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CKDu REVISITED R.M.G. Rajapakse (Ph.D., D.I.C., C. Chem., FNASSL) Senior Professor in Chemistry Department of Chemistry University of Peradeniya Peradeniya.

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Presentation on theme: "CKDu REVISITED R.M.G. Rajapakse (Ph.D., D.I.C., C. Chem., FNASSL) Senior Professor in Chemistry Department of Chemistry University of Peradeniya Peradeniya."— Presentation transcript:


2 CKDu REVISITED R.M.G. Rajapakse (Ph.D., D.I.C., C. Chem., FNASSL) Senior Professor in Chemistry Department of Chemistry University of Peradeniya Peradeniya

3 CONTENTS Chronic Kidney Diseases CKDu in Sri Lanka Geochemical Aspects and Chronic Renal Failure: F - Hypothesis Hypothesis of F - and Aluminofluorides Cd 2+ Hypothesis Cyanobacteria Hypothesis Arsenic Hypothesis Role of Glyphosate “Soft Ions” and Ionic Strength Dialysis Renal Substitutional Therapy Regenerative Medicine

4 Definition: Any condition that causes reduced kidney function over a period of time is a chronic kidney disease. CKD is present when a patient’s glomerular filtration rate remains below 60 mL min -1 for more than 3 months, or when a patient’s urine albumin-to-creatinine ratio is over 30 mg of albumin for each g of creatinine (30 mg g -1 ). CHRONIC KIDNEY DISEASES (CKDs) [1]: Glomerular filtration rate Glomerular filtration rate (GFR) is a test used to check how well the kidneys are working. Specifically, it estimates how much blood passes through the glomeruli each minute. Glomeruli are the tiny filters in the kidneys that filter waste from the blood (MEDLINE PLUS). Albumin is a protein found in the blood. A healthy kidney does not let albumin pass into the urine. A damaged kidney lets some albumin pass into the urine. The less albumin in your urine, the better. (National Kidney Disease Education Program of USA.) 1. Kidney Disease Statistics for the United States, National Kidney and Urologic Diseases Information Clearinghouse (NKUDIC), Accessed on July 5 th,

5 Creatinine is removed from the blood chiefly by the kidneys, primarily by glomerular filtration, but also by proximal tubular secretion. Little or no tubular reabsorption of creatinine occurs. If the filtration in the kidney is deficient, creatinine blood levels rise. Therefore, creatinine levels in blood and urine may be used to calculate the creatinine clearance (CrCl), which correlates with the glomerular filtration rate (GFR). Blood creatinine levels may also be used alone to calculate the estimated GFR (eGFR). SKIP

6 CHRONIC KIDNEY DISEASES (CKDs) ARE NOT UNCOMMON [1-5] One in 10 American adults, more than 20 million, have some level of CKD [1-3] due to diabetes or hypertension. Around 1.7 million Australians (1 in 10) aged ≥ 18 years have clinical evidence of CKD [4,5] due to diabetes or hypertension. [1] Kidney Disease Statistics for the United States, National Kidney and Urologic Diseases Information Clearinghouse (NKUDIC), kudiseases /pubs /kustats/#16. [2] National Kidney and Urologic Diseases Information Clearing House (NKUDIC), U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES. [3] Centers for Disease Control and Prevention, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES, NIH Publication No. 12–3895, November 15, [4] STATE OF THE NATION: CHRONIC KIDNEY DISEASE IN AUSTRALIA, MAY 2014, KIDNEY HEALTH AUSTRALIA: Accessed 03 rd July, [5] ANZDATA, Summary of Australian and New Zealand Dialysis and Transplantation Registry, South Australia, Adlaide, 2013.

7 CKDu IN SRI LANKA CKDu: Chronic Kidney Disease of unknown Aetiology Affected Areas Girandurukotte in the Badulla District Medawachchiya and Padaviya in the Anuradhapura District Medirigiriya and Hingurakgoda in the Polonnaruwa District Nickawewa in the Kurunagala District


9 FACTS ABOUT CKDu IN SRI LANKA Discovered since mid 1990’s. Affected patients are mostly rice paddy farmers. The estimated age-standardized prevalence of the disease is 15% [7]. According to the Ministry of Health, the estimated number of CKDu cases is 69,258 as of June 2014 [8, 9]. Estimated death toll is 20,000 [8]. Affected patients were of age categories over 55 years [10]. The age category is shifting towards younger ages [10]. It has been predicted that even the school children could be affected in the near future [10]. [7] N. Jayathilaka, S. Mendis, P. Maheepala and F.A. Metha, “Chronic Kidney Disease of Unknown Aeitiology: Prevalence and Causative Factors in a Developing Country”, BMC Nephrology, 14 (2013) doi: / [8]Channa Jayasumana, Sarath Gunathilaka and Priyantha Senanayake, “Glyphosate, Hard Water, and Nephrotoxic Metals: Are They the Culprit Behind the Epidemic of Chronic Kidney Disease of Unknown Etiology in Sri Lnka?”, International Journal of Environmental Research and Public Health, 11 (2014) [9] Private Communication with Dr. Channa Jayasumana [10] Channa Jayasumana, Lakbima, Sunday Edition, 22 nd June, 2014.

10 UNIQUE FEATURES OF CKDu It is said that CKDu aetiology does not include commonly known risk factors for a chronic kidney disease. No Diabetes mellitus No Hypertension No Glomerulonephritis [8, 11] SO, IT IS A VERY SPECIAL DISEASE!!! Glomerulonephritis, also known as glomerular nephritis, is a term used to refer to several renal diseases (usually affecting both kidneys). Many of the diseases are characterised by inflammation either of the glomeruli or small blood vessels in the kidneys, hence the name. [8] Channa Jayasumana, Sarath Gunathilaka and Priyantha Senanayake, “Glyphosate, Hard Water, and Nephrotoxic Metals: Are They the Culprit Behind the Epidemic of Chronic Kidney Disease of Unknown Etiology in Sri Lnka?”, International Journal of Environmental Research and Public Health, 11 (2014) [11] N.T. Athuraliya, T.D. Abeysekara, P.H. Amerasinghe, R. Kumarasiri, P. Bandara, U. Karunaratna, A.H. Milton and A. L. Jones, “Uncertain etiologies of Proteinuric Chronic Kidney Disease in Rural Sri Lanka”, Kidney International, 80 (2011)

11 CRITERIA FOR CASE DEFINITION OF CKDu [12] No past history or current treatment for diabetes mellitus, chronic or severe hypertension, snake bites, urological disease of known aetiology or glomerulonephritis. Glycosylated haemoglobin levels in blood (HbA1c) of < 6.5%. Blood pressure < 160/100 for untreated patients and < 140/90 for treated patients. The reference range of Glycosylated haemoglobin levels in blood is about mmol mol -1 (4 – 5.9 DCCT %) in a healthy patient. [12] Ministry of Health. Chronic Kidney Disease of Unknown Aetiology, Circular No /2009, Ministry of Health, Colombo, Sri Lanka (2009). CategorySystolic BP/mmHg Diastolic BP/mmHg Normal Prehypertension Stage 1 Hypertension Stage 2 Hypertension160100

12 A1c%Conc. Of Blood Sugar (Mean Value)/mg dl [13] Accessed 03 rd July, [14] Table 2: Iftikhar Ahmad Asim Syed and Waqar Ahmed Khan, “Glycated Haemoglobin � a Marker and Predictor of Cardiovascular Disease”, J. Pakistan Medical Association, 16 (July 2011) CORRELATION BETEEN GLYCATED HAEMOGLOBIN LEVEL IN BLOOD AND MEAN BLOOD SUGAR CONCENTRATION OVER THE PREVIOUS FOUR WEEKS [13] 6.5% Glycated haemoglobin means ~ 150 mg dL -1 blood sugar! SKIP

13 IMPORTANT POINTS TO CONSIDER (i)Patients are confined to a unique geographical area of the country. (ii)Disease was not found about 25 years ago. (iii) Number of patients is continuously increasing very rapidly now. (iv) Affected people are mostly rice paddy farmers. (v)Patients are mainly males. (vi) The disease is prevalent in the region where fluorosis is also prevalent. (vii)In general, drinking water has high salinity.

14 IMPLICATIONS! The aeitiology of the disease may be associated with local environmental and occupational conditions, food habits or may be due to genetic reasons. Occupation of the patients is already known. They are mostly rice paddy farmers. Look for unique environmental conditions of the region and consequences of farming. Farming communities in NCP and EP of Sri Lanka comprise of native people of the region plus those migrated from other areas, such as, Gampaha, Kurunegala, Kegalle, Galle, Matara, Hambantota, Kotmale etc. during the colonization period. Better to see whether there are any Genetic or Hereditary Factors such as the Patients are of Particular Tribe Living in this Area and so on. Better to investigate their food habits, habits such as chewing “BULATHWITA”. Consumption of illicit alcohols and inadequate water consumption during hard work of farming. Do they smoke? Smoking is the biggest source of cadmium intake.

15 GEOCHEMICAL ASPECTS-CHRONIC RENAL FAILURE [15] First Study by C.B. Dissanayake in Dental fluorosis and Kidney Diseases are commonly observed in Sri Lanka in the same geographical area. Chronic Renal Failure has been observed in the Areas of Anuradhapura and Polonnaruwa Districts. Geochemically, water in these areas are Na/K type with Cl - subtype predominating due to increasing drought conditions and consequent salt building up (This means water hardness is mostly due to permanent hardness and most of the compounds of sodium and potassium are water soluble!). The Total Dissolved Solids and Electrical Conductivity are high. Unique feature is that F - in ground waters of some areas is 10 mg L -1 which is much higher than maximum tolerable F - concentration in drinking water of 1.5 mg L -1 which has now reduced down to 0.5 mg L -1 (WHO Limit, vide infra). [15] C.B. Dissnayake, “Water Quality in the Dry Zone of Sri Lanka-Some Interesting Health Aspects”, J. National Science Foundation of Sri Lanka, 53 (2005)

16 The F - Map of Sri Lanka [16] [16] Rohana Chandrajith, J. P. Padmasiri, C.B. Dissanayake and K. M. Prematilaka, “SPAITIAL DISTRIBUTION OF FLUORIDE IN GROUND WATER OF SRI LANKA”, J. National Science Foundation of Sri Lanka, 40 (2012) Concentration of fluoride (mg/L = ppm) and Its Impact on Health 0.0 − 0.5 Limited growth and fertility, dental caries. 0.5 − 1.5 Promotes dental health, prevents tooth decay 1.5 − 4.0 Dental fluorosis (mottling of teeth) 4.0 − 10.0 Dental fluorosis, skeletal fluorosis (Pain in back and neck bones) > 10.0 Crippling skeletal fluorosis

17 CKDu STUDIES SECOND STUDY BY O. A. ILEPERUMA et al. [17, 18] Hypothesis on the connection to excess fluoride ions in drinking-water. Use of low-quality aluminium cooking pots. Possibility for the formation of water-soluble AlF 4 - and AlF 6 3- ions under cooking conditions. Subsequent intake of F - and fluoroaluminate ions. [17] O.A. Ileperuma, Keynote Speech: In Ileperuma, O.A., Priyantha, N., Chandrajith, R., Perera, A., Yatigammana, S.K. and Wijesundara, C. (Editors) (2013): Symposium Proceedings, Second International Symposium on Water Quality and Human Health: Challenges Ahead, March, PGIS, Peradeniya, Sri Lanka. [18] O.A.Ileperuma, H.A. Dharmagunawardhane and K.P.R.P Herath, “Dissolution of aluminium from sub-standard utensils under high fluoride stress: A possible risk factor for chronic renal failure in the North-Central Province, J. Natn. Sci. Foundation Sri Lanka, 37 (2009) 219—222

18 FLUORIDE IN DRINKING-WATER EXCELLENT REVIEW OF WHO [19] [19] J. Fawell, K. Bailey, J. Chilton, E. Dahi, L. Fewtrell and Y. Magara, Fluoride in Drinking-Water, World Health Organization, IWA Publishing, London, First Published SKIP


20 Fluoride in Australian Waters [20] Very Low. Fluoridization of drinking water is required to prevent dental caries among small and teenage children. The recommendations for fluoride levels in Australian water fluoridation programs should comply with those set out in the National Health and Medical Research Council’s public statement. The efficacy and safety of fluoridation is 0.5 ppm. [20] “Fluoride concentration in Australia’s Drinking Waters”, Victoria, Department of Health, 8/$FILE/US%20HHS%20proposal%20WEB.pdf, Accessed on 06/07/ /$FILE/US%20HHS%20proposal%20WEB.pdf SKIP

21 WHO STANDARDS OF FLUORIDE IN DRINKING-WATER: SKIP WHO noted that mottling of teeth (i.e. dental fluorosis) is sometimes associated with fluoride levels in drinking-water above 1.5 mg L –1 and crippling skeletal fluorosis can ensue when fluoride levels exceed 10 mg L –1. A guideline value of 1.5 mg L –1 was, therefore, recommended by WHO as a level at which dental fluorosis should be minimal (WHO, 1984). The 1.5 mg L –1 fluoride guideline value that was set in 1984 was subsequently re-evaluated by WHO and it was concluded that there was no evidence to suggest that it should be revised (WHO, 1996, 2004). The 1.5 mg L –1 guideline value of WHO is not a “fixed” value but is intended to be adapted to take account of local conditions (e.g. diet, water consumption, etc.).

22 FLUORIDE METABOLISM FLURIDE ABSORPTION BY THE HUMAN BODY Approximately 75–90% of ingested fluoride is absorbed. In an acidic stomach, fluoride is converted into hydrogen fluoride (HF) and up to about 40% of the ingested fluoride is absorbed from the stomach as HF (Discuss corrosiveness). Fluoride not absorbed in the stomach (60%) is absorbed in the intestine and is unaffected by pH at this site. Relative to the amount of fluoride ingested, high concentrations of cations that form insoluble complexes with fluoride (e.g. calcium, magnesium and aluminium) can markedly decrease gastrointestinal fluoride absorption.

23 FLUORIDE DISTRIBUTION IN THE HUMAN BODY Once absorbed into the blood, fluoride is readily distributed throughout the body. Approximately 99% of the body burden of fluoride is retained in calcium rich areas such as bones and teeth (dentine and enamel). Fluoride is then incorporated into the hydroxyapatite [Ca 10 (PO 4 ) 6 (OH) 2 ] lattice through ion-exchange. Porous Ca 10 (PO 4 ) 6 (OH) 2 (s) + xF - (blood) ⇌ Denser Ca 10 (PO 4 ) 6 (OH) 2-x F x (s) + xOH - (blood) Out of eleven important functions of bones, detoxification is one of the most important functions. Toxic anions such as aresenite and arsenate in the blood are adsorbed by the hydroxyl groups of hydroxyapatite and thereby the supply to other organs is prevented. These anions are then slowly released to the kidney for safe discharge. When the OH groups are substituted with F - anions this mechanism is not possible. Ca 2+ can be exchanged for Cd 2+ in Cd 2+ toxicity causing weak bones (osteoporosis) (vide infra)

24 STRUCTURE OF NATURAL BONE NANOTECHNOLOGICAL ARCHITECTURE OF BONES COMPACT AND SPONGY PARTS OF BONE A femur head (highest part of the thigh bone) with a cortex of compact bone and medulla of trabecular bone.

25 Tailor-made Prosthesis by the Bio-Chemical-Computer Science- Engineering- Medical Research Group of University of Peradeniya

26 EFFECTS OF FLUORIDE ON HUMANS SKIP A number of studies have reported on the acute effects of fluoride exposure following fluoridation overdosing. However, the effects of long-term exposure to naturally occurring fluoride from drinking-water and other environmental sources (Chronic effects) are the major concern with regard to human health. A large number of epidemiological studies have been conducted in many countries concerning the effects of long-term exposure to fluoride.


28 FACTS ON SKELETAL FLUOROSIS SKIP Paraestheria: Sensation of tingling, tickling, prickling, pricking, or burning of a person's skin with no apparent long-term physical effect. Poker Back: Arthritis and osteitis deformans involving the spinal column. EXOSTOSIS: Formation of new bone on the surface of a bone.

29 MUTAGENECITY AND RELATED END- POINTS. SKIP Studies have been carried out with bacteria, insects, laboratory animals and in vitro studies with human cells. Number of positive results in a variety of mammalian cell types for chromosome damage (USNRC 1993, WHO 1996, IPCS 2000) have been observed. Effects are due to interferences with protein synthesis rather than by any direct interaction between F - and DNA. THIS IS TRUE SINCE F - IS EXTREMELY INTERT IN ITS’ F - FORM.

30 FLUORIDE AND CANCER SKIP My Opinion: THE POSSIBILITY IS DUE TO VERY HIGH CORROSIVENESS OF HYDROGEN FLUORIDE SOLUTION. HF CAN ATTACK ANYTHING. Can Even Dissolve Glass! F - can precipitate certain important metal ions such as Ca 2+ as CaF 2 (s). F- can attack enzymes inhibiting their catalytic functions. In the body, hydrofluoric acid reacts with the ubiquitous biologically important ions such as Ca 2+ and Mg 2+. Formation of insoluble calcium fluoride is proposed as the aetiology for both precipitous fall in serum calcium and the severe pain associated with tissue toxicity [21]. [21] Hoffman, Robert S. et al. (2007) Goldfrank's Manual of Toxicologic Emergencies. New York: McGraw-Hill Professional, p. 1333, ISBN

31 ENZYME INHIBITION BY 1 ppm or less [22] Enzyme[F - ]/pp m % Inhibition Acetylcholinest earase 161% Glutamine synthetase 1100% DNA Repair Enzyme Systems 150% Lactoperoxidase150% Pterin deaminase 0.650% Alkaline phosphstase 0.352% [22] John Liyamouyiannis, “Fluoride: The Aging Factor, How to Recognize and Avoid Devastating Effects of Fluoride”, Health Action press, Ohio, United States, 3 rd Edition, 1993, 292 Pages. Organ[F - ] before fluoridation of drinking water in USA/ppm Before 1939 [F - ] after fluoridation of drinking water in USA/ppm Brain Heart Kidney Liver Lung Spleen PancreasNR ThyroidNR Accumulation of Fluoride Resulting From F - Exposure


33 UPTAKE OF FLUOROALUMINATE SPECIES BY BLUE-GREEN ALGAE [23] Have pH-dependent Mg 2+ and Ca 2+ -ATPase (Class of enzymes that catalyze the decomposition of ATP into ADP and a free phosphate ion) activities. Toxicity increases with increasing acidity. Toxicity of fluoroaluminate is reduced in the presence of EDTA and citrate (THESE ARE BETTER CHEALATING AGENTS THAN AlF 4 - ). AlF 4 − inhibits the ATPase activity by acting as a functional barrier without affecting the structure of the enzyme. This upsets the cell metabolism and the removal of toxins. [23] Yasmin Husaini, L. C. Rai, Nirupama Mallick,”Impact of aluminium, fluoride and fluoroaluminate complex on ATPase activity of Nostoc linckia and Chlorella vulgaris”,Biometals 9 (1996) SKIP

34 UPTAKE OF FLUOROALUMINATE SPECIES BY HUMANS Laboratory investigations have often used aluminofluoride complexes for stimulation of various guanine nucleotide binding proteins. These complexes form spontaneously in aqueous solutions containing fluoride and traces of aluminum and appear to act as phosphate analogous. In view of the ubiquity of phosphate in cell metabolism and together with the dramatic increase in the amount of reactive aluminum now found in ecosystems, aluminofluoride complexes represent a strong potential danger for living organisms including humans. Fluoride anions in the presence of aluminum affect the liver as an organ involved in glycogenolysis, fatty acid oxidation, and lipolysis. Enormous possibilities for multiple molecular interactions of aluminum and fluoride exist in the brain.

35 EFFECT OF FLUOROALUMINATE COMPLEXES ON KIDNEYS [24] SKIP The effects of aluminofluoride complexes on the kidneys have been studied using glomerular mesangial cells, proximal tubular cells, and inner medullar collecting tubule cells of rat kidneys. Fluoride and aluminum in kidney tubular cells affect the ion transporting processes. The kidneys from rats drinking water containing NaF exhibited glomerular hypercellularity, renal mesangial proliferation, and the deposition of proteins in the renal tubules. Mesangial proliferative glomerulonephritis (MPGN) is a rare kidney disorder characterized by blood in the urine caused by a particular type of inflammation inside the kidneys. [24] Anna Strunecká and Jiří Patočka, “PHARMACOLOGICAL AND TOXICOLOGICAL EFFECTS OF ALUMINOFLUORIDE COMPLEXES”, Fluoride, 32 (1999)

36 MY OPINION Fluoride or Fluoroaluminte complexes being directly responsible for CKDu is quite unlikely but blocking of glomeruli with precipitated CaF 2 and proteins is possible. However, fluorosis prohibits the detoxification mechanism of bones. Therefore, patients with fluorosis are more prone to get their kidneys damaged by other toxic chemicals such as cadmium and arsenite ions. F - can inhibit various enzymes and HF can etch anything causing cancers.

37 CADMIUM ION HYPOTHESIS [25-27] Hypothesis of Cd 2+ causing CKDu by Sarath Bandara et al. appears to the third such hypothesis. Elevated Cd 2+ Levels in rice and Freshwater Fish. Both Rice and Inland Fish are a popular food among the farming community in this area. Cd 2+ is a common poison present in TSP fertilizers. Atomic Absorption Spectroscopic determination of Cd 2+ in rice and freshwater fish. Concentration of Cd 2+ in roots of Nymphaea lotus L (Lotus roots) [25] J.M.R.S. Bandara et al. “Chronic Renal Failure among Farm Families in Cascade Irrigation Systems in Sri lanka Associated with Elevated Dietary Cadmium Levels in Rice and Freshwater Fish (Thilapia)”, Environmental Geochemistry and Health, 30 (2008) [26] J.M.R.S. Bandara et al., “Chronic Renal Failure in Sri Lanka caused by Elevated Cadmium Levels: Trojan Horse of the Green Revolution”, Toxicology Letters, 198 (2010a) [27] J.M.R.S. Bandara et al., “Pollution of River Mahaweli and Farmlands unde Irrigation by Cadmium from Agricultural Inputs Leading to a Chronic Renal Failure Among Farmers in NCP”, Environmental Geochemistry and Health, DOI /S

38 PROBLEMS OF THE ANALYTICAL TECHNIQUE Calibration plot drawn in a concentration range higher than those in the analyte samples. Calibration plot is in the Linear Dynamic Range. Beer-Lambert Law: A = εlc, where A is the absorbance, ε the molar absorption coefficient of the sample, l the path length and c is the concentration of Cd 2+. No guarantee for the holding of the law below or above the linear dynamic range. As such, the magnitudes of values may be different to actual values. This has been pointed out by Chandrajith et al. [28]. USE OTHER TECHNIQUES SUCH AS ANODIC STRIPPING VOLTAMMETRY! [28] Rohana Chandrajith, Shanika Nanayakkara, Kozuyoshi Itai, T.N.C. Aturaliya, C.B. Dissanayake, Thilak Abeysekara, Kouji Harada, Takao Watanabe and Akio Koishumi, “Chronic Kidney Diseases of Unknown Aeitiology in Sri Lanka, Geographical Distribution and Environmental Implications”, Environmental Geochemical Health, 33 (2011)


40 BIOACCUMULATION OF CADMIUM IONS SKIP Although Cd 2+ levels in surface water may be lesser than what Bandara et al. have reported, Cd 2+ has a notorious bioaccumulation and a very long biological half life (vide infra). Therefore, inland fish may have high concentrations of continually accumulated Cd 2+. Once Cd 2+ is ingested continuously, they remain in the human body for a long time causing chronic problems.

41 CADMIUM IN FISH Measured in flesh extract. Cd 2+ concentrates in the bones by exchanging for Ca 2+ ions. In this way, Cd 2+ accumulates in fish bones. Ca 10 (PO 4 ) 6 (OH) 2 (s) + yCd 2+ (blood) ⇌ Ca 10-y Cd y (PO 4 ) 6 (OH) 2 (s) + yCa 2+ (blood) Cd 2+ may be found in liver and kidneys as well. Mechanisms will be discussed later.

42 CADMIUM POISONING Cadmium toxicity occurs when a person breathes in high levels of cadmium from the air, or eats food or drinks water containing high levels of cadmium. Cadmium is found in Batteries: Ni/Cd Batteries: Major Cadmium Source. Pigments Metal coatings Plastics Some metal alloys Fertilizers: IMPORTED TSP: [Cd 2+ ] = 71.7 mg Cd 2+ /kg P 2 O 5 [29]. Since 1973 to 1984, 68.9 tonnes of Cd has been added to agricultural lands. EPPAWELA PHOSPHATE HAS VERY LOW Cd 2+ CONTAMINATION! Cigarettes Stabilizer in PVC

43 RISK FACTORS SKIP Smoking. Living near hazardous waste sites or industrial factories that emit cadmium into the air. Working in a metal smelting and/or refining plant. Working in a plant that produces cadmium products (e.g., batteries, coatings, plastics, pigments). Having a nutritional deficiency in calcium, iron, protein, and/or zinc [THESE PEOPLE USUALLY DO NOT TAKE A HEALTHY DIET].

44 SYMPTOMS OF CADMIUM POISONING SKIP Eating food or drinking water contaminated with high levels of cadmium can result in: Vomiting/nausea, Stomach cramps, Diarrhea, Kidney damage, Fragile bones and Death. SYMPTOMS OF THE ITAI-ITAI DISEASE Breathing in cadmium can result in: Lung damage (chest pain or shortness of breath), Kidney diseases, Fragile bones and Death.

45 DIAGNOSIS (SKIP DUE TO TIME LIMITATIONS) Your doctor will ask about your symptoms and medical history, and perform a physical exam. Tests may include the following: Blood tests Urine tests Hair or nail analysis Neutron activation analysis—a test to measure cadmium levels inside your liver and kidneys.

46 TREATMENT (SKIP DUE TO TIME LIMITATIONS) There is no effective treatment for cadmium toxicity. Avoid exposure. Your treatment will be designed to help manage and relieve your symptoms. You may be given vitamin D for the weak bones.

47 To help reduce your chances of getting cadmium toxicity, take the following steps: SKIP Do not smoke. Smoking is the single most important source of cadmium intake for most persons. Identify potential sources of cadmium in and around your home, at work, and where your children play. If you maintain a vegetable garden, consider having fertilizers tested for cadmium. Some fertilizers have been found to be high in cadmium, which may then concentrate in your vegetables. Avoid any use of cadmium containing fungicides near your vegetable gardens. Eat a balanced diet that provides enough calcium, iron, protein, and zinc. Take inventory of and properly store (out of the reach of children) cadmium-containing products in your home (e.g., fungicides, batteries, metals, fabric dyes, ceramic/glass glazes, fertilizer); check the label for cadmium.

48 Keep nickel-cadmium batteries out of the reach of small children and find out how to properly dispose of these batteries. Read instructions for safely using cadmium-containing fungicides or fertilizers on your lawn or garden. If you have a water well, have your water tested for the presence of cadmium. If cadmium is present in your well water, consider using bottled water for drinking or install a water filter that removes cadmium and other metals from drinking water. Do not allow young children to play in or around hazardous waste sites. SKIP

49 ITAI-ITAI DISEASE: DUE TO CADMIUM POISONING Itai-tai disease is caused by cadmium poisoning due to mining in Toyama Prefecture of Japan. Cd 2+ goes in through rice and cereals! This is a multi-factorial disease but cadmium is a necessary agent to result in severe kidney damage and osteomalacia (Osteomalacia is derived from Greek: osteo- which means "bone", and malacia which means "softness". ).

50 Cadmium ions have a high affinity to rice and cereals. Recent discovery of Cd 2+ accumulation in red onions is also a sensible finding. Allyl propyl disulfide can coordinate with Cd 2+. LIGNIN Latifoliate: Plants with broad leaves. Nelum roots have glucose, fructose, sucrose, mannitol, galacturonic acid, raffinose, 10 amino acids and fibers [29]. Cadmium ions can form coordination complexes with them. [29] S. Hujjatullah, A. K. Bloch and A. Jabbar, ” Chemical composition and utilisation of the roots of Nymphaea lotus L”, Journal of the Science of Food and Agriculture, 18 (1967)

51 Cadmium and the Kidney [30] SKIP Chronic cadmium exposed battery workers had major signs in kidneys: Tubular and Glomerular dysfunction with proteinuria. A high prevalence of kidney stones in Swedish and British Workers. Glomerular dysfunction: Damaging glomeruli and letting red blood cells leak into urine. Proteinuria: Presence of an excess of serum proteins in urine [30] L. Friberg, “Cadmium and the Kidney”, Environmental Health Perspectives, 54 (1984) History and Aetiology of Cadmium-induced Kidney Dysfunction and Related Disorders

52 Cadmium Metabolism, Metallothionein and Kidney Dysfunction After the ingestion, cadmium is transported to the liver where it stimulates the synthesis of metallothionein. Cadmium is then bound to metallothionein and transported via blood to kidneys. Metallothionein (MT) is a family of cysteine-rich, low molecular weight (MW ranging from 500 to 14,000 Da) proteins. In the human body, large quantities are synthesized primarily in the liver and kidneys. L-Cysteine

53 KIDNEY DAMAGE BY CADMIUM Cadmium bound to metallothioneins are filtered with the primary urine and reabsorbed into the tubular cells. A considerable catabolism of cadmium-metallothionein can take place. Complexes break and cadmium ions bind to newly formed metallothionein in the tubular cells. Kidney damage takes place when the kidney no longer produces metallothionein. The biological half-life of cadmium is very long: Years! Chronic cadmium exposures of 50 µg m -3 (10 times the new OSHA permissible exposure limit) for 10 years may cause kidney dysfunction with proximal tubular damage [30]. [30] Human Toxic Chemical Exposure-Cadmium, “Bulletin of Pacific Toxicology Laboratories”,

54 RENAL TOXICITY OF CADMIUM IONS The kidney is the major target organ of cadmium. The critical concentration for toxic effects appears to be between 200 and 250 µg/g of kidney tissue ( ppm) [31] When the renal tubular cells become saturated with cadmium (because the storage capacity of metallothionein is exceeded) free cadmium is released. It is widely believed that this free cadmium ion is the cause of the renal damage. Free cadmium ions may replace zinc in some systems, especially those enzymes involved in the reabsorption and catabolism of proteins. [31] Roels, H., Bernard, J., Goret, A., Lauwerys, R. Chettle, D., Harvey, T., Al Haddad, I., "Critical concentration of cadmium in renal cortex and urine" Lancet i, 221, 1979.

55 RENAL TOXICITY OF CADMIUM IONS SKIP This may cause the tubular damage and proteinuria characterized by an increased urinary excretion of low molecular weight proteins, which come from the glomerular filtrate but are not reabsorbed [32]. Some evidence suggests that cadmium- induced proteinuria may also be the result of glomerular damage [33]. The first detectable adverse effect of cadmium on the kidney is an increased excretion of specific proteins in the urine. [32] Bernard, A., Roels, H.A., Hubermont, G., Buchet, J.P. Masson, P.L., Lauwerys, R. "Characterization of the proteinuria in cadmium exposed workers" Int. Arch. Occup. Environ. Health 38 (1976) 19. [33] Kawada, T., Kayama, H. and Suzuki, S. "Cadmium, NAG activity and ß2-microglobulin in the urine of cadmium pigment workers" Brit. J. Ind. Med. 46 (1989) 52.

56 MITROCHONDRIAL DAMAGE [34] The nephrotoxic metal ion, Cd 2+, causes mitochondrial damage and apoptosis of kidney proximal tubular cells. [34] Wing-Kee Lee, Malte Spielmann, Ulrich Bork and Frank Th èvenod, “ Cd 2 +-induced swelling-contraction dynamics in isolated kidney cortex mitochondria: role of Ca 2+ uniporter, K + cycling and protonmotive force”, American Journal of Physiology: Cell Physiology, 289 (2005) C656-C664. SKIP DIAGRAM

57 In a variety of metalloenzymes, the active site contains a catalytically-essential, tetra-coordinated Zn 2+ ions. Three of the ligands to the metal ion are provided by amino acid side chains, generally in distorted geometrical relationships, while a water molecule serves as the fourth ligand. Chief examples of such enzymes are carboxypeptidase A, thermolysin, carbonic anhydrase, alcohol dehydrogenase and alkaline phosphatase. SUBSTITUTION OF Cd 2+ FOR Zn 2+ IN Zn 2+ -CONTAINING ENZYMES

58 The male farmers usually drink excessive amounts of illicit alcohol! The water used to dilute spirit may contain anything. Hint for male patients? They work hard under hot sun. Dehydration results in concentration of species present in blood. Alcohol consumption further aggravates dehydration. Cadmium ions, if ingested, can replace the Zn 2+ in alcohol dehydrogenase and thereby can affect detoxification mechanism of toxic ethanol from the liver. Cadmium ions bound to alcohol dehydrogenase can go to kidneys and damage them. Alcohol taking farmers who have got Cd 2+ through food and/or drinking water are more susceptible to several illnesses including kidney damage. Besides, male farmers are the people who spray toxic chemicals to paddy fields without having protective clothing and face masks.

59 ANALYSIS OF Cd 2+ -INDUCED RENAL TUBULAR DAMAGE Several proteins including retinol binding protein and NAG have been suggested as markers for proteinuria, but ß-2-microglobulin in urine appears to be the most widely used and best characterized analyte to evaluate cadmium-induced renal tubular damage [35]. [35] U.S. Department of Labor OSHA, 29 CFR Parts 1910, 1915, 1920 "Occupational Exposure to Cadmium; Final Rules" Federal Register vol. 57, no. 178, Monday, September 14, 1992, Rules and Regulations.

60 FOURTH HYPOTHESIS-CYANOBACTERIA By Dhammika M. Dissanayake et al. SKIP DESCRIPTIPON Cyanobacteria also known as Cyanophyta, is aphylum of bacteria that obtain their energy through photosynthesis. They release O 2 to the atmosphere an hence they have changed the early reducing atmosphere into oxidizing atmosphere for other biological species to evolve.

61 FACTS ABOUT CYANOBACTERIA: SKIP Aquatic cyanobacteria are known for their extensive and highly visible blooms that can form in both freshwater and marine environments. The blooms can have the appearance of blue-green paint or scum. These blooms can be toxic, and frequently lead to the closure of recreational waters when spotted. Marine bacteriophages are significant parasites of unicellular marine cyanobacteria.

62 WHAT CYANOBACTERIA BLOOMS LOOK LIKE: SKIP Look like foams, scum or mats of the surface of fresh water lakes and ponds. The blooms are blue, bright green, brown or red. They may look like paint floating on water. As algae in cyanobacterial blooms die, the water may smell bad.

63 CYANOTOXINS SKIP Some cyanobacteria produce toxins, called cyanotoxins. Among cyanotoxins are some of the most powerful natural poisons known, including poisons which can cause rapid death by respiratory failure. The toxins include potent neurotoxins,hepatotoxins, cytotoxins, and endotoxins.

64 Nephrotoxicity of Cyanobacteria [36] SKIP Nephrotoxic potential of laboratory cultures of freshwater cyanobacterium (blue-green alga) Microcystis aeruginosa PCC 7806 (Pasteur Institute) has been assessed in male rats. The animals have been injected intraperitoneally with 0.5, 1.0 and 2.0 LD50 doses of lyophilized cell extract. Elevated plasma urea and creatinine levels have been accompanied by decrease in protein and albumin levels, followed by hematuria, proteinuria and bilirubinuria. Also decrease in kidney lactate dehydrogenase and glutamic oxaloacetic transaminase indicated possible nephrotoxic potential of the cyanobacteria. The extract also produced various hematological changes associated with stagnant type of hypoxia (Deprived of adequate O 2 supply). [36] Bhattacharya R, Sugendran K, Dangi RS, Rao PV, “Toxicity evaluation of freshwater cyanobacterium Microcystis aeruginosa PCC 7806: II. Nephrotoxicity in rats.”, Biomedical and environmental sciences : BES 10: Mar pg

65 FURTHER STUDIES ON CYANOBACTERIAL TOXICITY TO KIDNEYS SKIP Some results suggest that kidney impairment from chronic exposure of toxic cyanobacterial blooms might be the first step, and then followed by hepatic failure [37]. Studies are also underway at the Institute of Fundamental Studies. Group Leader is D. N. Magana-Arachchi. [37] Qiu T, Xie P, Li L, Guo L, Zhang D, Zhou Q, “Nephrotoxic effects from chronic toxic cyanobacterial blooms in fishes with different trophic levels in a large Chinese lake.”, Environmental Toxicological Pharmacology, 33 (2012)

66 THE CYANOBACTERIAL TOXINS: A HIDDEN HEALTH HAZARD: By Dhammika M. Dissanayake [38] According to their ongoing research studies, it is substantiated that toxins produced by the cyanobacteria may have a role in the pathogenesis of CKDu in Sri Lanka. Further, they have found that the cyanobacterial toxins could be the causative agents for the nephropathy in Balkan countries because there are very close similarities between Balkan Nephropathy and CKDu in Sri Lanka. Environmental factors such as global warming and admixing of Nitrogen, Phosphate and Potassium of chemical fertilizers with run off water provide a favorable ecological background for the blooming and toxin production of cyanobacteria. The management of patients with renal disease is expensive and need long term care. This is an unbearable burden to the economy of the country. As such, short-term and long -term preventive strategies like introduction of new water sources, measures to control the usage of chemical fertilizers and control of global warming are mandatory to alleviate the disease in addition to early detection. THIS IS A GOLDEN SUGGESTION!!!!

67 BALTIC CDK [39] Fluoride in Drinking Water is Less. Fluoride is added to salt. Fluoride is added to drinking water. CYANOBACTERIA WORLD MAP Balken Nephropathy affects only certain endemic rural foci along tributaries of the Dangue River in Balken Countries of Bosnia, Bulgaria, Croatia, Rumania and Serbia [39]. IS IT GENETIC DISEASE THEN? [39] G. Bomias and J. Boletis, “Balken Nephropsthy, Evolution of Our Knowledge, American Journal of Kidney Disease, 52 (2008) SKIP

68 OBJECTIONS FOR CYANOBACTERIA AND SRI LANKAN CKDu Apparently, these farmers drink dug well (92%) and tube well (8%) waters [40]. The cyanobacteria levels in these waters are marginal [41]. [40] Priyani paranagama, “Potential Link between Ground Water Hardness, Arsenic and Prevalence of CKDu”, [Not a standard peer reviewed journal reference.] [41] CKDu and Arsenic by the Arsenic Group [Not a Standard Journal Article] COULD NOT FIND RELIABLE STANDARD JOURNAL ARTICLES! PLEASE SEE LATER: THIS DUG AND TUBE WELL WATERS CAN NOT HAVE GLYPHOSATE ALSO. THIS GOES AGAINST THEIR GLYPHOSATE HYPOTHESIS.

69 FIFTH HYPOTHESIS-ARSENIC SPECIS GROUP OF RESEARCHERS: “THE ARSENIC GROUP” From Kelaniya and Rajarata Universities and Government Hospitals Professor Nalin de Silva (Mathematician) Professor Priyani Paranagama (Organic Chemist) Professor Mala Amarasinghe (Botanist) Dr. Kithsiri Senanayake (MBBS) Dr. Kumudu Dissanayake (MBBS) Dr. Channa Jayasumana (MBBS) Dr. Chinthaka Wijayawardhana (MBBS) Dr. P. Mahamithawa (MBBS) Dr. L. Rajakaruna (MBBS) Dr. D. Samarasinghe (MBBS) Mr. S. Fonseka (University of Kelaniya) SKIP READING NAMES

70 LITTLE BIT OF ARSENIC CHEMISTRY Compounds of Arsenic exists in four Oxidation States; +5, +3, 0 and -3. As is not a true metal. It is a metalloid. In water, +3 and +5 Oxidation States DO NOT EXIST as As 3+ and As 5+ bare cations [That is why we write them as As(III) and As(V)] but they exist as oxyanions: arsenite (AsO 3 3- or AsO 2 - ) and arsenate (AsO 4 3- ). As such, Arsenic does not form coordination complexes as other cations do since As does not exist as a cation. Arsenes can act as ligands and can donate electrons to metal ions. Compounds of Arsenic are Covalent rather than Ionic. Arsenite salts are more soluble in water than arsenate salts. Solubility of Ca 3 (AsO 4 ) 2 in water is 0.13 g L -1 at 25 °C. Ca 3 (AsO 4 ) 2 is soluble in acids. If ingested, can dissolve in the liver. Ca 3 (AsO 3 ) 2 is soluble in water.

71 CKDu AND ARSENIC This group says: CKDu is NOT associated with known risk factors of CKD such as diabetes, hypertension and glorulonephritis. Clinically, the disease is associated with β-2- microglobulinuria. High levels of β-2-microglobulinuria is a feature of arsenic induced nephrotoxicity [43]. PLEASE SEE THE REFERENCE PROVIDED! [43] F. Homg, T. Jin, A. Zhan, “Risk Assessment on Renal Dysfunction caused by Co-exposure to Arsenic and Cadmium using Benchmark Calculation in Chinese Population”, BioMetals,17 (2004) All other articles in the same journal, same volume, are about β-2-microglobulinuria as a feature of Cd 2+ -induced nephrotoxicity [44]. [44] George Kazantzis, “Cadmium, Osteoporosis and calcium metabolism”, BioMetals, 17 (2004) Several other papers are about Cd 2+ poisoning, kidney dysfunction and β-2-microglobulinuria. We have described this previously [35]!

72 [ 45] Rodriguez J, Cortes J, Talpaz M, et al: Serum β-2-microglobulin levels are a significant prognostic factor in Philadelphia chromosome- positive chronic myelogenous leukemia. Clinical Cancer Research 6 (2000) Available from URL: full/6/1/147. As accessed February 10, [46] Henry JB: Clinical Diagnosis and Management by Laboratory Methods, 20th ed. Saunders, THERE ARE OTHER CAUSES ALSO SKIP

73 HYPER-PIGMENTATION AND KERATOSIS Photographs of patients Palms and Soles with hyper- pigmentation and keratosis are provided in an undated report. Photographs of the Full Bodies of the Patients are NOT provided. Dassanayake et al. have stated that arsenic-related skin changes were seen in 30 patients but they are NOT CKDu patients [47, 48]. [47] Noble, A.; Amerasinghe, P.; Manthrithilake, H.; Arasalingam, S Review of literature on chronic kidney disease of unknown etiology (CKDu) in Sri Lanka. Colombo, Sri Lanka: International Water Management Institute (IWMI). 41p. (IWMI Working Paper 158). doi: / [48] C. B. Dassanayake, S. Rajeewa Hulangamuwa and D.T.D.J. Abeysekara, “Arsenic-related Skin Manifestations in Patients with CKDu in the NCP of Sri Lanka, The Ceylon Medical Journal, Sri Lanka Medical Association, 57 (2012) Supplement 1.

74 JAYASUMANA et al.’s RESULTS [38, 40] SKIP Dermal ManifestationNo. of Patients % of PatientsNo. of Individuals in the Control Group %of Individuals in the Control Group Hyper-pigmentation of palms Hyper-pigmentation of soles Keratosis of palms Keratosis of soles [49] Noble, A.; Amerasinghe, P.; Manthrithilake, H.; Arasalingam, S Review of literature on chronic kidney disease of unknown etiology (CKDu) in Sri Lanka. Colombo, Sri Lanka: International Water Management Institute (IWMI). 41p. (IWMI Working Paper 158). doi: / [50] M.A.C.S Jayasumana., P.A.Paranagama, M.D. Amarasinghe, K.M.R.C. Wijayawardena, K.S. Dahanayake, S.I. Fonseka, K.D.L.M.P. Rajakaruna, A.M.P. Mahamithawa, U.D. Samarasinghe and V.K. Senanayake, “ Possible Link of Chronic Arsenic Toxicity with Chronic Kidney Disease of Unknown Etiology in Sri Lanka.”, J. Natural Science Research, 3 (2013) POOR RSEARCH PRESENTATION: AGE GROUPS OF PATIENTS AND OF CONTROL INDIVIDUALS ARE NOT GIVEN MAKING THE COMPARISON DIFFICULT IN A CHRONIC CASE!

75 DERMATALOGISTS VIEW FROM THE RESULTS OF CKDu PATIENTS DIAGNOSED, THERE ARE NO EVIDENCE TO INDICATE THE PRESENCE OF PIGMENTATION AND KERATOSIS ON THE SKIN. NO EVIDENCE FOR MEES’ LINES THAT ARE COMMON TO PERSONS SUFFERING FROM ARSENIC TOXICITY [49]. [49] Noble, A.; Amerasinghe, P.; Manthrithilake, H.; Arasalingam, S Review of literature on chronic kidney disease of unknown etiology (CKDu) in Sri Lanka. Colombo, Sri Lanka: International Water Management Institute (IWMI). 41p. (IWMI Working Paper 158). doi: / MEES’ LINES

76 MECHANISM OF CELL DEATH PROPOSED BY THE ARSENIC GROUP Binding of Ca 3 (AsO 4 ) 2 to arsenic transporters in the liver and transport to the kidneys. What are these transporters? Metallothinine is to transport Zn 2+ but Cd 2+ interferes strongly. Arsenate or arsenite can not bind to metallothionines. Deposition of Ca 3 (AsO 4 ) 2 crystals in the kidney tissues. Release of arsenate groups. This is an ionic compound. Release should depend on water solubility. This compound is NOT water soluble. Replacement of Phosphate Groups by Arsenate Groups and DNA Damage. THESE ARE ONLY SPECULATIONS WITHOUT ADEQUATE DATA OR LITERATURE SUPPORT! SOME ARE THERMODYNAMIC IMPOSSIBILITIES! Replacement of PO 4 3- by AsO 4 3- in DNA is thermodynamically forbidden and hence can not take place. DNA does not contain arsenic as an element even in “Arsenic Loving Bacteria” found in the California lake.

77 Chronic inhalation exposure to elevated levels of inorganic arsenic in humans is associated with irritation of the skin and mucous membranes (dermatitis, conjunctivitis, pharyngitis, and rhinitis). Chronic oral exposure to elevated levels of inorganic arsenic in humans has resulted in gastrointestinal effects, anemia, peripheral neuropathy, skin lesions, hyperpigmentation, gangrene of the extremities, vascular lesions, and liver or kidney damage. Some recent studies have reported an association between elevated arsenic levels in drinking water and neurocognitive or behavioral test results in school age children. SKIP

78 Ingestion of inorganic arsenic in humans has been associated with an increased risk of nonmelanoma skin cancer and also to an increased risk of bladder, liver and lung cancer. The Reference Dose (RfD) for inorganic arsenic is mg/kg/day (i.e ppm per day, very low, it is such a bad poison!) based on hyperpigmentation, keratosis, and possible vascular complications in humans. The RfD is an estimate (with uncertainty spanning perhaps an order of magnitude) of a daily oral exposure to the human population (including sensitive subgroups) that is likely to be without appreciable risk of deleterious noncancerous effects during a lifetime. SKIP

79 Arsenic-containing pesticides may still be found in some U.S. farms and homes [ATSDR 2007]. Gallium arsenide is used in integral components of discrete microwave devices, lasers, light-emitting diodes, photoelectric chemical cells, and semiconductor devices. Other industrial processes that use arsenic include coal-fired power plants, hardening metal alloys, and purifying industrial gases (removal of sulfur). Inorganic arsenic is found in industry, in copper chromated arsenate treated lumber, and in private well water in some parts of the USA. SKIP

80 Seafood (especially bivalves [clams, oysters, scallops, mussels], crustaceans [crabs, lobsters], and certain cold water and bottom feeding finfish, and seaweed/kelp. The organic forms of arsenic found in seafood (mainly arsenobetaine and arsenocholine, also referred to as “fish arsenic”) are generally considered to be nontoxic, and are excreted in urine within 48 hours of ingestion [ATSDR 2007]. However, inorganic forms of arsenic have been found in some types of seaweed. Recent literature suggests hijiki seaweed has very high levels of inorganic arsenic (MMA) [Rose et al. 2007]. SKIPARSENIC IN FOOD

81 Lead Arsenate Pesticides Both lead and arsenic can be toxic at high concentrations in soils. Lead: Preventing lead exposure in infants and young children is important because lead can affect their developing brain and nervous system. High levels of lead can adversely affect the nervous system and kidneys of adults and children. Lead hydrogen arsenate, also called lead arsenate, acid lead arsenate or LA, chemical formula PbHAsO 4, is an inorganic insecticide used primarily against the potato beetle. SKIP

82 Country2012 As 2 O 3 Production Belgium1,000 T Chile10,000 T China25,000 T Morocco6,000 T Russia1,500 T Other Countries 300 T World Total 44,000 T Much of the production is for the semiconductor industry, Beware of Cell Phone DANGER!! LEDs etc. Arsenobetaine or fish arsenic

83 There is much debate on Napoleon's death, whether he was poisoned by arsenic-tainted wine during his exile on the Island of St. Helena. More than 20 Arsenic Species are present in the Natural Environment and Biological Systems. Although the trivalent arsenic species, such as inorganic arsenite (As III ), monomethylarsonous acid (MMA III ), and dimethylarsinous acid (DMA III ) are highly toxic, arsenobetaine (AsB), the predominant arsenic species present in most crustaceans, is essentially non-toxic [51]. [51] X. Chris Lee, “Arsenic Exposure, Metabolism and Health Effects”, Home Page: Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, University of Alberta INORGANIC ARSENIC SPECIS ARE HIGHLY TOXIC SKIP

84 Biomethylation in the liver is the main mechanism for the metabolism of inorganic arsenic. It involves alternate steps of two-electron reduction followed by oxidative addition of a methyl group. [ 52] ARSENIC METABOLISM [52] SKIP Biomethylation was thought to be a detoxification mechanism of arsenic species. However, these organoarsene compounds are soluble in fat and in blood serum.

85 ARSENIC SPECIES ARE BOTH HEPATOTOXIC AND NEPHROTOXIC [53] [ 53] A.P. Singh, R.K. Goel and T. Kaur, “Mechanisms Pertaining to Arsenic Toxicity”, Toxicology International, (2011). OXIDATIVE STRESS AsO 2 - (aq) + 2H 2 O(l) + O 2 (aq) → AsO 4 3- (aq) + H 2 O 2 (aq) + 2H + (aq) H 2 O 2 → 2OH. Catalyzed by Fe(II), Mn(II) or Cd 2+ ROS ARSENIC SPECIES ARE MORE HEPATOTOXIC THAN NEPHROTOXIC.

86 GLYPHOSATE AND CKDu Glyphosate (N-(phosphonomethyl)glycine) is a broad- spectrum systemic herbicide used to kill weeds, especially annual broadleaf weeds and grasses known to compete with commercial crops grown around the globe. It was discovered to be a herbicide by Monsanto chemist John E. Franz in SKIP Monsanto brought it to market in the 1970s under the trade name "Roundup", and Monsanto's last commercially relevant United States patent expired in In alkaline conditions this exists as an anion.

87 Glyphosate kills plants by interfering with the synthesis of the aromatic amino acids phenylalanine, tyrosine and tryptophan. It does this by inhibiting the enzyme 5-enolpyruvylshikimate-3- phosphate synthatase (EPSPS), which catalyzes the reaction of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form 5- enolpyruvyl-shikimate-3-phosphate (ESP) [55]. [55] H.C. Steinrücken, N. Amrhein, "The herbicide glyphosate is a potent inhibitor of 5- enolpyruvyl-shikimic acid-3-phosphate synthase“, Biochemistry and Biophysics Research Communications, 94 (1980) 1207–1212. [56] Image : GLYPHOSATE ACTION SKIP

88 [57] J.P. Giesy, S. Dobson and K. R. Solomon, "Ecotoxicological Risk Assessment for Roundup® Herbicide“, Reviews of Environmental Contamination and Toxicology,167 (2000) 35–120. [58] Image: BREAKDOWN MECHANISMS OF GLYPHOSATE [57]

89 ENVIRONMENTAL FATE OF GLYPHOSATE [59 ] Glyphosate adsorbs to soil down to 6 inches of soil layer. Readily degraded by microbes to aminomethylphosphonic acid and carbon dioxide. Glyphosate and residues can not contaminate ground water because they do not move that down due to adsorption by surface soil. The Arsenic Group says that these patients drink/drank well waters {92% Dug Wells and 8% Tube Wells!}. Can contaminate with surface waters. The half-life of glyphosate in soil ranges between 2 and 197 days; a typical field half-life of 47 days has been suggested. The median half-life of glyphosate in water varies from a few days to 91 days. [ 59] National Pesticide Information Center

90 COORDINATION CHEMISTRY OF GLYPHOSATE [60-64] With divalent metal ions such as Cu 2+, Zn 2+, Mn 2+, Ca 2+ and Mg 2+, 1:1 adducts have been reported [60]. Further study revealed the possibility for glyphosate to form 1:1 and 2:1 metal complexes with divalent alkaline earth and transition metal cations and with trivalent cations [61]. Bis(glyphosate)metal(III) complexes have been reported. [63-64] Crystal structures were elucidated for 1:1 Ca 2+ and 1:1 Cu(II) complexes and shown them to be polymeric [65]. [60] Daniel Heineke, Jonya J. Franklin and Kinneth N. Raymond, “Coordination Chemistry of Glyphosate, Structural and Spectroscopic Characterization of Bis(glyphosate)metal(III) complexes”, Inorganic c Chemistry, 33 (1994) [61] H.E.L. Madsen, H.-H. Christensen and C. Gottlieb-Peterson, Chimica Scandinavia, Section A, (1978) A 32, 79. [62] R.J. Motekaitis and A.E. Martel, J. Coordination Chemistry, 14 (1985) 139. [63] P.H. Smith and K.N. Raymond, Inorganic Chemistry, 27 (1998) [64] P.R. Rudolf, E.T. Clarke, A.E. Martel and A. Clearfield, Inorganica Chimica Acta, 164 (1989) 58. SKIP

91 GLYPHOSATE AND CADMIUM Glyphosate reacts with Cd 2+ forming water soluble complexes [65]. Cd 2+ adsorbed on soil binds glyphosate through complexation [65]. [65] D.M. Zhou, Y.J. Wang, L. Chang, X.S. Hao and X.S. Luo, “Adsorption and cosorption of cadmium glyphosate on two soils with different characteristics, Chemosphere”, 57 (2004) Solid phase Cd 9 (Glyphosate) 6 (H 2 O) 10.6H 2 O crystals have been synthesized and their crystal structure has been determined. [66] [66] Medeleine Ranstedt, Caroline Norgren, Julia Sheals, Dan Bostöm, Steffan Sjöberg and Per Persson, “Thermodynsmic and Spectroscopic Studies of Cadmium(II)- N(Phosphomethyl)glycene Complexes”, Inorganica Chimica Acta, 357 (2004)

92 GLYPHOSATE AND ARSENIC THERE IS AN OPEN ACESS JOURNAL PAPER. [67] Channa Jayasumana, Sarath Gunathilaka and Priyantha Senanayake, “Glyphosate, Hard Water and Nephrotoxic Metals: Are They the Culprits Behind the Epidemic of Chronic Kidney Disease of Sri Lanka?”, International Journal of Environmental Research and Public Health, 11 (2014) This Glyphosate scandal came out due to THIS paper [67]!

93 They talk about glyphosate-divalent and trivalent metal complexes. The reference given is a DFT Computer Simulation Study. Only theoretical possibilities [68]. IMPORTANT POINT IS THAT “None of these complexes have arsenic even as arsenate. I have the paper. You know that arsenic does not form complexes by behaving as a metal ion. Replacement of phosphate by arsenate is not possible either! [68] Caetano, M.; Ramalho, T.; Botrel, D.; da Cunha, E.; de Mello, W., “Understanding the inactivation process of organophosphorus herbicides: A DFT study of glyphosate metallic complexes with Zn 2+, Ca 2+, Mg 2+, Cu 2+, Co 3+, Fe 3+, Cr 3+, and Al 3+.”, International Journal of Quantum Chemistry, 112 (2012) 2752–2762.

94 I have requested Dr. Udayanga Ranatunga to do the DFT study on possible complexes of metal ions, arsenic and glyphosate. Even if theoretically predicted, that does not mean that the compounds are practically synthesizable.

95 Other reference quoted for glyphosate-arsenic polymeric complex is 20for%20Dogs%20Booklet%20PD pdf 20for%20Dogs%20Booklet%20PD pdf THIS IS A BUSINESS REPORT ON ORGANIC DOG FOODS. AN ADVERTISEMENT! The polymeric complex in [67] is not contained in this pdf as well! SKIP

96 AS A CHEMIST’s POINT OF VIEW! At high pH, Glyphosate exists as anion with carboxylate and phosphate groups. Arsenate and Arsenite are also an anions. Naturally, they have no affinity to each other. In the high acidic environment of the liver, glyphosate carboxyl group and the phosphate group can get protonated. Arsenate or arsenite also get protonated. Some hydrogen bonding between them are possible in the liver. However, blood has almost neutral pH [pH = 7.4, tightly regulated between 7.35 and 7.45]. These hydrogen bonds are not stable then. So, I DO NOT understand how glyphosate is carryings arsenic species from liver to kidneys. Glyphosate can carry Cd 2+ by forming coordination complexes with metallothionines. However, glyphosate can make better coordination complexes with other metal ions such as Zn 2+, Mn 2+, Co 3+, Fe(II) and Fe(III) which are present in relatively higher concentrations when compared to that of Cd 2+ in human body. Therefore, glyphosate can induce essential metal ion deficiencies. It can be toxic.

97 IONIC STRENGTH THEORY Theory of Dharma-wardana et al. [68]. Increased ionicity due to all ionic species present in drinking water collectively responsible for denaturing of proteins of the kidneys through a Hofmeister- type mechanism is predicted. Hofmeister series: Cations: NH 4 + > K + > Na + > Mg 2+ > Ca 2+ Anions F - > H 2 PO 4 - > or = SO 4 2- > HCO 3 - > Cl - > NO 3 - Matches very well with existing data on drinking water quality. Fertilizer runoff through accelerated Mahaweli Programme has increased N- and P-containing ions. Increased drought conditions prevailing in these areas increase the concentration of everything dissolved in drinking water.. Dehydration increases the ionic strength in blood. Alcohol consumption can aggravate the problem. This could be substantiated with increased concentrations of nephrotoxic metal ions in blood. [68] M.W.L. Dharma-wardhana, Sarath L. Amarasiri, Nande Dharmawardana and C.R.Panabokke, “Chronic Kidney Disease of Unknown aetiology and ground-water ionicity, study based on Sri Lanka”, Environmental Geochemistry and Health(2014);

98 BETTER AVOID SYNTHETIC FERTILIZERS AND AGROCHEMICALS, ANYWAY! Whether they contribute to CKDu or not, they are poisons. It is indeed better to avoid them, if possible. Organic farming is good but the yields are less. Better to cultivate increased amounts. Cost of organically grown food stuffs are high though there is no certificate that these were grown without agrochemicals. Weed picking by hands as the way it was done in the past is NOT practical now. Feeding 21 million population in this country is also a prime concern or they will die due to starvation. If possible, better to grow vegetables and fruits in your own garden!

99 RENAL DIALYSIS A Patient Receiving Dialysis A process for removing waste and excess water from the blood. Used as an artificial replacement for lost kidney function in people with renal failure. The kidneys have important roles in maintaining health. When healthy, the kidneys maintain the body's internal equilibrium of water and minerals (sodium, potassium, chloride, calcium, phosphorus, magnesium, sulfate). The kidneys also function as a part of the endocrine system, producing erythropoietin and calcitriol. l Erythropoietin is involved in the production of red blood cells and calcitriol plays a role in bone formation. Dialysis is an imperfect treatment to replace kidney function because it does not correct the compromised endocrine functions of the kidney. Diffusion through concentration gradient and ultrafiltration are the principles involved. Semi - permeable membrane with different pore sizes is used.

100 ROLE OF NANOTECHNOLOGY IN RENAL SUBSTITUTION THERAPY Two Membranes called G Membrane and T Membrane. G membrane resembles the function of the glomerular basement membrane. G Membrane is a fully porous membrane which removes solute up to the molecular weight of albumin. Ultra-filtrate passing through the G Membrane passes through the T Membrane. T Membrane is meant to emulate the tubular membrane in the renal tubules. T membrane will reabsorb all of those substances we want to retain, some sodium, some potassium, calcium, a little bit of phosphorous, and so on. T Membrane is Nano-technologically Developed Membrane with very small pores ~ 1 nm length and the distance between pores is 1 – 5 nm. Dean G. Johnson , Tejas S. Khire, Yekaterina L. Lyubarskaya, Karl J.P. Smith, Jon- Paul S. DesOrmeaux, Jeremy G. Taylor, Thomas R. Gaborski, Alexander A. Shestopalov, Christopher C. Striemer, James L. McGrath, ‘Ultrathin Silicon Membranes for Wearable Dialysis”, Advances in Chronic Kidney Disease, 20 (2013)

101 [70] tethered-dialysis-machine DEVELOPING IMPLANTABLE ARTIFICIAL KIDNEYS

102 REGENARATIVE MEDICINE Current trend in Medical Research as an Alternative to Transplants. Recently, some heart cells of a monkey have been removed and human stem cells injected. After two weeks, monkey’s heart began to beat by regenerating it with human stem cells [71, 72]. [71] “Stem cell therapy regenerates heart muscle damaged from heart attacks in primates”, Science Daily, April 30, [72] Charles Murry et al., "Human embryonic-stem cell derived cardiomyocytes regenerate non-human primate hearts." Nature, April 30, 2014, Advanced Online Edition.

103 This image shows an implanted graft of cardiac cells derived from human stem cells (green) meshed and beat with primates' heart cells (red).Credit: Murry Lab/University of Washington MONKEY HEART REPAIRED [73] Murry et al., April 30, 2014, Advanced Online Edition of the Journal Nature in a Paper Titled, "Human Embryonic-Stem Cell Derived Cardiomyocytes Regenerate Non-human Primate Hearts."

104 Charles E. Murry Joint Professor of Pathology, Cardiology, and Bioengineering Phone: (206) South Lake Union campus: Office: 850 Republican St. Rm 453 RESEARCH INTERESTS Myocardial infarction Stem cells Tissue repair and regeneration Tissue engineering Angiogenesis SKIP

105 STEM CELL-BASED KIDNEY REPAIR The Harvard Stem Cell Institute is developing new therapies to repair kidney damage, reducing the need for dialysis and transplantation. The Harvard Stem Cell Institute (HSCI) Kidney Group has short, medium, and long-term strategies to develop new therapies for diabetes-related kidney damage (diabetic nephropathy). This multi-pronged approach aims to capitalize on promising translational achievements in the near future, while pursing potential drugs and the ultimate goal of creating an entirely artificial kidney using stem cells. HSCI Kidney Program Leader is Professor Benjamin Humphreys, MD, PhD.

106 STEM CELL-BASED KIDNEY REGENERATION [74] S. Yokote and T. Yokoo, “Stem cells in kidney regeneration”, Current Medicinal Chemistry, 19 (2012) [75] Brian A. Yeagy & Stephanie Cherqui, “Kidney repair and stem cells: a complex and controversial process”, Review, Pediatric Nephrology DOI /s x, Published Online 19 February, Kidney grown from stem cells by Australian scientists Australian scientists grow world's first kidney from stem cells in a breakthrough that could alleviate the demand for organ transplants, Journal Nature Cell Biology, "This is the first time anybody has managed to direct stem cells into the functional units of a kidney," Professor Brandon Wainwright, from the University of Queensland, told The Telegraph. [76] Kidney disease: how could stem cells help? Last updated: 12 Aug 2013 AND 100s MORE ARTICLES! THIS MIGHT BE THE WAY OUT FOR TREATING POOR PATIENTS!

107 SUMMARY CKDu is a serious epidemic in Sri Lanka. F - Hypothesis: HF can damage liver. Fluorosis prohibits bone’s detoxification mechanism. F - can cause kidney damage. Fluoroaluminate complexes are toxic. Reports on Renal Failure are available. Cd 2+ is present in TSP fertilizers and in some weed killers. Cd 2+ is a well known nephrotoxic ion. Cd 2+ is present in cigarette smokes. Arsenic species are poisons. Speculations are not scientific research. Metal ion-arsenate-glyphosate polymers have not been synthesized. No evidence to suggest connections between arsenic species and glyphosate being responsible for CKDu. This does not mean that they are not toxic. It is very likely that increased concentrations of all the ions in the blood is responsible for the denaturing of glomeruli proteins. Ions at the top of Hofmeister series are more dangerous than other ions. Research on Implantable Kidneys is important. Regenerative Medicine may be a possible way to treat CKDu Patients. However, it is still at the research level.


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