JOURNAL OF APPLIED TOXICOLOGY TOXICITY OF AFLATOXIN B1 AND T-2 TOXIN 139 J. Appl. Toxicol. 2006; 26: 139–147 Published online 17 October 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jat.1117 Comparative acute and combinative toxicity of aflatoxin B1 and T-2 toxin in animals and immortalized human cell lines Christopher McKean, Lili Tang, Madhavi Billam, Meng Tang, Christopher W. Theodorakis, Ronald J. Kendall and Jia-Sheng Wang* The Institute of Environmental and Human Health, Department of Environmental Toxicology, Texas Tech University, Box 41163, Lubbock TX 79409-1163, USA Received 27 September 2004; Revised 14 June 2005; Accepted 8 August 2005 ABSTRACT: Aflatoxin B1 (AFB1) and T-2 toxin (T-2) are important food-borne mycotoxins that have been implicated in human health and as potential biochemical weapons threats. In this study the acute and combinative toxicity of AFB1 and T-2 were tested in F-344 rats, mosquitofish (Gambusia affinis), immortalized human hepatoma cells (HepG2) and human bronchial epithelial cells (BEAS-2B). Preliminary experiments were conducted in order to assess the acute toxicity and to obtain LD50, LC50 and IC50 values for individual toxins in each model, respectively. This was followed by testing combinations of AFB1 and T-2 to obtain LD50, LC50 and IC50 values for the combination in each model. All models demonstrated a significant dose response in the observed parameters to treatment. The potency of the mixture was gauged through the determination of the interaction index metric. The results of this study demonstrate that these two toxins interacted to produce alterations in the toxic responses generally classifiable as additive; however, a synergistic interaction was noted in the case of BEAS-2B. It can be gathered that this combination may pose a significant threat to public health and further research needs to be completed addressing alterations in metabolism and detoxification that may influence the toxic manifestations in combination. Copyright © 2005 John Wiley & Sons, Ltd. KEY WORDS: aflatoxin; T-2 toxin; biotoxin; mycotoxin; combinative toxicity; cytotoxicity Introduction flavus, Aspergillus parasiticus and to a lesser extent Aspergillus nominus. Three strains of Aspergillus have Toxic fungal metabolites, mycotoxins, are structurally been found from which four major AFs (AFB1, AFB2, diverse compounds that represent the most important cat- AFG1 and AFG2) are produced. AFB1 is the most pre- egory of biologically produced toxins relative to human valent and toxic of the AFs, with acute toxicity demon- health and economic impact worldwide (Cole and Cox, strated in all species of animals, birds and fish tested −1 1981; Ciegler et al., 1981). Spurred by the discovery of resulting in LD50 values in the range 0.3–9.0 mg kg body aflatoxin in the 1960s the first cases of mycotoxicoses weight (bw). AFB1 is also known to be one of the most were noted, leading to the identification of more than 100 potent genotoxic agents and hepatocarcinogens identified toxigenic fungi and in excess of 300 mycotoxins world- (Busby and Wogan, 1984; Sharma and Salunkhe, 1991; wide (Sharma and Salunkhe, 1991; Miller and Trenholm, Miller and Trenholm, 1994; Wang et al., 1998). 1994). These mycotoxins display diverse chemical struc- The toxicity and carcinogenicity of AFB1 is thought tures accounting for their differing biological properties to be directly linked to its bioactivation, resulting and effects. Depending upon the toxins’ precise bio- in a highly reactive AFB1 8, 9-epoxide (AFBO). This chemical nature, they may have any of a number of toxic bioactivation of AFB1 occurs primarily by a microsomal properties including being carcinogenic, tetratogenic, cytochrome P450 (CYP450) dependent epoxidation of mutagenic, oestrogenic, neurotoxic or immunotoxic. the terminal furan ring of AFB1 and is responsible for Aflatoxins (AFs) represent a group of closely related binding to cellular macromolecules such as RNA, DNA difuranocoumarin compounds produced as secondary and other protein constituents (Massey et al., 1995). fungal metabolites of the common molds Aspergillus Damage to and necrosis of hepatocytes as well as other metabolically active cells is believed to be the result of this process (Eaton and Groopman, 1994). * Correspondence to: Dr Jia-Sheng Wang, The Institute of Environmental and Human Health and Department of Environmental Toxicology, Texas Tricothecenes are a group of mycotoxins produced Tech University, Box 41163, Lubbock TX 79409-1163, USA. by Fusarium species. One of the most important trico- E-mail: [email protected] thecenes is T-2 toxin, which is the common name for Contract/grant sponsor: Research Development and Engineering Command, U.S. Army; contract/grant number: DAAD13-00-C-0056; DAAD13-01-C- 4beta,15-diacetoxy-3alpha,dihydroxy-8alpha-[3-methyl- 0053. butyryl-oxy]-12,13-epoxytrichothec-9-ene. T-2 toxin is Copyright © 2005 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2006; 26: 139–147 140 C. MCKEAN ET AL. the product of F. sporotrichoides, F. poae, F. equiseti before experiments were initiated, and were housed and F. acuminatum and its production was usually en- individually in stainless steel cages under controlled tem- hanced by the unusual field conditions of prolonged wet perature (22° ± 1 °C), light (12 h light-dark cycle), and and cold weather during harvest. There are many diverse humidity (50% ± 10%). NIH open formula diet (NIH-07 mechanisms by which T-2 can produce toxicity and the Rat and Mouse Feed; Zeigler Bros., Inc.; Gamers, PA) relative importance of each in the production of the and distilled water were supplied ad libitum. The acute response is not fully understood (Coulombe, 1993). It toxicity study for individual mycotoxins was performed is thought that inhibition of protein synthesis and its using the method described by Horn (1956). Briefly, immunosuppressive properties are the most important F344 rats were randomly divided into 5–7 groups of human health impacts (Ueno, 1983, 1984; Yarom et al., five animals. One group was only given solvent vehicle 1984; Jagadeesan et al., 1982). (DMSO) and used as the control. The other groups were Co-exposure to multiple mycotoxins invokes cause orally administered mycotoxin at 1.0, 2.15, 4.64, 10.0 or for concern because so many have been shown to be 46.4 mg kg−1 body weight, respectively. The study was potent toxic agents with diverse effects and a synergetic performed over 7 days. Animals were carefully observed nature. It is logical to raise this issue because any single after treatment and symptoms of toxicity were recorded. compound may effect dissimilar reactions within a bio- Animals that died during the experiment or were logical system, while displaying antagonistic, additive, or euthanized by halothane (2-brome-2-chloro-1, 1, 1,- synergistic interactions with other compounds (Carpenter trifluoroethane) inhalation after the experiment were et al., 1998). However, little attention has been paid to necropsied. The major organs were excised and fixed in the study of combinative toxic effects of exposure to 10% buffered formalin for histopathological evaluations. multiple mycotoxins, which may be more potent and The combinative toxicity study for mycotoxin mixtures cause more damage to human health. The nature of was performed using the method described by Cornfield coexistence of many types of mycotoxins in complex (1964). In this study, F344 rats were randomly divided environmental samples, such as food and water, has been into 5–7 groups. Each group included 6–12 animals. One reported worldwide. How these mycotoxins affect human group was given only solvent vehicle (DMSO) and used health in combination is largely unknown. This study as the control. The other groups were administered by extended research efforts to test the toxicity of the gavage various fractions of the derived LD50 for each AFB1 and T-2 combination in animals (F344 rats and mycotoxin, respectively. The study was conducted over mosquitofish) and human cells (BEAS-2B and HepG2). 14 days. Animals were carefully observed after treatment and symptoms of toxicity were recorded. Animals that died during the 14-day experiment or were euthanized by Materials and Methods halothane (2-brome-2-chloro-1, 1, 1,-trifluoroethane) inha- lation after the experiment were necropsied. The major Materials organs were excised and fixed in 10% buffered formalin for histopathological evaluations. The mycotoxins selected for this study AFB1 and T-2 toxin were either purchased from Sigma Chemical Mosquitofish Co. (St Louis, MO) or were kindly provided by various research units of U.S. Food and Drug Administration. Mosquitofish (Gambusia affinis) (6 months old) were The purity of each toxin was tested with high per- purchased from Ken’s Hatchery & Fish Farm, Inc. formance liquid chromatography for AFB1 or gas chro- (Alapaha, GA) or Carolina Biological Supply Co. matography for T-2. Stock solutions were made with (Burlington, NC) 3 weeks before experiments were per- dimethylsulfoxide (DMSO) and kept under argon. The formed. After arrival, the fish were maintained in a 350– human hepatoma cell line, HepG2 and human bronchial 750 l aquaria (to maintain a minimal fish density), filled epithelial cell line, BEAS-2B, were purchased from with sea salt-buffered distilled water (60 mg l−1), equipped ATCC (Manassas, VA). All other chemicals and reagents with seasoned biological filters, and underwent quarantine were purchased commercially at the highest degree of procedures (treated once with trisulfa or every other day purity available. for 6 days with supersulfa). Goldfish flake fish food (Aquarium Pharmaceuticals, Inc., Chalfont, PA) was daily supplied ad libitum. After a week adaptation, the Animals and Treatment fish were further separated according to their gender into 40 l aquaria under similar conditions. Healthy fish (half F344 Rats male and half female) were randomly assigned into 3 l glass aquaria at the third week and were treated with Young male Fischer 344 rats (90–110 g) were obtained individual toxins or combinations at various concentra- from Harlan Lab Animals Inc.