FACTA UNIVERSITATISSeries: Medicine and Biology Vol.9, No 1, 2002, pp. 39 - 48 UC 616.61-004

ENVIRONMENT, GEOCHEMISTRY AND THE ETIOLOGYOF BALKAN ENDEMIC NEPHROPATHY: LESSONS FROM ROMANIA

William H. Orem1, Calin A. Tatu2*, Gerald L. Feder3, Robert B. Finkelman1, Harry E. Lerch1,Susan V.M. Maharaj4, Diana Szilagyi2, Victor Dumitrascu2, Virgil Paunescu2, Florin Margineanu5

1US Geological Survey, 12201 Sunrise Valley Drive, MS 956, Reston, VA 20192, U.S.A.2Department of Immunology, Clinical Laboratory No.1, Pta. E. Murgu No.2, RO-1900 Timisoara, Romania3Florida Community College at Jacksonville, Jacksonville, FL 32256,U.S.A.4Department of Environmental & Toxicologic Pathology, Armed Forces Institute of Pathology, 14th and Alaska Ave. N.W., Washington, D.C. 20306-6000, U.S.A5Center of Hemodialysis, County Hospital, 82 Unirii Str., RO-1500, Drobeta Turnu Severin, Romania * E-mail: cta@med.unc.edu

Summary. Balkan endemic nephropathy (BEN) is a fatal chronic kidney disease of unknown etiology geographicallyrestricted to several countries of the Balkan Peninsula and described for the first time almost 50 years ago. Alongtime, many factors have been proposed as etiological agents for BEN (including viruses, bacteria, mycotoxins,industrial pollution, radioactive compounds, etc.) but none of these have been confirmed. However, in the recentyears, based on field and laboratory investigations, an environmental etiology of the disease has become more widelyaccepted, with a prime role played by the geological background of the endemic settlements. In this regard, the mostincriminated are toxic organic compounds present in the drinking water, supposed to be leached by the groundwaterfrom low rank Pliocene lignite deposits adjacent to the endemic villages and transported into shallow household wellsor springs.We describe in our study several inorganic and organic geochemical features of water samples collected from theendemic villages, in comparison with water samples from nonendemic locations from Romania. Water samples wereanalyzed on site and in the laboratory using standard inorganic and organic geochemistry methods. Methanol andaqueous extracts of Pliocene lignite collected from the endemic areas were also analyzed by GC/MS.While no significant difference could be found in the inorganic parameters (i.e., pH, TDS, anions, alkalinity, oxygen,nitrates, etc.) of the endemic vs. nonendemic water samples, the organic content, as revealed by GC/MS, is morecomplex and much more higher in the endemic vs. the nonendemic water samples. Also, the Pliocene lignite from theendemic areas seems to have a particular geochemical composition, with many potentially nephrotoxic/carcinogenicaromatic and nonaromatic molecules, leachable into organic solvents as well as into water.Some of the molecules extracted from the endemic area Pliocene lignite are apparently present in the endemic watersamples, suggesting their origin in coal and sustaining the Pliocene lignite hypothesis for BEN etiology.

Key words: Balkan endemic nephropathy, medical geology, geomedicine, Pliocene lignite, GC/MS

IntroductionBalkan endemic nephropathy (BEN) is an irreversi-

ble, chronic, tubulo-interstitial nephropathy of unknownorigin, geographically confined to several rural regionsof the Balkan Peninsula. The first "official" observationof a disease resembling BEN was made almost six dec-ades ago in regions comprising the former Yugoslavia(1). Shortly thereafter, similar descriptions came fromBulgaria and Romania (2-4). The distribution of BENincludes regions of central and southeastern Serbia,southwestern Romania, northwestern Bulgaria, south-eastern Croatia, and parts of Bosnia and Kosovo (fig. 1).The geological setting of the endemic areas in the af-fected countries is similar. Most of the BEN villages are

located on alluvial plains of tributaries of the DanubeRiver, with older Tertiary layers (clays, sands and Plio-cene lignites) covered by Quaternary river sediments. Itis estimated that several thousand people in the affectedcountries are currently suffering from BEN and thatthousands more will be diagnosed with BEN in thecoming years.

Although BEN may have existed for centuries in theaffected areas, it was acknowledged to the medicalcommunity only in the 1950's when the first epidemicoutbreaks were reported. Prior to World War II, averagelife expectancy in the endemic areas was < 45 years,with major causes of short life expectancy being para-sitic or infectious diseases like malaria and tuberculosis.As BEN becomes manifest at older ages (40-50 years),

40 WH. Orem, CA. Tatu, GL. Feder, et. al.

it is possible that the shorter life expectancy in the en-demic areas before the war did not allow for a "criticalmass" of diseased people to accumulate, and an epi-demic kidney disease to be noted. After the war, im-provements in sanitation in the affected countries (in-cluding anti-malaria and anti-TB measures) coupledwith better diagnostic procedures in nephrology madepossible the description of BEN, as a distinct nosologi-cal entity. BEN was almost simultaneously recognizedin all affected countries (in Bulgaria, Tanchev and oth-ers in 1956 as reported in (5); in Yugoslavia, Danilovicet al. in 1957 (1); and in Romania, Fortza and Negoescuin 1961 (6)), and the endemic regions were relativelyclearly delimited. The geographic distribution of BENhas not changed significantly since the first descriptions,and villages afflicted in the past continue to be afflicted,while nonendemic villages (sometimes located only afew km from endemic villages) have remained free ofthe disease (7,8). Reports mentioning a kidney diseaseresembling BEN in the Balkans have been traced backto the early XXth century. For instance, German doctorsdocumented a large number of deaths from uremia inSerbia in 1915, while in the village of Bistretz (Bul-garia) a disease with the clinical features of BEN wasreported in 1910. In Romania, a possible link existsbetween BEN and familial kidney atrophy observedhere in 1905 (Susa, 1976, cited in (2)) raising the possi-bility of BEN having a genetic component.

Fig. 1. Map sketch showing the distribution of the BEN fociin the Balkan Peninsula.

Some of the medical and epidemiological features ofBEN are: focal occurrence of the clinical cases; familyaggregation without an obvious pattern of mendeliangenetic inheritance; long subclinical "incubation" periodwith a rapid onset of end-stage renal disease (ESRD); atleast 10-20 years residence in one or more of the en-demic villages; occurrence only in adults (age 30 to 50being most heavily affected, while almost no children orindividuals above 70 develop the disease); a slight sexdistribution (female:male ~1.5:1); a similar incidence indifferent ethnic and religious groups; restriction to a

rural, farming population; presence of high blood pres-sure in only 20% of the affected people; normochromicand normocytic anemia; a very high frequency of upperurinary tract tumors (UTT) in BEN patients. Most epi-demiological studies have revealed no obvious differ-ences in the nutritional habits or other significant differ-ences between the endemic and nonendemic householdsin the same village.

BEN is a slow-evolving disease, characterized by ir-reversible progression to renal failure and terminal ure-mia. Once end-stage renal disease is diagnosed in BENpatients, the therapeutical approaches are only sympto-matic. As renal transplantation is not a feasible optionfor most of the BEN patients, hemodialysis is the onlyapproach left. However, kidney transplantation has beenattempted in a few people with BEN and no recurrenceof the disease has been noted on the transplanted kid-neys. Due to adverse economic conditions of the coun-tries affected by BEN, kidney transplantation is not aviable option for the present and near future. The directconsequence of this situation is a poor quality of life forBEN patients in the ESRD phase.

Due to its medical and economical consequences,BEN has been a major research priority through theyears, involving multidisciplinary approaches and var-ied scientific disciplines (e.g., environmental medicine,microbiology, oncology, radiation biology, epidemio-logy, cytogenetics, geology, hydrogeology, biogeo-chemistry) and several international symposia have beendedicated to its medical aspects and origins. Despitethese research efforts by regional and international sci-entific groups, BEN is still a medical enigma in manyrespects, and its etiology a matter of contradiction andcontroversy. Many factors have been proposed as etio-logical agents for BEN, including: bacteria and viruses,heavy metals, radioactive compounds, trace elementimbalances in the soil, chromosomal aberrations, my-cotoxins (ochratoxin A), plant toxins (aristolochic acid),and industrial pollution (for a review see (9)). Ruralvillagers in the endemic areas often invoke astrologicalinfluences to explain the occurrence of BEN.

In recent years, field and laboratory investigationshave supported an environmental etiology for the dis-ease, with a prime role played by the geological back-ground of the endemic settlements (9-11). In this regard,there is a growing body of evidence suggesting the in-volvement of toxic organic compounds present in thedrinking water of the endemic areas. These compoundsare hypothesized to be leached by groundwater fromlow rank Pliocene lignite deposits topographicallylinked to the endemic villages, and transported intoshallow household wells or village springs (10,12,13).The population of the villages in the endemic areas useswell/spring water almost exclusively for drinking andcooking, and is therefore potentially exposed to anytoxic organic compounds found in the water. The pre-sumably low levels of toxic organic compounds presentwould likely favor relatively slow development of thedisease and over a time interval of 10 to 30 years or

ENVIRONMENT, GEOCHEMISTRY AND THE ETIOLOGY OF BALKAN ENDEMIC NEPHROPATHY 41

more. The frequent association of BEN with upper uri-nary tract (urothelial) tumors suggests the action notonly of a nephrotoxic, but also carcinogenic, factor.

Several inorganic and organic geochemical parame-ters of water and coal samples collected from a Roma-nian BEN endemic area were assessed in our study, inan attempt to find additional links between the environ-ment and BEN etiology.

Methods

Field investigations and waterand coal samples collectionLocation surveyWe performed two field trips in March and August

2000 in the main Romanian BEN endemic area (Dro-beta Turnu Severin), located in SW Romania. Ten (10)endemic locations and seven (7) non-endemic sites inthe area were visited on these occasions. We also visitedthree Pliocene lignite mines (Husnicioara, Pietris, andLivezile), located in the BEN endemic territory. Plio-cene coal was sampled from each coal mine and watersamples were also obtained from springs found at Hus-nicioara and Pietris mines.

Villages for well water sampling were selectedbased on medical records showing their endemic char-acter, which we obtained from the Drobeta TurnuSeverin County Hospital. Well water and spring siteswere classified as endemic (E) and non-endemic (NE).Endemic sites were defined as water sources used on aregular basis (at least 10-20 years) by a family or sev-eral families (if the water source is located outside theprivate household) with described BEN cases and/orfamily history of BEN. The non-endemic sites weredefined as water sources used (for 10-20 years) by afamily or families without any described BEN cases orfamily history of BEN.

Location, elevation above sea level, proximal presenceor absence of coal deposits and/or coal mines were notedfor each site. Observations of the surroundings wererecorded and the general placement of the villages withrespect to the adjacent forms of relief was also noted.

Water collection and analysisWater samples were collected at each site for both

inorganic and organics analyses. Standard "clean" pro-cedures for both inorganic and organic sample collec-tion were employed (14,15). For organics, water wascollected in solvent-cleaned, amber, glass bottles (2liters for each sample), after on site filtration throughpre-cleaned and pre-weight glass fiber filters. Methyl-ene chloride (60 ml) was added to each bottle to retardbacterial activity during storage and transport to USGSlaboratories in Reston, VA. Water samples were liq-uid/liquid extracted with 4 sequential volumes of 100 mlof methylene chloride to isolate the dissolved organiccompounds of interest. The combined extract (460 ml)was then concentrated to a volume of 50 µl by rotoe-vaporation and evaporation in nitrogen. 1 µl of each

sample was subsequently used for GC/MS analysis. Wepreviously attempted using solid phase extraction (SPE)columns to concentrate organic compounds from wellwater samples, however, this method proved untenablebecause of time constraints in the field and problemswith contaminants bleeding from the C18 SPE car-tridges interfering with GC/MS analysis. Despite theinconvenience of shipping large volumes of water be-tween the Balkans and the USA, liquid/liquid extractionprovided much more precise and accurate results for thisstudy.

The glass fiber filters used in the filtration of thewater samples were saved for analysis of particulateorganic compounds. The filters were dried at room tem-perature and reweighed to estimate the particulate mate-rial content of the water samples. The filters were thenextracted with sequential 100 ml volumes of GC/MSgrade methylene chloride in a clean (baked at 450°C)beaker with sonication. The combined extract was thenfiltered through a new clean glass fiber filter, and thefiltered extract processed in a manner identical to thatused for the water extracts for GC and GC/MS analysis.

Water samples for inorganic geochemical studieswere collected at each site in 60 ml plastic bottles. Sam-pling and analysis followed standard procedures forinorganics in water (16). Water samples for inorganicswere filtered at the sampling site using 0.45 µm Nu-clepore filters, and a plastic Millipore filtering appara-tus. All plasticware used for the inorganic analyses waspre-cleaned with acid using: (1) a 10% HCl soak anddistilled/deionized water rinse for anions, and (2) a 10%HNO3 soak, 1% ultrapure HNO3 soak, distilled/deionized water rinse for metals. Filtered water samplesfor metals analysis were stabilized with one drop ofconcentrated, ultrapure HNO3 for storage. Metals in thefiltered, acidified water samples were quantitativelydetermined by inductively coupled plasma massspectrometry (ICP-MS) (16). Filtered water samples foranion analysis were stored in clean, 60 ml plastic bottlesat room temperature; anions were analyzed by ionchromatography (17), and by colorimetry for phosphateand ammonium (18) at USGS labs in Reston, VA. Dis-solved oxygen, pH, conductivity, salinity, and total dis-solved solids were determined on site in unfilteredwell/spring water using portable meters and electrodes(18,19). Small water samples were collected in pre-cleaned bottles for the determination of sulfide and al-kalinity at the end of each day using standard electro-chemical methods (18).

Appropriate field and laboratory blanks were run toaccount for any operational or reagent contamination.

Coal analysisPliocene lignite samples were collected from the

three coal mines mentioned earlier. Methanol extracts ofthe lignite were made, using approximately 5 g of coaland 5 ml of chromatographic purity methanol. The ex-traction was performed in clean glass tubes for twodays. The extract was analyzed by GC/MS at the Clini-cal Laboratory No.1, Timisoara, Romania, using the

42 WH. Orem, CA. Tatu, GL. Feder, et. al.

same model instrument and conditions identical to thoseused at the USGS for well water studies. For compari-son, we also analyzed 17 Romanian nonendemic areaPliocene lignites, one North Dakota lignite, and Plio-cene lignites from two distinct endemic regions in Ser-bia (Bpesyn and Kostolac).

The same coal samples were also exposed to leach-ing by distilled water using both continuous soxhlet(80°C) and room temperature extraction. Leaching ex-periments were carried out on the coals to simulategroundwater leaching of coals in the endemic areas.Soxhlet extractions were performed for up to three days,and the room temperature extractions were carried onfor 1-4 months. The water extracts were liquid/liquidextracted with sequential volumes of methylene chlorideand analyzed by GC/MS under similar conditions atUSGS laboratories in Reston, VA.

Gas Chromatography-Mass Spectrometry(GC/MS)The concentrated extracts from both the dissolved

and particulate organics in the well/spring water sam-ples were analyzed first by GC with flame ionizationdetection (GC-FID), and then by GC/MS. The initialGC-FID analysis was used to screen the samples forcomplexity, and to determine if further separation byliquid chromatography (LC) was needed prior toGC/MS analysis. The GC-FID also provided initialquantification of peak area prior to identification of theorganic component in the chromatogram. A PerkinElmer (Boston, MA) model 8500 GC-FID with a 30 m× 250 µm × 0.25 µm J&W DB-5 column (95% dimethyl5% diphenyl polysiloxane) was used for the analysis.GC-FID conditions were as follows: 1.0 µl splitless in-jection, injector temperature of 150°C, detector tem-perature of 325°C, temperature program 70-325°C at4°C/min and a final hold at 325°C for 20 min, and acolumn pressure of 13 psig. GC/MS was used forstructural identification of the organic compounds andfor additional quantification. A Hewlett Packard (Ag-ilent Technologies, Palo Alto, CA, USA) 6890 seriesGC and a 5973 electron ionization (EI) mass selectivedetector (MSD) was used for GC/MS analysis. The GCmachine was fitted with a 30 m × 250 µm × 0.25 µmHP-5MS column (95% dimethyl 5% diphenyl polysi-loxane). The following conditions were used for theGC/MS run: 1.0 µl splitless injection, constant flow of1.0 ml/min, solvent delay of 6.0 min, injector tempera-ture of 150°C, interface at 300°C, temperature programof 70-300°C at 4°C/min and a hold at 300°C for 15 min,and mass scan from 50-550 Da. Individual organiccompounds in the sample chromatograms were identi-fied by comparison of mass spectral features to librariesof mass spectral data included in the GC/MS system(NIST98 and Wiley 7), by mass spectral identifications,and/or by comparison to standard compounds.

Previous experience showed that GC/MS is the bestmethod for determining low molecular weight (up to500-600 Da) organic compounds in the water. Only few

of the compounds present in the chromatograms, how-ever, are positively identified (>95% probability) usingthe available mass spectral libraries. This is not unusualin organic geochemical studies of environmental sam-ples. Our attention was primarily focused on aromaticcompounds and their functional derivatives: PAHs (an-thracene, pyrene, benzo[a]pyrene), aliphatics (alkanes,alkenes, and cyclics), and heterocyclic (especially O-,N- and S-containing) compounds.

Results

The setting of the endemic villagesAll endemic sites visited during the 1999 and 2000

fieldtrips are located at elevations below 250 m, in val-ley bottoms or on plains, and are surrounded by smoothhills. There is usually a main drainage (river or stream)passing through the village that collects smaller drain-ages originating from the hills. These main drainagesare tributaries of the Danube River. Field observationsand discussions with villagers indicated that Pliocenelignite deposits exist in the surrounding hills, and thatcoal mines are in close proximity (less than 1 km) toseveral endemic villages (Pietris, Erghevitza, Livezile,Prunisor, Husnicioara) (fig. 2).

Inorganic analysis of water samplesResults from inorganic analysis of water samples

collected from endemic and nonendemic sites in Roma-nia in March and August 2000 are shown in Figures 3-5.No consistent or significant differences for anions, nu-trients, alkalinity, sulfides, or the general inorganic pa-rameters measured were observed between samplesfrom the endemic and nonendemic sites. Phosphate wassomewhat elevated at one of the endemic sites and am-monium was elevated at two of the endemic locations(fig. 4). The relevance of this is unclear, but the highernutrient concentrations could be attributed to the usageof fertilizers in the area. Nitrate concentrations wereelevated in most of the water samples, reaching valuesas high as 225 mg/l, and exceeding USEPA guidelinesfor drinking water at all sites (fig. 5). Since no relevantdifference was observed between the endemic andnonendemic sites for nitrate, however, it cannot be di-rectly linked to BEN etiology. It is possible though thatnitrate is a factor contributing to disease causation in thepresence of toxic organic compounds in the water sam-ples. Nitrate can react with various functional groupsfound in organic molecules (e.g. amino-, hydroxy-,etc.), and could increase the toxicity and carcinogenicityof these compounds. High nitrate concentrations werealso found in endemic and nonendemic water samplesfrom Bulgaria (Dr. T. Voice -Michigan State University,personal communication), and likely originate from theinorganic and organic (manure) fertilizers used by thevillagers. Concentrations of metals in the water sampleswere also generally similar at endemic and nonendemicsites, except for zinc, which appeared to be deficient atthe endemic sites (data not shown).

ENVIRONMENT, GEOCHEMISTRY AND THE ETIOLOGY OF BALKAN ENDEMIC NEPHROPATHY 43

Fig 3. pH and the total dissolved solids (TDS) values for theendemic and the nonendemic water samples.

Fig. 4. The sulfate, phosphate and ammonium values for thewater samples. As for the pH and the TDS, nosignificant difference could be found between theendemic and the nonendemic locations.

Fig. 2. Map showing the distribution of the coal deposits in Romania. 1 and 2 are the two Romanian BEN areas, topographicallylinked to Pliocene lignite deposits.

44 WH. Orem, CA. Tatu, GL. Feder, et. al.

Fig. 5. The nitrate concentration shows high values for boththe endemic and the nonendemic water samples.Nitrate is also much higher in the wells, compared tospring water supplies.

Organic analysis of water and coalIn contrast to the inorganic geochemistry, dissolved

organic constituents in the well and spring methanolsamples collected from the main Romanian BENendemic area contain a greater number of differentcompounds (aliphatic and aromatic), and in much higherabundance (>10×), compared to water samples from thenonendemic sites (fig. 6). Some of the compoundsfound in the endemic area water samples were alsoobserved in the water extracts of the Romanian Pliocenelignites (see below), suggesting a possible connectionbetween leachable organics from the coal and organics

in the well water samples. The identification of thehundreds of individual compounds in the water samplesanalyzed by GC/MS is continuing. Preliminaryassessment shows the presence of both aliphatic andaromatic compounds, rich in oxygen (hydroxy, phenol,methoxy, aldehyde, keto, carboxy,etc.) and nitrogen(amino) containing functional groups. All the samples,without exception, contained various phthalates, whichare some of the most common man-made environmentalcontaminants. Pesticides were absent except for onenonendemic water sample where compounds from theatrazine family were found in µg/l concentration.

Compared to the water samples, the filter extractswere much "cleaner" in organic compounds, provingthat most of the organic contaminants are found in thedissolved phase rather than in the particulate matter. Nosignificant differences were found between the endemicand the nonendemic filter extracts.

Based on the hypothesis that water is the carrier ofthe BEN causing agent, we conducted laboratory waterextraction of different coals as an experimental modelfor their natural bioavailability. We demonstrated inearlier work (20,21) that methanol extracts of Pliocenelignites from the endemic area have a geochemical sig-nature distinct from that of higher rank coals, and arecharacterized by a much higher number and abundanceof organic compounds from GC/MS total ion currentplots. 13C-NMR experiments have provided similar re-sults, showing that the endemic area Pliocene lignitesare rich in aromatic and aliphatic components and highorganic functionality (11). Methanol extracts of thePliocene lignites from the endemic areas also appear tobe distinct from methanol extracts of other lignites col-lected from areas in Romania free of BEN. Total ioncurrents chromatograms of three endemic zone lignitesand one non-endemic area lignite of similar age, areshown in Figure 7. The complexity of the dissolvedorganics in methanol extracts of the endemic areaPliocene lignites is obvious, and it is not matched by

Fig. 6. Total ion current chromatograms of three water extractsshow a much higher organic contents in the endemicarea samples (A,B) vs. a nonendemic sample (C).

Fig. 7. Total ion current chromatograms of Pliocene lignitemethanol extracts show a higher organic content in thelignites collected from the endemic areas (Husnicioara,Kostolac and Bpesyn) compared to a lignite from aRomanian nonendemic area (Jilt Sud).

ENVIRONMENT, GEOCHEMISTRY AND THE ETIOLOGY OF BALKAN ENDEMIC NEPHROPATHY 45

any of the nonendemic area lignites analyzed. The totalion current of water extracts of the endemic area lignitesshow peaks corresponding to aliphatic (mainly cycloal-kanes/alkenes and steranic structures) and aromatic(mono- and polyaromatic terpanes, polycyclic aromatichydrocarbons) compounds. Many of these compoundshave attached oxygen-based functional groups (hy-droxy-, phenol-, keto-, methoxy-) and some of themcontain heterocyclic nitrogen or amino groups, struc-tural features that could make them nephrotoxic andcarcinogenic. Very few of these components are en-countered in the methanol extracts of lignite from thenonendemic areas. This difference could be due to dis-tinctive paleogeographic coal- forming conditions dur-ing the last period of the Tertiary in the correspondingBEN endemic and nonendemic areas (21). A lower ex-tractability in polar solvents of the organic compoundsfrom the nonendemic lignites can also explain the dif-ferences in the GC/MS data. We have demonstrated inprevious experiments (20) that a three minute methanolextraction of the endemic area Pliocene lignite is suffi-cient to give a high yield of organic components, thecompounds being loosely bound to the coal matrix andreadily extractable under the mild experimental condi-tions used. For higher rank coals and for the nonen-demic area lignites, a significantly longer period of time(hours→days) is needed to extract similar quantities oforganic compounds.

The endemic area Pliocene lignites also have muchmore water-soluble organic compounds compared tohigher rank coals (fig. 8). The room temperature waterextraction process seems also to be time dependent, the4 months water extract having a double amount of or-ganics compared to the one month extract. All of theindividual components have not yet been identified, butthey are mainly aromatic and aliphatic and it is likelythat many of them overlap with those from the methanolextracts. Similar experiments are underway usingnonendemic area Pliocene lignites.

DiscussionThe first etiological hypothesis for BEN, advanced

by Danilovic in Yugoslavia was that the disease is achronic intoxication with lead, originating from themilling wheels used to ground wheat and contaminatingthe flour consumed by the villagers in the endemic area(1). This suggestion was taken seriously by localauthorities from Serbia, who banned the use of the wa-termills in the area. This measure led to serious eco-nomic losses for the villagers who had to travel to otherlocalities to grind their grains, but no reduction in BENincidence was noted in the affected areas (2). Subse-quent laboratory investigations carried out in the coun-tries affected by BEN failed to confirm any role for leadin the causation of BEN. In addition, the clinical pictureof the BEN patients lacked the elements defining leadintoxication.

Measurements of other metals (Cu, Zn, Ni, Ti, Al,Mb, Co, W, Cd) in water and other environmental sam-ples from the endemic region and in BEN patients havegenerally produced values in the normal ranges, whichin general are in agreement with our recent data ob-tained from Romania. Some authors (22) have claimedhigher concentrations of Mn, Al, Cu and Fe, whichcould have significance in BEN etiology. According toBulgarian and Yugoslavian studies (23), silicon wasfound in high concentrations in drinking water fromendemic areas, suggesting a leaching process of thesediment and of the alluvium coming in contact with theground water. Silicon is known to be nephropathogenicbut it has not been found in significant concentrations inthe kidneys of BEN patients. Other trace elements likeselenium were found to be deficient in the endemic ar-eas, at least in the former Yugoslavia (24,25). The sig-nificance of selenium deficiency with respect to theonset of BEN, however, is unclear.

Fig. 8. Gas chromatograms of water extracts of two endemicarea Pliocene lignites and of a non-BEN area bitumi-nous coal. The organic content is much higher in thewater extract of the lignites compared to the high rankcoal.

Except for the nitrate concentrations that were foundto be high in both the endemic and the nonendemic wa-ter samples, all the other inorganic parameters measuredby us showed no strikingly high or low values for theendemic and the nonendemic locations. To some extentin contradiction with previous studies made in Yugosla-via, we found zinc to be in lower concentrations in theendemic vs. the nonendemic sites. Zn is a biologicallyessential element, and it is absolutely required for thedevelopment of normal immune response and defenseagainst cancer. Although more extensive geochemicalmapping in the affected countries is needed beforedrawing a final conclusion, it is possible that Zn-defi-ciency in the endemic region may have a role in BENcausation and in concert with other trace element imbal-ances (e.g. the selenium deficiency) be responsible, atleast in part, for the induction of the UTT's associatedwith BEN.

46 WH. Orem, CA. Tatu, GL. Feder, et. al.

The organic geochemical information obtained fromour study in the main Romanian BEN-endemic areaduring 1999-2000 is consistent with the Pliocene lignitehypothesis of BEN etiology, but it does not provide afinal confirmation to this theory. A "smoking-gun",imagined as an organic molecule with known nephro-toxic and/or carcinogenic properties, is still missing, butthe presence of much more potentially toxic organiccontaminants and in much higher concentrations in theendemic vs. nonendemic water samples is an encourag-ing finding, which by itself should justify further fieldwork not only in Romania, but in the other affectedcountries, too. In addition, we have demonstrated thatthe Pliocene lignites from the BEN areas produce amuch greater yield of organic substances in polar ex-tracts compared to higher rank coals and compared toother lignites from nonendemic areas in Romania. Thesecompounds include PAH's, and highly functionalized(O, N, and S) aliphatic and aromatic compounds. Someof these compounds were also identified in the watersamples collected from the endemic villages. It may bethat only a small number of compounds (perhaps evenonly one type of structure) out of the hundreds in thewater samples are responsible for BEN and the associ-ated UTT's. Moreover, the relatively low concentrationsof toxic organic compounds found in the drinking wa-ters (individual compounds generally <1 µg/l) in en-demic villages could explain the long period of expo-sure (decades) leading to BEN.

As the GC/MS method of identifying organic com-pounds is limited to molecular weights <600 Da, it islikely that some higher molecular weight compoundspresent in the water samples have remained undetected.Of particular interest from this standpoint are humicsubstances. There are some studies reporting a potentialtoxicity of humic substances present in ground waterand bioavailable to human populations. Certain humicacids have even been implicated in the etiology of blackfoot disease and Kashin-Beck disease in China (26,27),and a similar implication cannot be excluded in BEN,the more so as the presence of humic substances in theground water from the BEN areas is almost a certainty.Such compounds can also be leached from low rankcoals like Pliocene lignites and we plan to identify themby HPLC-MS in further experiments.

The clinical, scientific and environmental health im-portance of BEN is not limited by the relatively smallnumber of affected persons, or by the seemingly smallextent of the affected regions. As intriguing as its etiol-ogy and epidemiology is, BEN could still be considereda local problem, affecting just a discrete part of Europe.However, there are at least four reasons, advanced byRadovanovic et al. (2), why BEN has a universal rele-vance for the scientific and medical community:

1. The medical geography of BEN is not completelyclarified. Although traditionally described in only ina few Balkan countries, kidney diseases similar inetiology to BEN could occur unrecognized on amore or less endemic scale in other regions of the

world. This is true not only for regions with lowrank coal deposits such as the Pliocene ligniteslinked to BEN, but also for anywhere wheregroundwater used for drinking or cooking is inproximity to coal or coal waste stored near powerplants or mines. Since coal is a major source of en-ergy worldwide, there is a large potential for con-tamination of groundwater and surface water bytoxic organic compounds from coal (28). BEN-likekidney diseases could remain unrecognized due tofactors such as population mobility, short life ex-pectancy in an exposed population, or deficient localmedical systems. Also, point sources of contamina-tion, such as coal stored next to a power plant, mayaffect much smaller areas than those where BEN oc-curs.

2. Discovering the cause of BEN and revealing itsetiopathogenic mechanisms will lead to a better un-derstanding of other kidney diseases, such as analge-sic and cadmium nephropathies and of the end stagerenal disease. ESRD poses a significant economicburden in many countries, translated into costs forrenal dialysis and kidney transplantation. Accordingto a US Renal Data System (USRDS) report, due tothe recent growth in the ESRD patient population,total Medicare expenditures for the ESRD programhave been increasing steadily, from $5 billion in1991 to $12 billion in 1998. The total MedicareESRD program cost is projected to more than doublein the next ten years, surpassing $28 billion by theyear 2010. As for many ESRD patients the primarycause of the chronic renal failure is unknown, effec-tive preventive measures cannot be considered inthis case.

3. The emergence of UTT's in BEN patients has oftenbeen attributed to the same etiologic agent(s)causing BEN itself. Thus, identification of thisagent(s) may provide insight into the etiology ofcancer as well as ESRD.

4. From a humanitarian standpoint, identifying theBEN causal factor, will allow preventive measuresto be adopted in the directly afflicted countries, suchas by treatment or filtration of the drinking andcooking water or changing the main water supply ashas already been done in several endemic regions inSerbia. Such measures could prevent anothergeneration of BEN patients from developing.

A slight decrease in the incidence of BEN has beennoticed in the last two years in Serbia (29) however, thisseems to follow the usual oscillating pattern of the dis-ease, with low's and high's of incidence, and anotherepidemic outbreak is likely to occur in these areas in thefuture. It is interesting that a similar epidemiologicalpattern was noted in the incidence of diseases related toselenium deficiency in the endemic areas of China.Here, a peak incidence of Se deficiency (Keshan andKashin-Beck diseases) cases was noted in the late 1950s

ENVIRONMENT, GEOCHEMISTRY AND THE ETIOLOGY OF BALKAN ENDEMIC NEPHROPATHY 47

until the 1970s, followed by a reduction in the numberof cases (30). An epidemiological parallel can be drawnbetween BEN and Se-deficiency related diseases, assimilar factors (geological, environmental, social, die-tary, etc.) could control in a similar way the incidencesof these diseases. In both situations, the endemic out-breaks have occurred in stable rural populations, livingin similarly complex geological environments (charac-terized by inorganic as well as organically rich rocks)depending almost entirely on their immediate environ-ment for water and food and having little access togoods outside their areas. As the situation has graduallychanged after the end of WWII, with more food anddrink products coming into the areas from the outside,this would provide a partial explanation, at least in thecase of BEN, for the decreasing incidence of the dis-ease, without eliminating the original causative factorstill present in the area.

Although appealing, the Pliocene lignite implicationin BEN etiology is still a possibility rather than a cer-tainty. BEN cause probably resides in the complexity ofthe environment where the disease occurs and a combi-

nation of external factors acting together on a suscepti-ble genetic makeup should be considered. Organic com-pounds found in the groundwater are a potential majorculprit, but their source remains elusive; their origin inthe Pliocene lignite is likely but the fact that not all thedescribed endemic areas are topographically connectedto known Pliocene coal beds (i.e., one endemic area inSerbia and the endemic area in Bulgaria) rises furtherquestions about this possibility. Uncharted and of loweconomic importance Pliocene coal deposits could be anexplanation in this case, as is the situation of the smallerendemic area in Romania (Resita region). However,more extensive geochemical investigations and perhapssome novel methodological and conceptual approacheswill be needed in order to answer the main question,that of Balkan nephropathy etiology.

Acknowledgments. This work was supported through a grant from theUS Geological Survey and a collaborative linkage grant (CLG 975818)from NATO to W.H. Orem, an assistance award (00HQAG0213) fromthe US Geological Survey to C.A. Tatu and through funding from theRomanian Ministry of Health.

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