Multigenerational Neurobehavioural and Reproductive Toxicity of Arsenic in Zebrafish (Danio rerio)

Arsenic is a globally prevalent environmental toxicant known to affect neurological and reproductive functions, yet critical gaps remain in our understanding of its sex-specific toxicity and heritable effects across generations. This thesis investigated the multigenerational cognitive and reproductive toxicity of chronic dietary arsenic exposure in zebrafish (Danio rerio), with a focus on sex-specific susceptibility, epigenetic alterations, and lineage-dependent inheritance patterns. In the first study (Chapter 2), adult zebrafish were exposed to environmentally relevant dietary arsenic concentrations (30, 60, and 100 µg/g dry weight, as arsenite) for 60 days. A latent learning assay demonstrated significant impairments in cognitive performance at medium and high arsenic doses. These effects were associated with increased arsenic accumulation and dopamine levels in the brain, elevated oxidative stress, and altered expression of genes involved in dopaminergic signalling (drd1, th, and mao), neurotrophic support (bdnf), and purinergic signalling (entpd2_mg, entpd2_mq, and 5′-nucleotidase). These findings suggest that arsenic-induced cognitive dysfunction arises from oxidative imbalance and disruption of dopamine neurotransmission. Chapter 3 explored the intergenerational and transgenerational inheritance of these neurotoxic effects. Adult zebrafish (F0) exposed to arsenic were bred with unexposed partners to generate F₁ progeny via maternal and paternal lineages, which were further bred to produce F₂ offspring. Cognitive impairments were observed in both F1 and F2 generations, with maternal lineage effects emerging at lower arsenic doses compared to the paternal lineage. These behavioural deficits were associated with increased oxidative stress, dopaminergic dysregulation, and downregulation of drd1, mao, and bdnf in the brain. Promoter hypermethylation of these genes across F0 to F2 generations provided mechanistic evidence for epigenetic inheritance of arsenic-induced neurotoxicity. In Chapter 4, the sex-specific reproductive and developmental toxicity of chronic dietary arsenic exposure was examined. Adult male and female zebrafish were exposed separately to the same concentrations for 90 days and then bred with unexposed partners. Maternal arsenic exposure led to a dose-dependent decline in fecundity, fertilization, and hatching success, along with increased larval deformities and mortality. Paternal exposure also induced reproductive and developmental toxicity, though primarily at medium and high doses. Gene expression analysis revealed persistent downregulation of hypothalamus-pituitary-gonadal-liver (HPG-L) axis genes in females at all doses, whereas downregulation in males occurred only at higher doses. These results suggest that arsenic acts as an endocrine disruptor and causes more severe reproductive toxicity in females. Chapter 5 assessed the inheritance of arsenic-induced reproductive toxicity. F₁ offspring from maternal and paternal arsenic lineages were raised in clean conditions and assessed for reproductive performance in adulthood. Maternal lineage effects were evident even at the lowest arsenic dose, while paternal lineage effects appeared only at medium and high doses. Downregulation of sf-1 and cyp19a1a key regulators of the HPG axis was observed in both F0 and F₁ generations. Promoter hypermethylation of these genes in the brain and gonads further supported a role for epigenetic regulation in mediating multigenerational reproductive toxicity. Collectively, this thesis reveals that chronic dietary arsenic exposure causes sex-specific and heritable impairments in cognitive and reproductive function in zebrafish, driven by oxidative stress, endocrine disruption, and stable epigenetic modifications transmitted through both maternal and paternal lineages.

Name thesis:degree_name

Doctor of Philosophy (Ph.D.)

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Biology

Grantor

University of Saskatchewan

Year dc:date.issued

2025

Author dc:creator

• Rachamalla, Mahesh

Advisor dc:contributor.advisor

• Niyogi, Som

Committee members dc:contributor.committeemember

• Ham, Byung-Kook
• Moothathu, Suraj Unniappan
• Benson, James
• Gray, Jack
• Brinkmann, Markus

• Arsenic, zebrafish, reproductive toxicity, Cognitive impairment, multi and transgenerational toxicity

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10388/17089

OAI identifier oai:identifier

oai:harvest.usask.ca:10388/17089