MINOR

PROJECT
REPORT
SUBMITTED BYANCHAL GUPTA

UE1010

FALAK

UE1010

Carbon tetrachloride is the organic compound with the formula CCl4. It was formerly widely
used in fire extinguishers, as a precursor to refrigerants, and as a cleaning agent. It is a colorless
liquid with a "sweet" smell that can be detected at low levels. Both carbon tetrachloride and tetra
chloromethane are acceptable names under IUPAC nomenclature.
In the carbon tetrachloride molecule, four chlorine atoms are positioned symmetrically as corners
in a tetrahedral configuration joined to a central carbon atom by single covalent bonds. Because
of this symmetrical geometry, CCl4 is non-polar. Methane gas has the same structure, making
carbon tetrachloride a halomethane. As a solvent, it is well suited to dissolving other non-polar
compounds, fats, and oils. It can also dissolve iodine. It is somewhat volatile, giving
off vapors with a smell characteristic of other chlorinated solvents.

Uses for carbon tetrachloride

Most carbon tetrachloride is used to make chlorofluorocarbon propellants and refrigerants,
though this has been declining steadily. It has also been used as a dry cleaning agent and fire
extinguisher; in making nylons; as a solvent for rubber cement, soaps, insecticides, etc.

How are people exposed to carbon

tetrachloride?
Drinking/Eating: People are most often exposed to carbon tet in the environment by
drinking contaminated groundwater. Carbon tet may contaminate groundwater near
locations where the chemical was improperly disposed. Since the compound is heavy, some
of the spilled liquid will sink through soil and enter groundwater. Carbon tet does not move
easily with groundwater. Plants do not take up or store carbon tet when they grow in
contaminated soil.

Touching: Carbon tet can be absorbed through the skin if a person handles the chemical or
contaminated soil, or bathes in contaminated water.
Breathing: Carbon tet evaporates easily from water. Therefore, a person may be exposed
to its vapors when they bathe, cook, or wash with contaminated water.

TOXIC EFFECTS

Carbon tetrachloride is readily absorbed after ingestion and inhalation, but more slowly
through the skin .
Acute exposure to carbon tetrachloride can also cause central nervous system (CNS)
depression as well as gastrointestinal and neurological effects such as nausea, vomiting,
abdominal pain, diarrhoea, headache, dizziness, in-coordination, impairment of speech,
confusion, anaesthesia, and fatigue.
The liver and kidney are the major target organs for toxicity following acute inhalation or
ingestion exposure to carbon tetrachloride . Liver damage can occur after 24 hours and
in serious cases this can result in painful swollen liver, haemorrages, hepatic coma
and death. Kidney damage with an impairment in function normally occurs 2-3 weeks
after exposure, but in severe cases this can occur within 1-6 days in association with liver
failure.
Acute ocular exposure or skin contact can cause irritation of the eyes and skin. Direct
skin contact with undiluted carbon tetrachloride has been reported to cause a mild burning
sensation with mild redness. Some individuals may be hypersensitive and develop marked
swelling, itching and blisters following skin contact.
Chronic inhalation may result in liver and kidney toxicity and neurological effects from
depression of the central nervous system . Neurological and gastrointestinal symptoms
are similar to those for acute exposure, such as depression, nausea and other
gastrointestinal effects. In long term repeated dose studies in animals the liver has been
shown to be the most sensitive organ regarding toxicity.
The International Agency for Research on Cancer (IARC) concluded that there is inadequate
evidence for the carcinogenicity of carbon tetrachloride in humans. However, based on
evidence in animal studies, IARC has concluded overall that carbon tetrachloride is possibly
carcinogenic to humans (Group 2B). The doses inducing liver tumours in animal studies
are higher than those causing liver cell toxicity, and therefore are considered to arise

secondary to toxic effects on the liver .

Carbon tetrachloride does not have any significant mutagenic properties.
Data from animal studies indicate that carbon tetrachloride does not have any adverse
effects on development at dose levels below those producing toxicity to the maternal animals.
There are no adequate reproductive toxicity studies on carbon tetrachloride, but
limited data has suggested that exposure to relatively high concentrations of carbon
tetrachloride may impair fertility.

Health Effects of Acute / Single Exposure

Human Data
General toxicity

Acute exposure to carbon tetrachloride via any route of exposure can cause gastrointestinal
and neurological effects in the first 24 hours, such as nausea, vomiting, diarrhoea,
headache, dizziness, depression of conscious level and dyspnoea.
The liver and kidney are the major target organs for toxicity following acute inhalation or
ingestion exposure to carbon tetrachloride . Liver damage can occur after 24 hours and
in serious cases this can result in painful swollen liver, haemorrage, hepatic coma and death.
Kidney damage with an impairment in function normally occurs 2-3 weeks after
exposure, but in severe cases this can occur within 1-6 days in association with liver
failure.
Adverse effects on the liver can be markedly increased by the co-ingestion of alcohol,
due to hepatioc enzyme induction which results in increased production of toxic metabolites.
Inhalation

Inhalation of carbon tetrachloride may cause rapid depression of the central nervous system,
leading to headache, giddiness, weakness, lethargy and stupor. No effects were reported in
healthy volunteers following exposure to 50 ppm for 70 minutes or 10 ppm for 3 hours. A
review of a number of reports of carbon tetrachloride intoxication led to the conclusion that
no effects were noted up to 80 ppm for 3-4 hours. At higher concentrations nausea,
vomiting, headache, and more severe CNS effects have been noted . Headache and
dizziness was also reported following exposure to 250 ppm for 15 minutes.
Carbon tetrachloride is hepatotoxic, the principle features include swollen and tender liver,
elevated serum enzyme levels and jaundice as well as marked liver necrosis with steatosis.
CARBON TETRACHLORIDE TOXICOLOGICAL OVERVIEW
Ingestion

Ingestion of carbon tetrachloride can lead to hepatotoxicity. Single doses of approximately

90-180 mg kg-1 bw caused mild hepatotoxicity (fatty liver) and ingestion of 670 mg/kg
resulted in marked hepatotoxicity (severe necrosis) and also nephrotoxicity .
Ingestion of carbon tetrachloride can also result in CNS effects, with drowsiness being noted
after ingestion of 300 mg kg-1. No CNS effects were seen at lower concentrations, with only
nausea being reported following ingestion of 100 mg kg-1.

Dermal / ocular exposure

Carbon tetrachloride can cause irritation of the eyes and skin [4]. Direct skin contact with
undiluted carbon tetrachloride has been reported to produce a mild burning sensation with
mild redness . Some individuals may be hypersensitive and develop marked swelling,
itching and blisters following skin contact .
Acute toxicity of carbon tetrachloride is reported to be independent of the route of exposure,
therefore dermal exposure to relatively high concentrations may cause similar effects
as for oral and inhalation exposure.
Delayed effects following an acute exposure

Acute exposure to carbon tetrachloride via any route can cause liver damage after 24 hours
or more, renal dysfunction may occur in 1-6 days but may, in many cases, only be apparent
two to three weeks after exposure.

Animal and In-Vitro Data

General toxicity

The liver is the most sensitive target organ following inhalation or ingestion of carbon
tetrachloride in animals. Adverse effects on the kidney, central nervous system and lungs
also occur.
Inhalation

In animals, the hepatic effects of inhalation exposure to carbon tetrachloride are much the
same as in humans, such as elevated serum enzyme levels, steatosis, and centrilobular
necrosis progressing to fibrosis.
Changes in serum enzyme levels indicative of liver damage were seen in rats following 4hour exposure to 530 ppm or above. Liver necrosis has been reported following exposure to
4800 ppm . Signs of central nervous system depression, such as lack of
coordination, breathing difficulties and unconsciousness have also been observed in animals
exposed to approximately 7000 to 10500 ppm. In another study, rats exposed to <4600 ppm
for up to 8 hours showed signs of drowsiness; 7300 ppm caused uncoordination and
unconsciousness occurred at 12000 and 19000 ppm.
Ingestion

Adverse effects on the liver have been reported to be the major effect in rats following acute
ingestion of carbon tetrachloride. Rats given 20 mg kg-1 bw showed histopathological
evidence of liver toxicity and centrilobular vacuolisation. An increase in liver fat and liver
weight was observed after administration of 39.9 mg kg-1 and liver cell necrosis was reported
after ingestion of 80 mg kg-1 bw . In mice, a single oral dose of 32 mg kg-1 caused liver
necrosis.
Morphological changes and necrosis of Clara cells in the lungs of both mice and rats have
been reported after an acute oral dose of approximately 4000 mg kg-1 .
Dermal / ocular exposure

The irritant effects on carbon tetrachloride on skin have been investigated in guinea-pigs.
Carbon tetrachloride (513 mg cm-2) was applied under an occlusive dressing for 15 minutes
to 16 hours. Degenerative damage to the epidermal cells and oedema was reported within
15 minutes, which increased in severity over the following few hours. Liver necrosis was

seen approximately 16 hours after the initial contact .

Application of 0.1 ml into the eyes of rabbits in a Draize test produced mild irritation. The
response was evident at 24, 48 and 72 hours after exposure and recovery was complete
after 14 days .

Health Effects of Chronic / Repeated Exposure

Animal and In-Vitro Data
Inhalation

The liver and the kidneys are the principle target organs following chronic inhalation to
carbon tetrachloride in animals. The predominant signs of hepatotoxicity include steatosis,
elevated serum enzyme levels and centrilobular necrosis .
In a subchronic (13 week) study rats and mice were exposed to up to 800 ppm (5192 mg m3)
for 6 hours per day, 5 days per week. In both species microscopic changes were seen in the
liver at autopsy at the lowest dose level (10 ppm; 64 mg/m3). In rats, effects on the blood
were seen at 30 ppm (192 mg m3) and kidney damage at 270 ppm (1731 mg m3) and above.
In mice haematological effects were only seen at the top dose.
A 2-year inhalation study with rats exposed to approximately 0, 5, 25 or 125 ppm for 6 hours
per day, 5 days per week reported decreases in body weight, changes in haematology and
blood biochemistry including markers of hepatotoxicity and nephrotoxicity at 25 ppm and a
significant decrease in survival at 125 ppm, predominantly due to liver tumours and/or
chronic nephropathy. In a parallel study, mice exposed to the same concentrations had a
significantly decreased survival rate, mainly due to liver tumours, when exposed to 25 or 125
ppm. A decrease in body weight gain, changes in haematology and blood biochemistry were
also observed at 25 ppm.
Ingestion

The liver and kidneys are also target organs for chronic oral exposure to carbon tetrachloride
in animal studies .
Rats given carbon tetrachloride via gavage 5 days per week for 12 weeks had an increase in
serum liver enzymes and mild liver damage (vacuolation) at 10 mg kg-1 bw day-1 and
cirrhosis was observed at 33 mg kg-1 bw day-1. No effects were seen at 1 mg kg-1 bw day-1.
In mice given carbon tetrachloride in corn oil 5 days a week for 90 days, changes in serum
levels of liver enzymes were seen at 12 mg kg-1 bw day-1 and above, together with
histopathological evidence of liver damage (fatty infiltration and necrosis). Similar to the
previous study, no effects were seen at 1.2 mg kg-1 bw day-1.
Oral exposure has also been associated with suppression of the immune system . One
study with mice given 50 mg kg-1 bw day-1 of carbon tetrachloride for 14 days (sufficient for
liver toxicity) showed a reduced T-cell response to sheep red blood cells, and at 500 mg kg-1
bw day-1 a reduction in the absolute numbers of CD4+ and CD8- T-cells per spleen was
reported.
Genotoxicity

Carbon tetrachloride was not mutagenic in, but did induce DNA damage and mutations in
Escherichia coli . The results of in-vivo genotoxicity tests suggest that genotoxic effects
only occur at doses that produce cytotoxicity . Although carbon tetrachloride has produced
CARBON TETRACHLORIDE TOXICOLOGICAL OVERVIEW

some effects on genetic material, such as in mammalian cells, the effects are considered to
be secondary to carbon tetrachloride toxicity and not from direct interaction with DNA [2, 5].
Thus, overall carbon tetrachloride is not regarded as genotoxic .
Carbon tetrachloride has been extensively investigated in the Salmonella typhimurium invitro
assay for gene mutation and negative results have been consistently obtained.
Negative results have also been obtained in assays for chromosome damage in hamster
ovary cells, rat liver cells and human lymphocytes. DNA damage has been reported in E.
Coli and it has been shown to induce intrachromosomal and mitotic recombination in yeast.
When investigated in-vivo, carbon tetrachloride did not induce chromosome aberrations in
bone marrow of mice or liver of rats. Nor did it induce micronuclei or unscheduled DNA
synthesis in the liver of rats and mice. DNA binding has been reported in the liver of rats,
mice and hamsters.
It has been suggested that the positive effects on carbon tetrachloride in a few assays are
explicable in terms of damage to nuclear protein or to DNA damage induced as a secondary
effect to general toxicity .The weight of evidence indicates that carbon tetrachloride does not
have any significant genotoxic potential.
Carcinogenicity

Liver tumours have been produced in rats, mice and hamsters following carbon tetrachloride
exposure via oral, inhalation and subcutaneous administration . The doses inducing
liver tumours were higher than those causing liver cell toxicity, and therefore are considered
to arise secondary to toxic effects on the liver. IARC also noted one inhalation study in
mice that reported an increased incidence of a rare tumour in the adrenal glands. Overall,
IARC concluded that there is sufficient evidence for the carcinogenicity of carbon
tetrachloride in experimental animals.
Reproductive and developmental toxicity

There are a number of studies that have investigated the developmental toxicity of carbon
tetrachloride in pregnant rats, using the oral or inhalation route. There is also one study in
mice using the oral route. In all cases adverse effects (limited to fetotoxicity rather than gross
malformations) were seen only at dose levels associated with maternal toxicity. It was
concluded that the available data suggest that the fetus is not preferentially sensitive to
carbon tetrachloride and the effects on fetal development and post-natal survival are likely to
be secondary to maternal toxicity .
Rats and mice were given injections of carbon tetrachloride into the portal vein or
into the spleen, and the localization of the resulting hepatocellular lesions was
studied. In contrast to some statements in the literature, and confirming some older
reports, damage was found to be limited to the periportal areas. A range of changes
leading to cell necrosis was found, which was frequently associated with pronounced
vasodilatation. These features were considered to be evidence in favour of a direct
hepatotoxic effect of carbon tetrachloride, and are difficult to reconcile with the
view, formulated again recently by some workers, that carbon tetrachloride acts
indirectly on the liver cell by producing vasoconstriction.

TOXIC EFFECT OF CARBON TETRACHLORIDE

It is injected directly into the portal vein. Since a similar blanching effect was seen following the
injection of hypertonic saline or glucose-an observation which I can confirm he attributed this
change to a " non-specific response to a strong irritant." It is
of interest, for the purpose of this paper, to draw attention to the discrepancy
inherent in the fact that on the one occasion when the contraction of vessels, which
is postulated to cause the centrilobular changes, is known to occur, the pathological
changes, as described here, take place in the periportal and not in the central region
of the liver lobule. Since the nature and the localization of these changes are highly
suggestive of a direct hepatotoxic effect of carbon tetrachloride, at least if given
intraportally, it is questionable whether an indirect effect of such an agent can be
invoked.

OBJECTIVE
It is well established that carbon tetrachloride (CCl4) is a typical hepatotoxin causing centrizonal
necrosis . As a result of extensive studies, the initial event in the rat given CCl4 has been believed
to be lipid peroxidation of the endoplasmic reticulum of the liver cell initiated by trichloromethyl
radical generated by the reaction between CCl4 and cytochrome P450. Based on these results,
CCl4 has been assumed to be a typical poison causing severe oxidative stress and we will study
the oxidative stress produced by carbon tetrachloride by performing various enzyme and other
tests.
Oxidative stress induced by carbon tetrachloride in mice substantially decreases the increase in
body weight and relative testis weight. It also markedly increases the level of TBARS and
nitrites along with corresponding decrease in reduced glutathione and various antioxidant
enzymes in testis, i.e., catalase, peroxidase, superoxide dismutase and glutathione peroxidase.
Serum level of testosterone, luteinizing hormone and follicle stimulating hormone was
significantly decreased while estradiol and prolactin were increased with carbon tetrachloride
treatment.
CCl4-induced oxidative stress significantly cause reduced levels of glutathione (GSH), its
metabolizing enzymes and simultaneously cause the production of free radicals. This leads to
indirect genotoxicity and contribute to carcinogenicity.
the following tests were performed:1)
2)
3)
4)

Protein estimation by lowrys method

Lipid per oxidation
Catalase
Glutathione S transferase test

5) ELISA
It is reported that the level of specifically determined lipid hydroperoxides , which was shown
to be a good indicator of oxidative stress in typical pathologic conditions such as vitamin E
deficiency , iron overload , diabetes , and thioacetamide intoxication, was increased by CCl4 only
in mitochondria. Observations suggest that CCl4 does not cause extensive lipid peroxidation as
widely accepted. To evaluate the oxidative stress caused by CCl4, changes in the concentration of
antioxidative vitamins C and E in the liver and plasma were measured in this study. carbon
tetrachloride (1 and 4 mM) produces a small elevation in M1dG adducts and DNA strand breaks
and increases in 8-oxodG were observed at the threshold of, and concomitant with, cytotoxicity.
Also the administration of CCl4 increased lipid peroxidation and uric acid levels and inhibited
superoxide dismutase activity.
Protein concentrations were determined according to the method of Lowry et al. using bovine
serum albumin as the standard.

METHOD OF INJECTION
Carbon tetrachloride was tested for carcinogenicity in several experiments in mice
by oral and intrarectal administration and in rats by oral and subcutaneous
administration and by inhalation exposure; it was also tested in one experiment in
hamsters and one experiment in trout by oral administration. In various strains of
mice, it produced liver tumours, including hepatocellular carcinomas. In various
strains of rats, it produced benign and malignant liver tumours; and in one
experiment with subcutaneous injection, an increased incidence of mammary
adenocarcinomas was observed. In hamsters and trout, increased incidences of
liver tumours were observed s2e9
For the estimation of effect of carbon tetrachloride on mice we took 6 mice out of
which four were taken as test and the rest two were taken as control.the effect of
carbon tetrachloride was determined by us in various estimation methods.
For carbon tetrachloride to be injected in the mice olive oil is used as a carrier.100
microlitre of olive oil and carbon tetrachloride in 1:3 is injected in intraperitoneal
cavity of four mice taken as test and 100 microlitre of olive oil is inject in two mice
taken as control.
The mices are now kept for 24 hours.

Next day the mices are sacrificed. Mices are anesthetized with diethyl ether ans
then dissection is done and kidney, liver and testis are excised and the rest of the
part is disposed off.
The kidney, liver and testis need to be stored at low temperature so that their activity
is not lost. Now 200 mg of each sample is weighed. This sample is homogenized
using a homoge nizer with tris HCl buffer prepared with ph 7.4.
200mg of of sample and tris HCl buffer is homogenized and the volume is made
upto 2 ml. Now take about 10% of homogenizer and centrifuge it at 10,000 rpm for
about 30 minutes. Now discard the pellet and store the supernatant in eppendoffs in
freezers.

PROTEIN ESTIMATION BY LOWRYS METHOD

AIM-estimation of protein in the 17 samples using standard plot
MATERIAL REQUIREDA) APPARATUStest tubes, burette, pipette, measuring cylinder, cuvette, spectrophotometer

B) REAGENTS
A. 2% Na2CO3 in 0.1 N NaOH
B. 2% NaK Tartrate in H2O
C Copper sulphate solution-1%
D. Lowrys reagent-98:1:1(A:B:C)
E. Phenol Reagent - 1 part Folin-Phenol [2 N] : 1 part water
BSA Standard - 1 mg/ mL(prepare fresh)
Bovine Serum Albumin: 5 mg in 5 mL of water

PRINCIPLEThe Lowry protein assay is a biochemical assay for determining the total level of protein in a
solution. The total protein concentration is exhibited by a color change of the sample solution in
proportion to protein concentration, which can then be measured using colorimetric techniques.
It is named for the biochemist Oliver H. Lowry who developed the reagent in the 1940s. His
1951 paper describing the technique is the most-highly cited paper ever in the scientific
literature, cited over 200,000 times.
The principle behind the Lowry method of determining protein concentrations lies in the
reactivity of the peptide nitrogen[s] with the copper [II] ions under alkaline conditions and the
subsequent reduction of the Folin- Ciocalteay phosphomolybdic phosphotungstic acid to
heteropolymolybdenum blue by the copper-catalyzed oxidation of aromatic acid. The
concentration of the reduced Folin reagent is measured by absorbance at 750 nm.[
The Lowry method is sensitive to pH changes and therefore the pH of assay solution should be
maintained at 10 - 10.5. The Lowry method is sensitive to low concentrations of protein. The
major disadvantage of the Lowry method is the narrow pH range within which it is accurate.
However, we will be using very small volumes of sample, which will have little or no effect on
pH of the reaction mixture. A variety of compounds will interfere with the Lowry procedure.
These include some amino acid derivatives, certain buffers, drugs, lipids, sugars,
salts, nucleic acids and sulphydryl reagents . It is noted that ammonium ions, zwitter ionic
buffers, nonionic buffers and thiol compounds may also interfere with the Lowry reaction. These
substances should be removed or diluted before running Lowry assays.

PROCEDURE1) Prepare the chemicals as required.

2) Take 24 test tubes and label one as blank,6 as standard and 17 as test.
3) In test tubes labeled as standard add 10,2,30,40,50,60 microlitre of standard
prepared and make up the volume to about 1ml with distilled water
4) In blank take 1ml of distilled water
5) In test tubes labeled as test take add 10 microlitre of sample and make up the
volume to 1 ml with distillled water
6) Now add 3ml of lowrys reagent in each test tube and incubate it for 30 minutes at
37 degree Celsius.
7) Add 300 microlitre of follins reagent to each test tube and again incunbate it for
30 minutes at 37 degree Celsius.
8) Take reading at 620nm.
9) Plot the standard curve and estimate the amount of protein of tests from the
standard curve.

OBSERVATIONS-

S.
NO.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

Blank
S1
S2
S3
S4
S5
S6
T1 K
T1 L
T1 T
T2 K
T2 L
T2 T
T3 K
T3 L
T3 T
T4 K
T4 L
T4 T
C1 K
C1 L
C2 K
C2 L
C2 T

Std
&tes
t
(ul)
10
20
30
40
50
60
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

Distilled
water
(ul)

Lowrys
reagent(ml
)

1000
990
980
970
960
950
950
940
990
990
990
990
990
990
990
990
990
990
990
990
990
990
990
990

3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0

Follins
reagent
(ml)
Incubat
e
For
30
Minute
s
At 37
degree
celsius

0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3

O.D.

Incubat
e
For
30
Minutes
At 37
degree
celsius

0.0
0.120
0.162
0.450
0.192
0.226
0.270
0.411
0.339
0.335
0.542
0.275
0.197
0.254
0.332
0.189
0.428
0.273
0.193
0.422
0.678
0.093
0.336
0.201

From graphAmount of protein in each test sample is determined from graph

S.NO.

SAMPLE

PROTEIN(ug/ul)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17

T1 K
T1 L
T1 T
T2 K
T2 L
T2 T
T3 K
T3 L
T3 T
T4 K
T4 L
T4 T
C1 K
C1 L
C2 K
C2 L
C2 T

9.1
7.55
7.5
12
6.1
4.3
5.5
7.3
4.2
9.5
6.0
4.2
9.3
12.85
2.0
7.3
4.4

For 10ug
protein(ul)
1.098
1.324
1.33
.833
1.639
2.325
1.818
1.369
2.380
1.052
1.66
2.380
1.075
0.778
5.00
1.369
2.272

PECAUTIONS1) Warming up time of 15 minutes must be givenbefore taking the readings.

2) Samples should be kept in crushed ice while taking the readings.
3) New tips must be taken for every sample.

CATALASE TEST
AIM-To calculate the enzyme activity by catalase method
MATERIAL REQUIRED-

A) APPARATUStest tubes, burette, pipette, measuring cylinder, cuvette, spectrophotometer

B) REAGENTS
1)Phosphate Buffer, 50mM(pH 7)
KH2PO4 (50mM) + NA2HPO4 (50mM)
(40 ml)

(60 ml)

Set pH with KOH.

KH2PO4 (50mM) 50mM= .680gm/100ml OR 1.02gm/150ml
Na2HPO4(50mM) 50mM= .709/100ml OR 1.063gm/150ml
2)0.75M Hydrogen Peroxide

THEORYCatalase is a common enzyme found in nearly all living organisms exposed to oxygen.
It catalyzes the decomposition of hydrogen peroxide towater and oxygen.[1] It is a very important
enzyme in protecting the cell from oxidative damage by reactive oxygen species (ROS).
Likewise, catalase has one of the highest turnover numbers of all enzymes; one catalase
molecule can convert millions of molecules of hydrogen peroxide to water and oxygen each
second.[2]
The catalase enzyme serves to neutralize the bactericidal effects of hydrogen peroxide (13).
Catalase expedites the breakdown of hydrogen peroxide (H2O2) into water and oxygen (2H2O2 +
Catalase 2H2O + O2). This reaction is evident by the rapid formation of bubbles (2, 7).

PROCEDURE-

1) Prepare solution of .16 ml of H2O2 in 100 ml of phosphate buffer

2) Cover the bottle with foil.
3) Check the O.D. of H2O2 at 240 nm by taking 3ml in a cuvette. It should be around
0.5 and if it is less than that then add more of H2O2.
4) Take other test tubes and add 3ml of H2O2 in each test tube.

5) Now add 10 microlitre of sample in each test tube .

6) For each cuvette monitor the A240 untill constant and add 10 microliter of sample.
7) Take 3 readings after the interval of 1 minutes.
OBSERVATIONSReading of blank i.e. 3ml H2O2observed at 0.730
S. No. Samples(10
microliter)

H2O2(ml)

1.

T1 K

2.

O.D.

O.D.

O.D.

(Reading after 1
min)

(Reading
after 2 min)

(Reading after 3 min)

3.0

0.771

0.600

0.518

T1 L

3.0

0.528

0.349

0.302

3.

T1 Te

3.0

0.723

0.721

0.720

4.

T2 K

3.0

0.680

0.576

0.493

5.

T2 L

3.0

0.674

0.516

0.438

6.

T2 Te

3.0

0.825

0.783

0.744

7.

T3 K

3.0

0.749

0.642

0.604

8.

T3 L

3.0

0.655

0.519

0.450

9.

T3 Te

3.0

0.818

0.776

0.753

10.

T4 K

3.0

0.772

0.766

0.763

11.

T4 L

3.0

0.743

0.739

0.735

12.

T4 Te

3.0

0.799

0.765

0.753

13.

C1 K

3.0

0.854

0.679

0.578

14.

C1 L

3.0

0.654

0.495

0.578

15.

C1 Te

3.0

--

16.

C2 K

3.0

0.724

0.717

0.717

17.

C2 L

3.0

0.667

0.478

0.408

--

--

18.

C2 Te

3.0

0.829

0.811

0.795

RESULTSExtension coefficient=43.6mol-1cm-1
Enzyme activity=change in OD/min/ext coeff*mg of protein
Change in OD /min is calculated from the graph which is plotted between optical density and
time
S.NO.

SAMPLE

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17

T1 K
T1 L
T1 Te
T2 K
T2 L
T2 Te
T3 K
T3 L
T3 Te
T4 K
T4 L
T4 Te
C1 K
C1 L
C2 K
C2 L
C2 Te

CHANGE IN
OD/MIN
0.085
0.125
0.010
0.060
0.100
0.035
0.060
0.075
0.030
0.020
0.020
0.030
0.130
0.115
0.010
0.135
0.010

PROTEIN IN
MICROLITRE
91
75.5
75
120
61
43
55
73
42
95
60
42
32
128.5
20
73
44

ENZYME ACTIVITY
(mmol cm min-1 mg-1)
21.4
37
3.05
11.4
37
18
25
22
16
4.8
7.6
16.3
0.32
20.5
11.46
42
520

PRECAUTIONS1) Warming up time of 15 minutes must be givenbefore taking the readings.

2) Cuvette should be properly washed after each reading of sample.
3) New tips must be taken for every sample.

LIPID PEROXIDATION TESTAIM- To calculate the enzyme activity by LPO method

MATERIAL REQUIREDA) APPARATUStest tubes, burette, pipette, measuring cylinder, cuvette, spectrophotometer
B) REAGENTS
STANDARD Tetra Ethoxy Propane (TEP)( Range 2-10 nM)
22ul TEP 100ml (100nM)

1ml in 100 ml(10nM)

10% TCA 1.25g/12.5ml dW

TBA(Thio Barbituric Acid) 0.67% (670mg in 100ml dW)

THEORY- Lipid peroxidation refers to the oxidative degradation of lipids. It is the process in
which free radicals "steal" electrons from the lipids in cell membranes, resulting in cell damage.
This process proceeds by a free radical chain reactionmechanism. It most often
affects polyunsaturated fatty acids, because they contain multiple double bonds in between
which lie methylene bridges (-CH2-) that possess especially reactive hydrogens. As with any
radical reaction, the reaction consists of three major steps: initiation, propagation, and
termination.
Hepatic injury through carbon tetrachloride (CCl(4)) induced lipid peroxidation is well known
and has been extensively used in the experimental models to understand the cellular
mechanisms behind oxidative damage and further to evaluate the therapeutic potential of drugs
and dietary antioxidants
PROCEDURE1) Firstly prepare the solutions needed.
2) Then thaw the sample.

3) Now add 0.5ml supernatant sample and 0.5ml of Tris Hcl buffer in the 17 respective
eppendorfs.
4) Put these eppendorfs for 2 hour incubation at 370C.
5) Now add 750ul of TCA solution and 750ul of the above samples in another eppendorfs.
6) Spin the above samples at 3000rpm for 10 minutes.
7) Add 1ml of TBA solution and 1ml of supernatant from the centrifuged samples in the test
tubes.
8) Then put the test tubes in the water bath(preset at 1000C) for 30-45 minutes.
9) Now cool down the samples.
10) Observe the samples at 532nm under spectrophotometer and note O.D. of the samples.

OBSERVATIONS AND RESULTSExtension coefficient=1.56*105 mmol-1cm-1

Enzyme activity= OD(3ml)/min/ext coeff*mg of protein

S.No.

Samples

1.
2.
3.
4
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.

T1 K
T1 L
T1 Te
T2 K
T2 L
T2 Te
T3 K
T3 L
T3 Te
T4 K
T4 L
T4 Te
C1 K
C1 L
C2 K
C2 L
C2 Te

O.D.
(2ml Sample)
0.620
0.554
0.705
0.481
0.468
1.030
0.532
0.384
0.495
0.615
1.03
0.223
0.368
0.495
0.689
0.155
0.470

O.D.
(3ml Sample)

ENZYME ACTIVITY
(mmol cm mg-1)

0.401
0.391
0.410
0.300
0.295
0.597
0.310
0.167
0.245
0.340
0.51
0.140
0.176
0.315
0.356
0.091
0.256

0.56
0.66
0.70
0.32
0.62
1.76
0.72
0.28
0.74
0.44
1.08
0.426
0.242
0.30
2.28
0.44
0.26

PRECAUTIONS1) Warming up time of 15 minutes must be given before taking the readings.
2) Cuvette should be properly washed after each reading of sample.
3) New tips must be taken for every sample.

GLUTATHIONE S TRANSFERASE
AIM- Measurement of glutathione s transferase activity by using CDNB as the substrate.
MATERIAL REQUIREDA) APPARATUStest tubes, burette, pipette, measuring cylinder, cuvette, spectrophotometer
B) REAGENTS
1) CDNB(1 chloro 2,4-dinitrobenzene)=2mg/100ml of abs. alcohol
2)GSH(mM)= 3mg/10ml phosphate buffer {(1) & (2) to be prepared fresh.}
3)Phosphate Buffer[0.1M, pH6.5]
a)NaH2PO4.2H2O(0.1M)
b)Na2HPO4(0.1M) [128 ml of (a) + 72 ml of (b) & set pH 6.5]
THEORYGlutathione S-transferases in the small intestine function in detoxification of electrophilic
compounds ingested in foods, dietary supplements, and orally administered drug preparations.
Although the required substrate glutathione (GSH) is synthesized in the intestinal enterocytes,
the rate of synthesis is slow compared to both the maximal GST activity and the rate of uptake
of luminal GSH. GSH is supplied to the intestinal lumen in the bile, and normal luminal
concentrations in the rat are about 250 M. The 250 M of extracellular GSH stimulated
conjugation of 1-chloro-2,4-dinitrobenzene by approximately 300% in rat intestinal enterocyte
preparations. However, an unexpected finding was that most of this stimulated activity did not
depend upon uptake of GSH by the enterocytes but was due to glutathione S-transferase
associated with mucus. Immunohistochemistry of glutathione S-transferase in the intact small
intestine confirmed that a portion of the GST is present in the mucus layer. The presence of this

detoxication enzyme in the extracellular mucus layer provides a novel mechanism for
preventing direct contact of potentially toxic dietary electrophiles with the intestinal
enterocytes.
Glutathione (GSH) is supplied to the lumen of the small intestine from bile transported from the
liver . The concentration of GSH in bile is in the millimolar range and supply of GSH to bile
occurs via a specific transport system, which has been extensively characterized in bile
canalicular membranes. GSH can also be provided from the diet, with fresh fruits and
vegetables providing on average the highest concentrations . Direct measurements of luminal
(jejeunal) GSH in rat show a concentration range of 60200 M in fasted animals that increases
to 120300 M in fed animals .
Intestinal epithelial cells contain members of the GST family that catalyze the reaction of GSH
with electrophilic compounds
Chemical carcinogens are generally classified as genotoxic or non-genotoxic. However, weak
genotoxicity at high concentrations is sometimes observed and interpretation is often
problematic. In addition, certain rodent carcinogens exert their effects at doses associated with
cytotoxicity and compensatory hyperplasia may be a contributing factor to tumourogenesis. We
hypothesise that certain substances, at high concentrations, can induce an oxidative stress via
the depletion of glutathione (GSH) and other antioxidant defences and that this may lead to
indirect genotoxicity, that could contribute to carcinogenicity. In support of this, human HepG2
cells treated with buthionine sulphoximine (BSO) to deplete GSH, exhibited DNA strand
breaks alongside elevated 8-oxodeoxyguanosine (8-oxodG) and malondialdehyde
deoxyguanosine (M1dG) adducts under conditions associated with lipid peroxidation. In female
rat hepatocytes, chloroform treatment resulted in a small dose-dependent increase in M1dG
adducts (4 mM and above), DNA strand breakage (8 mM and above) and lipid peroxidation, in
the absence of any associated increase in DNA oxidation. GSH depletion only occurred in
association with cytotoxicity (20 mM; lactate dehydrogenase release). Alongside lipid
peroxidation, carbon tetrachloride (1 and 4 mM) produced a small elevation in M1dG adducts
and DNA strand breaks and increases in 8-oxodG were observed at the threshold of, and
concomitant with, cytotoxicity (4 mM). These effects may contribute to high dose genotoxicity
and carcinogenicity. Non-linearity in the dose response is expected on the basis of depletion of
antioxidants, and therefore, a pragmatic threshold for biologically relevant responses should
exist.
PROCEDURE1)
2)
3)
4)

Prepare the solutions as given in the requirements.

Take 17 test tubes and add 2.7 ml of phosphate buffer in each test tube.
Now add 0.1ml of CDNB
After this add about 0.1 ml of GSH in each test tube.

5) Now add this solution in the cuvette in spectrophotometer and add 10 microlitre of sample
6) Now take the reading at 340 nm.
OBSERVATIONSS. No.

Samples

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.

T1 K
T1 L
T1 Te
T2 K
T2 L
T2 Te
T3 K
T3 L
T3 Te
T4 K
T4 L
T4 Te
C1 K
C1 L
C2 K
C2 L
C2 Te

O.D.
(Reading after 1
min)
0.269
0.003
0.092
0.011
0.026
0.027
0.108
0.110
0.007
0.094
0.140
0.065
0.122
0.045
0.021
0.072
0.060

O.D.
(Reading after
2 min)
0.254
0.008
0.106
0.010
0.032
0.029
0.112
0.110
0.009
0.106
0.147
0.064
0.140
0.058
0.023
0.089
0.066

O.D.
(Reading after 3
min)
0.259
0.009
0.119
0.012
0.033
0.039
0.124
0.120
0.011
0.130
0.150
0.065
0.137
0.065
0.027
0.099
0.076

RESULTExtension coefficient=9.6 mmol-1cm-1

Enzyme activity=change in OD/min/ext coeff*mg of protein
Change in OD /min is calculated from the graph which is plotted between optical density and
time

S.NO.

SAMPLE

1
2
3
4
5

T1 K
T1 L
T1 Te
T2 K
T2 L

CHANGE IN
OD/MIN
0.020
0.002
0.010
0.001
0.020

PROTEIN IN
MICROLITRE
91
75.5
75
120
61

ENZYME ACTIVITY
(mmol cm min-1 mg-1)
22
2.7
13
0.86
34

T2 Te
T3 K
T3 L
T3 Te
T4 K
T4 L
T4 Te
C1 K
C1 L
C2 K
C2 L
C2 Te

6
7
8
9
10
11
12
13
14
15
16
17

0.0252
0.010
0.005
0.025
0.035
0.010
0.001
0.030
0.010
0.005
0.015
0.015

43
55
73
42
95
60
42
93
128.5
20
73
44

60.5
17
7.13
62
38
17
2.4
33
8.1
26
21
35

PRECAUTIONS1) Warming up time of 15 minutes must be given before taking the readings.
4) Cuvette should be properly washed after each reading of sample.
5) New tips must be taken for every sample.

ELISA
AIM-To perform ELISA
MATERIAL REQUIREDA) APPARATUStest tubes, burette, pipette, measuring cylinder, cuvette, spectrophotometer
B) REAGENTS
1) Phosphate Buffered Saline
PBS(pH 7.2 , 0.1 M)
a)NaH2 PO4 (M.Wt- 138) 0.1M=1.384 g/100ml
b)Na2HPO4 (M.Wt-178gm) 0.1M=1.78g/100ml

Nacl=0.9g/100ml. Add (a) to (b) to set pH

2) 0.06M Carbonate Buffer
a)NaHCO3= (8.4%) 0.1M
a(45.3)

b(18.0)

b)Na2CO3=(10.6%) 0.1M
Make upto 1 litre

For 100 mla)NaHCO3= 2.1 g/25ml

4.53 ml + b)Na2CO3= 2.65 g/25ml

1.82 ml

3) Citrate Buffer (pH 5, 0.1M)

a)Citric Acid C6H8O7.H2O (525mg/25ml)
b)Na2HPO4.2H2O ( 445 mg/25ml)
Add a & b in equal volume for pH 5
4) Substrate
ABTS= 0.5mg/ml of Citrate Buffer
H2O2= 5 ul/10ml
[Prepare this just before adding to the plates]
5)tween 80
6)Carbonate buffer

THEORYEnzyme-linked Immunosorbent Assays (ELISAs) combine the specificity of antibodies with

the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easilyassayed enzyme. ELISAs can provide a useful measurement of antigen or antibody
concentration. There are two main variations on this method: The ELISA can be used to detect
the presence of antigens that are recognized by an antibody or it can be used to test for
antibodies that recognize an antigen.
ELISAs are performed in 96-well plates which permits high throughput results. The bottom of
each well is coated with a protein to which will bind the antibody you want to measure. Whole
blood is allowed to clot and the cells are centrifuged out to obtain the clear serum with

antibodies (called primary antibodies). The serum is incubated in a well, and each well contains
a different serum (see figure below). A positive control serum and a negative control serum
would be included among the 96 samples being tested.
After some time, the serum is removed and weakly adherent antibodies are washed off with a
series of buffer rinses. To detect the bound antibodies, a secondary antibody is added to each
well. The secondary antibody would bind to all human antibodies and is typically produced in a
rodent. Attached to the secondary antibody is an enzyme such as peroxidase or alkaline
phosphatase. These enzymes can metabolize colorless substrates (sometimes called chromagens)
into colored products. After an incubation period, the secondary antibody solution is removed
and loosely adherent ones are washed off as before. The final step is the addition the enzyme
substrate and the production of colored product in wells with secondary antibodies bound.
When the enzyme reaction is complete, the entire plate is placed into a plate reader and the
optical density (i.e. the amount of colored product) is determined for each well. The amount of
color produced is proportional to the amount of primary antibody bound to the proteins on the
bottom of the wells.

TYPES OF ELISA
Direct ELISA
(1) Direct ELISAs involve attachment of the antigen to the solid phase, followed by an enzymelabeled antibody. This type of assay generally makes measurement of crude samples difficult,
since contaminating proteins compete for plastic binding sites.
Indirect ELISA
(2) Indirect ELISAs also involve attachment of the antigen to a solid phase, but in this case, the
primary antibody is not labeled. An enzyme-conjugated secondary antibody, directed at the first
antibody, is then added. This format is used most often to detect specific antibodies in sera.
Competitive ELISA
(3) The third type of ELISA is the Competition Assay, which involves the simultaneous addition
of 'competing' antibodies or proteins. The decrease in signal of samples where the second
antibody or protein is added gives a highly specific result.
Sandwich ELISA
(4) The last type of assay is the sandwich ELISA. Sandwich ELISAs involve attachment of a
capture antibody to a solid phase support. Samples containing known or unknown antigen are
then added in a matrix or buffer that will minimize attachment to the solid phase. An enzymelabeled antibody is then added for detection.
HMOX1 (heme oxygenase (decycling) 1) is a human gene that encodes for the enzyme heme
oxygenase 1 . Heme oxygenase is an essential enzyme in heme catabolism, it cleaves heme to
form biliverdin.

Heme oxygenase, an essential enzyme in heme catabolism, cleaves heme to form biliverdin,
which is subsequently converted to bilirubin by biliverdin reductase, and carbon monoxide, a
putative neurotransmitter. Heme oxygenase activity is induced by its substrate heme and by
various nonheme substances. Heme oxygenase occurs as 2 isozymes, an inducible heme
oxygenase-1 and a constitutive heme oxygenase-2. HMOX1 and HMOX2 belong to the heme
oxygenase family
The 70 kilodalton heat shock proteins (Hsp70s) are a family of conserved ubiquitously
expressed heat shock proteins. Proteins with similar structure exist in virtually all living
organisms. The Hsp70s are an important part of the cell's machinery for protein folding, and help
to protect cells from stress. In cooperation with other chaperones, Hsp70s stabilize preexistent
proteins against aggregation and mediate the folding of newly translated polypeptides in the
cytosol as well as within organelles. These chaperones participate in all these processes through
their ability to recognize nonnative conformations of other proteins. They bind extended peptide
segments with a net hydrophobic character exposed by polypeptides during translation and
membrane translocation, or following stress-induced damage. In case of rotavirus A infection,
serves as a post-attachment receptor for the virus to facilitate entry into the cell.
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) or ABTS is chemical compound used
to observe the reaction kinetics of specific enzymes. A common use for it is in the enzymelinked immunosorbent assay (ELISA) to detect for binding of molecules to each other.
It is commonly used as a substrate with hydrogen peroxide for a peroxidase enzyme or alone
with blue multicopper oxidase enzymes such aslaccase or bilirubin oxidase. Its use allows the
reaction kinetics of peroxidases themselves to be followed. In this way it also can be used to
indirectly follow the reaction kinetics of any hydrogen peroxide-producing enzyme, or to simply
quantify the amount of hydrogen peroxide in a sample.

PROCEDURE1) Coat the well with Ag diluted in carbonated buffer (100 ul/well).Keep it overnight at 40C in
humid chamber.
2)

Flick the plate to throw the Ag and blockthe unbound space with 1% BSA in 0.1M
PBS(100ul/well).

3) Incubate at 370C ,1 hour.

4)

Wash the plate (after flicking) with PBS-Tween 20 (125 ul/250ml of PBS) by filling, and
then flicking.[Each washing should be for atleast 3 minutes and give atleast 3-5
washings]

5) Dilute the primary Ab in PBS-Tween containing 1% BSA.

6) Add 100ul to each well, incubate for 2 hours at 370C .
7) Flick and wash with PBS_Tween.
8) Dilute the Secondary Ab in PBS-Tween containing 1% BSA & 100ul/well.
9) Keep at 370C for 2 hours and then flick & wash.
10) PBS-Tween act as inhibitor of ABTS ,therefore,give one last washing with ddH2O before
adding ABTS.
11) Prepare substrate, add100ul to each well.
12) Keep in dark for 30 minutes.
13) Quantitative colour reaction at 405 nm.
OBSERVATION-

S.No.

NAME

SAMPLE

1
2
3
4
5
6

C1 K
C2 L
C2 T
T4 K
T4 L
T4 TE

12.9
16.428
27.264
12.624
19.84
28.56

RESULTS-

BUFFER(micro
liter)
1200
1200
1200
1200
1200
1200