgesia, pain, inflammatory pain, obstructive hypertrophic cardiomyopathy, fatigue, Diamond-Blackfan anemia, and hypertrophic cardiomyopathy. Following exposure to 10 µg/L citalopram, several transcripts were linked to brain dysfunction like prostaglandin-endoperoxide synthase 2, microtubule associated protein tau, cathepsin B, and dystrophin. Genes related to cardiac dysfunction were altered in zebrafish following exposure to 1000 µg/L citalopram. Using literature and databases that describe gene interactions, molecular networks (clinical and disease networks) were constructed to understand effects of citalopram.
RESUMEN
Citalopram is a selective serotonin reuptake inhibitor used to treat depression and is often detected in aquatic environments. Here, we measured the acute toxicity of citalopram at environmentally relevant concentrations to zebrafish embryos/larvae and utilized RNA-seq to reveal potential mechanisms of toxicity. We also assessed behavioral outcomes in larval zebrafish. Zebrafish embryos were exposed continuously to embryo rearing medium (ERM), or one dose of 0.1, 1, 10, 100, and 1000 µg/L citalopram for 7 dpf. No acute toxicity was noted for citalopram over 7-days in developing zebrafish, nor were there any effects on hatch rates; however, exposure resulted in a dose-dependent decrease in heart rate at 2 dpf. Reactive oxygen species were also increased in 7-day old larvae zebrafish exposed to 100 µg/L citalopram. There were 29 genes differentially expressed in fish exposed to 10 µg/L citalopram [FDR < 0.05] and 79 genes differentially expressed in fish exposed to 1000 µg/L citalopram [FDR < 0.05]. In the 1000 µg/L citalopram treatment, there were several transcripts downregulated related to muscle function, including myhz2, myhz1, and myom1. Twenty-five gene set pathways were shared between exposure concentrations; for example, 'IL6 Expression Targets', 'Thyroid Stimulating Hormone (TSH) Resistance in Congenital Hypothyroidism', and 'GFs/TNF -> Ion Channels.' Enrichment of KEGG pathways revealed that 1000 µg/L citalopram altered processes related to the proteosome and cardiac muscle contractions. Larval zebrafish at 7 dpf showed hypoactivity with exposure to ≥10 µg/L citalopram. This may be related to the downregulation of transcripts involved in muscle function. Overall, our results show that citalopram as a pharmaceutical pollutant may have an adverse influence on aquatic species' ability to survive by reducing their abilities to elude predators. This study improves mechanistic understanding of the potential harm citalopram may cause fish and contributes to environmental risk assessments for SSRIs in aquatic species.
RESUMEN
Ifosfamide is an alkylating antineoplastic drug used in chemotherapy, but it is also detected in wastewater. Here, the objectives were to (1) determine teratogenic, cardiotoxic, and mitochondrial toxicity potential of ifosfamide exposure; (2) elucidate mechanisms of toxicity; (3) characterize exposure effects on larval behavior. Survival rate, hatch rate, and morphological deformity incidence were not different amongst treatments following exposure levels up to 1000⯵g/L ifosfamide over 7 days. RNA-seq reveled 231 and 93 differentially expressed transcripts in larvae exposed to 1⯵g/L and 100⯵g/L ifosfamide, respectively. Several gene networks related to vascular resistance, cardiovascular response, and heart rate were affected, consistent with tachycardia observed in exposed embryonic fish. Hyperactivity in larval zebrafish was observed with ifosfamide exposure, potentially associated with dopamine-related gene networks. This study improves ecological risk assessment of antineoplastics by elucidating molecular mechanisms related to ifosfamide toxicity, and to alkylating agents in general.
Asunto(s)
Antineoplásicos , Contaminantes Químicos del Agua , Animales , Pez Cebra/metabolismo , Ifosfamida/toxicidad , Ifosfamida/metabolismo , Frecuencia Cardíaca , Metabolismo Energético , Antineoplásicos/farmacología , Larva , Embrión no Mamífero , Contaminantes Químicos del Agua/metabolismoRESUMEN
Ifosfamide is a cancer-fighting chemotherapeutic that has been detected in aquatic ecosystems. Zebrafish larvae were exposed to either 0, 1 or 100 µg/L ifosfamide in the water for 7 days, and fish were subjected to total RNA extraction and RNA-seq analysis with the Illumina NovoSeq 6000 instrument. Raw sequence data were processed through fastp and clean reads obtained by removing adapter and poly-N sequences, as well as low quality reads. Differential gene expression was performed using the abundance of transcripts that mapped to the zebrafish genome. To uncover putative targets regulated by microRNAs, Pathway Studio 12.0 was used to conduct a subnetwork enrichment analysis. Expression data were used to predict which microRNAs were important for the response to ifosfamide exposure. There were 21 common microRNAs identified in both the "IFOS1" and "IFOS100" datasets. These were MIR150, MIR6515, MIR657, MIR216A, m_Mir741, MIRLET7E, miR-let-7, MIR2392, r_Mir3551, MIR181B1, MIR33A, MIR502, MIR193B, MIR146A, MIR431, MIR647, m_Mir1192, MIR297, MIR328, and MIR4717. Data can be re-used to advance adverse outcome pathways in regulatory toxicology and to refine biomarker discovery for antineoplastics in aquatic environments.