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Freshwater Cyanobacterial Blooms andPrimary Liver Cancer EpidemiologicalStudies in SerbiaZORICA SVIRČEV a , SVETISLAV KRSTIČ b , MARICA MILADINOV-MIKOV c

, VLADIMIR BALTIČ c & MILKA VIDOVIČ d

a Department of Biology and Ecology, Faculty of Sciences , Universityof Novi Sad , Serbiab Faculty of Natural Sciences , Institute of Biology , Skopje,Macedoniac Oncology Institute of Vojvodina , Sremska Kamenica, Serbiad Institute of Chemistry, Technology and Metallurgy , Belgrade,SerbiaPublished online: 09 Feb 2009.

To cite this article: ZORICA SVIRČEV , SVETISLAV KRSTIČ , MARICA MILADINOV-MIKOV ,VLADIMIR BALTIČ & MILKA VIDOVIČ (2009) Freshwater Cyanobacterial Blooms and PrimaryLiver Cancer Epidemiological Studies in Serbia, Journal of Environmental Science andHealth, Part C: Environmental Carcinogenesis and Ecotoxicology Reviews, 27:1, 36-55, DOI:10.1080/10590500802668016

To link to this article: http://dx.doi.org/10.1080/10590500802668016

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Journal of Environmental Science and Health Part C, 27:36–55, 2009Copyright C© Taylor & Francis Group, LLCISSN: 1059-0501 (Print); 1532-4095 (Online)DOI: 10.1080/10590500802668016

Freshwater CyanobacterialBlooms and Primary LiverCancer EpidemiologicalStudies in Serbia

Zorica Svircev,1 Svetislav Krstic,2 Marica Miladinov-Mikov,3

Vladimir Baltic,3 and Milka Vidovic4

1Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Serbia2Faculty of Natural Sciences, Institute of Biology, Skopje, Macedonia3Oncology Institute of Vojvodina , Sremska Kamenica, Serbia4Institute of Chemistry, Technology and Metallurgy, Belgrade, Serbia

A large part of Central Serbia experiences continual shortage of sufficient ground wa-ter resources. For that reason, more than 20 reservoirs serve as drinking water suppli-ers. Significant and persistent cyanobacterial “blooms” have been recognized in nine ofthem. Samples for cyanotoxin analyses were taken during and after “blooms” in CelijeReservoir and from Krusevac town-supplied tap water from that reservoir two dayslater. Concentration of microcystin-LR was 650 µgL–1 in the reservoir, while the tapwater contained 2.5 µgL–1.

In the two investigated periods, the high primary liver cancer (PLC) mortality of11.6 from 1980–1990 and extremely high PLC incidence of 34.7 from 2000–2002 wereobserved in the regions affected by heavy cyanobacterial “blooms.” In contrast, PLCmortality and incidence rates were substantially lower in the regions not affected bycyanobacterial blooms: in 1980–1990 the rate of PLC mortality amounted to 2.7 inKosovo, 7.6 in Vojvodina, and 8.3 in the non-affected regions of Central Serbia; whilein 2000–2002 PLC incidence amounted to 4.1 in Kosovo, 5.2 in Vojvodina, and 13.6in the non- or less-affected regions of Central Serbia. Keeping in mind that the mostaffected PLC regions in Central Serbia (Toplicki, Niski, and Sumadijski regions) havethe water supply systems based on six reservoirs found regularly in bloom duringsummer months and that some of the regions are also connected with two boundary“blooming” reservoirs, representing a total of eight of nine blooming reservoirs,

Received January 15, 2008; accepted October 25, 2008.The authors would like to acknowledge the funding of the Ministry of Science andEnvironmental protection of the Serbian Government (project number: 146021B). Theauthors received considerable scientific support for this study from Prof. Dr. GordanaSubakov and Prof. Dr. Slobodan Markovic. Mr Nebojsa Radeta (B.Sc.Comp.Sc) providedstatistical analyses, and Darko Petrovic provided significant technical support.Address correspondence to Zorica Svircev, Department of Biology and Ecology, Facultyof Sciences, University of Novi Sad, Novi Sad 21000, Serbia. E-mail: zoricas@ib.ns.ac.yu

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Freshwater Cyanobacterial Blooms and Primary Liver Cancer Studies 37

it is easy to presume that the PLC incidence could be related to drinking waterquality.

The uneven geographic distribution of liver cancer in Serbia is conspicuous and hotspots could be related to drinking water supply. It is very clear that the high-risk regionsfor PLC occurrence correspond with drinking water reservoirs continually found withcyanobacterial blooms, and the low risk regions correspond with water supplies notaffected by cyanobacteria.

Key Words: Cyanobacteria; blooms; cyanotoxins; PLC epidemiology; Serbia

INTRODUCTION

Influencing the formation of life on the whole planet, the cyanobacteria in-evitably have a profound, beneficial, and detrimental influence on human af-fairs (1). As primary producers and nitrogen-fixing organisms they globallycontribute to soil and water fertility (2) but also represent a potential sourcefor food production and solar energy conversion (3). However, mass develop-ment of cyanobacteria poses a considerable nuisance and even a threat in manysituations, especially in drinking water supply reservoirs. “Water blooms” oftoxin-producing species is an increasingly common feature of polluted inlandwaters but also in some coastal waters worldwide. Thus, cyanobacterial tox-ins have become a concern for the ecosystem and human health (4,5). Be-side a long list of toxins, cyanobacteria release other secondary metaboliteswith cytotoxic or cytostatic activity, some also with tumor-promoting activity(6).

Cyanotoxins are a very diverse group of toxins. They are either membrane-bound or exist free inside the cells. Release of toxins occurs during the cell lifebut mostly after cell death through passive flow out of the cellular content.Cyanobacterial toxins include neurotoxic alkaloids (anatoxin-a, anatoxin a(s),saxitoxins), hepatotoxic peptides (microcystins), and the hepatotoxic alkaloid(cylindrospermopsin) (7).

Hepatotoxic cyclic peptides include nodularin and different forms of mi-crocystins. Microcystins are a large group of cyclic peptides with variableamino acids at 7 different positions, representing about 60 different forms.The most toxic form is microcystin-LR. The toxicity of microcystins is due totheir strong binding and inhibition of protein phosphatases (4,8). After in-traperitoneal or intravenous injections, microcystins localize in the liver (9).Microcystins also promote the growth of tumors in experimental animals (10),although the significance of this in humans who may be subject to chronicexposure via drinking water is so far unclear. The inhibition of protein phos-phatase type 1 and type 2A activated by microcystin-LR is similar to thatof the known protein phosphatase inhibitor and tumor promoter, okadaicacid (11). Microcystins also have a genotoxic effect (12). The results after

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Ames testing (Salmonella typhimurium assay) indicated that microcystins hadstrong mutagenicity. Moreover, microcystins are able to induce DNA dam-age in primary cultured rat hepatocytes that are examined by cell-line assay(13).

The presence of toxic cyanobacterial blooms in waters used as drinkingwater reservoirs or for recreational purposes may represent serious healthrisks for the human population (14). A large number of intoxications not onlyof cattle (15), dogs (16), and waterfowl (17), but also of humans, have beenreported. The tragic deaths of 76 patients in a hemodialysis clinic in Brazilin 1996 was connected to the presence of cyanobacterial toxins in the wa-ter supply (18), and a high incidence of primary liver cancer in China hasbeen attributed to drinking water contaminated with cyanobacterial toxins(19,20,21).

The presence of cyanobacterial toxins in drinking water supplies posesa serious problem to water treatment facilities because not all technicalprocedures are able to effectively remove these toxins (22). Despite this,it is highly unlikely that lethal poisonings would occur following consump-tion of drinking water contaminated with cyanobacterial toxins. Of muchhigher concern are the low-level chronic exposures because the risks asso-ciated with long-term exposure have not been adequately described (7,14).Current drinking water treatment practices in Serbia do not regularly mon-itor or actively remove these toxins from the drinking water because this isa relatively new field of study and would involve extremely expensive mea-sures (23,24). Even with the treatment, low-level chronic exposure to the hep-atotoxins leading to tumor promotion or even carcinogenesis is possible inpersons who consume drinking water derived from surface water treatmentreservoirs.

The liver is the principal target organ for cyanobacterial hepatotoxins afteringestion (9,14). Primary liver cancer (PLC) is the fifth most common cancerin the world and is the fourth most common cause of cancer mortality. PLCrates have an extremely wide geographic variation; 80% of the cases arise indeveloping countries, particularly in Southeast Asia and sub-Saharan Africa(25). In these high-rate risk populations, chronic infection with hepatitis Bvirus (HBV) and contamination of foodstuffs with aflatoxin B1 are recognizedas major risk factors. Alcohol ingestion and hepatitis C virus (HCV) infectionsare more likely to occur in developed regions (25). It is generally consideredthat frequent hepatocyte irritation by chemicals, chronic or frequent digestivesystem infections, cirrhosis, or unhealthy diet, are the principal risk factors forliver cancerogenesis.

The aim of this study was to perform the epidemiological analysis of PLCincidence and occurrence due to detected prolonged blooming of drinking wa-ter reservoirs correlated with type of drinking water supply systems used indifferent regions of Serbia.

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Freshwater Cyanobacterial Blooms and Primary Liver Cancer Studies 39

MATERIAL AND METHODS

Study AreaAccording to the administrative division, Serbia consists of 30 regions. PLC

studies were conducted in three parts separately: Vojvodina with no reservoirsfor water supply, only deep wells (7 regions); Kosovo with a few high moun-tain reservoirs for water supply, no blooming occurred (5 regions); and CentralSerbia part (18 regions), which was divided during our epidemiological studiesinto regions with extremely high PLC incidence and regions with lower PLCincidence (Figure 1).

Data BasisData source for PLC incidence and mortality rates in Central Serbia and

Kosovo were found in publications by (a) Institute of Health Protection of Ser-bia Dr Milan Jovanovic Batut in Belgrade (26, 27); (b) KBC Bezanijska Kosain Belgrade (28); and (c) Local Administrative Health Institutions like Insti-tute of Public health in Zajecar. Regions in Vojvodina were used for comparisonwith data obtained by the Cancer Registry of Vojvodina, Oncology Institute ofVojvodina, Sremska Kamenica.

Research spanned from 1980–2003. Cyanobacterial blooming events wereobtained from the 25-year monitoring program in Serbia conducted by the Re-public Hydrometeorogical Institute and a series of published papers on thesubject (29). PLC mortality was conducted for the 1980–1990 period, whilePLC incidence and mortality were performed for the 2000–2002 period.

This study uses a descriptive epidemiological method. World standard pop-ulation and crude rates are used for indication of mortality and incidence. Ob-tained values for incidences are presented in diagrams and in colors on themap of Serbia. Maps also represent surface reservoirs found blooming that areused as water supply to determine and visualize eventual correlations betweenhigh PLC incidences and blooming reservoirs.

Microcystin-LR MeasurementsMicrocystin concentration was measured in only one water supply reser-

voir in Central Serbia (Celije) during the cyanobacterial bloom in July 2004.Colorimetric protein phosphatase inhibition assay (inhibition of enzyme

protein phosphatase 1-PP1) was used for the detection of microcystin-LR con-centration in water samples (30). Samples were concentrated by filtrationthrough 0.45 µm membrane filters and were extracted with 75% methanol(31). PP1 activity was determined by a measurement of the rate of color pro-duction from the liberation of p-nitrophenol from the substrate p-nitrophenilphosphate, which was measured at 405 nm using the microtiter plate reader.

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Figure 1: Administrative division of Serbia in regions. A total of 30 regions with 18 regions ofCentral Serbia:(1) Rasinski, (2) Zajecarski, (3) Macvanski, (4) Borski, (5) Podunavski, (6) Pcinjski, (7) Moravicki,(8) Zlatiborski, (9) Branicevski, (10) Pomoravski, (11) Beogradski, (12) Jablanicki, (13) Raski, (14)Kolubarski, (15) Pirotski, (16) Sumadijski, (17) Nisavski, (18) Toplicki.

The assay was carried out at 37◦C for two hours. Toxin concentrations were de-termined using a standard inhibition curve of microcystin-LR (SIGMA). Dueto a lack of relevant methods and conditions for the toxin analysis (apart of theabove-mentioned case), microcystin concentrations have never been detectedin surface water ecosystems in Serbia. In addition, there is no regular mon-itoring of the water quality—even the drinking water supply—regarding thecyanotoxin presence.

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Freshwater Cyanobacterial Blooms and Primary Liver Cancer Studies 41

Statistical AnalysisStatistical analysis was performed using data from regional and country

databases and registers. Significant threshold between the two groups of re-gions with low (“light grey” regions) and increased (“dark grey” regions) PLCincidence and mortality were calculated by grouping values above and bellowthe mean value on which the standard deviation was added (x+ standard de-viation). Within the two groups of “light gray” and “dark grey” regions, meanvalues and deviations were also performed.

RESULTS

According to our previous results (29), a large number of water ecosystems inSerbia were found blooming since 1980 (Figure 2A). Among 83 examined waterecosystems, 58 were found in blooming condition during past 2.5 decades. Partof Central Serbia has been found to be very risky for surface reservoir watersupply since more than 20 reservoirs serve as drinking water suppliers and 9 ofthese were detected in severe and prolonged cyanobacterial blooming (Figure2B).

Dominant blooming cyanobacterial taxa belonged to Microcystis, Aphani-zomenon, Anabaena, and Oscillatoria (Planktothrix) genera, which are repre-sented by the most frequently observed Microcystis aeruginosa, M. flos-aquae,Aphanizomenon flos-aquae, Anabaena flos-aquae, A. spiroides, and Plank-tothrix agardhii taxa, all of which are well-known toxin producers.

In July 2004, Celije reservoir, used as drinking water supply for the cityof Krusevac and its surroundings, was found blooming with taxa that belongto Aphanizomenon, Anabaena, and Microcystis (Plate 1). This mass develop-ment of toxic cyanobacteria resulted in concentration of microcystin-LR 650µgL−1 in reservoir water, while in the drinking (tap) water of Krusevac citythe concentration was 2.5 µgL−1.

From 1980–1990, the standardized PLC mortality rate in Central Serbiawas high in 9 regions (Moravski, Sumadijski, Raski, Branicevski, Rasinski,Zajecarski, Niski, Toplicki, and Pirotski) (Figure 3), with a rate of 11.6, whilein the other 9 regions it was slightly lower at 8.3. In this period, the PLCmortality rate in Vojvodina was 7.6 and only 2.7 in Kosovo.

Blooming drinking water reservoirs are located in the regions where higherPLC standardized rates were recorded (Figure 4).

By comparing the first published results on PLC mortality in CentralSerbia for 1980–1990 with data from Vojvodina and Kosovo for the same period(Figure 4), a hypothesis has been postulated that the higher PLC occurrencein Central Serbia might be correlated with the presence of cyanotoxins in thedrinking water. Further investigations have been conducted on the base of allindices and rates obtained from the Central register database of Serbia.

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Figure 2: (A) Freshwater ecosystems monitored in the past 25 years in Serbia with detected“blooming” events; (B) Drinking water supply reservoirs in Central Serbia with “blooming”ones are marked by dark grey dots (29).1. Bajina Basta, 2. Barje, 3. Batlava, 4. Borkovac, 5. Borsko jezero, 6. Bovan, 7. Bresnica, 8.Brestovac, 9. Bukulja, 10. Celije, 11. -Derdap I, 12. -Derdap II, 13. Garasi, 14. Gazivode, 15.Gracanka, 16. Grliste, 17. Grosnica, 18. Gruza, 19. Kokin Brod, 20. Kamenica, 21. Krajkovac,22. Kudos, 23. Lazic, 24. Lisina, 25. Ljukovo, 26. Mees, 27. Meuvrsje, 28. Moharac, 29. OvcarBanja, 30. Potpec, 31. Pridvorica, 32. Prilepnica, 33. Radojina, 34. Radonji, 35. Sokolovica, 36.Sot, 37. Selovrenac, 38. Tavankut, 39. Tisa, 40. Uvac, 41. Tisa-Novi Knezevac, 42. Vlasina, 43.Vrla 2, 44. Vrutci, 45. Zavoj, 46. Zlatibor, 47. Zobnatica, 48. Zvornik, 49. Pek-Blagojev kamen,50. Sava Litije-Ostruznica, 51. Kanal Odzaci-Sombor, 52. Dunav-Apatin, 53. Vrla1, 54. MrtvaTisa-Mol, 55. Rakina bara, 56. Zapadna Morava-Cacak, 57. Opovacki Dunavac, 58.Ponjavica, 59. Veliki Zaton, 60. Bosut, 61. Studva, 62. DTD-Novi Sad, 63. Jegricka, 64.DTD-Vrbas, 65. DTD-Backo Gradiste, 66. DTD-Bac, 67. DTD-Srpski Miletic, 68. Zlatica, 69.Krivaja, 70. Keres, 71. Palic, 72. Ludos, 73. Carska bara, 74. Koviljski rit, 75. Obedska bara, 76.Zasavica, 77. Provala, 78. Ecka, 79. Stari Begej-Srpski Itebej, 80. Tamis-Botos, 81. Ribnica, 82.Divcibare, 83. Pustinjac.

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Freshwater Cyanobacterial Blooms and Primary Liver Cancer Studies 43

Figure 2: (Continued)

Regions in Central Serbia mostly have surface drinking water supplyreservoirs that are blooming during summer months, compared with Vojvodinaand Kosovo that have underground water supply systems. Consequently,the differences in drinking water quality in these regions might be a re-sult of the presence of hepatotoxins as a product of many cyanobacteriablooming.

Since the detected differences among regions in Central Serbia, Vojvodina,and Kosovo in the first investigated period were not highly significant (Figure4), epidemiological analysis for 2000 and 2002 was performed (Figures 5 and6). Results clearly point to differences between Central Serbia and the “control”

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Figure 3: Standardized PLC mortality rates per 100,000 inhabitants in Central Serbia from1980–1990. Dark grey columns represent the rate above 10 (9.63 + 0.77, 95% significancethreshold); light grey is below this value. Numbers indicate regions in Central Serbia (seeFigure 1).

Figure 4: Standardized PLC mortality rates per 100,000 inhabitants in Serbia connected withthe “blooming” reservoirs in 1980–1990.

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Freshwater Cyanobacterial Blooms and Primary Liver Cancer Studies 45

Figure 5: PLC incidence per 100,000 inhabitants in Central Serbia in 2000–2002. Dark greycolumns represent the rate above 24 (17.09 + 6.84, 95% significance threshold); light grey isbelow this value. Numbers indicate regions in Serbia (see Figure 1).

regions in Vojvodina and Kosovo, leading to a statistically significant increaseof PLC in Central Serbia.

In 2000, a significant increase of PLC incidence in Central Serbia has beennoticed (incidence rate was 16); the trend also continued in 2002 (incidencerate 18.8), while in Vojvodina in the same period a stagnation was clearly de-tected (incidence rates 6.6 and 6.2 for 2000 and 2002, respectively).

According to statistical analyses, increasing of PLC incidence in CentralSerbia in 2000–2002 (Figure 5) was extremely significant in three regions(Sumadijski, Niski, and Toplicki) and was rated 34.7, while in the rest of Cen-tral Serbia regions the mean value was 13.6. PLC incidence in Vojvodina forthat period was 5.2.

Keeping in mind that the three most affected PLC regions in CentralSerbia have water supply systems based on reservoirs found to be regularlyblooming during summer months (Bovan, Gruza, Grosnica, Garasi, Pridvorica,Bresnica) and that some of them are also connected with boundary “blooming”reservoirs (Celije Plate 1. and Brestovac), it is easy to presume that the PLCincidence could be connected with the drinking water quality.

DISCUSSION

There is no regular monitoring of the presence of potentially toxic, toxiccyanobacteria, and cyanotoxins in drinking and surface waters in Serbia. Spe-cific regulations and legislation on maximum permitted levels of cyanotoxinsin water do not exist. The World Health Organization (WHO) has addressedhealth hazards cyanotoxins present as a part of the WHO Guidelines for Drink-ing Water Quality. WHO Guideline Value (GV) for total microcystin-LR indrinking water is 1 µgL–1 (5,32)—a limit that is already being used in somecountries (e.g., Australia, United Kingdom) (33).

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Figure 6: PLC incidence rates per 100,000 inhabitants in Serbia connected with the“blooming” reservoirs in 2000–2002.

Presented results on drinking water reservoirs in Serbia (Figure 2A) thatwere found blooming during the investigated period clearly point to the rela-tionship with increased PLC incidence rates in these regions compared withthe control regions of Kosovo and Vojvodina and unaffected regions in CentralSerbia.

Since cyanotoxins were detected in the majority of monitored bloomingsamples from Great Britain, Germany, Finland, and Australia (4,5,34,35), itseems that occurrence of blooming is sufficient enough for the risk analysis.Based on a geographical information system (GIS) map comparison of bloom-ing reservoirs without actual toxin measurements, Fleming et al. (36) havealso shown similar patterns of primary hepatocellular carcinoma increasedrisk within the population in areas with surface drinking water supplycompared with randomly selected ground water treatment service or the cu-mulative incidence rate for the investigated period in Florida. Regardless, thepriority of the future research in Serbia will inevitably be based on cyanotoxin

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Freshwater Cyanobacterial Blooms and Primary Liver Cancer Studies 47

Plate 1: Cyanobacterial “blooming” in Celije reservoir in July 2004.

concentration monitoring in all drinking water reservoirs. Nevertheless,toxicity not accounted for by the currently known cyanotoxins is observedfrequently, particularly in surveys that apply both chemical analysis andbioassays. These facts indicate the existence of additional (not yet identified)cyanotoxins and/or possible synergistic effects with other substances duringthe blooms (4). In these cases, the detection of blooms could be of greaterimportance than cyanotoxin measurements per se.

Although toxin concentrations in the Celije reservoir were quite high(650 µgL–1), their presence in tap water (2.5 µgL–1) was nearly withinthe WHO recommended limits. Since the Rasina region belongs to thelow PLC incidence group, this result suggests that drinking water treat-ment was efficient regarding cyanotoxin removal. But if the excess chlo-rination was used in the treatment, it could pose another problem sinceincreased concentrations of trihalomethanes are detected as one of thepossible risk factors for developing colon, rectal, and bladder cancers(http://www.epa.gov/OGWDW/MDbp/dbpfr.html). According to the Cancer Reg-ister for Central Serbia (26,27) significantly higher incidences of colon cancerwere found in three affected regions together with Zajecarski and Beogradskiin 2000–2002 (colon incidence rates were 75–90 compared with the Central

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Serbia mean value of 57). Additionally, Zhou et al. (37) have positively relatedmicrocystins in drinking water with colorectal cancer incidence and have sug-gested further studies for resolving microcystins’ role in colon and rectum can-cerogenesis.

Another reservoir in which cyanobacterial blooms occur followed by a lowPLC incidence is Grliste in the Zajecarski region. In this case, PLC occur-rence analysis was performed for Zajecar city in 2000, revealing a significantlyhigher number of PLC cases in correlation to the rest of the region. These re-sults suggest that hepatotoxin presence in the Grliste reservoir might be thecause for increased PLC incidence after all, but the statistical presentation ofPLC occurrence per capita in the region is “masked” by the number of peoplewho do not drink water from the Grliste reservoir. This methodological prob-lem has been termed “the Zajecar nonsense.” In order to resolve the doubtsregarding the connection of hepatotoxins and PLC incidence in this case, it isnecessary to separate people who drink water from the reservoir and those whodrink it from other water supplies in the Zajecar region. It is also noteworthythat colon cancer incidence in 2000–2002 increased at 75 compared with theCentral Serbia mean value of 57 (26,27).

The estimations of the worldwide incidence of liver cancer in 1990 showedthat for individuals living in developed countries versus developing countries,the age-standardized incidence rates were 10.2/100,000 and 24.1/100,000, re-spectively (38). For Serbia, in the period 2000–2002, the standardized inci-dence rate was 17.8. In Southeast China, rates of less than 15 incidents per100,000 people were seen in some districts, compared with over 60 incidentsper 100,000 people in adjacent localities (39). PLC incidence rate in an urbanChinese population affected by cyanotoxins in the period 1981–2000 was 38.9(40), while the same PLC incidence “dark grey regions” rate in Serbia in 2000–2002 was 34.7. The fact that the unaffected regions in Serbia for water supply,like Vojvodina, belong to the part of world with lower PLC incidence has beenconfirmed in the period 1988–1992 (25).

A possibility of PLC triggering by the presence of hepatotoxins in drink-ing water and the relationship of surface drinking water reservoirs to PLChave been reported in very few papers regarding epidemiological analysis forregions in China and Florida, where people drink water from cyanobacteriablooming reservoirs (20,36,39).

Hepatotoxins cause liver tumor promotion, especially in the case of pro-longed cumulative influence (41,42); it is easy to presume that the presence ofthese toxins in drinking water reservoirs has caused their penetration intodrinking water systems due to inconvenient or low-efficiency treatment ofcyanotoxins removal by the drinking water industry purification processes.

In relation to very scarce epidemiological results in the literature, mainlycoming from China or Florida, there was an increasing opinion that the ex-posure to some other risk factors (other than cyanotoxins) is detrimental for

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inducing the PLC (43,44), such as viral hepatitis B or C, aflatoxins in food,or alcohol abuse (20,25,39). Investigations in China have clearly emphasizedthe presence of aflatoxins as a primary risk factor for PLC, triggered by mi-crocystin (39). Microcystin in the pond-ditch water is a hepatotoxin that caninduce hepatitis and can promote the development of hepatocellular carci-noma. The combined effect of microcystin, HBV, and aflatoxin may be re-sponsible for the endemicity of PLC (45). However, epidemiological analysesperformed in our study have so far failed to expose any other possible riskfactors in Serbia. Standardized death rates of chronic liver diseases and cir-rhosis (all ages per 100,000 inhabitants) in Serbia and Monte Negro in rela-tion to Europe has revealed a much lower average death rate of 9.3 correlatedto all European Union countries of 21.6, for the period 1992–2002. Accord-ing to this risk factor, Serbians have a 20% possibility of liver cirrhosis in re-lation to Europeans (data analyzed from European Health for All Databasehttp://data.euro.who.int/hfadb/). Viral hepatitis B incidence data analysis per100,000 inhabitants also shows a much lower average infection rate in Serbia(4.9) compared with Europe (14.2) for 1993–2004 period. Again, less than 20%of Serbians are at risk for infection compared with Europeans. The very sameresults are obtained for hepatitis C infection rate for the 1997–2004 period.The analysis of pure alcohol consumption, liters per capita in Serbia and MonteNegro for 1992–2001 period, shows lower average consumed alcohol quantitiesin Serbia related to all European countries (7.2 and 8.8, respectively). Accord-ing to this risk factor, Serbia is ranked among countries with less than 50%alcohol consumed per capita. Aflatoxin occurrence in food is not recorded inthe specific statistical and epidemiological databases.

Other possible factors that might influence the obtained PLC mortalityrates and incidences, such as smoking habits or migration patterns of the pop-ulation between and among the regions around, were deliberately omitted dur-ing this study because of lack of scientific significant evidences, and lack ofrelevant data. There are no evidences in the literature so far to connect thenicotine and PLC genesis directly. Additionally, the latest investigations (46)have failed to reveal any difference in GSTM1 and GSTP1 gene interactionsin non-tumorous liver tissue between smoking and non-smoking groups of pa-tients. Major immigration in Serbia happened during 1993–1996; the majorityof new inhabitants settled in Vojvodina and Belgrade. There are no publisheddata about the demographic changes in Central Serbia. There are even lessdata available in relation to the health conditions of the people immigratingfrom Bosnia and Croatia.

In connection with the previously stated comments on the other possiblerisk factors for PLC incidence in Serbia, we would strongly like to emphasizethe evident causality between the blooming drinking water reservoirs and in-creased PLC rates in the “hot spot” regions. At this point of investigations,we do consider the data for the blooming events even more important than

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measurements of microcystins in the water per se, because their presence ina reservoir does not implicate that there are no any other hepatotoxins and/orcarcinogens present as well. On the other hand, the mere presence of micro-cystins in a reservoir does not pose an immediate threat to humans, especiallyif there is an effective drinking water purification system in place.

Despite the opinion that exposure to other risk factors is detrimental for in-ducing PLC, some studies implicated that microcystin may also be a potentialcarcinogen because repeated injections of microcystin could induce neoplasticnodules in mouse liver without an initiator, and it could also induce oxidativeDNA damage in human hepatoma cell line HepG2 (47,48). Moreover, it wasfound that microcystin-LR induced base substitution mutation at K-ras codon12 in human Rsa cells (49). Hu et al. (50) clearly stated that microcystins canstrongly promote liver tumor genesis and that changes of bcl-2 and bax geneexpression possibly play an important role in the microcystin-induced liver tu-mor formation.

Provoked by the accumulated evidences, 19 scientists from eight coun-tries met at the International Agency for Research on Cancer (IARC) in Lyon,France, to assess the carcinogenicity of the ingested cyanobacterial peptide tox-ins microcystin-LR and nodularins (51,52). The IARC conclusions were madebased on the following evidence: tumor-promoting mechanisms, via PP’s 1 and2A inhibition in rodent liver and hepatocytes (53) induced by these toxins, wasstrongly supported; these toxins modulate the expression of oncogenes, early-response genes, and tumor necrosis factor α, thus affecting cell division, cellsurvival, and apoptosis (42,54,55,56). The Working Group conclusion was thatmicrocystin-LR is “possibly carcinogenic to humans” (Group 2B) (52).

Monto and Wright (57) clearly stated that the PLC incidence is higher incountries where hepatitis B is endemic, and that PLC is substantially a compli-cation of liver cirrhosis. HBV and HCV are the predominant causes of cirrhosis,and as such, of PLC as well. Other less common forms of chronic liver diseasecan also lead to PLC (57). Keeping in mind that cyanotoxins, via cumulativeexposure activity, may gradually lead to liver damage even in small doses (58),cirrhosis could occur as the consequence. In that case, the liver cancer genesiscan be triggered by the same risk factor, making it possible to presume thatlow toxin doses can generate PLC over time. The cumulative effect of cyan-otoxins could be considered a prerequisite for PLC occurrence. Consequently,we do not state that the presence of high cyanotoxin concentrations in a spe-cific drinking water reservoir may lead to PLC and that the detected values areimmediate proof of it. Presented results, which enable the link and correlationof drinking water reservoir blooming to regions in Serbia where PLC incidenceis the highest just serve as a warning that cyanotoxins as risk factor shouldbe considered as potential cause of PLC in the future. The direct evidence andconclusion that will inevitably link liver cancer promotion or even carcinogene-sis in humans with the cyanotoxin presence in drinking water is impossible in

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this type of study, but probably even in much more detailed studies as well. Itis therefore important to stress the possibility of such an outcome, even morein light of everyday, uncontrolled decreasing water quality, prolonged presenceof cyanotoxins in such water bodies throughout the year, and cyanotoxin re-moval methods that are not used regularly and universally in the water fac-tories while the time needed for scientifically sound evidence and proof of thishypothesis may be very long. Based on this and the present time distance,there is only one important question to answer: Will the cost of ignorance andcarelessness be high?

CONCLUSIONS

The increasing PLC incidence in Central Serbia in 2000–2002 was extremelysignificant in 3 regions (Sumadijski, Niski, and Toplicki) at 34.7, while in therest of Central Serbia, the mean value was 13.6. PLC incidence in Vojvodinafor that period was 5.2.

Presented results enable the conclusion that the observed “uneven” geo-graphical distribution of PLC may be a consequence of hepatotoxin presencein drinking water systems and that the “dark grey regions” may be related tocyanobacterial blooms in drinking water reservoirs.

Detected differences between regions that use surface reservoirs as drink-ing water supplies and those where the drinking water reservoirs do not bloommay be related to increased risk for primary liver cancer in Central Serbia.These conclusions are also supported by epidemiological studies performed inregions that have underground water supplies, such as Vojvodina and Kosovo,where significantly lower PLC incidence was detected.

As an ecological and epidemiological study, this article can only be consid-ered to be hypothesis generating and cannot prove or disprove any etiologicalassociation. In order to obtain much more reliable conclusions on statisticallysignificant correlations, it is necessary to examine the presence of cyanotoxins(not only microcystins) in water supplying reservoirs and the tap water, and toresolve the stated methodological problems.

Because microcystins have been shown to promote liver tumors and sinceevidence exists that they can also initiate tumors, supported with our andother epidemiological analyses, it is likely that microcystins can act as car-cinogens; this underlines the importance of monitoring and eliminating micro-cystins during drinking water treatment.

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