INTRODUCTION DRUGS INDUCED HEPATOTOXICITY TYPES OF HEPATOTOXICITY

1. Hepatoprotective
Daksha Gupta

2. Introduction
 Hepatotoxicity (from hepatic toxicity) implies
chemical-driven liver damage.
 Can be caused by-
1. Certain medicinal agents.
2. Chemical agents used in labs and industries
3. Natural chemicals, For e.g. Microcystins.
4. Herbal remedies.
 Chemicals that cuses liver injury- Hepatotoxins.
 Hepatic reaction to a drug could be
hepatocellular necrosis, fatty change and a
mixed or cholestatic hepatitis.

3. Drug induced Hepatotoxicity

4. Types of Hepatotoxicity
 Symptomatic-
Have predictable dose-response curves.
Higher concentrations cause more liver damage.
Well characterized mechanisms of toxicity.
 Idiosyncratic-
Does not have a clear dose-response or temporal
relationship.
Causes liver damage in only a small fraction of
the population that is exposed to the agent.
Most often do not have predictive models.
Led to withdrawl of drugs like-
Troglitazone (Rezulin®), Ranitidine (Zantac®),
and trovafloxacin (Trovan®)

5. Classification of Hepatotoxic drugs
 A] Natural origin or hepatotoxic herbs:
Eg. Tannic acid, pyrrolizidine alkaloids,
gyrometrin, amatoxins etc.
 B] Synthetic origin:
Therapeutically important hepatotoxin:
Eg. Paracetamol, isoniazide, sulphonamides,
para- amino salicylic acid, ethanol, rifampicin
etc.
Hepatotoxin used for experimental purpose:
Eg. Carbon tetrachloride, chloroform,
thioacetamide etc.

6. Mechanism of Hepatotoxicity
 Paracetamol- Symptomatic hepatotoxin
Hepatotoxic in overdose
Paracetamol  metabolism by glucuronidation
and sulfation  non-toxic metabolite  excreted
in urine.
Small portion of drug  oxidation by cytochrome
P450  highly reactive metabolite N-acetyl–P-
benzoquinoneimine [NAPQI]  sulfhydryl group
of protein  cell necrosis and lipid per oxidation
in the liver.
NAPQI is in cell detoxified by reacting with
glutathione. When the amount of active molecule
formed exceeds the glutathione available for
binding, it causes necrosis.

7.  Carbon tetra chloride CCL4
Mechanism is complex, multifactorial, and not
completely understood.
CCl4 hepatotoxicity has two distinct
manifestations-
Fat accumulation and hepatic necrosis.
 Thioacetamide
It interferes with the movement of RNA from
nucleus to cytoplasm which further leads to
membrane injury.

8. Drugs used as Hepatoprotective
 Guava- Psidium
guajava Linn.
 Nyctanthes
Arbortristis
 Yellow Sweet Clover
(Melilotus officinalis)
 SILYBUM MARINUM

9.  Kalamegha-
Andrographis
paniculata
 Olive leaves- contains
oleanolic acid
 Kakamachi (Black
night shade) Solanum
nigrum
 Vitex negundo against
carbon tetrachloride-
induced liver damage

10. Screening
 Induce liver damage in experimental animals.
 The test substance is administered prior to or
after the toxin treatment.
 If the hepatotoxicity is prevented or reduced, the
test substance is effective.
 There are various models of inducing
hepatotoxicity in rodents (rats and mice).

11. Screening Methods
 Hepatitis in Long Evans Cinnamon rats-
Useful model to study genetically transmitted
hepatitis and chronic liver diseases.
Underlying cause is thought to be due to
excessive copper accumulation in liver of Long
Evans Cinnamon rats, thus making this animal a
model for Wilson’s disease.

12.  Procedure-
Male Long Evans Cinnamon rats (age- 5 weeks)
are housed in temperature and humidity
controlled rooms at 12:12 light/dark cycle.
↓
Groups of 6-10 rats are given different diets
based on a 15% purified egg protein diet and
supplemented with vitamins or drugs (applied
with minipumps intraperitoneally implanted under
anaesthesia).
↓
Jaundice is observed when ear and tail turns
yellow and urine- bright orange, staining fur in
lower abdominal region.
↓
Jaundice worsens, ending in death of the animal
within about a week.
↓
Incidence of jaundice and mortality vs. time are
used as parameters to measure the extent of
hepatoprotective activity.

13. Allyl alcohol induced liver necrosis in rats
Procedure-
Albino rats (weight 120-150g) are used.
↓
Food but not the water is withdrawn.
↓
After 6 hr test sample (for protective activity) is
administered i.p. or orally.
↓
1 hr later, 0.4 ml/kg of 1.25% solution of allyl
alcohol in water is dosed orally to animals.
↓
Next morning, the treatment is repeated with
potentially protective drugs.
↓

14. Food but not water is withheld until the 3rd day.
↓
Next morning animal is sacrificed and liver is
removed.
↓
The parietal sides of the liver are checked with
stereomicroscope (dissecting microscopes) with
25 times magnification.
↓
Focal necrosis is observed as white-green or
yellowish haemorrhagic areas clearly separated
from unaffected tissue.
↓
The diameter of the necrotic areas is determined
using an ocular micrometer.
↓
These values are added for each animal to obtain
an index for necrosis.

15. Carbon tetrachloride induced hepatotoxicity
Procedure-
Rats are fasted for 16 hr. then divided into
several groups.
↓
One group (control group)- normal saline only.
↓
Animal of other group- CCl4 [3 mg/ kg body
weight in 50% olive oil solution] is administered
to by subcutaneous [S.C.] injection at the back.
↓
The test drug at varying concentration depending
on the design of the experiment is administered.
↓

16. Reference drug is administered to different
groups, at 2 hr before and 24 & 48 hr after CCl4
administration. In the last two groups, CCl4 and
the vehicle are given with no additional treatment
↓
All animals are killed 72 hr after CCl4
administration, whole blood is drawn from the
carotid artery and serum is separated for the
different assays.
↓
Liver tissues are removed, and then liver is fixed
in 10% neutral formalin.

17. Paracetamol [acetaminophen] induced
hepatotoxicity
Procedure-
Rats are fasted for 16 hrs. , then divided into
several groups.
↓
One group served as control, receiving normal
saline only.
↓
Paracetamol [5oo mg/kg, dissolved in 25% PEG
400, i.p.] is administered to animals of the other
group.
↓
The test drug at varying concentration depending
on the design of the experiment is administered.
↓

18. Reference drug are administered to different
groups, 2 hr before and 2 or 6 hr after
paracetamol administration.
↓
In the last two groups paracetamol and the
vehicle are given with no additional treatment.
↓
All animals are killed 24 hr after paracetamol
administration, whole blood is drawn from the
carotid artery and serum is separated for the
different assays.
↓
Liver tissues are removed, and then liver is fixed
in 10% neutral formalin.

19. Country Made Liquor (CML) model
CML (containing 28.5% alcohol) is used to
produce hepatotoxicity in this model.
 Procedure-
Rats are divided into groups of 8 each.
↓
The control group receives 1 % gum acacia as
vehicle, corn oil (1 ml/100 g/day) and glucose
isocaloric to the amount of alcohol.
↓
The positive control group receives CML (3
ml/100 g/day) in two divided doses and corn oil
(1 ml/100 g/day) in a single dose.
↓
Other test groups receive drugs in respective
doses along with CML (3 ml/100 g/day) and corn
oil.
↓

20. After 21 days, the blood is withdrawn for analysis
of SGOT, SGPT, alkaline phosphate, serum
cholesterol, albumin, total proteins, bilirubin,
glucose, and creatinine.
↓
The rats are sacrificed and livers dissected out for
Histopathological analysis.
↓
The value of the test is compared with the control
using suitable statistical analysis.

21. Thioacetamide induced hepatotoxicity [TAA]:
Adult female Wistar rats 180-200 gm are kept in
wire bottomed cages at controlled temperature
[210C ± 20C] with a 12 hr light-dark cycle for at
least a week before the treatment.
↓
The animals are given ad libitum access to food
and tap water, the food is to be withdrawn 18 hr
before the intoxication.
↓
The animals are randomly divided into several
groups. These groups are pretreated with
different antihepatotoxic agents [test drug at
various concentrations].
↓
All agents were dissolved in saline and
administered i.p. 24 hr before TAA intoxication.
↓

22. The blank and TAA groups [groups B &T,
respectively] received the same volume of vehicle
as the treated groups.
↓
Hepatotoxicity is induced in all except for the B
group, by oral administration of saline –dissolved
in TAA.
↓
After 24 hr of TAA intoxication, the rats are i.p.
anaesthetized with 1.25 g/ kg body wt. of
urethane.
↓
Blood is collected from the abdominal aorta in
hepatinized tubes, and centrifuged at 3000 rpm
for 15 min. at 40 C to obtain plasma
↓
Livers are removed, washed with cold saline,
weighed and divided into several slices, which are
immediately frozen and stored at -800C until
analyzed.

23. Alcohol and Carbon Tetrachloride induced
hepatotoxicity
After acclimatization, the animals are divided into
five groups of 8 mice each.
↓
Group 1 animals are given distilled water per os
[p.o.] at 1 ml/kg body weight once daily for
successive seven days [normal group].
↓
The animals of group 2 to 5- 40 % v/v ethanol
[10 ml/kg body weight, p.o.] once daily for seven
consecutive days.
↓
On the 7th day, each animal received 0.2 ml of
20 % v/v CCl4 in olive oil by stomach tube 1 hr
after ethanol administration. In addition to these
treatments, the animals of group 3, 4, 5 are
administered with varying concentration of the
test drugs once daily 1 hr following ethanol or
CCl4 treatment from 5th to 7th day.

24. ↓
The animals from each group are fasted overnight
prior to sacrifice.
↓
24 hr after the last treatment with distilled water
or CCl4, all mice are sacrificed under light
anesthesia.
↓
Blood is to be collected directly through heart
puncture before scarify and centrifuge at 2000
rpm for 10 min after keeping it at room
temperature for 1 hr.
↓
The separated serum is collected and kept at 4O
C for biochemical estimation.
↓
 Livers are promptly excised, washed with chilled
normal saline and quickly homogenized in ice
cold 0.15 KCL to yield a 10 % w/v liver
homogenate.

25. Parameters for evaluation
 The serum ALT & AST values are measured. The
changes in the serum AST & ALT after test drug
administration on the liver injury model are
observed. For the hepatotoxic drug there are
significant increased in serum AST & ALT, SGOT,
SGPT, ALP and bilirubin values compared with the
control groups.

26. Histopathological observation
 In the histopathological observation of liver
tissues after various treatments following
parameters are studied-
1. Gross necrosis in the centribular area
2. Sinusoidal congestion
3. Infiltration of the lymphocytes
4. Kuffer cells around the central vein and
5. Losses of cell foundries are observed.

27. REFERENCES
 Vogel, H Herald in, ‘Drug Discovery &Evaluation:
Pharmacological Assays’.
 S. S. Aagrawal, M. Paridhavi in ‘Herbal Drug
Technology’

28. 