ACUTE ORAL TOXICITY OF DEOXY- CYLINDROSPERMOPSIN IN MALE BALB/C MICE

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1 ACUTE ORAL TOXICITY OF DEOXY- CYLINDROSPERMOPSIN IN MALE BALB/C MICE Ian Stewart 1,2 Wasantha Wickramasinghe 2 Steve Carter 3 Glenn McGregor 4 Brad Davis 2 Alan Seawright 2 1: Food & Water Toxicology Consulting 2: The University of Queensland, National Research Centre for Environmental Toxicology (EnTox) 3: Queensland Health Forensic and Scientific Services 4: Queensland Department of Science, Information Technology and Innovation

2 CYLINDROSPERMOPSIN (CYN) Palm Island mass poisoning, 1979 Hawkins PR, Runnegar MT et al 1985 Appl Environ Microbiol 50: Historical acute enteric illnesses in outback northern & central Australia posited: Barcoo Sickness Hayman J 1992 Med J Aust 157:794-6 Confirmed mortalities in Queensland cattle Stewart I, Seawright AA, Shaw GR 2008 Adv Exp Med Biol 619: CYN found in haemodialysis clinic filters, Caruaru, Brazil, 1996 Carmichael WW, Azevedo SM et al 2001 Environ Health Perspect 109:663-8 NHMRC s Australian Drinking Water Guidelines, 2011 edition: Health Alert for CYN at 1μg/L Genotoxic, protein synthesis inhibitor Falconer IR, Humpage AR 2006 Environ Toxicol 21:

3 CYLINDROSPERMOPSIN (CYN) LD 50 (i.p. mouse): 2.1mg kg -1 at 24 hrs 200μg kg -1 at 5-6 days Ohtani I, Moore RE, Runnegar MT 1992 J Am Chem Soc 114:7941-2

4

5 Discovered at EnTox/FSS Reported as non-toxic in vivo: n = 3 outbred 800μg kg -1 i.p. Norris RL, Eaglesham GK et al 1999 Environ Toxicol 14:163-5 BUT: subsequent in vitro work showed D-CYN and CYN inhibit protein synthesis at equimolar concentrations Looper RE, Runnegar MTC, Williams RM 2005 Angew Chem Int Ed Engl 44: AND: D-CYN and CYN suppress proliferation in various cell lines at equimolar concentrations Neumann C, Bain P, Shaw G 2007 J Toxicol Environ Health A 70:

6 Filamentous form (benthic, epiphytic): Microseira wollei [formerly Lyngbya wollei] Seifert M, McGregor G et al 2007 Harmful Algae 6:73-80

7 Extracted from M. wollei Purified through C 18 SPE, then C 18 preparative HPLC Eluted with 10% MeOH % formic acid

8 Study Design Outcome measures of core hypothermia by intermittent rectal thermometry, observation and recording of sickness behaviour (piloerection, grip strength, social interaction, mobility, eyelid ptosis) Hypothermia threshold: 35 O C Experiment concluded when sustained hypothermia and sickness behaviour recorded, or x6 days elapsed, whichever came first for each treatment group On delivery, mice randomly allocated to three treatment groups: Test group (D-CYN, 6mg kg -1 mouse bodyweight), n = 6 Positive control (CYN, 6mg kg -1 mouse bodyweight), n = 6 Negative control (prep HPLC eluate, reconstituted in water, 6mL kg -1 mouse bodyweight), n = 4

9 Study Design

10 Study Design Test group (D-CYN) and negative controls: five consecutive daily doses by oral gavage, unless and until signs of acute toxicity observed Positive controls (CYN): single oral gavage dose at Day 0 Investigator-blind dosing, temperature measurement and observation from Day 0 to Day 1 Mice housed in metabolic cages for 8 hours after dosing, for collection of urine and faeces at 4 & 8 hrs At necropsy: whole animal weight, then intact liver and left kidney weights for relative organ weights. Gross examination of organ pathology; harvest & fix organs for histopathology

11

12

13 Necropsy Images CYN D-CYN Negative control

14 Unilateral peri-orbital haemorrhage: CYN-dosed mouse

15 Relative liver weights

16 Relative kidney weights

17

18

19

20 RSD = 1.7% RSD = 1.8%

21 In vitro toxicology of DCYN (Neumann, Bain and Shaw 2007)

22 In vitro toxicology of DCYN (Neumann, Bain and Shaw 2007)

23

24 Negative controls

25 CONCLUSIONS No evidence for acute toxicity of D-CYN at five consecutive oral doses of 6mg/kg D-CYN quantified by HPLC-MS/MS D-CYN purity confirmed by NMR, uracil moiety in keto form D-CYN recovered in liver, urine & faeces Regulated hypothermia and observation of sickness behaviour appears to be a useful non-lethal endpoints model for acute toxicity of cyanotoxins (and other HAB toxins) Stewart I, McLeod C 2014 J AOAC Int 97(2):356-72

26 FURTHER QUESTIONS? Why is the solubility of d-cyn so different to CYN? What of protein synthesis inhibition? other mechanism/s of toxicity? psi in vitro but not in vivo? psi in vivo but inconsequential? What of discordant findings from earlier work? contamination? stereochemistry?

27 Acknowledgements: Tony Carroll, School of Environment, Griffith University Geoff Eaglesham, UQ EnTox Staff at Herston Medical Research Institute

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