Exposure to the antineoplastic ifosfamide alters molecular pathways related to cardiovascular function, increases heart rate, and induces hyperactivity in zebrafish (Danio rerio).

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 µg/L ifosfamide over 7 days. RNA-seq reveled 231 and 93 differentially expressed transcripts in larvae exposed to 1 µg/L and 100 µg/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.

In silico microRNA network data in zebrafish after antineoplastic ifosfamide exposure.

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.

Disease network data for the pesticide fipronil in rat dopamine cells.

Transcriptome data were collected in rat dopamine cells exposed to fipronil for 24 h using microarray analysis. Fipronil is a phenylpyrazole pesticide that acts to inhibit gamma-aminobutyric acid (GABA), blocking inhibitory synaptic transmission in the central nervous system. Transcriptome data were subjected to pathway analysis and subnetwork enrichment analysis. We report that 25 µM fipronil altered transcriptional networks in dopamine-synthesizing cells that are associated with Alzheimer's Disease, Huntington Disease, and Schizophrenia. Data analysis revealed that nerve fibre degeneration, nervous system malformations, neurofibrillary tangles, and neuroinflammation were all disease processes related to the transcriptome profile observed in the rat neuronal cells. Other disease networks altered by fipronil exposure at the transcript level were associated with the mitochondria, including mitochondrial DNA depletion syndrome and mitochondrial encephalomyopathies. These data, along with those presented in Souders et al. (2021), are significant because they increase understanding into the molecular mechanisms underlying human disease following exposures to neuroactive pesticides. These data can be reused to inform adverse outcome pathways for neurotoxic pesticides.

Elucidating Conserved Transcriptional Networks Underlying Pesticide Exposure and Parkinson's Disease: A Focus on Chemicals of Epidemiological Relevance.

While a number of genetic mutations are associated with Parkinson's disease (PD), it is also widely acknowledged that the environment plays a significant role in the etiology of neurodegenerative diseases. Epidemiological evidence suggests that occupational exposure to pesticides (e.g., dieldrin, paraquat, rotenone, maneb, and ziram) is associated with a higher risk of developing PD in susceptible populations. Within dopaminergic neurons, environmental chemicals can have an array of adverse effects resulting in cell death, such as aberrant redox cycling and oxidative damage, mitochondrial dysfunction, unfolded protein response, ubiquitin-proteome system dysfunction, neuroinflammation, and metabolic disruption. More recently, our understanding of how pesticides affect cells of the central nervous system has been strengthened by computational biology. New insight has been gained about transcriptional and proteomic networks, and the metabolic pathways perturbed by pesticides. These networks and cell signaling pathways constitute potential therapeutic targets for intervention to slow or mitigate neurodegenerative diseases. Here we review the epidemiological evidence that supports a role for specific pesticides in the etiology of PD and identify molecular profiles amongst these pesticides that may contribute to the disease. Using the Comparative Toxicogenomics Database, these transcripts were compared to those regulated by the PD-associated neurotoxicant MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). While many transcripts are already established as those related to PD (alpha-synuclein, caspases, leucine rich repeat kinase 2, and parkin2), lesser studied targets have emerged as "pesticide/PD-associated transcripts" [e.g., phosphatidylinositol glycan anchor biosynthesis class C (Pigc), allograft inflammatory factor 1 (Aif1), TIMP metallopeptidase inhibitor 3, and DNA damage inducible transcript 4]. We also compared pesticide-regulated genes to a recent meta-analysis of genome-wide association studies in PD which revealed new genetic mutant alleles; the pesticides under review regulated the expression of many of these genes (e.g., ELOVL fatty acid elongase 7, ATPase H+ transporting V0 subunit a1, and bridging integrator 3). The significance is that these proteins may contribute to pesticide-related increases in PD risk. This review collates information on transcriptome responses to PD-associated pesticides to develop a mechanistic framework for quantifying PD risk with exposures.