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[](https://)Serum microcystin levels as an indicator to Primary Liver cancer occurrence at the Jaramogi Oginga Odinga Teaching and Referral Hospital, Kenya 
Evaline Chemutai1, David M. Onyango 1, Cyrus Ayieko 1, Ngwena G. Magak 2
Affiliations:
1.  Maseno University, School of Physical and Biological Sciences, Department of Zoology.
P. O. Box 333-40105, Maseno
2. Maseno University, School of Medicine, Department of physiology.
P. O. Box 333-40105, Maseno
Corresponding   Author. David M. Onyango. Maseno University, Department of Zoology.
P. O. Box 333-40105; Email.dvdonyango7@gmail.com. Tel. +254722660647
Abbreviations: PLC: Primary Liver Cancer; MCs: Microcystin; WHO: World Health
Organization; JOOTRH: Jaramogi Oginga Odinga Teaching and Referral Hospital; PP1: Protein
Phosphatase 1; PP2A: Protein phosphatase 2A; TDI; Total Daily Intake
Abstract	
Microcystin (MCs) is known to promote primary liver cancer (PLC) through inhibition of serine-threonine protein phosphatases (PP) 1 and 2A.   There are limited reports from other parts of the world to suggest that MCs could be a factor in PLC development. However, no follow up studies have been conducted in Kenya, especially among the population living around Lake Victoria that has high levels of MCs. To establish this association, a case control study was designed with the aim to investigate: prevalence and levels of microcystin in PLC cases; the levels of serum serine-threonine phosphatases (PP 1 and PP 2A) in PLC cases; and the association between serum-microcystin and the occurrence of PLC.  A total of 20 PLC cases and 20 controls were recruited from Jaramogi Oginga Odinga Teaching and Referal Hospital (JOOTRH) oncology department. Approximately, five milliliters of blood samples were collected and analyzed for serum microcystin and the total serum-serine-threonine Protein Phosphatase 1 and 2A levels using enzyme-linked immunosorbent assay. The association between serum microcystin and PLC occurrence was determined using chi-square test. A total of 28 samples (71.43% cases and 28.57% controls) confirmed the presence of microcystin. The serum MCs median (interquartile range) of PLC cases and controls were recorded. PLC patients had higher levels of serum MCs; 0.524µg/ml (1.34µg/ml-0.051µg/ml) than controls 0.111µg/ml;  (0.84µg/ml-; -024µg/ml). Pearson’s chi-squared test showed a strong association between serum-microcystin and PLC development (X²=6.321, df=1, P=0.012); the association was statistically significant p<0.05. Similarly, higher levels of PPs activities were expressed in PLC patients (103.9 ±17.83µg/ml) than in controls (25.91± 3.342µg/ml). The independent t-test showed that the variations of microcystin levels were statistically significant (P<0.05; t38=4.298;). These findings indicate that continuous exposure to MCs may lead to development of PLC and should be considered as one of the cancer causative agents along cyanobacteria contaminated water bodies. This therefore poses a health risk to communities who depend on lake water not only for domestic use but also for recreational activities. 
Keywords.  Microcystins-Leucine Arginine (MC-LR), Primary Liver Cancer (PLC), Hepatitis B and C infection, Serine threonine protein phosphatase(1 , 2 A), Gastrointestinal tract (GIT)
1.0 Introduction.
Primary Liver Cancer (PLC) is a global public health problem with little or no- proper medical attention in developing countries (28). This malignancy is reported to affect hepatocyte through the organic anion-transporting polypeptides (OATPS 1/2). The malignancy is ranked second among the leading cancer-related deaths worldwide (4). Majority of the incidence rates occurred in regions such as Asia and Africa. Currently, in Kenya, there is an upward surge of primary liver cancer cases with no confirmed associated risk factors along the lake region of Nyanza (8). The high number of cases (5.2%) reported in Nyanza region, is as a result of a number of counties along the lake region recording high prevalence rate of PLC respectively (8). Among the predisposing risk factors in Kenya to PLC are chronic hepatitis B and C virus infection, exposure to afflatoxin and excess alcohol intake (30). However, little is known regarding the link between PLC prognosis and prolonged exposure to microcystin through either consumption of contaminated drinking water and food (e.g. fish and other animal products), or through recreational facilities (swimming pools). 
Effect of climate change together with increased anthropogenic activities along water bodies has led to eutrophication resulting to cyanobacterial blooms(3) , in the world, such as in the Baltic Sea in Europe, Lake Erie in America, and Lake Victoria in Africa (34). The cyanobacterial bloom results in release of cyanotoxins (36), microcystins being one of them.  Recent reports indicate increased cyanobacterial bloom in Lake Victoria, Nyanza bay, with the levels of microcystin up to 21.4µg/L (31), which is high above the World Health Organisation (WHO) recommended levels (35). This poses a health risk to communities who depend on lake water for domestic use (36). Microcystins (MCs) is a type of water-borne toxin that is secondary metabolite from cyanobacterial blooms (34). MCs are usually contained within the cyanobacterial cells and are released to the environment during cell lysis (27). Therefore, their primary route of exposure to mammals is via oral consumption of contaminated water, vegetables and aquatic organism such as (fish, crusteaceans and molluscs) (26). The toxin is reported to accumulate in aquatic wildlife and in water bodies where it is transferred to higher trophic levels with the risk of bio-accumulating in their systems and may lead to PLC (10). Hence this study envisaged to determine the association between exposure to microcystin toxin and occurrence of PLC disease in communities living along Lake Victoria, Kenya.
Microcystin primary target organ in animals are the liver and kidneys (38), though the liver is the most affected (1). MCs within the gastrointestinal tract (GIT) is transported via the liver hepatic portal vein into the liver (16). Once inside the hepatocytes, MC exhibit toxicity via inhibition of a number of enzymes including the protein phosphatases 1 (PP1) and 2A (PP2A) (18). These two enzymes catalyze the de-phosphorylation of the serine/threonine amino acids from proteins and are significant in many signal transduction pathways in the cellular processes such as cytoskeletal rearrangement, cell movement and apoptosis (1). Consequently, inhibition of these enzymes leads to uncontrolled cell proliferation and hence cancer (12). Indeed, MCs have also been described as possible tumor promoters (23,14). However, there has been little focus on the involvement of MCs in PLC (39). For instance, Chen et al., (9) reported a positive correlation between microcystin serum concentrations in local fishermen from Lake Chaohu and several liver function enzymes. In addition, Li., et al (21), observed that children who consumed drinking water collected from microcystin contaminated lakes had a significantly higher level of liver enzymes e.g. alkaline phosphatase (ALP), aspartate aminotransferase(AST) than the control population. While exposure to MCs has also been linked to increased incidence of Primary Liver Cancer in studies done in Serbia, United States and China (15), PLC cases decreased after cyanotoxin-free drinking water was provided into a population (16). There is no record of follow-up studies in sub-Saharan Africa, where changes in human activities have led to increased algal bloom hence MC in water systems in the recent past.  Therefore there was need to investigate the association between exposure to microcystins toxin in Lake Victoria and occurrence of primary liver cancer.
Consequently, a case control study that aimed at determining the association between microcystin and PLC occurrence was carried out among patients attending Jaramogi Oginga Odinga Teaching and Referral Hospital Kisumu Western Kenya who happened to have had an earlier or recent exposure to microcystin contaminated water from the either the Lake, borehole or swamps as their source of water. 
2.0 Materials and Methods
2.1 Sampling site and study population.
This was a clinical case-control study design, conducted at Jaramogi Oginga Odinga Teaching and Referral Hospital (JOOTRH). The hospital in found in Kisumu county within latitude -0.08842° or 0° 5' 18" south and longitude 34.7704° or 34° 46' 14" east, open location code 6GFPWQ6C+J5, Open¬Street¬Map ID node 8190853318. The study was approved by ethics committee of Maseno University (Msu/drpi/muerc/00753/19), JOOTRH (ierc/jootrh/179/20) and National Commission for Science, Technology and Innovation (NACOSTI) (NACOSTI/p/20/4122). Informed written consent was obtained from each study participant as well as permission to undertake the study within the hospital premises by the hospital ethical scientific board.  Newly diagnosed PLC cases who underwent hepatectomy at JOOTRH and confirmed positive pathologically were recruited in the study.  The patients were to residents of Nyanza region for at least five years. The controls who were enrolled to the study had other predisposing risk factors for PLC and were referred to the liver clinic for the respective risk management, however they did not have PLC despite the displaying PLC risk factor symptoms. The controls had risk factors for PLC, such as hepatitis B and C infection, Diabetes and alcohol use but negatively tested of PLC. They were recruited from blood transfusion services, medical outpatient department, liver clinic and medical wards. The hall mark of development of PLC is inflammation of the liver; hence, the controls with risk factors were the best group since they had a factor which can cause liver inflammation. Exclusion criteria entailed patients who failed to answer the questions in the questionnaire: who had other types of tumors; decline to participate; had severe digestive system diseases. A total of 40 participants were enrolled into the study: 20 PLC patients and 20 controls. 

2.2 Sample collection and analysis
About five milliliters of venous blood sample were collected from each study participant by a trained phlebotomist and aliquoted into anticoagulant free vacutainer tubes. The collected blood was then stored in a cool box at 4ᵒC and transported to the laboratory at Maseno University Zoology department for processing within 6 hours of collection. At the laboratory, the blood samples were processed to obtain serum. The serum was aliquoted into cryovials tubes and stored at -20ᵒC for batch analysis.	
2.2.1 Determination of microcystin levels in serum 
MCs levels in serum were determined using direct competitive Enzyme-linked Immunosorbent assay kits (20-0068; Beacon Analytical Systems Inc.), following the manufacturer’s instructions. Briefly, 50 µL of microcystin–horseradish peroxidase enzyme conjugate solution was added to each well, and then 50 µL of negative control, standard solution (serial dilutions of 0.1, 0.3, 0.8, 1.0, and 2.0 ng/mL) and 50 µL of each sample were added into the assigned well in duplicate. The plates were lightly swirled to mix the contents thoroughly. The mixture was then incubated at 37ᵒC for 30 minutes in the dark. The mixture was then washed five times with wash buffer. Then the plate was inverted and lightly patted on absorbent paper towels to remove remaining solution in wells. This was, followed by addition of 100 μL of substrate solution to each well and then shaken lightly and incubated. After incubation for 30 minutes at 37ᵒC in the dark, 100 µL of stop solution was added to each well. The plate was read on a microtiter plate reader (Beacon Analytical Systems Inc.), at 450 nm wave length. 
2.2.3 Determination of serine-threonine protein phosphatase 1 and 2A using para-Nitro phenyl phosphate (p-NPP)
Total levels of serine-threonine PP 1 and 2A was determined using p-NPP alkaline protein phosphatase kit (Biovison/Abcam company), following manufacturer’s instructions. Alkaline phosphatase dilution buffer was prepared for protein phosphatase using the following reagents :( 20mM Tris HCL, pH=7.5,5 mM MgCl2, 1mM EDTA, 0.02% DTT 3). Alkaline phosphatase standards (10µg/ml-comp E) were then diluted to 0.2µg/ml (1:50) using dilution buffer to make 2-fold serial dilutions of the following concentration; 100, 50, 25, 12.5, 6.2, 3.1 and 0 µg/ml, and were added into each well. Serum sample from each participant was aliquoted and diluted with dilution buffer in a ratio of 1:100 and added into each well (clear 96-well plate) in duplicates. A 100µl of each serially diluted standard concentration were also added into their specific wells in duplicates. A non-phosphates- containing sample was included as a negative control. 50µl of p-NPP substrate solution were added into each well. The reagents were mixed gently by rocking the plate for 30 seconds. The reaction mix was incubated at room temperature (25ᵒC) for 30 minutes. After incubation, 50 µL of stop solution were added into each well (96-well plate), the plates were then shaken on a plate shaker for 1 min before the reading. The absorbance was then measured at 405 nm using ELISA reader plate.
2.3.0 Statistical analysis
Statistical analysis was performed using GraphPad Software, LLC Prism for windows vs 8.3.0 (538). The levels of serine-threonine protein phosphatase liver enzymes between the PLC cases and normal individuals were analyzed using independent t-test. The association between the presence of microcystin and PLC occurrence were determined using Chi-square test. 
3.0 RESULTS
3.1 Socio-demographic characteristics of the study participants.
A total of 40 participants were enrolled into the study, 20 PLC cases and 20 controls. Out of these 23 (57.5%) were male and 17(42.5%) were female. Of the PLC patients, 14 (70%) were males while 6 (30%) were females. Through open ended questionnaire we were able to ascertain that all our study participants (PLC cases and controls) had lived within Nyanza region for a period of at least five years. In addition most of them  were involved in fishing as their source of economic livelihood while the rest had come in contact with the Lake Victoria water in their lifetime for bathing, fishing and other recreational activities (Table1).
Table 1: Socio-demographic characteristics of PLC Cases and Controls
Characteristic		Total N (%)	Plc Cases  N (%)	Controls N (%)
Participants		40	20	20
Gender	Male	23 (57.5%)	14 (70%)	9 (45%)
	Female	17 (42.5%)	6 (30%)	11 (55%)
Age group	15-30yrs	11 (27.5%)	5 (25%)	6 (30%)
	31-49yrs	17 (42.5%)	8 (40%)	9 (45%)
	>50yrs	12 (30%)	7 (35%)	5 (25%)
Source of drinking water	Lake	17 (42.5%)	8 (40%)	9 (45%)
	Tap Water	11 (27.5%)	5 (25%)	6 (30%)
	Well	12 (30%)	7 (35%)	5 (25%)

3.2.0 Identification and level of microcystin in serum of PLC patients and controls.
Out of the 40 participants, 28 were reported to be microcystin positive.  Of the 28 participants, 17 had microcystin levels above WHO recommended value per weight of 1.0µg/ml. The highest microcystin level reported was 5.36µg/ml and 0.2µg/ml as lowest. However, only 8 participants confirmed the presence of microcystin in the control group. The highest microcystin value in this group was 3.34µg/ml and 0.32µg/ml as lowest level.  Statistically, PLC cases had higher levels of serum MCs than controls by Pearson’s chi-squared test (P-value< 0.05; P=0.0119) comparison. The serum MCs median (interquartile range) of 2.10µg/mL for PLC cases and -0.01µg/mL for controls was recorded.  However, the PLC cases had a mean of 2.403µg/ml and 0.382 for the controls was equally recorded. Table 2.
Table 2: Summary of cases/controls identified with varying MCs levels and prevalence
 Levels of MCs
                                    conditions	Mean values
 of MCs level
 (µg/ml)	Median 
Values of  Mcs
µg/ml	Variance
Values 
of   Mcs 
	Standard
Deviation
values

	Prevalence of 
samples with 
MCs	Prevalence of samples without MCs
PLC Cases (n=20)	0.60 	0.53	0.13	± 0.36	20 (50%)	0%
N-PLC Controls (n=20)	0.28 	0.11	0.08	± 0.28	8 (20%)	12 (30%)
3.3.0 serum-microcystin on total levels of serine-threonine protein phosphatase 1 and 2A among PLC cases and controls
The results indicate that the serine-threonine protein phosphatase activities in liver cancer patients were highly expressed (Table 3.0; Fig. 2.0). The mean concentration levels of serum to total serine-threonine proteins phosphatases (PP1/PP2A among liver patients was observed to be statistically significant (P<0.05; t 38 =4.298). These levels varied among cases and controls in that PP1/PP2A among the liver cancer patients was 103.9 ±17.83µg/ml and that of controls was 25.91±3.342µg/ml.
Table 3.0: Summary of total levels of serine-threonine protein phosphatases among cases/controls
Conditions/Levels	Mean values 
 µg/ml	Median values µg/ml	Variance
	 Standard
deviation
	LOW (below
20µg/ml)	NORMAL
 (20-125
ug/ml)	HIGH (above 125
ug/ml)
PLC Cases(n=20)	103.9 	90.22	317.91	± 17.83	3 (15%)	8 (40%)	9 (45%)
Controls(N-PLC)(n=20)	25.91	33.94	11.16	±3.34	5 (25%)	15 (75%)	0
Legend: A summary of the data obtained from the total serine-threonine protein phosphatase of
PLC cases and controls PLC; Primary Liver cancer; N-PLC; Negative Primary Liver
cancer; n=sample size; Normal range of serine-threonine protein phosphates (20-125µg/ml).

Fig 2.0 A bar-graph representation of the mean concentration variation of Serine-Threonine protein phosphatase among the study participants. The mean concentration variation was statistically significant; **p-value = 0.0001; Y-axis represent. TSTP>Total threonine protein phosphatase whereas the X-axis represent the condition of study participants; PLC>Primary liver cancer cases and controls (non-primary liver cancer patients- N-PLC
3.4.0 Association between microcystin presence and occurrence of PLC.
The figures 3,4 and 5 below show the results   of serum microcystin and its association with PLC occurrence. The logistic regressions analysis shows a perfect prediction that microcystin was an
independent risk factor for PLC occurrence in this study. Statistical analysis using pearson’s chi-
squared test with Yates’ continuity correlation showed a significant correlation between serum-
microcystin and PLC development (X²=6.321, df=1, P=0.01193).

Fig.3.0: The brown and blue colors depicted negative and positive correlations respectively 

Fig 4.0 A correlogram showing the correlation between MCs levels and PLC occurrence.	

Fig 5.0: This means that the model predicts the occurrence of PLC.
4.0 DISCUSSION.
In the present study, we report for the ﬁrst time the presence of MCs in serum samples of PLC cases and controls who were chronically exposed to microcystin along Lake Victoria, Kenya. Total amount of PLC serum-MCs had a mean value of 2.403µg/ml, which were beyond the WHO recommended standard values of 1µg/ml. So far, there is paucity of information regarding human serum MCs concentration. In previous studies from the Caruaru hemodialysis patients who had suffered from acute lethal exposure, serum-microcystin concentration was estimated to be 2.2 ng/ml (17 samples from 22 victims) (2,7). This depicted a lower mean MC concentration found in serum samples as compared to this study. Zheng et al.,(39)  in their study also reports a median serum-MC concentration of 0.59ng/mL, which is lower when compared to this study where it was 2.095µg/ml. These differences among the studies could be attributed to various factors such as methods of analysis, duration of exposure of individual to a risk factor or individual variation in toxicokinetics of microcystin. However, the difference in data is not that large in order to adjudicate among the studies. 
Several studies have reported levels of serum microcystin in humans to be within a mean of 1.8ng/ml, (9,19,40)  illustrating a systemic exposure in different population as compared to the results in the current study. This support the attributing factor of the duration of exposure as an indicator that those populations who have got prolonged exposure are likely to have a higher level of serum microcystin than those of short time exposure. For instance, a study in fishermen exposed to Total Daily Intake (TDI) levels of Microcystin-leucine arginine (MC-LR) (~0.04 µg/kg) reported an average MC-LR serum concentration of 0.39 ng/Ml (9). In relation to other MC-LR studies, the data presented in this study indicate the levels of concentration accumulating in human serum over a different period of exposure, since the time of exposure was not taken into consideration. However compared to other two studies in which time of exposure was considered;(Oral exposure in fishermen versus intravenous exposure routes in rats;20µg/kg MC-LR in 120min)(11);the level of serum concentration between the two studies was only of a 500-fold differences, yet it was hypothesized that oral exposure would have a lower level of serum concentration(9).In contrast a pig study used a dose 50 times above the TDI(2µg/kg), but on the contrary, no serum microcystin concentration was detected, thus suggesting that animals could be having lower intestinal absorption compared to humans.
The high levels of microcystin detected were imperatively hypothesized to be associated to the levels of protein phosphatase which was expected to have some effect on liver cancer development. In these regard, the study reports an increase in levels of serine-threonine protein phosphates among PLC patients in relation to inhibitory effects of MC that was present in serum sample of PLC. The results therefore, indicates that the serine-threonine protein phosphatase activities in liver cancer patients were highly expressed above the normal range of a healthy individual (20-140U/L) as compared to controls. In addition, we further report that, there was a significant increase in the mean concentration of serum levels of total serine-threonine proteins phosphatases among the Liver cancer patients as compared to the controls. This is an indication of existence of a risk that directly affects the physiological expression of serine-threonine protein phosphatases and their modes of action given that the other clinical factors were under medical control. Therefore, it is inferred that the increase in the levels of serine-threonine PP1/2A in these patients is attributed to the confirmed presence of microcystin in the serum of these patients. These results are in consistent with those of Liang et al., (22) who reported increase in PP2A activities in human amnion FL cells treated with low-dose treatment of MCLR for a period of 6 h, whereas high-dose treatment of MCLR for 24 h decreased the activity of PP2A. Moreover, studies by Guo et al., (17) and He et al., (18) where exposure to MC-LR and bloom extract increased the protein levels of the A subunit of PP2A in vivo and in vitro, supports the findings of this study. This therefore, supports a direct correlation between serum microcystin to activities of PP1/PP2A that serves to regulate cell hemostasis, proliferation, metabolism and cell death (27). According to Liang et al., (22), hyper-phosphorylation of serine-threonine PP2A/PP1 by microcystin induces a cascade of negative effects on cellular functions such as de-regulation of phosphoproteins (P53, DNA-PK, & amp; MAPK) resulting in to tumor promotion and apoptosis among others. In this regard, hyper-phosphorylation of serine threonine phosphatases (PP 1 and 2A) caused by the inhibition activities of  MC as  reported in this study, is suggestive of its contribution to development of Primary liver cancer among the participants(17,22,37).To further confirm the above results, the study analyzed the association between serum microcystin and PLC occurrence in a case control population; Based on the results of 20 cases and 20 controls in this study. There was a signiﬁcant association between serum-microcystin and PLC occurrence, regardless of the confounding effects of known risk factors such as HBV, aﬂatoxin, alcohol drinking and smoking. These results were in consistent with those of Zheng et al., (40), who reported that serum-microcystin was significantly associated with tumor differentiations. However, in previous studies where association between microcystins and PLC, were done through estimations of drinking MCs-contaminated water (13,36,37) there were no direct determinants of serum microcystins reported. Microcystins are considered to act predominantly as tumor promotors through inhibition of eukaryotic serine/threonine protein phosphatases 1 and 2A, thereby increasing the overall level of phosphorylation/dephosphorylation in hepatocytes (24,25). In addition, in vitro and in vivo studies have shown that MCs exert their Hyper-phosphorylation effect on cytoskeletal proteins that results in alteration of cytoskeleton, loss of cell shape, induction of hepatocyte deformation and trigger of apoptosis due to oxidative stress which may result into primary liver cancer development (26) as is herein reported.
Therefore, cellular toxicity in response to acute and chronic MC exposure can be assessed by monitoring the activities of PP2A/PP1 (6). This is inferred through Mitogen-activated protein kinases (MAPKs) that regulates the expression of proto-oncogenes such as c-Jun, c-Fos, sand c-Myc which are responsible in regulation of the transcription of genes involved in the growth and differentiation (27).Expression of MAPKs is mediated by PP2A (6) and therefore inhibition of PP2A by MC-LR will result to activation of MAPK which will activate proto-oncogenes responsible for initiating transcription of growth and differentiation genes. Based on this study findings, in vitro cell culture studies are needed in order to ascertain the molecular involvement mechanism of microcystin toxins on hepatocytes physiology.
5.0 Conclusion
This being a first of its kind study within the lake region, it suffices to note that continuous exposure to MCs,  poses a great health risk challenge to the communities that depend on  lake, boreholes, wells, swamps and streams as their water sources for domestic use. In conclusion, this study found out that: serum microcystin levels detected among study participants were beyond the recommended WHO guideline levels; the increased levels of serine-threonine PP1/2A in PLC patients could be attributed to the presence of microcystin in the serum; the logistic regressions analysis shows a perfect prediction that microcystin was an independent risk factor for PLC occurrence in this study that should be considered during patient management. The study also recommends that in vitro cell culture studies should be picked to ascertain the molecular involvement mechanism of microcystin toxins on hepatocytes physiology and its presentation.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.
Author’s contributions
Evaline Chemutai;, Investigation, Methodology, Software analysis; writing original draft :David M. Onyango-:Conceptualization, funding acquisition, project administration, supervision, validation,: Cyrus Ayieko-;Investigation, Methodology and Data Curation; Ngwena G. Magak-;Supervision, Visualization, Writing and Editing
Acknowledgement
I wish to acknowledge my entire family for their moral and financial support not to forget Mr.Philip Ochieng and Mr. Joseph Muga for their academic support.
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