Teratogenic, bioenergetic, and behavioral effects of exposure to total particulate matter on early development of zebrafish (Danio rerio) are not mimicked by nicotine

Abstract

Cigarette smoke has been associated with a number of pathologies; however, the mechanisms leading to developmental effects are yet to be fully understood. The zebrafish embryo is regarded as a ‘bridge model’; however, not many studies examined its applicability to cigarette smoke toxicity. This study examined the effects of total particulate matter (TPM) from 3R4F reference cigarettes on the early development of zebrafish (Danio rerio). Zebrafish embryos were exposed to two concentrations of TPM (0.4 and 1.4 μg/mL equi-nicotine units) or nicotine at equivalent doses. The exposures began at 2 h post-fertilization (hpf) and lasted until 96 hpf. Several physiological parameters were assessed during or after the exposure. We show that TPM increased mortality, delayed hatching, and increased the incidence of deformities in zebrafish. TPM exposure also increased the incidence of hemorrhage and disrupted the angiogenesis of the major vessels in the brain. Moreover, TPM exposure reduced the larval body length, decreased the heart rate, and reduced the metabolic rate. Biomarkers of xenobiotic metabolism and oxidative stress were also affected. TPM-exposed zebrafish also differed behaviorally: at 24 hpf the embryos had a higher frequency of spontaneous contractions and at 144 hpf the larvae displayed swimming hyperactivity. This study demonstrates that TPM disrupts several aspects of early development in zebrafish. The effects reported for TPM were not attributable to nicotine, since embryos treated with nicotine alone did not differ significantly from the control group. Collectively, our work illustrates the utility of zebrafish as an alternative model to evaluate the toxic effects of cigarette smoke constituents.

Glutathione and zebrafish: Old assays to address a current issue

“…Specifically, oxidative stress induction is supported by an increased ratio of GSSG to TGSH (or GSH), or a decreased ratio of TGSH (or GSH) to GSSG. While only four of the aforementioned zebrafish studies reported the ratios, the ratios supported the induction of oxidative stress in all four studies by Arnold et al (2016), Jaja-Chimedza et al (2012), and Massarsky et al (2013; 2015). Notably, the studies by Massarsky et al (2013; 2015) and Arnold et al (2016) used the methods described in the present manuscript.…”

“…In the studies reporting decreased GSH concentration, the concentration of GSSG was increased (Liu et al, 2015 - PCB 126), not significantly affected (Massarsky et al, 2013 – silver nanoparticles; Arnold et al, 2016 – selenomethionine), or decreased (Wu et al, 2011 - bisphenol A and nonylphenol). In the studies reporting increased GSH concentration, the concentration of GSSG was not significantly affected (Jaja-Chimedza et al, 2012 - bacterial lipopolysaccharides) or increased (Massarsky et al, 2015 – total particulate matter). It is noteworthy that the methods described herein allow for the concentrations of GSH and GSSG to be derived from the same sample, which is efficient in terms of cost, reagents, and time.…”

“…For example, exposure to antidepressant amitriptyline resulted in a U-shape response, such that lower concentrations decreased GSH concentration, while higher concentrations increased GSH concentration (Yang et al, 2014). Exposures to bacterial lipopolysaccharides (Jaja-Chimedza et al, 2012) and total particulate matter from cigarette smoke (Massarsky et al, 2015) also appeared to elevate GSH, while exposures to β-naphthoflavone and α-naphthoflavone (Timme-Laragy et al, 2009) did not significantly affect GSH concentrations.…”

“…The activity of GR is expected to increase with oxidative stress in order to recycle the generated GSSG. The activity of GR was increased when zebrafish were exposed to various toxicants, including zinc oxide nanoparticles (Zhao et al, 2013), di-n-butyl phthalate and diethyl phthalate (Xu et al, 2013), and total particulate matter (Massarsky et al, 2015). Some studies reported no significant difference in GR activity when zebrafish were exposed to cyanobacterial lipopolysaccharides (Jaja-Chimedza et al, 2012) and silver nanoparticles (Massarsky et al, 2013).…”

“…With regards to oxidative stress, various parts of the zebrafish antioxidant system, including antioxidant enzyme activities and glutathione concentrations, were assessed previously (e.g. Massarsky et al, 2013; 2015). Given the importance of glutathione within the antioxidant defense system, this manuscript aims to outline detailed protocols to assess GSH and GSSG concentrations, as well as GPx and GR activities in zebrafish larvae.…”