Cyanobacterial Toxins -- Microcystin-LR

Health Effects

Blue-green algae have been known to cause animal and human poisoning in lakes, ponds and dugouts in various parts of the world for over 100 years.

Effects in Humans

Recreational Water

Incidences of human illness have been linked to the recreational use of water contaminated by cyanobacterial blooms, including Anabaena and Microcystis, in North America, the United Kingdom, the Netherlands and Australia. No associated fatalities have been reported. In Canada, illnesses have been reported in Saskatchewan, and symptoms have included stomach cramps, vomiting, diarrhoea, fever, headache, pains in muscles and joints and weakness.⁶³ Similar symptoms as well as dermal irritation, sore red eyes, sore throat and allergic responses have also been reported elsewhere.⁶ The reported instances of illnesses are few; however, because they are difficult to diagnose, such illnesses may be more common than it appears.⁶⁴ Exposure has been mainly through skin contact and some inadvertent ingestion of the water containing dispersed cyanobacteria. Despite the high toxicity of cyanobacterial toxins in animals, serious acute human illness due to these toxins has been reported only rarely. This is probably because humans have an aversion to ingesting algal scum.

In one recent incident in the United Kingdom, 10 of 18 military recruits on an exercise in a reservoir with a bloom of Microcystis aeruginosa suffered abdominal pain, nausea, vomiting, diarrhoea, sore throat, dry cough, blistering at the mouth and headache. Two were hospitalized and developed an atypical pneumonia, although it is possible that the pneumonia was caused by inspiration of algal material that may have also contained lipopolysaccharides. Serum enzymes indicating liver damage were elevated. Microcystin-LR was identified in the bloom material.⁶⁵

Drinking Water

In the United States and Australia, several different cyanobacterial toxins have been implicated in human illness, often after algal blooms in certain municipal water supplies had been treated with copper sulphate.^6,66,67 Although a direct cause-and-effect relationship was not established in most of the outbreaks, there has been strong circumstantial evidence that cyanobacterial blooms were present at the water intake area or in open reservoirs. Although in most cases the cyanobacteria involved and sometimes the toxins involved have been identified, the levels of toxin associated with illness have not been established in any of the outbreaks. In at least one outbreak in Palm Island, Australia, in 1979,⁶⁸ acute copper poisoning has been suggested as an alternative cause,⁶⁹ although further study has shown that a toxin from a species of blue-green algae (Cylindrospermopsis raciborskii) may have been the causative agent.⁷ In this case, complaints of bad taste and odour in a water supply, which were attributed to a cyanobacterial bloom, resulted in authorities treating the reservoir with copper sulphate. Within one week, numerous children developed severe hepatoenteritis, and 140 children and 10 adults required hospital treatment. No deaths were reported.

Possible liver damage, as evidenced by significant increases in --glutamyl transferase, was seen in persons drinking water from supplies containing blooms of Microcystis after treatment with copper sulphate (Malpas Dam, Armidale, Australia) compared with persons drinking uncontaminated water.⁷⁰

To date, the most lethal outbreak attributed to exposure to cyanobacterial toxins in drinking water occurred in Brazil in 1988.⁷¹ An immense cyanobacterial bloom developed in a newly flooded dam, resulting in more than 2000 cases of gastroenteritis, with 88 deaths reported (mostly children) over a 42-day period. It appears that the cyanobacterial proliferation resulted from the decomposing biomass and other conditions that prevailed in the newly flooded reservoir area.

More recently, in February 1996, liver failure and death were reported in haematolysis patients of a Brazil-ian dialysis clinic where the dialysate was found to be contaminated with fragmented microalgal and cyanobacterial cells and probably the microcystin-LR toxin.^72,73 Death was reported in approximately 50% of the dialysis patients exposed to the contaminated dialysate; however, no information was available on the type, abundance or toxicity of the cyanobacteria in the reservoir that was the source of the water during the period in question. Liver histology confirmed the presence of acute toxic hepatitis similar to that observed in animals exposed to microcystins. Analytical examination of liver tissue and serum samples of patients and the carbon filter from the dialysis unit confirmed the presence of three microcystin derivatives (YR, LR and AR). It was concluded that inadequate additional treatment of the water used in the dialysis process at the clinic was most likely responsible for the toxins in the dialysate and that intravenous exposure to these microcystins was a major contributing factor in the deaths of these patients.⁷⁴ From further analysis of the phytoplankton from the dialysis centre and tissue and serum samples from the 76 victims and other affected patients, it was estimated that the water used in the dialysis treatment contained 19.5 µg microcystin/L.⁷⁴ Because of the susceptibility of dialysis patients to contaminated dialysate, dialysis centres should be informed if the source water from their local treatment plant is prone to blue-green algal blooms, so that they may provide additional treatment of the water, if necessary. Continuous monitoring of treatment plant performance and equipment is also necessary to ensure an adequate water supply.

Zilberg⁷⁵ postulated that seasonal acute childhood gastroenteritis observed during 1960-1965 in Salisbury, Rhodesia (now known as Harare, Zimbabwe), might be linked to annual algal blooms in the lake serving as the water supply. An adjacent water supply was not similarly affected and was not associated with this disease.

El Saadi and Cameron⁷⁶ reported on 26 cases (aged 1-64 years) with a variety of symptoms associated with exposure, during 1991-1992, to river water or rainwater (River Murray, Australia) that was stored in open tanks and contained Anabaena blooms. Symptoms following oral intake (drinking water) included diarrhoea, vomiting, nausea, muscle weakness, sore throat, respiratory difficulties and headaches. Complaints following skin contact (recreational activities) or oral mucosal contact included rash, itching, mouth blistering and eye irritation. Further case-control studies in the same area are ongoing.

There have been reports of cyanobacterial blooms in surface water used for drinking purposes in China, where there is a high incidence of primary liver cancer; however, data are lacking.⁶ In an epidemiological study by Yu⁷⁷ of human primary liver cancer in Qidong county in China, the incidence of liver cancer was about eight times higher in people who drank pond and ditch water than in people who drank well water (no levels of algal toxins were determined). Further analytical epidemio-logical studies are required to elucidate a possible (additional) role of microcystins in this disease of multi-factorial aetiology. Hepatitis B infection and dietary exposure to aflatoxin B₁ are two known risk factors for liver cancer and are present in the same area of China.

A similar relationship was not observed in a larger epidemiological study of primary liver cancer in 65 Chinese counties reported by Junshi.⁷⁸ In this study, the use of deep well water was directly associated with liver cancer, which is contrary to the findings of Yu.⁷⁷ In a more recent epidemiological survey in Haimen City (Jian-Su province) and Fusui county (Guangxi province) in China, Ueno et al.⁷⁹ found a close relationship between the incidence of primary liver cancer and the use of drinking water from ponds and ditches. A combination of ELISA and affinity column chromatography was used to detect (detection limit 0.05 µg/L) very low levels of microcystins in the water samples without cleanup and concentration procedures (for method, see Nagata et al.⁸⁰). In September 1993, three of 14 ditch water samples contained microcystins, in a concentration range of 0.09-0.46 µg/L. Following this, samples were collected from five ponds/ditches, two rivers, two shallow wells and two deep wells monthly throughout 1994. The data showed that the highest concentrations of microcystins occurred from June to September, with a range of 0.058-0.296 µg/L. A third trial on the 989 water samples collected from the different water sources in July 1994 revealed that 17% of the pond/ditch water, 32% of the river water and 4% of the shallow well-water contained microcystins, with average concentrations of 0.1, 0.16 and 0.068 µg/L, respectively. Microcystins were not detected in deep well-water. A similar survey on 26 drinking water samples in the Guangxi province showed a high frequency of micro-cystins in the water of ponds/ditches and rivers, but no microcystins were found in shallow or deep wells.

Pilotto et al.⁸¹ examined the relationship between potential exposure to cyanobacterial toxins in drinking water during pregnancy and birth outcomes. The study examined >32 000 singleton live births between 1992 and 1994 in 156 Australian communities. Although significant differences were observed between exposure to cyanobacteria (estimated as cyanobacterial occurrence and cell density in the source drinking water) during the first trimester and the incidences of low and very low birth weights, the results do not suggest a causal link to cyanobacteria; no dose-response relationships were observed. The authors concluded that the study did not provide clear evidence of an association between cyano-bacterial contamination of drinking water supplies and adverse pregnancy outcomes.

Since cyanobacterial blooms tend to occur repeatedly in the same water supply, some human populations are at risk of repeated ingestion of cyanobacterial toxins.

Effects on Animals

Kinetics and Metabolism

Although the most likely route of exposure to cyanobacterial toxins is via ingestion, there have been few pharmacokinetic studies with orally administered microcystins. After intravenous or intraperitoneal injection of sublethal doses of variously radiolabelled micro-cystins in mice and rats, about 70% of the toxin is rapidly localized in the liver,^82-87 whereas oral administration resulted in less than 1% uptake into the liver of mice.⁸⁷ Although microcystin-LR does not readily cross cell membranes and does not enter most tissues, micro-cystins appear to be transported into hepatocytes and into the cells of the intestinal lining via the bile acid transport system^88,89; microcystin-LR has also been found to cross the ileum through the multispecific organic ion transport system.⁹⁰ In hepatocytes, micro-cystin-LR is covalently bound to a 40 000-dalton protein (protein phosphatase 2A and possibly protein phos-phatase 1) in the cytosol⁹¹ (for a review, see Fujiki and Suganuma⁹²). Microcystin congeners that are more hydrophobic than microcystin-LR may cross cell membranes by other mechanisms, such as diffusion.^7,13

Plasma half-lives of microcystin-LR, after intravenous administration, were 0.8 and 6.9 minutes for the alpha and beta phases of elimination, but the concentration of radioactive (³H-microcystin-LR) label in the liver did not change through the six-day study period; about 9% of the dose was excreted early via the urinary route, with the remainder being excreted slowly (~1% per day) via the faecal route.⁸⁵ Based on the protective effect of microsomal enzyme inducers, it is evident that the liver plays a large role in the detoxification of microcystins.⁸³ Time-dependent appearance and disappearance of additional peaks, thought to represent detoxification products, were seen in urine, faeces and liver cytosol fractions,⁸⁵ but these products have not been structurally identified. Three metabolic products have been identified in rats and mice following intraperitoneal injection of microcystin-LR, including glutathione and cysteine conjugates and a conjugate with the oxidized ADDA diene.⁹³

Acute Toxicity

Microcystin-LR is extremely toxic after acute exposure. Fatalities have been observed after animals consumed water containing large numbers (>10⁶/mL) of cyanobacterial cells.¹

The LD₅₀ by the intraperitoneal route is approximately 25-150 µg/kg bw in mice; the oral (by gavage) 94,95 LD₅₀is 5000 µg/kg bw in mice and higher in rats.This indicates that, even by the oral route, micro-cystin-LR is extremely toxic in mice following acute exposure; intraperitoneal injection is 30-100 times more toxic. Thus, a significant amount of microcystin-LR escapes the effects of peptidases in the stomach and is absorbed. The oral LD₅₀ of a toxic Anabaena extract in mice was also reported to be at least 170 times higher 96 than the intraperitoneal LD₅₀ of the same extract.

Yoshida et al.97 reported that the LD₅₀ for orally (gavage) administered (10.0 mg/kg bw) microcystin-LR in six-week-old mice was 167 times higher than the intraperitoneal value (65.4 µg/kg bw). Histologically, both routes of administration resulted in similar types of injuries to hepatocytes, including haemorrhage and necrosis.

The intraperitoneal LD₅₀s of several of the commonly occurring microcystins (microcystin-LA, -YR and -YM) are similar to that of microcystin-LR, but the intraperitoneal LD₅₀for microcystin-RR is about 10-fold higher.^98,99 However, because of differences in lipo-philicity and polarity between the different microcystins, it cannot be presumed that the intraperitoneal LD₅₀will predict toxicity after oral administration.

The microcystins are primarily hepatotoxins. After acute exposure by intravenous or intraperitoneal injection of microcystins, severe liver damage is characterized by a disruption of liver cell structure (due to damage to the cytoskeleton), a loss of sinusoidal structure, increases in liver weight due to intrahepatic haemorrhage, haemodynamic shock, heart failure and death. Other organs affected are the kidney and lungs.¹⁰⁰ Intestinal damage is a consequence of the transport of micro-cystins through the lining cells, which are damaged in a similar manner to hepatocytes.⁸⁹

Subchronic and Chronic Toxicity

In a study conducted at Quintiles by WRc in the United Kingdom, microcystin-LR was administered orally by gavage to groups of 15 male and 15 female mice at 0, 40, 200 or 1000 µg/kg bw per day for 13 weeks. The no-observed-adverse-effect level (NOAEL) for liver toxicity was 40 µg/kg bw per day. At the next highest dose level, there was slight liver pathology in one male and two female mice. At the highest dose level, all mice showed liver changes, which included chronic inflammation, degeneration of hepatocytes and haemosiderin deposits. In male mice at the two highest dose levels, alanine and aspartate aminotransferases were significantly elevated, serum --glutamyl transferase was slightly reduced and there were small but significant reductions in total serum protein and serum albumin; alkaline phosphatase was also significantly increased at the highest dose. In female mice at the highest dose level, only increases in alkaline phosphatase and alanine aminotransferase were observed. Male mice exhibited reduced body weight gain in all treatment groups, but there was no dose-response relationship, and the final body weight was depressed by only 7%.⁹⁴

Another oral repeated-dose study was conducted with Microcystis aeruginosa extract supplied to mice (410 in total) at six concentrations (control, one-sixteenth dilution, one-eighth dilution, one-fourth dilution, one-half dilution and undiluted toxic extract; undiluted extract had a toxin concentration of 56.6 µg/mL, estimated by LD₅₀value, which can be calculated to be approximately equivalent to a dose of 11 300 µg/kg bw per day) in their drinking water for up to one year. The mortality rate increased with dose at the two highest doses. At the highest dose, body weight was reduced in both sexes at nine weeks, and liver weight was increased in females at five weeks; males showed significantly increased liver weight as a percentage of body weight at the second highest dose but not at the highest dose, owing to high mortality and loss of body weight. At the two highest concentrations, alanine aminotransferase levels were elevated at five and nine weeks, and chronic active liver injury was apparent following exposure for up to 13 weeks; after longer periods of exposure to lower doses, no pathological changes in the liver were found that were directly related to the effect of toxin on hepatocytes, and no hepatic neoplasms were noted. There was also some evidence for increased bronchopneumonia with increased concentrations of extract.¹⁰¹

Ito et al.¹⁰² studied the effects of age on the liver of young and aged mice orally administered microcystin-LR. Twenty-nine 32-week-old (aged) and 12 five-week-old (young) male ICR mice received 500 µg/kg bw via gastric intubation; each group had three unexposed controls (aged and young). Animals from each group were sacrificed after two, five and 19 hours, and their livers and small intestines were examined. Sixty-two percent of the aged mice showed hepatic injury that could not be distinguished pathologically from hepatic injuries caused by intraperitoneal administration, indicating that microcystin-LR was incorporated into the liver following oral administration. The most severe damage in the small intestine of the aged mice was observed in the duodenum. In contrast, no effects, in either the liver or intestine, were observed in the young (five-week-old) mice. No significant differences were observed in either biochemical tests (glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase) or morphological examination of the livers of non-treated aged mice and young mice, indicating that the livers of the aged mice were healthy. Further testing in aged and young mice suggests that the uptake of the toxin via the oral route is related to the condition of the surface epithelial cells and the permeability of the capillaries in the villi of the small intestine and is strongly related to aging.

Heinze¹⁰³ studied the toxicity of pure microcystin-LR toxin in the drinking water of rats (10 animals/ group) exposed to approximately 50 or 150 µg/kg bw per day for 28 days. Dose-dependent increases in relative liver weight and serum enzymes (lactate dehydrog-enase and alkaline phosphatase) were observed. Liver damage, defined as "toxic hepatosis," was clearly indicated by histological examination of the tissues; damage was more severe at the higher dose.

In a poorly described study, Fitzgeorge et al.¹⁰⁴ reported that intranasal instillation of microcystin-LR in mice resulted in extensive necrosis of the nasal mucosa epithelium in both the olfactory and respiratory zones. The necrosis progressed to destruction of large areas of mucosa down to the level of deep blood vessels. The reported LD₅₀ for this route was the same as the LD₅₀ for intraperitoneal administration (250 µg/kg bw), and dose-dependent liver lesions were observed. The authors also reported cumulative liver damage following repeated intranasal dosing. Although no increase in liver weight was observed following a single dose of 31.3 µg/kg bw, repeated daily administration of this same dose for seven days resulted in a 75% increase in liver weight, which was very near the effect observed from a single intranasal dose of 500 µg/kg bw (87% increase in liver weight).

In a subchronic study, Microcystis aeruginosa extract was given to groups of five pigs in their drinking water for 44 days at microcystin dose levels calculated to be equivalent to 0, 280, 800 and 1310 µg/kg bw per day (the potency of the extract used was based on its intraperitoneal LD₅₀in mice). The extract contained at least seven microcystin variants, with microcystin-YR tentatively identified as the major peak. Liver function (as evidenced by changes in --glutamyl transpeptidase, alkaline phosphatase, total bilirubin and plasma albumin) was affected at the two highest doses, whereas visible liver injury was observed at all three doses; only one pig was affected at the lowest dose level. Thus, it may be appropriate to consider the 280 µg/kg bw per day dose level as a lowest-observed-adverse-effect level (LOAEL). LOAELs of a similar order of magnitude (ranging from 90 to 270 µg/kg bw per day) can be calculated using toxin contents of the dried cyanobacterial scum as determined by other laboratories.¹⁰⁵

In a chronic toxicity study, three vervet monkeys were dosed intragastrically (three times per week) with microcystin-LA for 46 weeks; the dose levels were gradually increased from 20 to 80 µg/kg bw over the duration of the experiment. No statistically significant alterations in clinical or haematological parameters or serum enzyme levels were observed in treated animals compared with controls, and there were no histopatho-logical changes in the liver or other organs of treated animals.¹⁰⁶ The results, although preliminary, suggest that the NOAEL of microcystin-LA in the vervet monkey is no less than the NOAEL of microcystin-LR observed in mice and may be higher.⁹⁴ Ueno et al.¹⁰⁷ exposed BALB-c female mice to 20 µg microcystin-LR/L in their drinking water for seven days per week ad libitum for 18 months (567 days); control mice received water alone. Animals were sacrificed at three, six, 12 and 18 months. Mean cumulative microcystin-LR intake after 18 months of exposure was estimated at 35.5 µg per mouse. No chronic toxicity or accumulation of the toxin in the liver was observed, nor was there any absorption from the intestines in the study. Neither water nor food consumption was affected by the treatment, and there were no treatment-related changes in a wide range of test parameters.

Reproductive and Developmental Toxicity

To investigate the effects of microcystin-LR on the embryonic and foetal development of the mouse, four groups of 26 time-mated female mice of the Cr1:CD-1(ICR)BR strain were dosed once daily by oral gavage with aqueous solutions of microcystin-LR from days 6 to 15 of pregnancy, inclusive. The dose levels were 0, 200, 600 and 2000 µg/kg bw per day. On day 18 of pregnancy, the females were killed and a necropsy was performed. The foetuses were weighed, sexed and subjected to detailed external, visceral and skeletal examinations for abnormalities. Only treatment at 2000 µg/kg bw per day was associated with maternal toxicity and mortality; seven of the 26 females died, and two were sacrificed prematurely during the dosing period because they showed signs of distress. There was no apparent effect of treatment at any dose level on litter size, incidence of resorption or the sex distribution of the live foetuses. Mean foetal weight was significantly lower in the high-dose group, and there was an increased incidence of foetuses with delayed skeletal ossification; both are common findings associated with maternal toxicity. Otherwise, there was no increased incidence of foetal abnormalities at any dose. The no-effect level for any aspect of developmental toxicity was 600 µg/kg bw per day.^94,95

To examine the effect of toxic Microcystis aeruginosa extract on reproduction in mice, Falconer et al.¹⁰¹ exposed male and female parents to a one-fourth dilution of extract (approximately 2800 µg/kg bw per day) in drinking water for 17 weeks prior to mating and through pregnancy and early lactation. No effects on fertility, embryonic mortality or teratogenicity were observed, other than reduced brain size in about 10% of the neonatal mice, compared with controls.

Tumour Promotion

There has been some evidence of tumour promotion in animal studies. In a modified two-stage carcino-genesis mouse skin bioassay, dimethylbenzanthracene (DMBA) (500 µg) in acetone was applied to the skin of four of six groups of 20 three-month-old Swiss female mice. After one week, the DMBA-treated mice received 1) drinking water, 2) Microcystis extract in drinking water (actual microcystin-YM dose not provided), 3) croton oil (as a positive control) applied to the skin (0.5% in 0.1 mL acetone twice a week) plus drinking water or 4) croton oil plus Microcystis extract; the control mice received drinking water or Microcystis extract in drinking water. After 52 days from initiation, substantial skin tumours and ulcers were visible on the DMBA-treated mice consuming Microcystis extract. Tumour growth was less substantial in the other three groups of DMBA-treated mice. The mean weight of skin tumours per mouse was significantly higher in DMBA-treated mice given the Microcystis extract than in the DMBA-treated mice given water. The actual number of tumours per mouse and the weights of the tumours in relation to the weights of the animals were not provided. It was concluded by the authors that Microcystis extract consumed in drinking water may act as a promoter.⁹⁶ However, the mechanism of action is not clear, as microcystins have difficulty penetrating epidermal cells.¹⁰⁸ The tumour weight per mouse in DMBA-treated mice given both croton oil and the algal extract was slightly lower than in those given croton oil and drinking water. These latter findings could not be explained by the author.⁹⁶

In a two-stage carcinogenicity bioassay, groups of 9-15 seven-week-old male Fischer 344 rats were initiated by intraperitoneal injection with diethylnitrosamine (200 mg/kg bw), followed by partial hepatectomy at the end of the third week. Tumour promotion was assessed by intraperitoneal injection of microcystin-LR at 1 or 10 µg/kg bw twice per week from the third week of the experiment. Tumour promotion, as indicated by an increase in glutathione S-transferase placental form (GST-P) positive liver foci, was seen after eight weeks in animals dosed with microcystin-LR at 10 µg/kg bw.¹⁰⁹ Microcystin-LR had no effect when given to non-initiated rats; as well, treatment with 1 µg/kg bw did not show any significant increase of foci. To confirm the tumour-promoting activity of microcystin-LR, the same authors administered microcystin-LR dose levels of 10 µg/kg bw before partial hepatectomy and 10, 25 or 50 µg/kg bw twice a week after partial hepatectomy to groups of 14-19 male rats. It was found that the increase in GST-P-positive foci following repeated intraperi-toneal injections of microcystin-LR was dose related. According to the authors, the results suggest that microcystin is the strongest of the liver tumour promoters found to date. Even though the study involved intra-peritoneal dosings, the authors suggested that tumour promotion by microcystin should be considered possible in humans as well.

Ito et al.¹¹⁰ compared the formation of hepatic neoplastic nodules in mice exposed to microcystin-LR by intraperitoneal and oral routes without pretreatment with initiators. Multiple neoplastic nodules (up to 5 mm in diameter) were observed in all mice (13/13) receiving intraperitoneal injections of 20 µg microcystin-LR/kg (five times per week) for a total of 100 injections over 28 weeks. Five mice were sacrificed immediately following the last injection, and the remaining eight were allowed to recover for two months prior to sacrifice.

Liver weights in these two groups were 9.0% and 6.8% of total body weights, compared with 4.7% in controls. The same researchers exposed 22 mice to repeated intragastric intubation of 80 µg microcystin-LR/kg for 80 or 100 treatments over 28 weeks; seven mice were allowed a two-month withdrawal prior to sacrifice. Although there were injuries to the hepatocytes of some of the animals, there were no characteristic chronic injuries to the liver, such as fibrous changes and nodule formation, as were observed in the intraperitoneal study; mean liver weights were not significantly different from the controls.

In another tumour initiation and promotion assay aimed at evaluating possible tumour-promoting effects in the upper small intestine, Falconer and Humpage¹¹¹ orally administered two doses (40 µg/kg bw each) of the initiator N-methyl-N-nitrosourea to C57 black mice, one week apart, followed by drinking water containing various levels of Microcystis extracts (0, 10 or 40 mg Microcystis toxins per litre), estimated to be equivalent to 0, 1.2 or 4.2 mg microcystins/kg bw per day, for up to 154 days. No primary liver tumours were seen in any group, and there was no evidence of microcystin-induced promotion of lymphoid or duodenal tumours.

Microcystin-LR was found to be a potent inhibitor of eukaryotic protein serine/threonine phosphatases 1 and 2A both in vitro^112,113 and in vivo,¹¹⁴ and this effect has become the basis of one of the bioassays to detect its presence. Such substances are considered to be 12-O-tetradecanoylphorbol-13-acetate (TPA) type tumour promoters. For the tumour promoter TPA, the mechanism of action is attributed to its activation of protein kinase C. Other substances that act similarly to microcystins are okadaic acid, nodularin, tautomycin and calyculin A (for a review, see Fujiki and Suganuma⁹²). The protein phosphatases serve an important regulatory role in maintaining homeostasis in the cell. They slow down cell division by counteracting the effects of various kinases through dephosphorylation of proteins. Protein phosphatase inhibition results in a shift in the balance towards higher phosphorylation of target proteins. This is a major post-translational modification. It can result in excessive signalling and may lead towards cell proliferation, cell transformation and tumour promotion. In liver cells, cytoskeletal components (intermediate filaments followed by microfilaments) are affected, which may result in reduced contact with other cells.^100,115 The inhibition of protein phosphatase 2A by microcystin-LR can be effectively reversed in the presence of polyclonal antibodies against microcystin-LR.¹¹⁶ The implications for low-level chronic exposure to microcystins in humans are not known.

Genotoxicity

No mutagenic response was observed for purified toxins derived from Microcystis in the Ames Salmonella

assay with or without S9 activation. The Bacillus subtilis multigene sporulation test was also negative with regard to mutagenicity using both the 168 and hcr-9 strains.In contrast, results of a study in which the purified toxins were tested against human lymphocytes suggested that the toxins may be clastogenic, as indicated by increased and dose-related chromosomal breakage.¹¹⁷ More recently, Ding et al.¹¹⁸ reported that a microcystic cyanobacterial extract (extract prepared was derived from >90% Microcystis aeruginosa) showed a strong mutagenic response in the Ames test (strains TA97, TA98, TA100 and TA102; with or without S9 activation), induced significant DNA damage in primary cultured rat hepatocytes (comet assay) and produced bone marrow micronucleated polychromatic erythrocytes in mice.