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Author Topic: Toxic effects of Chloroquine on the liver and kidney  (Read 10321 times)
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Francis Umeoguaju
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« on: September 29, 2011, 09:15:54 am »


Approximately 50-70% of chloroquine in plasma is bound to plasma proteins. The tissues exhibit particularly high binding to chloroquine especially those containing melanin, for example the retina. Significant binding also occurs in the liver, kidney and spleen. Chloroquine (Resochin,Avloclor, Nivaquine, Arelen) C18H26CIN3 7- Chloro -4- (4’- diethlyamino-1’-methylamino0 quinoline.

Chloroquine is a white powder with a bitter taste, prepared by chemical synthesis .it is available as sulphate and phosphate salts. The sulphate (1 in 3) and the phosphate (1 in 4) are soluble in water. Chloroquine is best known as an antimalarial agent but it is also used in the treatment of rheumatoid arthritis. Chloroquine is effective against the erythrocytic stages of all four plasmodium species which cause human malaria with the exception of matured plasmodium falciparum gametocytes. The exact mechanisms of the action of chloroquine against malaria parasites are not fully understood.
Parasitized red cells accumulate approximately 100-600 times as much chloroquine. The concentration of chloroquine in malaria parasite requires energy and is thought to require a membrane. There are three theories on the way state as that chloroquine, being a basic compound, is protonated in the lysosomes thus raising lysosomal pH. This effect may raise the intralysosomal pH above a critical level all bring about loss lysosomal function. This would reduce the parasite’s digestion of heamoglobin, and thus prevent its growth.

Chloroquine intercalates into double stranded DNA and inhibits both DNA and  RNA synthesis. The intercalation theory suggests that chloroquine may be bound with increased affinity by certain parts of the genome and be toxic to the malaria parasite by selective accumulation in specific genes, inhibiting their expression. The ferriprotorphyrin IX (FP) which inhibits sequestration of FP into malaria pigment. This could impair heamoglobin degradation and permits damage to the food vacuole sufficient to discharge its Ph gradient.antimalaria activity is possessed equally by the enantiomers of chloroquine and the main metabolite desethlychloroquine is also active against chloroquine- sensitive Plasmodiam. Chloroquine also has anti- inflammatory activity. The concentrations of chloroquine or hydrochloroquine found in serum in the treatment of rheumatoid disease raise the pH of acid vesicles in mammalian cell within 3-5 min in vitro. This and the observation that the view that chloroquine and hydoxychloroquine act in the rheumatic disease by raising the pH of acid vesicles. Effects of raised vesicle pH include inhibition lysosomal proteolysis, interference with the targeting of acid proteases and inhibition of cellular maturation .raise pH in the macrophage vesicle can interfere with antigen processing. This is thought to be the explanation for the impaired antibody response to pre-exposure to human diploid cell rabies vaccine found in individual receiving concurrent chemoprophyaxis with chloroquine. In addition, chloroquine inhibits the chemotactic response of mononuclear cells and suppresses lymphocytes transformation.
It is therefore very important to study the effects of chloroquine on the liver, kidney and spleen.

Liver
The liver is a vital organ present in vertebrates and some other animals. It has a wide range of functions, including detoxification, protein synthesis, and production of biochemicals necessary for digestion. The liver is necessary for survival; there is currently no way to compensate for the absence of liver function long term, although liver dialysis can be used short term.
This organ plays a major role in metabolism and has a number of functions in the body, including glycogen storage, decomposition of red blood cells, plasma protein synthesis, hormone production, and detoxification. It lies below the diaphragm in the abdominal-pelvic region of the abdomen. It produces bile, an alkaline compound which aids in digestion via the emulsification of lipids. The liver's highly specialized tissues regulate a wide variety of high-volume biochemical reactions, including the synthesis and breakdown of small and complex molecules, many of which are necessary for normal vital functions.[2]
Medical terms related to the liver often start in hepato- or hepatic from the Greek word for liver, h?par (Huh?).

Anatomy of the liver
The liver is a reddish brown organ with four lobes of unequal size and shape. A human liver normally weighs 1.44–1.66 kg (3.2–3.7 lb),[3] and is a soft, pinkish-brown, triangular organ. It is both the largest internal organ (the skin being the largest organ overall) and the largest gland in the human body. It is located in the right upper quadrant of the abdominal cavity, resting just below the diaphragm. The liver lies to the right of the stomach and overlies the gallbladder. It is connected to two large blood vessels, one called the hepatic artery and one called the portal vein. The hepatic artery carries blood from the aorta, whereas the portal vein carries blood containing digested nutrients from the entire gastrointestinal tract and also from the spleen and pancreas. These blood vessels subdivide into capillaries, which then lead to a lobule. Each lobule is made up of millions of hepatic cells which are the basic metabolic cells.

Kidney
The kidneys, organs with several functions, serve essential regulatory roles in most animals, including vertebrates and some invertebrates. They are essential in the urinary system and also serve homeostatic functions such as the regulation of electrolytes, maintenance of acid-base balance, and regulation of blood pressure (via maintaining salt and water balance). They serve the body as a natural filter of the blood, and remove wastes which are diverted to the urinary bladder. In producing urine, the kidneys excrete wastes such as urea and ammonium; the kidneys also are responsible for the reabsorption of water, glucose, and amino acids. The kidneys also produce hormones including calcitriol, erythropoietin, and the enzyme renin.
Located at the rear of the abdominal cavity in the retroperitoneum, the kidneys receive blood from the paired renal arteries, and drain into the paired renal veins. Each kidney excretes urine into a ureter, itself a paired structure that empties into the urinary bladder.

Histology
Histology (compound of the Greek words: Huh?? "tissue", and -?Huh? -logia) is the study of the microscopic anatomy of cells and tissues of plants and animals. It is performed by examining a thin slice (section) of tissue under a light microscope or electron microscope. The ability to visualize or differentially identify microscopic structures is frequently enhanced through the use of histological stains. Histology is an essential tool of biology and medicine.

Stereology
Stereology (from Greek stereos = solid) was originally defined as "the spatial interpretation of sections". It is an interdisciplinary field that is largely concerned with the three-dimensional interpretation of planar sections of materials or tissues. It provides practical techniques for extracting quantitative information about a three-dimensional material from measurements made on two-dimensional planar sections of the material (see examples below). Stereology is a method that utilizes random, systematic sampling to provide unbiased and quantitative data. It is an important and efficient tool in many applications of microscopy (such as petrography, materials science, and biosciences including histology, bone and neuroanatomy). Stereology is a developing science with many important innovations being developed mainly in Europe. New innovations such as the proportionator continue to make important improvements in the efficiency of stereological procedures.
In addition to two-dimensional plane sections, stereology also applies to three-dimensional slabs (e.g. 3D microscope images), one-dimensional probes (e.g. needle biopsy), projected images, and other kinds of 'sampling'. It is especially useful when the sample has a lower spatial dimension than the original material. Hence, stereology is often defined as the science of estimating higher dimensional information from lower dimensional samples.

Stereology is based on fundamental principles of geometry (e.g. Cavalieri's principle) and statistics (mainly survey sampling inference). It is a completely different approach from computed tomography.
Classical examples
Classical applications of stereology include:
•   calculating the volume fraction of quartz in a rock by measuring the area fraction of quartz on a typical polished plane section of rock ("Delesse principle");
•   calculating the surface area of pores per unit volume in a ceramic, by measuring the length of profiles of pore boundary per unit area on a typical plane section of the ceramic (multiplied by 4 / ?);
•   calculating the total length of capillaries per unit volume of a biological tissue, by counting the number of profiles of capillaries per unit area on a typical histological section of the tissue (multiplied by 2).
The popular science fact that the human lungs have a surface area (of gas exchange surface) equivalent to a tennis court (75 square meters), was obtained by stereological methods. Similarly for statements about the total length of nerve fibres, capillaries etc. in the human body.
 
Effects Of Chloroquine On The Liver And Kidney
In an experiment using Sprague Dawley rats as a mammal case study. Ten rats were exposed to chloroquine once a day for three days. The treated rats received the 0.125ml/100g body weight of chloroquine phosphate injection intraperitineally. Control rats received the same amount of normal saline intraperitoneally.
The histology of the chloroquine treated  liver and kidney was also compared with controls. It was observed that chloroquine caused malformation in these tissues.
Stereologically, the parameters measured for the liver and kidney was also compared with controls. The estimated absolute volume V =Vv (structure) x v (ref) of the blood vessels and renal corpuscles of the fractions were determined and compared. For the liver chloroquine caused a reduction in the absolute volume of the blood vessels when compared with controls. For the kidney, chloroquine caused a reduction in the absolute volume of the kidney renal corpuscles when compared with control rats.

EXPERIMENTAL PROCEDURE
The twenty male Sprague- Dawley rats were collected from the Animal House of the College of Medicine University of Lagos Akoka, Lagos State.
They weighed between 100-150g and were fed with the normal rat feed from Pfizer PLC Ikeja Lagos. Weight of animals was taken twice daily throughout the duration of the experiment. Ten male rats were used as controls. The remaining ten male rats were labelled by ear puncture as treated rats and kept in cages. Administration of drug was 0.125ml of chloroquine /100g body weight for 3 days intrapertoneally. Chloroquine phosphate injection was obtained from the community pharmacy of the Lagos university teaching hospital (40mg/ml chloroquine phosphate injection).The control received the same quantity of normal saline.

Histological Analysis
The twenty male rats were sacrificed as discussed earlier after treatment with the chloroquine phosphate injection .The liver and kidney were removed and fixed in Bouin’s fluid. Each specimen of equal length was cut transversely and longitudinally into serial cross sections of 3µm normal thickness with Reichert Jung Supercut Mictrotome for control and treated rats. The tissues were sectioned using tissues preparation tissues method with heamatoxylin and eosin stains and examined the light binocular microscope at a magnification of 100 and 400 respectively.

Stereological Analysis
The vertical sections of the histochemical preparation of stratum length of 0.5cm from 10 controls and 10 treated rats liver and kidney were made at a final print magnification of 100 and 400 respectively.
5 slides will be obtained from the control and 5 slides from the treated rats.
For each of the fractions, the NT/A ? number of test point per unit area of blood vessels and renal corpuscles of the fractions were estimated by point counting method using the forbidden rule Hans Gundersen,1977) which states that any structure that touches the forbidden line must not be counted. The reference volume V(ref) of blood vessels and renal corpuscles  were estimated by point counting (Wiebel, 1979, Gundersen et al, 1988).
At Magnification (M) = 100 final magnification using a Square Grid of test point diameter (d) =1.2cm apart. The test system used in the light microscopic analysis within a square frame measuring 20cm x 20cm onto which microscopic image was projected using a wild Leitz microscope equipped with a mirror at a magnification of 25 on a white screen.

Estimated V(ref) = (stratum length) x d2 x mean NT/A (structure).
                                                                   M2                
d= diameter of test grid
M=magnification of projection
The relevant volume density of blood vessels and renal corpuscles of the fractions Vv (structure) were estimated on the same section at a final magnification of 100. Each field was projected onto a test system consisting of three sets of points with numerical densities in the ratio 1:4:16. The corresponding distance between the test points of each set were 4.8, 2.4 and 1.2cm respectively.
The criteria for test point design and allocation were based on efficiency considerations; thus approximately the same number of test points (which does not need to exceed 200) should be in each structure within each organ (Gundersen and Jensen, 1987; Gundersen et al ., 1988; Cruz Orive and Wiebel; 1990). The required volume density of the fractions were estimated as follows:

Estimated Vv(structure) = NvR  x  NT/A( structure)
Vv = volume density NvR = numerical density ratio
Finally, the absolute volume of blood vessels and  renal corpuscle within each organ were estimated using this equation.
V(structure) =Vv (structure) x V (ref)
V(structure) = Absolute volumes of structure
Vv(ref)         = Reference volume of structure
 
RESULT AND FINDINGS
Chloroquine caused defects in the microscopic structure of the liver and kidney of the Sprague-Dawley rats. Renal corpuscles were few and deformed with noticeable patches in the kidney when compared with controls. Blood vessels in the treated rats compared with controls were few in the liver when compared with controls. Stereologically, the individual estimated absolute volume of fractions were determined and compared. For the liver there was a reduction in the absolute volume of the blood vessels when compared with controls. For the kidney, Chloroquine caused a reduction in the absolute volume of the renal corpuscles when compared with controls.

Histological findings
This study focused on the microscopic structures of the liver and kidney of animals treated with chloroquine once a day for three days. The investigation confirmed defects in microscopic structures. For the kidney, there were few renal corpuscles with noticeable patches in the treated rats compared with controls. For the liver there were few blood vessels in the treated rats compared with controls. (Patricia & Nathan, 1981).
Sterelogical findings
This study focused on the morphometric investigations. Absolute volumes of the liver and kidney special components were stereologically estimated after treatment with chloroquine. The investigation confirmed that chloroquine has deleterious effects on the quantitative analysis of these important tissues of the body (Ausburger & Arnold 1991).There was a reduction in the absolute volume of the blood vessels present in the liver after treatment with chloroquine compared with controls. There was a reduction in the absolute volume of the renal corpuscles after chloroquine treatment compared with controls. (Cruz-Orive et al 1993).

]SUMMARY AND CONCLUSION
In summary the present study has demonstrated that chloroquine though an antimalaria drug when taken in the rightful dosage have deleterious effects on some vital organs in the body. Chloroquine has deleterious effects on the microscopic structures of the liver and kidney on the morphometric /quantitative analysis of the liver and kidney vital components. However, further research are necessary on these findings.

MEDICAL ADVICE
The drug should therefore be taken only if it is prescribed by a doctor and people in the society should not abuse the use of this drug except it has been confirmed by the doctor that they have malaria fever.
Article authored by  Akinribido F.A(She’s a researcher affiliated with the Dept of Anatomy and Biochemistry, Bells University of technology Ota, Ogun State)
Contact details; e-mail fumibos@yahoo.co.uk Tel:08023654975

« Last Edit: September 29, 2011, 09:18:36 am by Francis Umeoguaju » Logged

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