Biology subjects

Salanga, M. C.

Publications and source records attributed to Salanga, M. C..

4 recordsLinked to original sources

bioRxiv2026

Environmentally relevant depleted uranium exposure damages mitochondria, decreases cytosolic reductive capacity, and increases global DNA damage accumulation through a ROS-independent mechanism involving slingshot protein phosphatase 1b enrichment.

Depleted uranium (DU) is an environmental contaminant with a 30 {micro}g/L (ppb; parts per billion) EPA maximum contaminant level (MCL) for drinking water. The mining of uranium and use of DU in modern weapons underly human exposure that disproportionally impacts military and tribal communities in the United States. Uraniums radiotoxic characteristics are understood, but its chemical hazards much less so. In zebrafish (Danio rerio) and human cell cultures we test the hypothesis that exposure to DU negatively impacts cellular function and development through disruption of mitochondrial metabolism. Using a novel shrapnel model with TEM/SEM+EDS, we showed uranium microparticles caused proximity-dependent mitochondrial disruption. In waterborne exposure paradigms, larval movement was reduced and hatching delayed as a result of reduced movement and not enzyme deficiencies in response to 18 ppb DU, below the MCL. Increased DNA damage accumulation was detected in exposed larva and cells. DNA-damage quantitative PCR of DU-exposed larvae showed increased damage in the ahr1 locus (nuclear gene) and decreased mitochondrial DNA (mtDNA) copy number, but mtDNA damage levels varied across experiments. Mitochondrial function was assessed using a resazurin-based assay in the presence and absence of antioxidants and showed diminished cytoplasmic reductive capacity. DU exposure alone did not enrich antioxidant gene expression, contrasting with arsenic exposure, a known ROS-inducer and Nrf2-activator. Sulforaphane (SFN), a potent Nrf2-activator, did not blunt the effects of DU exposure, despite activation of antioxidant response element (ARE) genes (gstp and gss), but did blunt the effects of arsenic exposure. The most enriched transcript in DU-exposed larvae coded for slingshot protein phosphatase (ssh), further exploration revealed ssh1b as the zebrafish-specific ortholog activated in response to DU, and inhibition using an identified SSH1 inhibitor, Sennoside A, partially rescued the metabolic and hatching defects observed. Our data points to a cytotoxic mechanism in which DU disrupts mitochondrial function through ssh1b enrichment that impairs normal mitophagy, leading to decreased cellular reductive potential independent of either ROS production or ARE-activation. Our results suggest that health impacts from DU exposure may be directly linked to impaired mitochondrial functions.

pharmacology and toxicology↗

bioRxiv2021

Orphan cytochrome P450 20A1 CRISPR/Cas 9 mutants and neurobehavioral phenotypes in zebrafish

Orphan cytochrome P450 (CYP) enzymes are those for which biological substrates and function(s) are unknown. Cytochrome P450 20A1 (CYP20A1) is the last human orphan P450 enzyme, and orthologs occur as single genes in every vertebrate genome sequenced to date. The occurrence of high levels of CYP20A1 transcripts in human substantia nigra and hippocampus and abundant maternal transcripts in zebrafish eggs strongly suggest roles both in the brain and during early embryonic development. Patients with chromosome 2 microdeletions including CYP20A1 show hyperactivity and bouts of anxiety, among other conditions. Here, we created zebrafish CYP20A1 mutants using CRISPR/Cas9, providing vertebrate models with which to study the role of CYP20A1 in behavior and other neurodevelopmental functions. The homozygous cyp20a1 null mutants exhibited significant behavioral differences from wild-type zebrafish, both in larval and adult animals. Larval cyp20a1-/- mutants exhibited a strong increase in light-simulated movement (i.e., light-dark assay), which was interpreted as hyperactivity. Further, the larvae exhibited mild hypoactivity during the adaptation period of the optomotor assays. Adult cyp20a1 null fish showed a pronounced delay in adapting to new environments, which is consistent with an anxiety paradigm. Taken together with our earlier morpholino cyp20a1 knockdown results, the results described herein suggest that the orphan CYP20A1 has a neurophysiological role.

pharmacology and toxicology↗

bioRxiv2021

Developmental exposure to domoic acid disrupts startle response behavior and circuitry

Harmful algal blooms produce potent neurotoxins that accumulate in seafood and are hazardous to human health. Developmental exposure to the harmful algal bloom toxin, domoic acid (DomA), has behavioral consequences well into adulthood, but the cellular and molecular mechanisms are largely unknown. To assess these, we exposed zebrafish embryos to DomA during the previously identified window of susceptibility (2 days post-fertilization) and used the well-known startle response circuit as a tool to identify specific neuronal components that are targeted by exposure to DomA. Exposure to DomA reduced the probability of eliciting a startle after auditory/vibrational or electrical stimuli and led to the dramatic reduction of one type of startle, short latency c-start (SLC) responses. Furthermore, DomA-exposed larvae had altered kinematics of both SLC and long latency c-start (LLC) startle responses, exhibiting shallower bend angles and slower maximal angular velocities. Using vital dye staining, immunolabelling, and live imaging of transgenic lines, we determined that while the sensory inputs were intact, the reticulospinal neurons required for SLC responses were absent in most DomA-exposed larvae. Furthermore, axon tracing revealed that DomA-treated larvae also showed significantly reduced primary motor neuron axon collaterals. Overall, these results show that developmental exposure to DomA leads to startle deficits by targeting specific subsets of neurons. These findings provide mechanistic insights into the neurodevelopmental effects of excess glutamatergic signaling caused by exposure to DomA. It further provides a model for using the startle response circuit to identify neuronal populations targeted by toxin or toxicant exposures. Summary statementWe used the zebrafish startle response as a tool to identify sensory-motor deficits and the loss of specific neural populations after developmental exposure to the harmful algal bloom toxin domoic acid. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/425996v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1aac99aorg.highwire.dtl.DTLVardef@199cb73org.highwire.dtl.DTLVardef@1fc1a00org.highwire.dtl.DTLVardef@f1dff_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2019

CRISPR-Cas9 mutated pregnane x receptor (pxr) retains pregnenolone-induced expression of cytochrome p450 family 3, subfamily A, polypeptide 65 (cyp3a65) in zebrafish (Danio rerio) larvae.

Pregnane x receptor (PXR) is a nuclear receptor that regulates transcriptional responses to drug or xenobiotic exposure in many vertebrate species. One key response is the induction of cytochrome P450 3A (CYP3A) transcription. PXR is a promiscuous receptor activated by a wide range of ligands that can differ across species, making functional studies on its role in the chemical defensome, most relevant when approached in a species-specific manner. Genetic knockout studies in mammals have shown a requirement for PXR in ligand-dependent activation of CYP3A expression or reporter gene activity. Transient knockdown in zebrafish revealed a similar requirement, however it is not known what the effect of a genetic knockout would be in the zebrafish model. Here, we report on generation of two zebrafish lines each carrying a genetic deletion in the pxr coding region, predicted to result in loss of a functional gene product. To our surprise zebrafish larvae, homozygous for either of the pxr mutant alleles, retain their ability to induce cyp3a65 mRNA expression following exposure to the established zebrafish Pxr ligand, pregnenolone (PN). Thus, zebrafish carrying pxr alleles with sizable deletions in either the DNA binding or the ligand binding domains do not yield a loss-of-function phenotype, suggesting that a compensatory mechanism is responsible for cyp3a65 induction. Two alternative possibilities are that Pxr is not required for the effect or that truncated yet functional mutant Pxr is sufficient for the effect.

pharmacology and toxicology↗