Portfolio on toxicology about Microcystin cyanotoxins

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BACHELOR OF PHARMACY (HONOURS)
PPT4452 TOXICOLOGY
SEMESTER 5 (BP15)
PORTFOLIO
Title : The Danger of Microcystin Cyanotoxins
to Human Health in Taking Algae Dietary Supplement
Name : Lam Hoi Sun
Student ID : 'confidential'
Tutor’s name : Mr. Tan K. S
Date : 2rd
October 2017

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Contents
Abstract......................................................................................................................................3
1.0 Introduction..........................................................................................................................3
2.0 The Toledo “Do not drink or boil” Water Advisory and its management...........................4
3.0 Cyanobacteria ......................................................................................................................5
4.0 Algae based supplements.....................................................................................................5
5.0 Microcystins.........................................................................................................................6
5.1 Chemical and Physical Properties....................................................................................6
5.2 Biosynthesis and Detection Methods...............................................................................7
5.3 Routes of Exposure..........................................................................................................7
5.4 Mechanism of action........................................................................................................8
5.5 Toxicokinetics..................................................................................................................8
6.0 Health Effects on Humans and Animals..............................................................................8
6.1 Acute or chronic toxicity reported in humans from contaminated water ........................9
6.2 Chronic toxicity reported in animal from taking algae supplement ................................9
6.3 Impact on human health from taking algae supplements...............................................10
7.0 Suggestions and Implications ............................................................................................10
8.0 Role of Health Care Professionals in health policy, regulation and guideline ..................11
9.0 Conclusion .........................................................................................................................12
Appendix..................................................................................................................................13
References................................................................................................................................17

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The Danger of Microcystin Cyanotoxins to Human Health
Abstract
Microcystins are highly potent cyanotoxins produced by blue-green algae or cyanobacteria in
freshwater. The algae contaminated the water body at Lake Erie with substantial amount of
microcystins and this affected ten-thousands of people in Toledo having no clean water
sources for two days. Microcystins-LR is the most potent microcystins congeners among
others. They demonstrated significant hepatotoxic actions in animal or human after exposure.
Acute and chronic intoxication of microcystins may ultimately cause severe liver impairment,
intrahepatic hemorrhage as well as hypovolemic shock. Blue-green algae are now marketed
as health dietary supplements such as Spirulina and Aphanizomenon flos-aquae (AFA).
However, risk of water contamination by microcystins raised the public concern on safety of
algae supplements for human health. Lastly, various fields of healthcare professionals play
the important roles in ensuring the safety of algae supplements in the market to the public by
taking certain measures.
1.0 Introduction
Water is the main natural source to living organisms for survival. People in Ohio from Toledo
had no clean water for two days after their government issued ‘DO NOT DRINK OR BOIL
water’ advisory in year 2014. Their main water source originated from Lake Erie has been
contaminated by mass of algae bloom with toxins producing cyanobacteria (1). They
synthesized potent cyanotoxins which are life-threatening to human and animals especially
microcystins.
Surprisingly, blue-green algae are commercially available as health dietary supplements in
the markets typically with Spirulina sp. and Aphanizomenon flos-aquae (AFA) algae (2).

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Most of the health-orientated people believed that Spirulina is one of the superfoods in the
21th century with its high nutritional values. However, recent study (3) have detected
microcystins in Spirulina-based and AFA-based algae supplements from German market
which are exceeding the tolerable daily intake (TDI) value of 0.04μg/kg proposed by WHO
(2,3). This raises the safety issue of taking algae supplements for human after the water
contamination crisis happened.
Therefore, these review focuses on the effects of microcystins from contaminated water to the
humans’ health whom taking algae-based dietary supplements.
2.0 The Toledo “Do not drink or boil” Water Advisory and its management
On 2 August 2014, more than 400,000 people in Ohio experienced life without clean water
source from Lake Erie for two days after the local government issued emergency water
advisory (4). They warned people not to drink and boil water after they detected water sample
collected from Lake Erie was at dangerously high level of microcystins at 2.47 ppb from
acceptable value of 1.0 ppb proposed by WHO (5,6).
Formation of algae blooms are the main contributor for water pollution in Lake Erie. In 2014,
they grew extensively with substantial amount in a shocking growth rate. It was primarily
started when excessive level of phosphorus flow into the lake watershed (7). Horstman (8)
stated that tons of phosphorus load into Lake Erie were emerged from agricultural fertilizers
used for farming (Figure 1) and manure runoff effect which caused sudden spike of algal
bloom growth with high nutrients load available.
In order to prevent further growing of algae bloom in Lake Erie, U.S. and Canada
governments implemented Lakewide Action and Management Plan (LAMP) that aims to
reduce phosphorus content in lake water by 40 % in 2016 (9). Besides, U.S. Department of

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Agriculture invested $41 million over a three-year period to support new conservation
farming practices such as subsurface injection of fertilizers (7) and agricultural best
management practices (BMPs) (10). BMP is proved to be minimally effective in reducing
nutrient and sediments loads to Lake Erie despite on different factors and it should be widely
implemented (10).
3.0 Cyanobacteria
Cyanobacteria are groups of autotroph bacteria that obtain energy through photosynthesis
(11,12). They are known as blue-green algae which derived from their color appearance.
They appear in spiral shape and arranged either in unicellular, colony or multicellular
filamentous forms (12,13). They are Gram-negative bacteria with their thick peptidoglycan
wall membrane as shown in Figure 2.
They are asexually reproduce through binary fission and grow rapidly to form bloom at their
favorably aquatic environment with high nutrients concentration, optimum temperature
(>25°C), water with long hydraulic retention time (> 1 month) and stable water body
stratification. (14)
In this case, the nutrients availability from dissolved phosphorus and nitrogen loaded into
Lake Erie from fertilizers contribute the rapid growth of cyanobacterial bloom. Besides,
optimum climate temperature like summer can increase the rate of microcystins biosynthesis
because it may enhance mineralization of nutrients that is beneficial to cyanobacteria (14).
4.0 Algae based supplements
Blue-green algae are well recognized health supplements by the public with rich source of
proteins, phytonutrients, minerals and vitamins (15). They used to treat and prevent from
malnutrition, rheumatism and cancers with their anti-inflammatory, anti-cancer and anti-

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oxidant properties. Besides, they are good in lowering cholesterol levels for hyperlipidaemic
patients (16) and enhancing the expression of β-cells in the islets of Langerhans in diabetic
patients(17).
5.0 Microcystins
5.1 Chemical and Physical Properties
Microcystin is a large monocyclic heptapeptide that contains 7 amino acids joined together as
a cyclic compound (12,13) with approximately 100 types or congeners of it based on their
amino acid composition listed in Table 1. The most frequently studied congener is
Microcystin-LR (MCLR) with Leucine (L) and arginine (R) amino acids in their cyclopeptide
structure as shown in Figure 3 (11,17–19). Microcystin congeners with hydrophobic L-amino
are more toxic than hydrophilic R-amino acids (21). The physical and chemical properties of
Microcystin-LR are summarized in Table 2.
Microcystins are water soluble compounds from cyanobacterial blooms commonly found in
freshwaters as described in the case (7). They are stable in aquatic environments because they
tend to locate within cyanobacterial cell (12). Fang Li et. al (22) observed that the boiling of
contaminated water can precipitate the cyanotoxins concentration in water especially at
boiling point of water for 20-minutes. They may persist for more than 3-weeks to 3-months in
the water even in dark (12). That’s reason on why people in Ohio not to use contaminated
water for drinking, boiling and showering with risk of microcystins exposure. In nature,
microcystins are degraded by aquatic bacteria in water reservoirs such as Arthrobacter spp
(12).

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5.2 Biosynthesis and Detection Methods
Microcystins are released in substantial amount from cyanobacterial cytoplasm into aquatic
environments at bloom upon cells lysis (1,12). The rate of microcystins biosynthesis is
mainly regulated by environmental and nutritional factors such as light intensity, nutrients
availability and temperature. In this case, the nutrients availability and temperature change
are main factors of forming cyanobacterial blooms as well as the production rate of
microcystins.
Adda ELISA assay is the most frequently technique to detect presence of microcystins
because the prepared polyclonal antibodies have strong binding affinity against Adda moiety
in microcystins (2,18,23). Besides, High Resolution Melting (HRM) analysis is highly
sensitive, cost-effective and rapid method to detect cyanotoxins in Spirulina dietary
supplements (24). The identity of microcystin congeners can be identified using liquid
chromatography tandem mass spectrometry (LC-MS/MS) (25).
5.3 Routes of Exposure
The City of Toledo advised their people not to use the contaminated water for drinking,
boiling and showering (1). This is because humans and animals commonly expose to
microcystins from direct ingestion, inhalation and skin contact of contaminants (12,18,20,26).
Moreover, microcystins can present in aerosol form from evaporation of contaminated water
which can contact the respiratory epithelium cells (20) especially during showering or
swimming. However, the highest risk of microcystins intoxication documented in living
organisms are either from ingestion of suspected food (18) or consumption of contaminated
water (20,27).

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5.4 Mechanism of action
Upon exposure of microcystins, they are actively absorbed into portal circulation from the
digestive tract and rapidly cleared from the plasma through active transport into hepatocytes
(28). Microcystins are tightly bound to serine/threonine protein phosphatase 1 (PP1) and
protein phosphatase 2A (PP2A) in hepatocytes and inhibited their action as shown in Figure 4
(28). These could result in hyperphosphorylation of hepatocytes cellular proteins, causing
morphologic change of hepatocytes and sinusoids (28). They also induce disruption of
cellular organelles especially mitochondria that functions to produce ATP energy. Acute
exposure of microcystins could severely damage the hepatocyte cytoskeleton and ultimately
lead to intrahepatic hemorrhage with hypovolemic shock (28).
5.5 Toxicokinetics
Microcystins are transported into hepatocytes through passive diffusion using Organic Acid
Transporter polypeptide (OATp). MCLR are potent hepatotoxins with oral bioavailability of
more than 95% (28). Moreover, microcystins are metabolized either through glutathione or
cysteine conjugations and excreted as faeces through biliary route (28). Furthermore, MCLR
has nephrotoxic effect because small fraction of it accumulated in kidneys and excreted in
urine. At last, the severity of microcystins intoxication is largely depending on the amount of
microcystins being exposed. (28)
6.0 Health Effects on Humans and Animals
Several cases of acute and chronic microcystins intoxication in humans and animals have
been reported throughout the years (18,20,27). Majority of them were exposed to
microcystins either from direct contact or ingestion of contaminated water (20,27). There is
few microcystins intoxication cases reported from taking contaminated algae supplements
only in animal but not in human(18,26).

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6.1 Acute or chronic toxicity reported in humans from contaminated water
Acute poisoning of cyanobacterial from contaminated water is common in most cases. Firstly,
an acute microcystin intoxication case has reported on 19 years-old man who exposed
cyanotoxins from swimming at a recreational water park in Salto Grande. The water has been
contaminated with high concentration of MCLR at 48.9 μg/dL and he have direct contact,
oral ingested as cyanotoxins from contaminated water for at least 2-hours (20). He initially
has mild gastrointestinal symptoms such as vomiting and diarrhea, followed by pulmonary
illness until diagnosed with hepatotoxicosis and multiple organ failure (20).
Moreover, parental exposure of cyanotoxins is more severe than oral administration. This
could be explained in the outbreak of severe hepatitis happened at a Brazilian hemodialysis
center from Caruaru in year 1996 (29). 100 patients were developed with acute liver failure
from this incidence on 50% mortality rate from initially 86% of patients complained of
‘Caruaru syndromes’ including painful hepatomegaly, nausea and vomiting after undergo
hemodialysis treatment. This is caused by defect of dialysis water treatment system that fails
to filter the contaminated reservoir water with microcystins for dialysis treatment (29).
6.2 Chronic toxicity reported in animal from taking algae supplement
The first case of microcystins intoxication from taking algae supplement was reported to 11
years-old female spayed Pug dog in year 2015 who is fed with 1g of AFA-based algae
supplement once daily for 3 ½ weeks (18). Upon powder analysis, four specific microcystin
monogeners have been detected including 166 ng/g of MCLR and 962 ng/g of MC-LA. (18)
This dog was diagnosed with coagulopathy disease with increased liver enzymes level which
indicating to hepatic impairment (18). The autopsy of its liver tissues is characterized by
severe gross hepatomegaly with progressive centrilobular hepatocytes necrosis by the actions

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of microcystins (18,27). From the case, study suggested the lethal dose of microcystins is
ranging from 43 μg/kg to 11 mg/kg, depending on which congeners.(18)
6.3 Impact on human health from taking algae supplements
How about the human health after taking contaminated algae supplements? Currently, there is
no such mortality case reported in humans. However, majority of cyanobacterial intoxication
are associated with gastrointestinal and hepatic illnesses (15,19,20,27–29) especially during
outbreak of cyanobacterial bloom and artificial bloom lysis from chemicals runoff (1).
Microcystins-infected patients may experience loss of body weight with increased of liver
damage, liver weight and ATP enzymes levels (20,29). Besides, microcystins toxicity is also
associated with centrilobular hepatic necrosis, destruction of sinusoidal endothelium,
disruption of bile canalicular function, intrahepatic hemorrhage, loss of microvilli and bleb
formation in hepatocytes (14,20,28,29). Microcystins also may induce tumor necrosis factor-
α and leads to primary liver cancer as reported in China (30).
Contrary to microcystin-LR, algae supplement may contain other cyanotoxins such as
neurotoxic alkaloids anatoxin–α and saxitoxins which worsen the toxicity due to synergetic
effects (14,31). The toxicity of Spirulina has been studied in various aspects and been
summarized as shown in Table 3 (15).
7.0 Suggestions and Implications
The risk of health following exposure of microcystins from contaminated drinking water has
widely recognized nowadays. However, people whom taking blue-green algae dietary
supplements have greater potential of exposing to microcystins especially to infants and
elderly(28). This is because daily intake of cyanotoxins is limited by water consumption rate
but no limitation for taking algae supplement proposed by Gilroy (19).

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Most of the algae plantation was located at open or controlled aquatic environment. The
supplements were mostly harvested on bloom season for high productivity which feasible to
cyanotoxins contamination if there are no proper management and functioning water
treatment system (32). Every water body for algae plantation should be tested with routine
toxin analyses to ensure the cyanotoxins are under tolerable concentration (31).
Routine product testing is required for every algae supplement products before release to the
markets. There is a need for quality control system for monitoring cyanobacterial populations
intended for dietary supplements (27). FDA has approved all the algae dietary supplements in
the markets without strict regulations as they are considered to be safe for intended use. They
only take actions against the products if there is significant or unreasonable risk of health and
injury.
8.0 Role of Health Care Professionals in health policy, regulation and guideline
Pharmacists play crucial roles in educating public on nutritional supplements especially to
health-orientated people. Pharmacists should counsel patients on selection of algal
supplements that are safe to taken and licensed by local authority such as National
Pharmaceutical Regulatory Agency (NPRA). Pharmacists should be knowledgeable about the
products information and details of manufacturers and distributors prior to public concerns.
The pharmaceutical manufacturers have responsibility on manufacturing nutritional products
that complying the regulations and guidelines from FDA and WHO if applicable to ensure
nutritional products safety and efficacy. In April 2017, FDA issued a new regulation on any
algae nutritional products should be negative to microcystin toxins after analysis (33).
Environmental Health Officers should inspect recreational parks, swimming pools and
agricultural water reservoirs regularly for water analysis. WHO established Guideline Value

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(GV) derived from toxicity data and TDI value of 0.04μg/kg for microcystin is 1μg of
microcystins in 1L of drinking water which used as investigational marker for the needs of
any intervention (27,31). There was lack of evidence on microcystins intoxication in human
from consumption of contaminated algae supplements. Even through Microcystin-LR has the
most potent hepatotoxic effect, further studies are needed to study properties of others
microcystin congeners to improve the accuracy and consistency of the findings.
9.0 Conclusion
In conclusion, microcystins are highly poisonous hepatotoxin synthesised by blue-green algae.
They are the main causative agent of water pollution in Toledo Water Crisis. People have to
aware of the potential risk of microcystins toxicity on taking algae supplements especially the
sources of algae production. People infected with microcystins could develop with mild
gastrointestinal symptoms to severe liver damage. Therefore, it is important to select certified
algae supplements in the markets which are safe to be taken and free from cyanotoxins
contamination. Lastly, government should encourage the implementation of new farming
practices and improvement of water management to prevent the prognosis of cyanobacterial
blooms growth.

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Appendix
Figure 1: Total Phosphorus Load into Lake Erie from Various Sources (34)
Figure 2: Cross-section of a Typical Cyanobacterial Cell. (35)

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Figure 3: Structure of Microcystin-LR. (36)
Table 1: Amino Acid Compositions of Various Microcystin Congeners. (12)
Table 2: Physical and Chemical Properties of Microcystin-LR. (12)

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Figure 4: Effects of microcystins in hepatocytes (H) and sinusoidal capillary structure (SC)
by inhibiting protein phosphatases PP1 and PP2A enzymes. (28)
Figure 5: Toxicokinetic properties of microcystins (28).

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Table 3: Toxicity of Spirulina demonstrated from Various Tests. (15)

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px