DOI: 10.4231/hgw3-es04
title: Multigenerational Lipid Alterations in Zebrafish Larvae (120 hpf) Following Parental Embryonic Exposure to Low-Dose Lead (Pb)

description: &lt;p style="text-align:justify; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;b&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;Description&lt;/span&gt;&lt;/b&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p style="text-align:justify; text-indent:.5in; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;This study was based on the hypothesis that low doses of lead (Pb) during embryonic development can induce behavioral, biochemical, and lipid alterations in zebrafish (Danio rerio) offspring across generations. To test this, wild-type (5D strain) adult zebrafish were bred, and fertilized eggs were collected and transferred to Petri plates (50 embryos per plate). Embryos were exposed to 0, 0.01, 0.1, or 1 ppb Pb (ng/L) in embryo water (20 mL) from 1 to 72 hours post-fertilization (hpf). At 72 hpf, Pb solutions were removed by rinsing embryos three times with embryo water. Fish were then reared to 4 months of age, at which point they were sex-segregated and bred at a 1:3 male-to-female ratio. Fertilized eggs from Pb-exposed parents (F0) were designated as the F1 generation. F1 embryos (50 per plate) were collected and assessed for behavioral endpoints including sociability, anxiety-like behavior, learning, decision-making, and exploration. A subset of F1 fish were reared to 4 months of age and bred in the same manner to produce the F2 generation. Since no behavioral alterations were observed in F2 offspring, only the F1 generation was analyzed further for targeted MRM profiling at 120 hpf, as described below.&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p style="text-align:justify; text-indent:.5in; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;For lipidomic profiling, one larva from each Petri dish was collected at 120 hpf and transferred into individual Eppendorf tubes. A total of eight larvae per treatment group were obtained from eight independent biological replicates (n = 8). Lipid analysis was carried out using multiple reaction monitoring (MRM) profiling following previously established methods (Reis et al., 2023; Xie et al., 2021). After transfer, larvae were placed on ice until movement stopped, residual water was carefully removed, and 50 &micro;L of ultrapure water was added. The samples were flash-frozen in liquid nitrogen and kept at &ndash;80 &ordm;C for no longer than one week before processing.&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p style="text-align:justify; text-indent:.5in; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;For extraction, frozen samples were thawed and homogenized in ultrapure water for 30 s using pellet pestles (Kimble Kontes). Lipids were isolated according to the Bligh and Dyer method, as described in Lima et al. (2018). Briefly, 90 &micro;L of HPLC-grade methanol was first added, followed by 50 &micro;L of HPLC-grade chloroform, with mixing after each addition to ensure a monophasic solution. Samples were incubated at room temperature for 15 min, after which 50 &micro;L of ultrapure water was added, mixed, and then followed by the addition of 50 &micro;L chloroform. The mixtures were centrifuged at 5000 &times; g for 5 min at room temperature, and the lower organic phase was transferred to clean tubes and dried in a SpeedVac concentrator for 12 h at room temperature. Dried extracts were resuspended in 200 &micro;L of acetonitrile:methanol:ammonium acetate (3:6.65:0.35, v/v/v), adjusted to a final concentration of 10 mM ammonium acetate.&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p style="text-align:justify; text-indent:.5in; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;Before acquisition, extracts were diluted 1:7 in the same solvent mixture. Lipid detection was performed using flow injection without chromatographic separation, as described by Edwards et al. (2021). In short, 10 &mu;L of each diluted extract was introduced into the electrospray ionization (ESI) source of an Agilent 6410 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) via a G1377A micro-autosampler. The system operated with a capillary pump at 8 &mu;L/min and 150 bar. Instrument settings included a capillary voltage of 5 kV and a drying gas flow of 5.1 L/min at 300&deg;C.&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p style="text-align:justify; margin-bottom:11px"&gt; &lt;/p&gt;

&lt;p style="text-align:justify; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;b&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;Notes:&lt;/span&gt;&lt;/b&gt;&lt;span new="" roman="" style="font-family:" times=""&gt; Data were generated on an Agilent 6410 QQQ system. Raw MRM spectra were processed with an in-house script to extract MRM transitions. Only ions with intensities at least 30% greater than blanks were retained for downstream analyses. Ion intensities were normalized to the Total Ion Current (TIC) within each lipid class.&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p style="text-align:justify; margin-bottom:11px"&gt; &lt;/p&gt;

&lt;p style="text-align:justify; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;b&gt;&lt;span lang="PT-BR" new="" roman="" style="font-family:" times=""&gt;References&lt;/span&gt;&lt;/b&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p class="MsoBibliography" style="text-align:justify; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span lang="PT-BR" new="" roman="" style="font-family:" times=""&gt;Reis LG, Casey TM, Sobreira TJ, Cooper BR, Ferreira CR, 2023. &lt;/span&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;Step-by-Step approach to build multiple reaction monitoring (MRM) profiling instrument acquisition methods for class-based lipid exploratory analysis by mass spectrometry. Journal of Biomolecular Techniques: JBT 34&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p class="MsoBibliography" style="text-align:justify; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;Xie Z, Ferreira CR, Virequ AA, Cooks RG, 2021. Multiple reaction monitoring profiling (MRM profiling): Small molecule exploratory analysis guided by chemical functionality. Chemistry and Physics of Lipids 235, 105048&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p class="MsoBibliography" style="text-align:justify; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span lang="PT-BR" new="" roman="" style="font-family:" times=""&gt;de Lima CB, Ferreira CR, Milazzotto MP, Sobreira TJP, Vireque AA, Cooks RG, 2018. &lt;/span&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;Comprehensive lipid profiling of early stage oocytes and embryos by MRM profiling. Journal of mass spectrometry: JMS 53, 1247&ndash;1252&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p class="MsoBibliography" style="text-align:justify; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;Edwards ME, Marasco Jr CA, Schock TB, Sobreira TJ, Ferreira CR, Cooks RG, 2021. Exploratory analysis using MRM profiling mass spectrometry of a candidate metabolomics sample for testing system suitability. International Journal of Mass Spectrometry 468, 116663&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;
description: Description of data: Targeted-MRM profiling of whole zebrafish larvae at 120 hours post-fertilization (hpf), derived from parents developmentally exposed to low Pb concentrations (0, 0.01, 0.1, or 1 ppb) during 1–72 hpf.