Adult-Onset Transcriptomic Effects of Developmental Exposure to Benzene in Zebrafish (Danio rerio): Evaluating a Volatile Organic Compound of Concern.

Urban environments are afflicted by mixtures of anthropogenic volatile organic compounds (VOCs). VOC sources that drive human exposure include vehicle exhaust, industrial emissions, and oil spillage. The highly volatile VOC benzene has been linked to adverse health outcomes. However, few studies have focused on the later-in-life effects of low-level benzene exposure during the susceptible window of early development. Transcriptomic responses during embryogenesis have potential long-term consequences at levels equal to or lower than 1 ppm, therefore justifying the analysis of adult zebrafish that were exposed during early development. Previously, we identified transcriptomic alteration following controlled VOC exposures to 0.1 or 1 ppm benzene during the first five days of embryogenesis using a zebrafish model. In this study, we evaluated the adult-onset transcriptomic responses to this low-level benzene embryogenesis exposure (n = 20/treatment). We identified key genes, including col1a2 and evi5b, that were differentially expressed in adult zebrafish in both concentrations. Some DEGs overlapped at the larval and adult stages, specifically nfkbiaa, mecr, and reep1. The observed transcriptomic results suggest dose- and sex-dependent changes, with the highest impact of benzene exposure to be on cancer outcomes, endocrine system disorders, reproductive success, neurodevelopment, neurological disease, and associated pathways. Due to molecular pathways being highly conserved between zebrafish and mammals, developmentally exposed adult zebrafish transcriptomics is an important endpoint for providing insight into the long term-effects of VOCs on human health and disease.

Evaluating Phenotypic and Transcriptomic Responses Induced by Low-Level VOCs in Zebrafish: Benzene as an Example.

Urban environments are plagued by complex mixtures of anthropogenic volatile organic compounds (VOCs), such as mixtures of benzene, toluene, ethylene, and xylene (BTEX). Sources of BTEX that drive human exposure include vehicle exhaust, industrial emissions, off-gassing of building material, as well as oil spillage and leakage. Among the BTEX mixture, benzene is the most volatile compound and has been linked to numerous adverse health outcomes. However, few studies have focused on the effects of low-level benzene on exposure during early development, which is a susceptible window when hematological, immune, metabolic, and detoxification systems are immature. In this study, we used zebrafish to conduct a VOC exposure model and evaluated phenotypic and transcriptomic responses following 0.1 and 1 ppm benzene exposure during the first five days of embryogenesis (n = 740 per treatment). The benzene body burden was 2 mg/kg in 1 ppm-exposed larval zebrafish pools and under the detection limit in 0.1 ppm-exposed fish. No observable phenotypic changes were found in both larvae except for significant skeletal deformities in 0.1 ppm-exposed fish (p = 0.01) compared with unexposed fish. Based on transcriptomic responses, 1 ppm benzene dysregulated genes that were implicated with the development of hematological system, and the regulation of oxidative stress response, fatty acid metabolism, immune system, and inflammatory response, including apob, nfkbiaa, serpinf1, foxa1, cyp2k6, and cyp2n13 from the cytochrome P450 gene family. Key genes including pik3c2b, pltp, and chia.2 were differentially expressed in both 1 and 0.1 ppm exposures. However, fewer transcriptomic changes were induced by 0.1 ppm compared with 1 ppm. Future studies are needed to determine if these transcriptomic responses during embryogenesis have long-term consequences at levels equal to or lower than 1 ppm.

Degron capability of the hydrophobic C-terminus of the polyglutamine disease protein, ataxin-3.

Ataxin-3 is a deubiquitinase and polyglutamine disease protein whose cellular properties and functions are not entirely understood. Mutations in ataxin-3 cause spinocerebellar ataxia type 3 (SCA3), a neurodegenerative disorder that is a member of the polyglutamine family of diseases. Two major isoforms arise from alternative splicing of ATXN3 and are differently toxic in vivo as a result of faster proteasomal degradation of one isoform compared to the other. The isoforms vary only at their C-termini, suggesting that the hydrophobic C-terminus of the more quickly degraded form of ataxin-3 (here referred to as isoform 2) functions as a degron-that is, a peptide sequence that expedites the degradation of its host protein. We explored this notion in this study and present evidence that: (a) the C-terminus of ataxin-3 isoform 2 signals its degradation in a proteasome-dependent manner, (b) this effect from the C-terminus of isoform 2 does not require the ubiquitination of ataxin-3, and (c) the isolated C-terminus of isoform 2 can enhance the degradation of an unrelated protein. According to our data, the C-terminus of ataxin-3 isoform 2 is a degron, increasing overall understanding of the cellular properties of the SCA3 protein.

Interaction of the polyglutamine protein ataxin-3 with Rad23 regulates toxicity in Drosophila models of Spinocerebellar Ataxia Type 3.

Polyglutamine (polyQ) repeat expansion in the deubiquitinase ataxin-3 causes neurodegeneration in Spinocerebellar Ataxia Type 3 (SCA3), one of nine inherited, incurable diseases caused by similar mutations. Ataxin-3's degradation is inhibited by its binding to the proteasome shuttle Rad23 through ubiquitin-binding site 2 (UbS2). Disrupting this interaction decreases levels of ataxin-3. Since reducing levels of polyQ proteins can decrease their toxicity, we tested whether genetically modulating the ataxin-3-Rad23 interaction regulates its toxicity in Drosophila. We found that exogenous Rad23 increases the toxicity of pathogenic ataxin-3, coincident with increased levels of the disease protein. Conversely, reducing Rad23 levels alleviates toxicity in this SCA3 model. Unexpectedly, pathogenic ataxin-3 with a mutated Rad23-binding site at UbS2, despite being present at markedly lower levels, proved to be more pathogenic than a disease-causing counterpart with intact UbS2. Additional studies established that the increased toxicity upon mutating UbS2 stems from disrupting the autoprotective role that pathogenic ataxin-3 has against itself, which depends on the co-chaperone, DnaJ-1. Our data reveal a previously unrecognized balance between pathogenic and potentially therapeutic properties of the ataxin-3-Rad23 interaction; they highlight this interaction as critical for the toxicity of the SCA3 protein, and emphasize the importance of considering protein context when pursuing suppressive avenues.

The deubiquitinase ataxin-3 requires Rad23 and DnaJ-1 for its neuroprotective role in Drosophila melanogaster.

Ataxin-3 is a deubiquitinase and polyglutamine (polyQ) disease protein with a protective role in Drosophila melanogaster models of neurodegeneration. In the fruit fly, wild-type ataxin-3 suppresses toxicity from several polyQ disease proteins, including a pathogenic version of itself that causes spinocerebellar ataxia type 3 and pathogenic huntingtin, which causes Huntington's disease. The molecular partners of ataxin-3 in this protective function are unclear. Here, we report that ataxin-3 requires its direct interaction with the ubiquitin-binding and proteasome-associated protein, Rad23 (known as hHR23A/B in mammals) in order to suppress toxicity from polyQ species in Drosophila. According to additional studies, ataxin-3 does not rely on autophagy or the proteasome to suppress polyQ-dependent toxicity in fly eyes. Instead this deubiquitinase, through its interaction with Rad23, leads to increased protein levels of the co-chaperone DnaJ-1 and depends on it to protect against degeneration. Through DnaJ-1, our data connect ataxin-3 and Rad23 to protective processes involved with protein folding rather than increased turnover of toxic polyQ species.