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Kidneys have been recognized as an easy target for the toxicity of a number of chemicals and paharmacologically active substances. Over the last few decades it has become increasingly evident that kidneys may be exposed to harm by a variety of substances, including environmental, industrial and naturally occurring chemicals as well as an array of medicines which are used for different ailments. The dose of the chemical may be small and for a prolonged period leading to chronic nephrotoxicity, or it may be heavy and for a short period leading to acute nephrotoxicity(WHO 1991). Small size of the kidneys relative to their blood supply and diversity of their functions makes them easy targets not only for the harmful effects of toxic chemicals, but also for the pharmacologically active substances.
Most of the cases of end-stage renal disease brought to nephrology and dialysis units may be the consequence of chemical exposure(WHO 1991). Epidemiological evidence indicates that nephrotoxicity leading to acute and/or chronic renal failure represents a substantial financial burden to society(Nuyts, Elseviers et al. 1989). If we need to avoid this burden, we must assume some prophylactic measures to avoid injury to the kidneys in the first place. This may be achieved by avoiding exposure to toxic substances. However, if the exposure is unavoidable, or we have already been exposed to the culprit agent, then we need to use something which can protect us from the harmful effects of the toxic substance. The present study focuses at finding the ameliorating effects of ginkgo biloba extract on lead induced nephrotoxicity.
Among the pharmacologically active chemicals responsible for nephrotoxicity are the non-steroidal anti-inflamatory drugs (NSAIDS), antibiotics like aminoglycosides, cephaloceporins, amphotericin-B and tetracyclines, penicillamine, lithium, urograhic contrast media, anti- cancer drugs like cisplatin, adriamycin, and immune-suppressants like cyclosporin A. Other chemicals concerned with nephrotoxicity include ethylene glycol, organic chemicals and solvents like volatile hydrocarbons, chloroform, halogenated alkenes, hydrocarbons etc., silicon and mycotoxins.(WHO 1991)
Heavy metals are also among the important offending agents capable of producing nephrotoxicity. People continue to be exposed to heavy metals in the environment. In many parts of the world, food and water are contaminated with heavy metals. Metal leaches from cooking utensils and is added to our food(Kumar, Srivastava et al. 1994). Modern era of industries and lot of mining of heavy metals has resulted in a great deal of environmental pollution, which is responsible for many occupational diseases due to various chemical toxins(Klaassen 2001). Some of the most important sources of heavy metal poisoning in this modern era are from burning of fossil fuels containing heavy metals, tetra-ethyl lead addition to gasoline, and increased industrial uses of metals(Klaassen 2001). Toxic metals are widely distributed in our environment, and humans are exposed to the toxic effects of these metals from water, soil, air and food contamination(Ercal, Gurer-Orhan et al. 2001).
Damage to the kidneys is one of the primary toxic effects of metals, and heavy metal induced nephrotoxicity has been extensively studied. Studies show that low dose mercury exposure can result in degenerative changes in mice kidney(Afonne, Orisakwe et al. 2002). Nephritic syndrome as well as membranous nephritis may occur in humans who are exposed to mercury and its salts for a long time.(Aymaz, Gross et al. 2001; Li, Zhang et al. 2010) Another study has shown glomerular and tubular alterations in workers who are chronically exposed to mercury, particularly its fumes.(Roels, Hoet et al. 1999) Similarly, cadmium toxicity in sufficient cumulative dosage leads to the development of Fanconi syndrome which is characterized by generalized proximal tubular reabsorptive defect related to both ATP production and Na-K-ATPase activity.(Gonick 2008) Another study has shown that histological examination of kidneys of rats treated with cisplatin revealed significant proximal tubular lesions varying from minimal changes to severe toxicity.(Bompart and Orfila 1990) When more than one toxic agents are involved, the damaging effects are exaggerated. For example the histopathologic changes in kidneys were aggravated when lead was administered concomitantly with cadmium.(Prasada Rao, Jordan et al. 1989)
Lead is a ubiquitous metal present everywhere on the earth. It is found in all soils, rivers, seawater, even in sea spray and dust in the air.(Dharmananda 2001) Lead toxicity may affect virtually every organ of the body, but the effects on kidneys are the most insidious. Acute nephrotoxicity may result in cytosolic and nuclear inclusion bodies which can be seen microscopically. The intracellular lead is associated with high affinity proteins and may combine with metallothionein. Acute exposure is associated with proximal tubular dysfunction with Fanconi type syndrome, and alterations in the structure of mitochondria(Nolan and Shaikh 1992).
Metals and other substances that are considered to be toxic to humans produce their toxic effects by virtue of producing oxidative stress. This involves the production of reactive oxygen species which can overwhelm cell's intrinsic antioxidant defense mechanisms. Cells which undergo oxidative stress display dysfunctions of lipid, proteins and DNA(Ercal, Gurer-Orhan et al. 2001). Oxidative stress is involved in acute renal toxicities induced by substances like lead, gentamicin, cisplatin, glycerol, cyclosporine and others(Baliga, Ueda et al. 1999).
Ginkgo biloba extract is well known for its free radical scavengering ability. Many studies have shown the anti-oxidant effect of ginkgo biloba extract. It has been proved to be effective in preventing ethanol induced gastric ulcer, which is most probably due to its inhibitory effect on lipid peroxidation and cell apoptosis(Chen, Liang et al. 2005). Zhu and others have conducted a study in which they proved protective anti-oxidant effect of ginkgo biloba extract on early diabetic nephropathy(Zhu, Shi et al. 2005). In another study, it has shown a delaying effect on the development of glomerulosclerosis in diabetic nephropathy(Wang, Yin et al. 2006; Lu, Yin et al. 2007).
Present study deals with the ameliorating effect of ginkgo biloba extract on lead-induced nephrotoxicity. Until now, both the anti-oxidant effects of ginkgo biloba extract and lead-induced nephrotoxicity have been studied separately several times, but this study is unique in a sense that this combination has never been studied before to the best of our knowledge.
REVIEW OF LITERATURE
Lead has been known as a poison since ancient times. It is classified as one of the most serious threats to human wellbeing particularly in developing countries like ours. Greek poet and philosopher Nikander was the first who discovered the clinical syndrome of lead poisoning in 200 BCE. The high lead concentration in Roman wine was claimed to be one of the important factors which contributed to the downfall of Roman Empire(Gilfillan 1965). Portuguese wine was once used to be transported with lead bars submerged in it to increase its flavor and durability. The use of this was responsible for several incidences of lead poisoning among the English aristocracy(Kathuria, Fasn et al. 2003).
Although use of lead has been known since ancient times, its massive consumption came into existence in the 20th century. More than 300 million tons of lead has been mined out between 1920 and 2000, and has been added to the atomosphere due to combustion activities and applied to the surfaces in the form of leaded paints.
Lead is the heaviest of all non-radioactive metals which are found in significant quantities in earth's crust(Dharmananda 2001). It is bluish gray in color and has a low melting point. It is usually found combined with other elements to form lead compounds.
Sources of lead
Lead compounds are used as a pigment in paints, dyes, and ceramic glazes and in caulk. Lead, being a ubiquitous metal, is found everywhere. Even natural soil is not totally lead free(Dharmananda 2001). Soil is a major source of lead, and blood lead levels are higher in children living in areas having lead contaminated soil(Ranft, Delschen et al. 2008).
The most obvious and hazardous spread of lead in the environment has been due to the use of leaded gasoline, lead being in the form of tetraethyle and tetramethyle lead, starting from 1923, that has caused release of billions of tons of lead into the environment from vehicle exhausts. Lead was used in gasoline to increase octane rating and to act as anti-knocking agent. In most of the developed countries, use of lead has been phased out starting from 1980, and it was banned for use in vehicles in U.S.A starting from January 1991. In Pakistan, however, lead has been phased out from gasoline staring from October 2001 and was completed in July 2003(Rabinowitz and Needleman 1982). This has led to decrease in blood levels of lead in Pakistani children, but not to the desired level. It will take time to get the desired level because lead once emitted in the environment is not destroyed and remains in soil for long times. Moreover, it has been observed that even when lead additives are removed from gasoline, smaller amounts of lead continue to be emitted from automobiles, trucks, and other vehicles. In addition to some natural lead in gasoline, the gradual loss of fine particles of metal, rubber, and other components that contain lead contribute to pollution, particularly in urban areas with high traffic density(Dharmananda 2001).
Lead-glazed ceramics are also important source of lead. Lead exposure due to use of lead-glazed ceramics can result in elevated blood lead levels in people of relatively high socio-economic status(Hernandez Avila, Romieu et al. 1991). Acidic foods and beverages like tomato juice, fruit juice, carbonated drinks, cider, michels etc, that are contained in improperly glazed containers, can absorb lead and lead to subsequent lead poisoning in the consumers(Klaassen 2001).
An earlier nationwide survey in UK has shown that 10% of the urban population was exposed to lead levels in excess of 2,000 µg /g in house-hold dust. Approximately 50% of lead intake in 2 year old children was from dust ingested as a result of hand-to-mouth activity. Important sources of dust lead included lead-based paint, road dust and soils. It appears that although lead of minewaste origin may be present at elevated levels in dusts and soils, it does not necessarily contribute to elevated blood lead levels when the lead is present in relatively insoluble form.(Thornton, Watt et al. 1994) The dust in houses which are close to roads may be contaminated with metals. Metals in houses may travel from the roads, through the windows and balconies. The houses where windows and doors are kept closed, and there is regular sweeping and vacuum cleaning, levels of contaminants are low. The color of the wall paint used in the house may be another factor influencing the contamination levels(Tong and Lam 2000). . While adults are exposed particularly due to their profession, children are usually affected by their exposure to the lead dust and paint flakes from old houses, especially during renovation(Bellinger, Hu et al. 2005).
A study conducted in Karachi showed that 80% of the children had a blood lead concentration of more than 10µg/dl. Father's education and his exposure to lead at his workplace, the child's habit of eating food from street vendors, the child's hand-to-mouth activity, the use of metal cooking utensils and living in "west-open" houses were identified as independent factors associated with elevated blood lead concentrations in children in Karachi(Rahbar, White et al. 2002).
Lead used in ammunition is a significant non-battery end-use and has been quite a common source of lead in recent years(Lewis, Sjostrom et al. 2010). Shooting range soils have been investigated for lead contamination from Pb bullets and the soil has been found to be heavily contaminated(Hardison, Ma et al. 2004). Spitz et al have described a case of bullet retention in which symptoms of lead toxicity in the form of choreoathetosis were seen after 7 years of initial exposure(Spitz, Lucato et al. 2008). Firearm projectiles have been found to be rare cause of lead toxicity. Spitz et al have described a case of bullet retention in which symptoms of lead toxicity in the form of choreoathetosis were seen after 7 years of initial exposure(Spitz, Lucato et al. 2008).
Lead is also an important component of batteries and the workers in battery manufacturing factories and repair shops are easy victims of lead exposure(Kasuba, Rozgaj et al. 2010). People living near such plants are also at risk of getting exposed to high levels of lead(Dartey, Adimado et al. 2010). Children of the workers in battery manufacturing factories and workshops are particularly liable to get exposed to the high levels of lead which their fathers bring home in their clothes(Morton, Saah et al. 1982). Sathye et al have reported a case of a child who suffered from lead poisoning, the source of lead being the lead dust on clothes of his father who worked in a brass industry where brass alloys contained lead in substantial amounts (Sathaye and Javadekar 2000).
Kohl and surma are eye cosmetics which are commonly used in India, Pakistan, Near East, Middle East and North Africa. Their use has even spread to the western countries. The main ingredient of these cosmetics is lead sulphide, and their use is associated with increased blood lead levels particularly in children, who are the main users of these cosmetics(De Caluwe 2009). However, according to another study, "relation between Kohl and toxicity or increased blood lead concentration upon its application to eyes as reported elsewhere is likely to be more of theoretical nature rather than a practical health hazard"(Mahmood, Zoha et al. 2009).
Kushta is a traditional medicine used as an aphrodisiac in the indo-pak subcontinent, and contains harmful metals like mercury, lead and antimony mixed with certain traditional herbs. Consumption of such medicines is also associated with the risk of getting exposed to lead and other metal poisoning(Haq and Asghar 1989).
Absorption of lead
Elemental lead and non-organic lead compounds are usually absorbed either through ingestion or inhalation. Organic lead like tetraethyl lead as well as inorganic lead oxide can be absorbed through skin as well (Filon, Boeniger et al. 2006). Rate of absorption of lead is more in children as compared to adults. When ingested, lead is absorbed primarily in the duodenum. Zinc, iron and calcium are competitors of lead for the shared receptors for absorption in the duodenal epithelium. Thus the absorption of lead is reduced in the presence of these substances. Iron deficiency and periods of rapid growth in children are associated with greater absorption of lead (Barton, Conrad et al. 1978; Conrad and Barton 1978) and children who have iron deficiency anemia are more prone to have subsequent lead toxicity when exposed(Wright, Tsaih et al. 2003). Low calorie and high fat intake is also associated with enhanced absorption of lead(Kathuria, Fasn et al. 2003). Dietary phosphate deficiency leads to increased absorption of lead in the intestine and hence increased body burden of lead in bones(Barton and Conrad 1981). Rate of absorption of lead particles also depends upon the size of the particles. Smaller particles that are dispersed in the air and are therefore respireable, are absorbed 10 times faster as compared to larger ingestable particles(Hodgkins, Robins et al. 1991). The respirable particles are more numerous in industrial areas, and people, particularly children living nearby, are more prone to inhale these particles and get affected by the harmful effects of lead(Zelikoff, Parsons et al. 1993). Workers of a lead factory were investigated for blood lead levels. It was found that those who were exposed to lead fumes, had higher blood lead levels than those who were simply involved in handling lead materials(Khan, Malik et al. 1994).
Distribution and excretion of lead
Once absorbed, about 99% of lead binds to hemoglobin in erythrocytes. Only about 1% remains free in serum. Inorganic lead is first distributed to soft tissues like the tubular epithelium of kidneys and liver. Eventually however, most of lead is redistributed and deposited in bone, teeth and hair. Bone is the major store house of lead and contains about 95% of body lead burden. Small amounts of inorganic lead are also stored in gray mater and basal ganglia of brain(Klaassen 2001).
Factors which govern the distribution of lead are similar to those for calcium. High phosphate intake is associated with increased deposition of lead in bone and low concentration in soft tissue. Conversely, low intake of phosphate results in mobilization of lead from bone to the soft tissues. Lead is deposited in bone as tertiary lead phosphate, which does not contribute to toxicity. High calcium intake in the presence of low phosphate results in mobilization of lead from bone into soft tissues. Vitamin D tends to enhance the deposition of lead into bone in the presence of sufficient phosphate. However, where there is deficiency of phosphate, calcium competes for the available phosphate, and lead is mobilized to the soft tissues. Parathyroid hormone leads to mobilization of lead into soft tissues, and enhances its excretion in urine(Klaassen 2001). Deficiency of micronutrients like zinc may result in increased retension of lead in tissues(Bushnell and Levin 1983).
The excretion of lead is limited, therefore it tends to accumulate in the body, particularly in bones(Conrad and Barton 1978). In experimental animals the route of excretion is mainly bile and hence the feces(Klaassen and Shoeman 1974). In humans however, urinary excretion is more important as compared to bile(Kehoe 1987). Since most of lead in circulation is in erythrocytes, only small quantities are filtered in urine. Other routes of excretion include sweat and milk, and placental transfer is also observed(Klaassen 2001).
The biological half-life of lead in erythrocytes is about 35 days. In soft tissues like liver, kidney and brain it is about 40 days, whereas in bone it ranges between 20 to 30 years(Ellenhorn and Barceloux 1988).
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