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c mice.
Toxicon 43 (2004) 251–254 www.elsevier.com/locate/toxicon
The effect of intraperitoneally administered microcystin-LR on the gastrointestinal tract of Balb/c mice. Nicolette Bothaa, Maryna van de Ventera, Tim G. Downinga, Enid G. Shephardb, Michelle M. Gehringerc,* a
Department of Biochemistry and Microbiology, University of Port Elizabeth, P.O. Box 1600, Port Elizabeth 6000, South Africa b MRC/UCT Liver research centre, University of Cape Town, Groote Schuur Hospital, Old Main Building, Rondebosch, Cape Town, South Africa c School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia Received 5 November 2003; revised 25 November 2003; accepted 26 November 2003
Abstract Microcystin-LR (MCLR) has been associated with the development of gastrointestinal complaints in people ingesting cyanobacterial bloom contaminated water. The symptoms usually present a day or two after exposure raising questions as to the toxic effects of MCLR on the gastrointestinal tract. This study investigated the apoptotic effect of ip administered MCLR over time on the duodenum, jejenum and ileum of mice receiving a single 75% LD50 dose. The apoptotic index was significantly raised in all sections at 8 h post exposure (pe) and continued to rise for the 16, 24 and 32 h pe groups, while the glycogen levels were normal at 24 h pe. The duodenum exhibited the most significant increase in apoptotic index overall, followed by the jejenum and ileum. Immunohistochemistry indicated the presence of MCLR in the lamina propria suggesting a role for MCLR in the induction of apoptosis in the GIT of mice exposed to a single sublethal dose of MCLR. q 2003 Elsevier Ltd. All rights reserved. Keywords: Microcystin-LR; Apoptosis; Gastrointestinal tract
1. Introduction Cyanobacterial blooms are on the increase in water bodies worldwide as are the ailments that are brought about by the ingestion of the cyanobacteria and/or their toxins (Falconer, 2001; Codd et al., 1997). The most commonly occurring cyanobacterial toxin is microcystin-LR (MCLR), which induces severe damage in the liver of exposed individuals (Kuiper-Goodman et al., 1999). Recently there have been increased reports of gastrointestinal ailments resulting from ingestion of the cyanobacterium, Microcystis aeruginosa and/ or MCLR (Kuiper-Goodman et al., 1999; El Saadi et al., 1995). Orally administered MCLR is readily taken up via the bile transport system and transported to the liver from where it * Corresponding author. Tel.: þ61-2-9385-3235; fax: þ 61-29385-1591. E-mail address: [email protected] (M.M. Gehringer). 0041-0101/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2003.11.026
can be reintroduced into the small intestine by entero-hepatic recirculation (Falconer et al., 1992). Falconer and Humpage (1996) showed that MCLR did not promote duodenal tumor formation in mice previously treated with a tumour initiator, N-methyl-N-nitroso-urea, when compared to controls not receiving toxin. Falconer et al. (1994) failed to demonstrate any damage to the cells of the gastrointestinal tract of pigs repeatedly dosed with bloom material containing microcystin in their water. This is contradictory to the evidence that MCLR induces gastroenteritis in humans (Kuiper-Goodman et al., 1999; El Saadi et al., 1995). Ito et al. (2000) demonstrated that orally administered MCLR was primarily taken up via the small intestine of dosed mice. The villi that stained strongly positive for MCLR in the surface epithelium and lamina propria had severely eroded tips (Ito et al., 2000). MCLR was also excreted in the mucous of goblet cells in both the small and large intestine. Intratracheally administered toxin was detectable in the liver and small intestine of mice
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14 days after toxin exposure (Ito et al., 2001). There was a significant dose-dependent increase in the size of aberrant crypt foci in the colons of mice given a tumour initiator, azoxymethane, prior to oral administration of microcystin in their drinking water (Humpage et al., 2000). The authors suggested that the cells of the intestinal lining were at risk of damage by MCLR that was recirculated into the small intestine and that MCLR may stimulate preneoplastic colon tumour growth. MCLR is known to induce apoptosis in vitro in the enterocyte cell line Caco2 in a time-dependent manner (Botha et al., 2004) starting at 24 h post exposure (pe) and increasing to 84 ^ 5% SD at 72 h exposure. The apoptosis was preceded by increased production of reactive oxygen species and activation of micro and milli calpain. The question was thus raised as to the induction of apoptosis by MCLR in vivo over time in the gastrointestinal tract. The aim of this study was to investigate the apoptotic effect of intraperitoneally (ip) administered MCLR in vivo. The jejenum, ileum and duodenum were obtained from animals used in a previous study (Gehringer et al., 2004) and investigated for apoptosis, glycogen content and presence of MCLR.
Elizabeth (permit number AEC 2000/2) (Gehringer et al., 2004). Toxin was administered ip in 200 ml sterile saline solution. Seven-week-old female special pathogen free Balb/c mice, obtained from the Animal Unit of the South African Institute of Medical Research, were used in the study. Fifteen mice, 3 per group, were administered a 75% LD50 dose of pure MCLR ip and sacrificed at 8, 16, 24 and 32 h pe, with control mice receiving saline (Gehringer et al., 2004). Duodenal, jejunal and ileal sections were obtained at sacrifice and washed thoroughly in PBS. 2.2. Histology Small pieces of intestinal tissue were fixed in 25 volumes of 10% formalin in PBS (pH 7) and processed within 24 h. Sections (4 – 5 mm thick) were stained with Haematoxylin and Eosin (H&E), and also with Periodic Acid Schiff stain (PAS) for glycogen, and viewed using an Olympus BX 60 Microscope under the 40 £ objective. Photographs representative of the pathology were taken using an Olympus Camedia C-3040ZOOM digital camera. Apoptotic indices were determined from these H&E stained sections by averaging the results from 10 fields of view for each section from each mouse in the group.
2. Methods and materials 2.3. Immunohistochemistry General reagents were purchased from Merck (South Africa). Mouse anti-MCLR was bought from Alexis Biochemicals (Germany). MCLR, goat anti-mouse IgG Fc specific antibody and anti-mouse FITC conjugated antibody were obtained from Sigma (South Africa). 2.1. Animals Mice were kept in accordance with the stipulations of the Animal Ethics Committee of the University of Port
Sections were hydrated through a series of alcohols and placed in sterile saline. Sections were blocked in 5% BSA for 20 min. Endogenous mouse IgG was blocked with 48 mg/ml goat anti-mouse IgG (Fc specific) antibody for 2 h at room temperature. Mouse anti-MCLR (83.25 mg/ml) antibody was applied for 1 h at room temperature followed by an overnight incubation at 4 8C. Sections were allowed to reach room temperature before the secondary goat antimouse FITC conjugated antibody (42 mg/ml) was added
Fig. 1. Apoptotic indices of sections of the small intestine from mice receiving a single dose of MCLR. Mice received a single ip dose of 75% LD50 MCLR and were sacrificed at 8, 16, 24 and 32 h pe. Bars represent (A) duodenum, ( ) jejunum and ( ) ileum. * indicates a significant increase in apoptotic cell numbers from the control sections at 0 h as determined by Newman–Keuls (p , 0:05; N ¼ 3).
N. Botha et al. / Toxicon 43 (2004) 251–254
followed by incubation at room temperature for 30 min. Slides were washed and examined under the microscope at 420– 490 nm. Representative photographs were taken.
3. Results and discussion Fig. 1 indicates that apoptosis was time-dependent, with more apoptotic cells observed in the duodenal and jejunal sections, followed by the ileal sections. The apoptotic index was higher (4.25 ^ 0.125%) in the duodenum than in the jejunum (2.5 ^ 0.15%). These results agree with in vitro studies, which
253
demonstrated increased apoptosis in an enterocyte cell line, Caco2, exposed to MCLR (Botha et al., 2004). The data would suggest that recirculated ip administered MCLR primarily damages the duodenum, followed by the jejunum and ileum. Apoptotic cells were observed in the villi (Fig. 2), which confirmed the results of Ito et al. (2000) who found that orally administered toxin damaged both the villi and the individual cells of the gastrointestinal lining of exposed mice. The presence of MCLR in the lamina propria of the duodenum, jejunum and ileum was clearly visible in toxin-treated mice (Fig. 2g,h and i, respectively). The toxin presumably reached the gastrointestinal tract via the bile system as demonstrated for intratracheally administered toxin (Ito et al., 2001).
Fig. 2. Stained sections of the small intestine from mice receiving a single dose of MCLR. Mice received a single ip dose of 75% LD50 MCLR and were sacrificed at 8, 16, 24 and 32 h pe. Photographs a, d, g and j represent the duodenum; b, e, h and k the jejenum and c, f, i and l the ileum. Panels a –c are control sections stained with H&E; d–f are H&E stained sections from mice sacrificed at 24 h pe; g–i are sections from mice sacrificed 24 h pe showing the presence of MCLR; j –i sections are stained with PAS to visualize the glycogen content of the sections obtained from mice sacrificed at 24 h pe. Arrows indicate apoptotic bodies. Panels a –i: bar ¼ 20 mm, panels j –l: bar ¼ 60 mm.
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The levels of glycogen at 24 h pe approached those of the control sections (Fig. 2). This is in agreement with glycogen levels in the livers of these mice, which showed near normal glycogen levels 24 h pe (Gehringer et al., 2004). Of interest is the fact that the liver histology showed most damage at 8 and 16 h pe accompanied with significantly elevated serum ALT and LDH levels. Sections of liver from the 24 h pe mice indicated extensive repair with two of the three mice in the 32 h pe group appearing normal (Gehringer et al., 2004). The apparent delayed appearance of damage in the gastrointestinal tract when compared to the liver may be a reflection of the detoxification function of liver tissue, allowing it to remove both the toxin and its oxidative stress by-products more efficiently than cells lining the gastrointestinal tract and/or a delay in the initial biliary excretion of the toxin into the gastrointestinal tract. In conclusion, this study clearly illustrates that ip administered toxin induces apoptosis of the cells of the gastrointestinal tract. The increase in MCLR-induced apoptosis in cells of the duodenum, jejenum and ileum was found to be time-dependent after administration of a single ip non-lethal dose of pure MCLR. The greatest increase was seen in the duodenum, reaching a maximum of 4.25 ^ 0.125% by 32 h pe compared to the controls. The presence of MCLR in the villi would suggest that the damage was toxin mediated. It is reasonable to conclude that the delayed gastrointestinal ailments seen in cases of MCLR toxicosis could result from apoptosis of the cells of the small intestine.
References Botha, N., Van de Venter, M., Downing, T.G., Shephard, E.G., Gehringer, M.M., 2004. The role of Microcystin-LR in
the induction of apoptosis and oxidative stress in Caco2. Toxicon 43, 85–92. Codd, G.A., Ward, C.J., Bell, S.G., 1997. Cyanobacterial toxins: occurrence, modes of action, health effects and exposure routes. In: Seiler, J.P., Vilanova, E. (Eds.), Applied Toxicology, Suppl. 19. Springer, Berlin, pp. 399– 410. El Saadi, O., Esterman, A.J., Cameron, S., Roder, D.M., 1995. Murray river water, raised cyanobacterial counts and gastrointestinal and dermatological symptoms. Med. J. Aust. 162, 122–125. Falconer, I.R., 2001. Toxic cyanobacterial bloom problems in Australian waters: risks and impacts on human health. Phycologia 40, 228 –233. Falconer, I.R., Humpage, A.R., 1996. Tumour promotion by cyanobacterial toxins. Phycologia 35, 74–79. Falconer, I.R., Dornbusch, M., Moran, G., Yeung, S.K., 1992. Effects of the cyanobacterial (blue-green algal) toxins from Microcystis aeruginosa on isolated enterocytes from chicken small intestine. Toxicon 30, 790–793. Falconer, I.R., Burch, M.D., Steffensen, D.A., Choice, M., Coverdale, D.R., 1994. Toxicity of the blue-green alga (cyanobacterium) Microcystis aeruginosa in drinking water to growing pigs, as an animal model for human injury and risk assessment. Environ. Toxicol. Water Qlty 9, 131 –139. Gehringer, M.M., Shephard, E.G., Nielan, B.A., Downing, T.G., Wiegand, C., 2004. An investigation into the detoxification of Microcystin-LR by the glutathione pathway in Balb/c mice. Int. J. Biochem. Cell B. in press. Humpage, A.R., Hardy, S.J., Moore, E.J., Froscio, S.M., Falconer, I.R., 2000. Microcystins (cyanobacterial toxins) in drinking water enhance the growth of abberant crypt foci in the mouse colon. J. Toxicol. Environ. Health 61, 155– 165. Ito, E., Kondo, F., Harada, K., 2000. First report on the distribution of orally administered microcystin-LR in mouse tissue using an immunostaining method. Toxicon 38, 37– 48. Ito, E., Kondo, F., Harada, K., 2001. Intratracheal administration of microcystin-LR, and its distribution. Toxicon 39, 265 –271. Kuiper-Goodman, T., Falconer, I.R., Fitzgerald, J., 1999. Human health aspects. In: Chorus, I., Bartram, J. (Eds.), Toxic Cyanobacteria in Water, E&FN Spon, London, pp. 113–153.
The effect of intraperitoneally administered microcystin-LR on the gastrointestinal tract of Balb/c mice. Nicolette Bothaa, Maryna van de Ventera, Tim G. Downinga, Enid G. Shephardb, Michelle M. Gehringerc,* a
Department of Biochemistry and Microbiology, University of Port Elizabeth, P.O. Box 1600, Port Elizabeth 6000, South Africa b MRC/UCT Liver research centre, University of Cape Town, Groote Schuur Hospital, Old Main Building, Rondebosch, Cape Town, South Africa c School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia Received 5 November 2003; revised 25 November 2003; accepted 26 November 2003
Abstract Microcystin-LR (MCLR) has been associated with the development of gastrointestinal complaints in people ingesting cyanobacterial bloom contaminated water. The symptoms usually present a day or two after exposure raising questions as to the toxic effects of MCLR on the gastrointestinal tract. This study investigated the apoptotic effect of ip administered MCLR over time on the duodenum, jejenum and ileum of mice receiving a single 75% LD50 dose. The apoptotic index was significantly raised in all sections at 8 h post exposure (pe) and continued to rise for the 16, 24 and 32 h pe groups, while the glycogen levels were normal at 24 h pe. The duodenum exhibited the most significant increase in apoptotic index overall, followed by the jejenum and ileum. Immunohistochemistry indicated the presence of MCLR in the lamina propria suggesting a role for MCLR in the induction of apoptosis in the GIT of mice exposed to a single sublethal dose of MCLR. q 2003 Elsevier Ltd. All rights reserved. Keywords: Microcystin-LR; Apoptosis; Gastrointestinal tract
1. Introduction Cyanobacterial blooms are on the increase in water bodies worldwide as are the ailments that are brought about by the ingestion of the cyanobacteria and/or their toxins (Falconer, 2001; Codd et al., 1997). The most commonly occurring cyanobacterial toxin is microcystin-LR (MCLR), which induces severe damage in the liver of exposed individuals (Kuiper-Goodman et al., 1999). Recently there have been increased reports of gastrointestinal ailments resulting from ingestion of the cyanobacterium, Microcystis aeruginosa and/ or MCLR (Kuiper-Goodman et al., 1999; El Saadi et al., 1995). Orally administered MCLR is readily taken up via the bile transport system and transported to the liver from where it * Corresponding author. Tel.: þ61-2-9385-3235; fax: þ 61-29385-1591. E-mail address: [email protected] (M.M. Gehringer). 0041-0101/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2003.11.026
can be reintroduced into the small intestine by entero-hepatic recirculation (Falconer et al., 1992). Falconer and Humpage (1996) showed that MCLR did not promote duodenal tumor formation in mice previously treated with a tumour initiator, N-methyl-N-nitroso-urea, when compared to controls not receiving toxin. Falconer et al. (1994) failed to demonstrate any damage to the cells of the gastrointestinal tract of pigs repeatedly dosed with bloom material containing microcystin in their water. This is contradictory to the evidence that MCLR induces gastroenteritis in humans (Kuiper-Goodman et al., 1999; El Saadi et al., 1995). Ito et al. (2000) demonstrated that orally administered MCLR was primarily taken up via the small intestine of dosed mice. The villi that stained strongly positive for MCLR in the surface epithelium and lamina propria had severely eroded tips (Ito et al., 2000). MCLR was also excreted in the mucous of goblet cells in both the small and large intestine. Intratracheally administered toxin was detectable in the liver and small intestine of mice
252
N. Botha et al. / Toxicon 43 (2004) 251–254
14 days after toxin exposure (Ito et al., 2001). There was a significant dose-dependent increase in the size of aberrant crypt foci in the colons of mice given a tumour initiator, azoxymethane, prior to oral administration of microcystin in their drinking water (Humpage et al., 2000). The authors suggested that the cells of the intestinal lining were at risk of damage by MCLR that was recirculated into the small intestine and that MCLR may stimulate preneoplastic colon tumour growth. MCLR is known to induce apoptosis in vitro in the enterocyte cell line Caco2 in a time-dependent manner (Botha et al., 2004) starting at 24 h post exposure (pe) and increasing to 84 ^ 5% SD at 72 h exposure. The apoptosis was preceded by increased production of reactive oxygen species and activation of micro and milli calpain. The question was thus raised as to the induction of apoptosis by MCLR in vivo over time in the gastrointestinal tract. The aim of this study was to investigate the apoptotic effect of intraperitoneally (ip) administered MCLR in vivo. The jejenum, ileum and duodenum were obtained from animals used in a previous study (Gehringer et al., 2004) and investigated for apoptosis, glycogen content and presence of MCLR.
Elizabeth (permit number AEC 2000/2) (Gehringer et al., 2004). Toxin was administered ip in 200 ml sterile saline solution. Seven-week-old female special pathogen free Balb/c mice, obtained from the Animal Unit of the South African Institute of Medical Research, were used in the study. Fifteen mice, 3 per group, were administered a 75% LD50 dose of pure MCLR ip and sacrificed at 8, 16, 24 and 32 h pe, with control mice receiving saline (Gehringer et al., 2004). Duodenal, jejunal and ileal sections were obtained at sacrifice and washed thoroughly in PBS. 2.2. Histology Small pieces of intestinal tissue were fixed in 25 volumes of 10% formalin in PBS (pH 7) and processed within 24 h. Sections (4 – 5 mm thick) were stained with Haematoxylin and Eosin (H&E), and also with Periodic Acid Schiff stain (PAS) for glycogen, and viewed using an Olympus BX 60 Microscope under the 40 £ objective. Photographs representative of the pathology were taken using an Olympus Camedia C-3040ZOOM digital camera. Apoptotic indices were determined from these H&E stained sections by averaging the results from 10 fields of view for each section from each mouse in the group.
2. Methods and materials 2.3. Immunohistochemistry General reagents were purchased from Merck (South Africa). Mouse anti-MCLR was bought from Alexis Biochemicals (Germany). MCLR, goat anti-mouse IgG Fc specific antibody and anti-mouse FITC conjugated antibody were obtained from Sigma (South Africa). 2.1. Animals Mice were kept in accordance with the stipulations of the Animal Ethics Committee of the University of Port
Sections were hydrated through a series of alcohols and placed in sterile saline. Sections were blocked in 5% BSA for 20 min. Endogenous mouse IgG was blocked with 48 mg/ml goat anti-mouse IgG (Fc specific) antibody for 2 h at room temperature. Mouse anti-MCLR (83.25 mg/ml) antibody was applied for 1 h at room temperature followed by an overnight incubation at 4 8C. Sections were allowed to reach room temperature before the secondary goat antimouse FITC conjugated antibody (42 mg/ml) was added
Fig. 1. Apoptotic indices of sections of the small intestine from mice receiving a single dose of MCLR. Mice received a single ip dose of 75% LD50 MCLR and were sacrificed at 8, 16, 24 and 32 h pe. Bars represent (A) duodenum, ( ) jejunum and ( ) ileum. * indicates a significant increase in apoptotic cell numbers from the control sections at 0 h as determined by Newman–Keuls (p , 0:05; N ¼ 3).
N. Botha et al. / Toxicon 43 (2004) 251–254
followed by incubation at room temperature for 30 min. Slides were washed and examined under the microscope at 420– 490 nm. Representative photographs were taken.
3. Results and discussion Fig. 1 indicates that apoptosis was time-dependent, with more apoptotic cells observed in the duodenal and jejunal sections, followed by the ileal sections. The apoptotic index was higher (4.25 ^ 0.125%) in the duodenum than in the jejunum (2.5 ^ 0.15%). These results agree with in vitro studies, which
253
demonstrated increased apoptosis in an enterocyte cell line, Caco2, exposed to MCLR (Botha et al., 2004). The data would suggest that recirculated ip administered MCLR primarily damages the duodenum, followed by the jejunum and ileum. Apoptotic cells were observed in the villi (Fig. 2), which confirmed the results of Ito et al. (2000) who found that orally administered toxin damaged both the villi and the individual cells of the gastrointestinal lining of exposed mice. The presence of MCLR in the lamina propria of the duodenum, jejunum and ileum was clearly visible in toxin-treated mice (Fig. 2g,h and i, respectively). The toxin presumably reached the gastrointestinal tract via the bile system as demonstrated for intratracheally administered toxin (Ito et al., 2001).
Fig. 2. Stained sections of the small intestine from mice receiving a single dose of MCLR. Mice received a single ip dose of 75% LD50 MCLR and were sacrificed at 8, 16, 24 and 32 h pe. Photographs a, d, g and j represent the duodenum; b, e, h and k the jejenum and c, f, i and l the ileum. Panels a –c are control sections stained with H&E; d–f are H&E stained sections from mice sacrificed at 24 h pe; g–i are sections from mice sacrificed 24 h pe showing the presence of MCLR; j –i sections are stained with PAS to visualize the glycogen content of the sections obtained from mice sacrificed at 24 h pe. Arrows indicate apoptotic bodies. Panels a –i: bar ¼ 20 mm, panels j –l: bar ¼ 60 mm.
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N. Botha et al. / Toxicon 43 (2004) 251–254
The levels of glycogen at 24 h pe approached those of the control sections (Fig. 2). This is in agreement with glycogen levels in the livers of these mice, which showed near normal glycogen levels 24 h pe (Gehringer et al., 2004). Of interest is the fact that the liver histology showed most damage at 8 and 16 h pe accompanied with significantly elevated serum ALT and LDH levels. Sections of liver from the 24 h pe mice indicated extensive repair with two of the three mice in the 32 h pe group appearing normal (Gehringer et al., 2004). The apparent delayed appearance of damage in the gastrointestinal tract when compared to the liver may be a reflection of the detoxification function of liver tissue, allowing it to remove both the toxin and its oxidative stress by-products more efficiently than cells lining the gastrointestinal tract and/or a delay in the initial biliary excretion of the toxin into the gastrointestinal tract. In conclusion, this study clearly illustrates that ip administered toxin induces apoptosis of the cells of the gastrointestinal tract. The increase in MCLR-induced apoptosis in cells of the duodenum, jejenum and ileum was found to be time-dependent after administration of a single ip non-lethal dose of pure MCLR. The greatest increase was seen in the duodenum, reaching a maximum of 4.25 ^ 0.125% by 32 h pe compared to the controls. The presence of MCLR in the villi would suggest that the damage was toxin mediated. It is reasonable to conclude that the delayed gastrointestinal ailments seen in cases of MCLR toxicosis could result from apoptosis of the cells of the small intestine.
References Botha, N., Van de Venter, M., Downing, T.G., Shephard, E.G., Gehringer, M.M., 2004. The role of Microcystin-LR in
the induction of apoptosis and oxidative stress in Caco2. Toxicon 43, 85–92. Codd, G.A., Ward, C.J., Bell, S.G., 1997. Cyanobacterial toxins: occurrence, modes of action, health effects and exposure routes. In: Seiler, J.P., Vilanova, E. (Eds.), Applied Toxicology, Suppl. 19. Springer, Berlin, pp. 399– 410. El Saadi, O., Esterman, A.J., Cameron, S., Roder, D.M., 1995. Murray river water, raised cyanobacterial counts and gastrointestinal and dermatological symptoms. Med. J. Aust. 162, 122–125. Falconer, I.R., 2001. Toxic cyanobacterial bloom problems in Australian waters: risks and impacts on human health. Phycologia 40, 228 –233. Falconer, I.R., Humpage, A.R., 1996. Tumour promotion by cyanobacterial toxins. Phycologia 35, 74–79. Falconer, I.R., Dornbusch, M., Moran, G., Yeung, S.K., 1992. Effects of the cyanobacterial (blue-green algal) toxins from Microcystis aeruginosa on isolated enterocytes from chicken small intestine. Toxicon 30, 790–793. Falconer, I.R., Burch, M.D., Steffensen, D.A., Choice, M., Coverdale, D.R., 1994. Toxicity of the blue-green alga (cyanobacterium) Microcystis aeruginosa in drinking water to growing pigs, as an animal model for human injury and risk assessment. Environ. Toxicol. Water Qlty 9, 131 –139. Gehringer, M.M., Shephard, E.G., Nielan, B.A., Downing, T.G., Wiegand, C., 2004. An investigation into the detoxification of Microcystin-LR by the glutathione pathway in Balb/c mice. Int. J. Biochem. Cell B. in press. Humpage, A.R., Hardy, S.J., Moore, E.J., Froscio, S.M., Falconer, I.R., 2000. Microcystins (cyanobacterial toxins) in drinking water enhance the growth of abberant crypt foci in the mouse colon. J. Toxicol. Environ. Health 61, 155– 165. Ito, E., Kondo, F., Harada, K., 2000. First report on the distribution of orally administered microcystin-LR in mouse tissue using an immunostaining method. Toxicon 38, 37– 48. Ito, E., Kondo, F., Harada, K., 2001. Intratracheal administration of microcystin-LR, and its distribution. Toxicon 39, 265 –271. Kuiper-Goodman, T., Falconer, I.R., Fitzgerald, J., 1999. Human health aspects. In: Chorus, I., Bartram, J. (Eds.), Toxic Cyanobacteria in Water, E&FN Spon, London, pp. 113–153.