Functional Tests Conducted on Animals | Toxicological Tests | Toxicology

The following points highlight the three main functional tests conducted on animals. The tests are: 1. Respiratory Functional Tests 2. Kidney Functional Tests 3. Assessment of Liver by Liver Functional Test.

1. Respiratory Functional Tests:

Tests on these functions are often carried out because of their greater sensitivity than morphological changes and also because of their ability to detect reversible effects. Consequently, these tests are more useful in human studies to provide more valid comparison between humans and the experimental animals with respect to their relative susceptibility to the toxic effects of the substances being tested.

Respiratory frequency is a sensitive indicator of local irritation and is often related to concentration. Certain gases, e.g., ozone and NO₂, increase the frequency whereas others, e.g., SO₂ and HCHO, decrease it.

Respiratory efficiency can be estimated by measuring O₂ and CO₂ in the blood or by measuring the rate at which inhaled carbon monoxide is taken into the blood.

2. Kidney Functional Tests:

Nephrotoxins can exert adverse effects on various parts of the kidney (Fig. 16.1A and 16.1B) resulting in alterations of different functions. Luwe (1981) reported that urinary concentrating ability, and kidney weight were the most sensitive and consistent indicators of nephrotoxicity. Goldstein et al (1981) observed that urine osmolarity was another most sensitive indicator of the nephro­toxicity of a platinum complex, whereas GFR and ERPF were affected only later and at higher doses.

Hence functional examinations of the kidney are routinely carried out as an integral part of short and long-term toxicity studies and may be done in a number of ways:

(a) Urine Analysis:

(i) Proteinuria:

Because of the size of their molecules, a very small amount of proteins of low molecular weight pass through the glomerular filter. The low molecular weight protein are readily reabsorbed by the proximal tubules. The occurrence of large amounts of protein in the urine is thus an indication of a loss of integrity of glomeruli. It is to be noted that normal rat urine may contain some protein. A critical comparison of the treated animals with that of the control is, therefore, important.

(ii) Glycosuria:

Glucose in the glomerular filtrate is completely reabsorbed in the tubules, provided the amount of glucose to be reabsorbed does not exceed the transport maximum (Tm.). Glycosuria in the absence of hyperglycemia thus indicates tubular dysfunction.

(iii) Urine Volume and Osmolarity:

These two values are usually inversely related and are useful indicators of renal function in concentration test, wherein water is with­held from the animal, and also in dilution test, wherein a large amount of water is given to the animal.

The osmolarity can be estimated from the specific gravity, but the freezing point of urine provides a more accurate measurement. A toxicant may cause high output renal failure or, on the other hand, oliguria or even anuria, resulting from tubular injury with concomitant interstitial edema and intraluminal pigment or debris.

(iv) Acidifying Capacity:

This can be assessed from the pH of urine, titratable acids, and NH₄⁺. The capacity is reduced when there is distal tubular dysfunction.

(v) Enzymes:

Enzymes such as maltase in urine may indicate destruction of proximal tubules. Lysozyme level in urine is greatly increased following intoxication with chromium, but only moderately so after mercury poisoning. Urine alkaline phos­phatase, on the other hand, may be renal or hepatic in origin.

Plummer (1981) suggests that the levels of urinary enzymes not only are useful indicators of renal damage but also indicate the subcellular site of origin e.g., alkaline phosphatase is located in endoplasmic reticulum, glutamate dehydrogenase in mitochondria, and lactate dehydrogenase in cytoplasm.

(vi) Proteins:

High molecular weight proteins are normally not filtered through the glomeruli. Their appearance in urine, therefore, indicates glomerular damage. On the other hand, low molecular weight proteins do pass through the glomerular filter, but they are reabsorbed by the proximal tubules. If this tubular function is impaired, such proteins will appear in the urine, e.g., in cadmium poisoning.

(b) Blood Analysis:

(i) Blood Urea Nitrogen (BUN):

Blood urea nitrogen is derived from normal metabolism of protein and is excreted in the urine. Elevated BUN usually indicates glomerular damage. However, its level can also be affected by poor nutrition and hepatotoxicity, which are common effects of many toxicants.

(ii) Creatinine:

Creatinine is a metabolite of creatinine and is excreted completely in the urine via glomerular filtration. An elevation of its level in the blood is thus an indication of impaired function. Furthermore, data on its level in blood and its amount in urine can be used to estimate the glomerular filtration rate; however, one drawback of this method is that some creatinine is secreted by the tubules.

Special Renal Functional Tests:

(i) Glomerular Filtration Rate (GFR):

The glomerular filtration rate can be more accurately determined by the clearance of inulin, a polysaccharide. It is diffused into the glomerular filtrate and is neither absorbed nor secreted by the tubules.

(ii) Renal Clearance:

This is the volume of plasma that is completely cleared of a substance in a unit of time. The renal clearance of p-aminohippuric acid (PAH) exceeds that of inulin because it is not only filtered through the glomeruli but also secreted by the tubules. A reduction of PAH elimination without a concomitant decrease of GFR indicates tubular dysfunction.

PAH is nearly completely (up to 90%) removed from the blood in one passage. The rate of its clearance is, therefore, useful in determining the effective renal plasma flow (ERPF). The renal blood flow can also be determined by the use of radio-labelled microspheres or an electromagnetic flowmeter.

(iii) PSP Excretion Test:

The rate of excretion of phenolsulphonphelein is related to the renal blood flow. It is, therefore, often used in the assessment of renal function. However, a reduced secretion rate can also result from cardiovascular diseases.

Morphological Examination as Functional Test of Kidney:

(i) Gross Examination:

Weight of the kidney in term of body weight of the animal, as a rule, is routinely determined at the end of short-term and long term toxicity studies. Alteration in its weight, when compared to that of control, often suggests kidney lesion. A number of other pathological lesions can also be detected on gross examination.

(ii) Light Microscopy:

Histopathological examinations can reveal site, extent and morphological nature of renal lesions. Sharrat and Frazer (1963) found that the histopathological examinations were more sensitive than the functional test used in assessing acute and chronic glomerular or tubular injuries. However, some of the newer functional tests (in vitro studies) appear more sensitive.

(iii) Electron Microscopy:

From the functional test viewpoint, this technique is useful in assessing ultrastructural change in the cells, such as mitochondria, other organelles, basement membranes and brush border. For example, prolonged exposure to methyl mercury increased the volume density of mitochondria and lysosomes.

In Vitro Studies:

Kidney Slices:

Tissue slices are a useful tool for the study of renal tubular function.

In assessing the effects of a toxicant on kidney, external factors that might affect the blood volume or blood pressure should be taken into consideration since they may indirectly impair renal functions. Furthermore, kidney diseases, such as those associated with ageing, may be prevalent and should also be considered.

3. Assessment of Liver by Liver Functional Test:

Following specific functional tests are carried to evaluate the liver function:

i. Serum Bilirubin and Van den Bergh Reaction:

This test is done for the differential diagnosis of prehepatic, hepatic, and post-hepatic jaundice.

Determination of total serum bilirubin is a good index of determining the severity of jaundice. Its level in the normal adults may vary from 0.4-0.8 mg/100 ml. In acute, infective and toxic hepatitis, the values may increase upto 10 mg/100 ml. In haemolytic jaundice, the values may range from 2-3 mg/100 ml.

ii. Van den Bergh Test:

When freshly prepared diazotised sulphanilic acid reagent is added to serum, the coagulated bilirubin, if present, gives a reddish- violet colour within a minute. This bilirubin is called direct reacting bilirubin (Direct van den Bergh reaction). The unconjugated bilirubin does not give any colour within one minute. If alcohol is added to the mixture, indirect bilirubin becomes soluble and colour is developed.

This fraction of bilirubin is called indirect bilirubin, and the test is known as indirect van den Bergh reaction. Formation of a faint pink colour after one minute and deepening of the colour in 2-3 minutes occurs when serum contains both conjugated as well as unconjugated bilirubin. This is called biphasic or delayed van den Bergh reaction.

A positive direct van den Bergh reaction with raised serum bilirubin indicates that the liver is functionally normal but the biliary tract is obstructed. Positive indirect van den Bergh test indicates hemolytic as well as hepatic jaundice. In hepatic jaundice both conjugated as well as unconjugated bilirubins are found to be raised, hence delayed van den Bergh reaction is often observed.

Functional Tests Based on Carbohydrate Metabolism:

Galactose and fructose are converted into glycogen in the liver alone, and, hence, if the liver is damaged due to any hepatic toxicant, these sugars are found in the circulation in high amounts.

(i) Galactose Tolerance Test:

Fasted individual is given 40 g galactose dissolved in 300 ml water. Blood is drawn at half an hour intervals for 2 hrs after administration of galactose. Galactose content of the four samples is determined after removing glucose by fermenting with yeast. Then galactose index is calculated which is the sum of the galactose values of the four samples. McLagan used a figure of 160 mg as the upper limit of normal galactose index. In infective and toxic hepatitis and in cirrhosis of liver, values up to 500 mg were obtained.

(ii) Fructose Tolerance Test:

To the fasted individual, 50 g fructose dissolved in 300 ml water is administered. Fasted blood sample is taken after every half an hour up to 2 hrs. The total sugar content of blood (glucose and fructose) is determined. In normal subjects, the highest blood sugar should not exceed the fasting level by more than 30 mg per 100 ml. Higher blood sugar level is obtained in the patients with infective hepatitis.

Test Based on Detoxicating Function of the Liver:

The most sensitive and best test is the detoxication of benzoic acid by liver to hippuric acid by conjugating it with glycine. It is known as the hippuric acid test.

Test:

Three hours after light breakfast and emptying the urinary bladder, the patient is given to drink 6 g sodium benzoate dissolved in 200 ml. water. Subsequently, Urine is collected for 4 hours. The urine should have 4.5 g hippuric acid. Smaller quantities are excreted in acute and chronic liver diseases.

Test Based on Excretory Function of the Liver:

Liver can remove a dye BSP (Bromsulphthalein) from its combination with plasma albumin and excrete it in the bile. When the dye is injected, it immediately combines with the plasma albumin and circulates in blood. Liver removes this dye and excretes it in the bile.

Test:

5 mg/kg body weight of dye is injected. The blood retains less than 10% of the dye in 30 minutes and 7% in 45 minutes. At 60 minutes, no dye is found to be retained. This is considered to be the most sensitive and reliable liver function test.

If the liver function is impaired due to any toxicant/drug, the dye is excreted very slowly. About 50% dye is retained at the end of 45 minutes. The test loses its value in obstructive jaundice. The test is found to be very useful in the diagnosis of liver cell damage without showing clinical jaundice in chronic hepatitis and in cirrhosis of liver.

Serum Enzymes in Determination of Liver Functions:

The level of SGPT (Serum Glutamic Pyruvic Transaminase), SGOT (Serum Glutamic-Oxaloacetic Transaminase), Isocitrate dehydrogenase and lactic dehydrogenase increases in circulation in the damage of liver cells due to toxicants.

Example:

In normal case SGOT and SGPT activity remain 4-20 (I.U./liter) whereas in toxic liver necrosis this value may be 15-100 (I.U./liter) times of normal value.

Blood Ammonia as an Index of Liver Function:

Actually, liver is the only organ which converts ammonia into urea. In advanced liver damage or in hepatic coma itself, due to damage of the parenchymal cells of the liver, it is not able to convert ammonia into urea. Thus, the level of ammonia rises from the normal range of 40-70 µg/100 ml to even up to 250 µg/100 ml (measured in terms of nitrogen).