Fluoranthene (FLA) is Cardiotoxic and Induces Cytochrome P450 Type 1A (CYP1A) mRNA Expression in Xenopus laevis Embryos

Madison Sestak, Caithlin Lamoureux, Jeffrey Hall, Jillian Coburn, and Susan Whittemore, Ph.D, Keene State College 03435 (3162-422)

Introduction

MethodsExposure Protocols: Embryos were obtained by breeding albino adults from the Whittemore lab’s X. laevis colony, exposed to 0.02-0.04% DMSO, FLA, or BaP as described in figure legends, and maintained at 22°C. Media was renewed daily. Cardiac Function (HR, AV): Prior to recording atrial and ventricular heart rates in beats/minute, embryos were acclimated to RT for 1 h and anesthetized with MS-222. 20 second videos were taken using Leica imaging™ and Camtasia ™ editing software.  Atrioventricular block was determined as atrial > ventricular HR and full block as an absence of ventricular contractions for the 20 s period. Pericardial edema and snout to vent length: Exposed embryos were assessed for pericardial edema (mm2) and snout-to-vent length (mm) using ImageJ software.Statistics: All data was analyzed using one way ANOVA followed by Dunnett’s multiple comparison test, ; * indicates a difference, p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001, from the vehicle control. Gene Expression Assays: Purified RNA was obtained from whole embryos (NF stage 42, 72 hpe) using an RNaqueous™ protocol followed by TURBO DNA-free™ treatment (both Life Technologies, Carlsbad CA, USA). mRNA was reverse transcribed using a High Capacity RNA to cDNA™ kit (Life Technologies) and quantitative RT-PCR was performed using a StepOnePlus™ system (Applied Biosystems, Inc., ABI) and Taqman® Gene Expression Assays (ABI) for cytochrome P450 type 1A6 (cyp1a6), cytochrome P450 type 1A7 (cyp1a7), T-box transcription factor (tbx5)12, NK2 Homobox 5 (Nnkx2.5)13 and ornithine decarboxylase (odc1, endogenous control) mRNA14.

Figure 1. (a) Ventricular and atrial heart rates in beats/min for vehicle control (DMSO) and PAH-treated animals at four stages of heart development (stage 37 [48 hpe], stage 42 [72 hpe], stage 45 [96 hpe], and stage 46/47 [120 hpe]). Means ± S.E.M for n=6 batches/treatment (20-53 animals). * indicates a difference p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 from the vehicle control (b) Percent of animals that exhibited AV block with area in black indicating incidence of full ventricular block. 7Nieuwkoop PD, Faber J, (Eds): Normal Table of Xenopus laevis (Daudin). 1994, New York: Garland Publishing

Results

Gene ExpressionWindows of Exposure

Acknowledgments: This project is supported by the New Hampshire IDeA Network of Biological Research Excellence (NH-INBRE) NIH Grant Number 1P20RR030360-01 from the INBRE program of the National Center for Research Resources. An additional thank you to Julia Pinette for her work on the heart rates and Jessica Dude for her help on the mortality data.

ConclusionsAnimals exposed from 0-24 h exhibited tachycardia at 72 h suggesting that pacemaker activity can be altered with an early limited FLA exposure In contrast, AV block (120 hpe) was only evident for those animals exposed to FLA at the time

of measurementFLA exposure was associated with increased expression of cyp1a suggesting that FLA may act as an AHR agonist in Xenopus Increased expression of the cardiac transcription factors tbx5 and nkx2.5 with exposure to 0.25 and

2.5, but not 25, μM FLA, suggests a possible low dose effect. Among other impacts, over expression of nkx2.5 has been shown to induce enlarged hearts in Xenopus19, while increased tbx5 expression is associated with advanced cardiomyocyte development20

Differences in FLA and BaP-induced effects on cardiac function between the early life stages of fish and frog indicate species specificity regarding impacts of developmental PAH exposure

Future DirectionsEROD assays to determine if CYP1A enzyme activity is induced with FLA Examine the long-term cardiac impacts, including heart size, of low

dose exposures to FLA Determine cardiac or cyp1a expression responses to mixtures of

FLA and BaPPreliminary findings:

VentricleAtrium

Mortality Increased mortality with highest dose of FLA (83% mortality at day 8, 94% mortality at day 10)Growth 6.6% decrease in snout-to-vent length at stage 46/47 with highest dose of FLAPericardial Edema No pericardial edema was observed

F, forward primer; P, probe; R, reverse primer10Fujita et al.(1999) Arch. Biochem. Biophys. 371(1): 24-2811Zhou et al. (2009) Mol. Cell Biol. 29(7): 1786-179512Brown et al. (2005) Development 132(3): 553-56313Keren-Politansky et al. (2009) Dev. Biol. 335(2): 374-384.14Sindelka et al. (2006) Dev. Dyn. 235 (3): 754-8

FLA

BaP

Biotransformation Pathway

Polycyclic aromatic hydrocarbons (PAHs) are present in air, soil, water, and food.1 Their cardiotoxicity has been extensively investigated in fish. 2-5 To expand existing understanding of mechanism(s) of toxicity for certain priority PAHs, shown to cross the human placenta, we studied the effect of exposure to pyrene, PYR; phenanthrene, PHE; fluoranthene, FLA; and benzo(a)pyrene, BaP on cardiac function. We used a model system of human heart development, the early life stages of Xenopus laevis, that has low sensitivity to aryl hydrocarbon receptor (AHR) agonists (e.g. TCDD),6 to compare with results derived from fish (Fig 1).

With the notable exception of BaP, exposure to the other priority PAHs was associated with abnormal cardiac pacemaker and conduction activity. One of the more surprising findings was the severity of cardiotoxicity associated with FLA exposure. To our knowledge, exposure to FLA as a single compound does not affect normal cardiac function in fish.8,9

ObjectivesTo examine this finding further, we investigated:

whether FLA exposure affected survival, growth, or incidence of pericardial edema the impact on cardiac function after varying the duration and timing of FLA exposurethe effect of FLA on expression of cytochrome P450 type 1A1 (cyp1a1) and two transcription factors

essential to heart development (tbx5 and nkx2.5)

Figure 4. Incidence of atrioventricular block measured as a percentage of PAH exposed animals for stage 46/47 (n=5 batches, 60-70 animals). Data were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test, ‘a’ represents a difference (p ≤ 0.05) from DMSO (vehicle control) .

Recreated from:Castell et al. (2005) 57: 189-204

NF stage 46/47: Mature heart formed

NF Stage 45: Atrial Partitioning almost complete

NF Stage 42:Valve formation

NF Stage 37: Looping of heart complete

Figure 3. Fold change in mRNA expression, relative to vehicle control (=1), for (a) cytochrome P450 type 1a (cyp1a6 and cyp1a7) and for (b) the cardiac transcription factors tbx5 and nkx2.5 following 72 h exposure to fluoranthene (FLA) and benzo(a)pyrene (BaP). Values were calculated using the ΔΔCT method and are expressed as a range (incorporating the standard deviation).18 Each sample represents a pool of 10-11 whole embryos (NF stage 42) (n= 4-6 samples/treatment; 3 batches). * indicates a difference, p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001, from the vehicle control.18Livak and Schmittgen (2001). Methods in Mol Biol 25: 402-408.

Exposure Protocol

1Agency of Toxic Substances and Disease Registry (1995). USPHS HHS; 2Incardona et al. (2004) Toxicol App. Pharacol 196(2):191-205; 3Incardona et al. (2005) Environ Health Persp 113(2):1755-1762; 4Scott et al. (2011) Aquat Toxicol 101:165-1745;5Hatlen et al. (2010) Aquat Toxicol 99:56-64; 6Zimmermann et al. (2008) Toxicol Sci 104(1):124-134 The cytochrome P4501A (cyp1a 6 and 7 for X. laevis) genes are induced with AHR activation6.

The cardiotoxicity associated with certain PAHs is mediated through the AHR2-5.

The two cardiac transcription factors nkx2.5 and tbx5 are essential for normal cardiac development. Exposure to certain PAHs has been shown to alter their gene expression15-17.

15Zhang et al. (2012) Aqu Toxicol: 119-124, 16Zhang et al. (2013) Aqu Toxicol: 26-32, 17Bartlett et al. (2007) Dev Dyn 236: 2475-2484

Timing and duration of exposure to25μM FLA

Atrioventricular Block NF Stage 46/47

Target cDNA (GenBank #) Primer/Probe Sequencecyp1a6 (NM_1172237)10 F: 5’–GCAGTATTGGCTTAACTCCTGG-3’

P: 5’-TGTTGCAAAAGTCTTGGTTCCCCAG-3’R: 5’-CCCTTTCTATTCTCTGCCCTG-3’

cyp1a7 (NM_1097072)10 F: 5’–GTTCTAGTTACTTCCCTGCCC-3’P: 5’-TTGTTTCCCCTTGCACTTTTGGTTCC-3’R: 5’-ATAGTTTGTACATTTGCTCTTGCC-3’

odc1 (NM_1086698)11 F: 5’–CTGTTAGGAGCTTTGTCAGAGG-3’P: 5’-TGGTTATTTGTGGCCTCGATGGGT-3’R: 5’-ACAAGTCCTACGCTCTCAATG-3’

tbx5 (NM_001085701.1)12 F: 5’-GCCAGCTGAGCATTCCTATAA-3’P: 5’-TTCACCAAGCGAAGAGGACCCTTT-3’R: 5’-AGAGGTGGTAGATGAGGAAGAG-3’

nkx2.5 (NM_001086721.1)13 F: 5’-GAAATGGGTTGCAGGGATTTG-3’P: 5’-AGCTACAGTTGGGTGTGTGTGGTT-3’R: 5’-GACATCTGACCGTCGCATATT-5’

(b)

(a)

(b)

8VanTiem and DiGiullo (2011) Toxicol Appl Pharmaco 254(3):280-287. 9Jayasundara et al. (2015) Toxicol Sci 143(2):469-481.

(a)7

Figure 2. (a) Atrial and ventricular heart rates in beats/min for two stages of heart development (NF stage 42 and 46/47) where the timing and duration of FLA (25 μM) was varied. Means ± S.E.M for n=4 batches(23-42 animals).. * indicates a difference, p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001, from the vehicle control. (b) Percent of animals that exhibited AV block with area in black indicating incidence of full ventricular block.

19Cleaver et al. (1996) Development 122:3549-56, 20Herrmann et al. (2011) Dev Dyn. 240: 2634-2645

FLA BaP