Toxicology Reports 2 (2015) 976–983 Contents lists available at ScienceDirect Toxicology Reports j ournal homepage: www.elsevier.com/locate/toxrep Butafenacil: A positive control for identifying anemia- and variegate porphyria-inducing chemicals ∗ Jessica K. Leet, Rachel A. Hipszer, David C. Volz Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, SC 29208, USA a r t i c l e i n f o a b s t r a c t Article history: Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPOX), an enzyme that catalyzes Received 20 April 2015 oxidation of protoporphyrinogen IX to protoporphyrin IX during chlorophyll and heme biosynthesis. Received in revised form 3 June 2015 Based on a high-content screen, we previously identified butafenacil as a potent inducer of anemia in Accepted 6 July 2015 zebrafish embryos. Therefore, the objective of this study was to begin investigating the utility of butafe- Available online 13 July 2015 nacil as a positive control for identifying anemia- and variegate porphyria-inducing chemicals. Static exposure to butafenacil from 5 to 72 h post-fertilization (hpf) in glass beakers resulted in a concentration- Keywords: dependent decrease in arterial circulation at low micromolar concentrations. At 72 hpf, the magnitude Butafenacil Zebrafish of butafenacil-induced anemia was similar when embryos were exposed in the presence or absence of Anemia light, whereas protoporphyrin accumulation and acute toxicity were significantly lower or absent when Porphyria embryos were exposed under dark conditions. To identify sensitive developmental windows, we treated Drug development embryos to butafenacil from 5, 10, 24, or 48 hpf to 72 hpf in the presence of light, and found that ane- mia and protoporphyrin accumulation were present at 72 hpf following initiation of exposure at 5 and 10 hpf. On the contrary, protoporphyrin accumulation – but not anemia – was present following ini- tiation of exposure at 24 hpf. Lastly, protoporphyrin accumulation at 72 hpf after exposure from 24 to 48 hpf suggests that protoporphyrin was not eliminated over a 24-h recovery period. Collectively, our data suggests that butafenacil may be a reliable positive control for identifying anemia- and variegate porphyria-inducing chemicals. © 2015 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction As butafenacil targets PPOX in both plants and animals, expo- sure of rodents (rats and mice) and aquatic plants (algae, diatoms, Butafenacil is a broad-spectrum imide herbicide that inhibits and duckweed) to butafenacil results in hematologic abnormalities protoporphyrinogen oxidase (PPOX), an enzyme that catalyzes oxi- and inhibition of growth, respectively, at relatively low concentra- dation of protoporphyrinogen IX to protoporphyrin IX – the last tions [14]. However, due to low application rates (one application common step of chlorophyll and heme biosynthesis in plants and of <50 g ai/ha per year), rapid environmental degradation (t1/2 < 5 animals, respectively. Similar to other post-emergent herbicides, days), and little to no potential for bioaccumulation (t1/2 < 3 days), butafenacil is phytotoxic on contact and provides rapid knock- butafenacil is not expected to pose a significant human health nor down of broadleaf and grass weeds [14]. Butafenacil has been ecological risk following agricultural use according to the label registered for agricultural use within Australia, Argentina, Brazil, [14]. Based on a high-content screen using zebrafish embryos, we Japan, Switzerland, and Thailand since the early 2000s, and, within previously revealed that exposure to butafenacil from 5 to 72 h these countries, is sold as a pre-mixed formulation containing other post-fertilization (hpf) abolished blood cell and hemoglobin pro- post-emergent herbicides such as triasulfuron and glyphosate [14]. duction in the absence of effects on survival, body length, cardiac function, and blood vessel development [13]. Therefore, based on this study, we identified butafenacil as a potential positive con- trol for identifying chemicals that induce blood disorders such as Abbreviations: ai, active ingredient; DMSO, dimethyl sulfoxide; EM, embryo media; hpf, hours post-fertilization; MS-222, tricaine methanesulfonate; PPOX, pro- anemia and variegate porphyria. toporphyrinogen oxidase; RO, reverse osmosis. Anemia is an inherited or acquired disorder that is character- ∗ Corresponding author at: University of South Carolina, Department of Envi- ized by (1) abnormally low levels of circulating red blood cells due ronmental Health Sciences, 921 Assembly St Public Health Research Center 401, to decreased red blood cell production, blood loss, or hemolysis Columbia, SC 29208, USA. Fax: +1 803 777 3391. or (2) normal levels of circulating red blood cells that are micro- E-mail address: [email protected] (D.C. Volz). http://dx.doi.org/10.1016/j.toxrep.2015.07.006 2214-7500/© 2015 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). J.K. Leet et al. / Toxicology Reports 2 (2015) 976–983 977 cytic and/or hypochromic. As a result of decreased oxygen delivery a five-shelf stand-alone system (Aquatic Habitats, Inc., Apopka, FL, throughout the body, patients with anemia experience a variety USA) containing photoperiod light-cycle enclosures and recircu- of symptoms (the most common being fatigue) and, in severe and lating conditioned reverse osmosis (RO) water. Dissolved oxygen, chronic cases, irreversible damage to vital organs [7]. Within the pH, conductivity, salinity, alkalinity, and temperature within recir- United States, anemia is the most prevalent blood disorder across culating water were maintained at 4-6 mg/L, 6.5–7.5, 425–475 ␮S, ◦ all age, racial, and ethnic groups. Iron-deficiency anemia is the most <1 ppt, 50–100 mg/L, and 27–28 C, respectively; in addition, lev- common form of anemia within American children, where preva- els of ammonia, nitrite, and nitrate within recirculating water lence is as high as 14% within children between 1 and 2 years were consistently below 0.1 mg/L, 0.05 mg/L, and 2 mg/L, respec- of age (http://www.cdc.gov/nchs/fastats/anemia.htm). Anemia can tively. Adult females and males were bred directly on-system also be caused by chronic exposure to certain drugs used to treat using in-tank breeding traps suspended within 3-L tanks. For all cancer and rheumatoid arthritis [11]. experiments described below, newly fertilized eggs were staged Porphyrias are a rare class of disorders caused by abnormal according to previously described methods [10]. All fish were han- heme production, leading to accumulation of porphyrins or por- dled and treated in accordance with approved Institutional Animal phyrin precursors within the liver and other organs [17]. Within the Care and Use Committee protocols at the University of South Car- United States, less than 200,000 people are diagnosed with acute olina – Columbia. or cutaneous porphyria, with the most prevalent form (porphyria cutanea tarda) resulting from chemically-induced deficiencies in 2.2. Chemicals uroporphyrinogen decarboxylase [1]. Variegate porphyria is a hep- atic porphyria caused by a human PPOX mutation that decreases Butafenacil (99.3% purity) was purchased from Sigma–Aldrich oxidation and conversion of protoporphyrinogen IX to proto- (St. Louis, MO). Stock solutions of each chemical were prepared by porphyrin IX [2]. During acute attacks, patients with variegate dissolving chemicals in high performance liquid chromatography- porphyria can experience abnormal skin reactions to sunlight (or grade dimethyl sulfoxide (DMSO) (50 mM), and then performing photodermatitis) due to reactive singlet oxygen formation and two-fold serial dilutions into DMSO to create stock solutions oxidative stress following photooxidation of protoporphyrinogen for each working solution. All stock solutions were stored at to protoporphyrin [2]. Moreover, certain drugs (e.g., barbiturates) room temperature within 2-mL amber glass vials containing and antibiotics (e.g., sulfonamides) can trigger acute symptoms of polytetrafluoroethylene-lined caps. For each exposure, working porhyrias such as photodermatitis, underscoring the importance solutions of all treatments were freshly prepared by spiking stock of screening for potential effects on heme production during drug solutions into embryo media (EM) (5 mM NaCl, 0.17 mM KCl, development. 0.33 mM CaCl2, 0.33 mM MgSO4), resulting in 0.1% DMSO within As the identification of potential adverse hematologic effects is all vehicle control and treatment groups. an important consideration during drug development, the objective of this study was to begin investigating the utility of butafe- 2.3. Embryonic exposures nacil as a positive control for identifying anemia- and variegate porphyria-inducing chemicals. Our working hypothesis was that, Newly fertilized eggs were collected immediately after spawn- similar to variegate porphyria, butafenacil-mediated inhibition of ing and placed in groups of approximately 100 per petri dish within PPOX in transparent zebrafish embryos results in accumulation of a light- and temperature-controlled incubator until 5 hpf. Prior to protoporphyrin and increased acute toxicity when reared under each experiment, 50-mL glass beakers were thoroughly rinsed with light