Title page for ETD etd-05122008-100402

Document Type Master's Dissertation
Author Masango, Mxolisi Goodwill
Email masangom@arc.agric.za
URN etd-05122008-100402
Document Title A comparative analysis of the cytotoxicity of cyanotoxins using in vitro (cell culture) and in vivo (mouse) assays
Degree MSc (Paraclinical Studies)
Department Paraclinical Sciences
Advisor Name Title
Dr J G Myburgh Co-Supervisor
Dr D Naicker Supervisor
  • protein phosphatase inhibition
  • cytotoxicity of cyanotoxins
  • toxin profile
  • catfish primary hepatocytes
  • mouse bioassay
  • toxicity
Date 2007-11-23
Availability unrestricted
The main objective of this study was the application and comparison of different assays in assessing toxicity of cyanobacterial samples, and also characterizing toxicity of the field samples. Therefore, toxicity of purified microcystin-LR (MC-LR) and cyanobacterial samples collected from the Hartbeespoort (HBP) Dam (winter and summer seasons of 2005/2006) and Kruger National Park (KNP) were investigated and compared using the ELISA, mouse bioassay, catfish primary hepatocytes (in vitro assay) and protein phosphatase inhibition (PPi) assays.

During sampling in the summer season at the HBP Dam, the dam surface was covered with a thick-green layer of cyanobacterial scum and a foul smell coming from the water surface was always present. Only blue-green streaks of cyanobacteria covered the dam surface during the winter season. All HBP Dam samples (winter and summer samples) and KNP samples (Nhlanhanzwani Dam, Mpanama Dam and Sunset Dam) were dominated by Microcystis aeruginosa with the exception of Makhohlola Dam samples which were found to have no cyanobacteria.

The World Health Organization (WHO) has proposed a guideline value for human use of 1.0 g/L (0.001 mg/L) for MC-LR, the most common microcystin (MC) variant, in drinking water (WHO 1998), whereas 2 000 Microcystis cells/mL have been recommended as the limit of cyanobacteria in drinking water for animals (DWAF 1996). Cyanotoxin concentrations exceeding the prescribed guideline value were detected in all HBP Dam samples (ELISA results ranging between 3.67 to 86.08 mg/L; PPi results ranging between 2.99 to 54.90 mg/L) and KNP samples (ELISA results ranging between 0.1 to 49.41 mg/L; PPi results ranging between 0.006 to 10.95 mg/L) using both the ELISA and PPi assays.

In the current study, a dose of about 175 g/kg of purified MC-LR was demonstrated to be lethal in male CD-1 SPF mice. The HBP Dam summer samples and Nhlanganzwani Dam samples were the only cyanobacterial samples that resulted in death (acute toxicity) of mice.

In order to be able to investigate further the in vivo effects of cyanotoxins, transmission electron microscopy (TEM) was used to complement results obtained from the in vivo assay. Ultrastructural changes of varying degree were observed in livers of mice exposed to both the HBP Dam winter and summer samples. Early stages of hepatocyte to hepatocyte disassociation, slight vesiculation of endoplasmic reticulum (ER) and swollen mitochondria were the most significant ultrastructural changes produced in mouse hepatocyte tissues by the HBP Dam winter samples. The most significant ultrastructural changes produced in mouse hepatocyte tissues by the HBP Dam summer samples were massive hepatic haemorrhage indicated by the appearance of erythrocytes between hepatocytes and the extensive vesiculation of ER.

This is the first time that the African sharptooth catfish primary hepatocyte model has been used to assess the hepatotoxicity of purified MC-LR and cyanotoxin-containing water samples. In this study, the toxicity of cyanobacterial samples and purified MC-LR to cause hepatotoxicity in mice was confirmed in vitro using the catfish primary cell line. A comparison among the cyanobacterial samples using EC50 showed the following hepatotoxicity trend in the catfish primary cell line: HBP Dam summer samples > Nhlanganzwani Dam samples > HBP Dam winter samples > Mpanama Dam samples > Sunset Dam samples > Makhohlola Dam samples. The HBP Dam samples were the most hepatotoxic and Makhohlola Dam samples were the least hepatotoxic. The EC50 for purified MC-LR using the catfish primary hepatocytes was about 91 nM.

A statistical comparison of the assays used in this study (i.e. ELISA, PPi, mouse test and cytotoxicity [catfish primary hepatocyte] assays) was performed based on the Kappa coefficient (K). An almost perfect agreement (K > 0.80) was observed between the mouse test and cytotoxicity assay; mouse test and ELISA; cytotoxicity assay and ELISA; and ELISA and PPi assay.

In conclusion, field samples collected during the summer season were found to have very high levels of toxins and a higher degree of toxicity when compared to the winter samples. The cytotoxicity assay using African sharptooth catfish (Clarias gariepinus) primary hepatocytes has been shown for the first time to produce results similar to those observed when using the mouse bioassay in assessing cyanobacterial toxicity. Therefore, this primary cell line may be used as a potential alternative to the mouse assay in toxicity testing of cyanotoxins. Three KNP dams (Nhlanganzwani Dam, Mpanama Dam and Sunset Dam) investigated in this study were found to contain Microcystis aeruginosa. All four KNP dams (Nhlanganzwani Dam, Mpanama Dam, Makhohlola Dam and Sunset Dam) had cyanotoxin levels above the prescribed guideline value, which is of concern and warrants further investigations to the effects on wildlife in the park.

Future studies will include use of High Performance Liquid Chromatography (HPLC) to investigate the toxin profile of the field samples in order to fully describe the different classes/or types of toxins present in the samples. More validation studies that could give a more comprehensive understanding about the sensitivity of the catfish primary cell line for microcystins will also be undertaken.

University of Pretoria 2007

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