LAPORAN PRAKTIKUM 6

TOXICOLOGY

CYANIDE INTOXICATION

Dosen: Drh Diah Nugrahani Pristihadi, MSi

Kelompok 3/ Paralel 4

Heng Xin Mei B04188002

Lynette Ong Huey B04188008

Keertana a/p Silvarajoo B04188028

Cassie Angeline Tanjaya B04188029

Yogi Nikmatul Maula B04188033

Pharmacology and Toxicology Division

Department of Anatomy, Physiology and Pharmacology

Faculty of Veterinary Medicine

IPB University

2021
 INTRODUCTION

Cyanide is a chemical compound contains C≡N which is cyano group

made up of a carbon atom with triple-bond to nitrogen atom. Cyanide is
rapid-acting and can exist in many forms. Hydrogen cyanide (HCN) or cyanogen
chloride (CNCl) exist as colorless gas, whereas sodium cyanide (NaCN) or
potassium cyanide (KCN) are in crystal form. Natural substances such as almonds
contain cyanide, as well as manufacturing and industrial sources such as
insecticides or jewelry cleaners (Graham 2021). Cigarette smoke or smoke
inhalation from fire, or plants such as apricot pits, cassava etc. contains cyanide.
Cyanide has been used as an agent of genocide in gas chambers during the WWII
(Parker-Cote et al. 2018).

Cyanide poisoning is rare but it can be deadly. Cyanide is able to inhibit

oxidative phosphorylation, in which cells use enzymes to oxidize nutrients,
releasing the chemical energy stored within the nutrients in order to produce
adenosine triphosphate/ATP. Cyanide binds to the Fe3+ of the cytochrome C
oxidase enzyme which then blocks the transport chain in mitochondria. This leads
to cellular hypoxia as well as ATP depletion. Utilization of oxygen by tissue
occurs, followed by vital function impairment (Pauluhn 2016). The prolonged
lack of oxygen causes accumulation of lactic acid in the body as the end product
of glycolysis. Excess H+ ions further lead to metabolic acidosis (Fitzgerald 2013).
Besides, there will be a lack of ATPs as well since the oxidative phosphorylation
process is disturbed, the body will start to break down other tissues to convert
either protein or lipids into energy. Lipid breakdowns often lead to ketoacidosis
which can be life threatening if left untreated. Some of the clinical manifestations
of cyanide poisoning include general weakness, confusion, bizarre behavior,
excessive sleepiness, coma, shortness of breath, headache, dizziness, vomiting,
abdominal pain, and seizures.

LITERATURE REVIEW

2.1 Cyanide

Cyanide can be found in natural sources or manufactured sources. It is a

chemical compound that has CN group, also known as cyano group, which can be
found in many forms; from simple to complex cyanide, cyanogens, cyanide ions
to nitrile compounds that can be in solid, liquids or gas form (Schnepp 2006).
Natural resources of cyanide include hydrogen cyanide and cyanogenic glycosides
in more than 200 types of plants. The hydrogen cyanide is ubiquitous in nature,
which is released into the atmosphere by biogenic processes, biomass burning,
and volcanos. Cyanogenic glycosides are found in many plants such as cassava,
linseeds, stone fruits, lima beans, bamboos and others. Anthropogenic sources of
cyanide can be derived from metal mining processes, organic chemical industries,
iron and steel plants or manufacturers, vehicle exhaust and cyanide containing
pesticides. The most common forms of these cyanides are calcium cyanides,
potassium cyanide, and hydrogen cyanide (Kuliahsari et al. 2021).

Although there are many forms of cyanide, the toxicity mechanisms are
similar, which is the cyanide ions that are being absorbed to the body by
inhalation, ingestion or dermal exposure. The primary toxic agent is HCN, which
interferes with the aerobic metabolism of the body. Once the cyanide is absorbed,
cyanide is transferred in the blood, which can be metabolized in the body to be
excreted. Most cyanide is converted to thiocyanate, which is formed primarily in
the liver and then excreted by the kidneys. A large amount of cyanide intake will
lead to toxicity. The cyanide inhibits cytochrome oxidase, specifically the a3-CuB
binuclear center of cytochrome c oxidase. This enzyme is functioned on cellular
respiration by binding with haem iron, thereby inhibiting the respiratory process
which eventually leads to histotoxic hypoxia. Other than that, cyanide also binds
with the terminal enzyme of the mitochondrial electron transport chain, impaired
the function of tissues to carry out oxygen exchange. According to WHO (2004),
cyanide can also inhibit approximately 40 enzymes such as metalloenzymes
containing iron, copper, or molybdenum.

The sign of cyanide intoxication includes excitement, dizziness, nausea

and vomiting, then progressing to tachycardia, chest tightness and dyspnoea,
cyanosis, convulsions, coma and eventually death. In acute poisoning, dyspnea,
tachycardia, “bitter almond” breath smell, salivation, excess lacrimation, voiding
of urine and feces, vomiting may occur. Animals may stagger and struggle before
collapse. Venous blood is classically described as “cherry red”. Ruminant animals,
especially cattle and sheeps are more susceptible than monogastric animals.
Chronic exposure may lead to headaches, fatigue, eye problems, thyroid
dysfunction and other health issues. Hypothyroidism with or without goiter,
chronic cyanide and plant cyanide metabolite–associated neuropathy toxidromes
may occur. Posterior ataxia, flaccid paralysis, cystitis secondary to urinary
incontinence, late-term abortion and musculoskeletal teratogenesis may also
occur. Treatment of acute large amounts of cyanide can be done by administering
the antidotes. The antidotes of cyanide poisoning include sodium thiosulfate, amyl
nitrite, sodium nitrite, 4-dimethylaminophenol, hydroxocobalamin, dicobalt
edetate, and others.
2.2 Cassava

Cassava (Manihot esculenta) is a plant containing cyanogenic glycosides,

known as linamarin and lotaustralin. It is an edible tuberous root, and acts as a
major source of carbohydrates in tropical areas. This crop is resistant to drought,
pests, and disease, which makes this crop to become one of the staple food in
tropical areas.

In general, the cyanogenic glycosides in plants are relatively nontoxic. The

toxicity shows when free hydrogen cyanide is produced from the glycoside by two
processes. When plant cells are damaged or stressed through crushing, chewing,
frost, drought, and other conditions, the glycoside in the plants contact with the
enzymes such as glycosidases and lyases. This will increase the HCN level, which
is highly poisonous to all animals after being absorbed through the gut. It may
cause acute or chronic toxicity in animals. The cyanide levels in cassava tubers
can be reduced by using simple traditional methods such as boiling, roasting,
drying, grating, soaking and fermentation (Gupta 2019).

2.3 Antidotes for Cyanide

Antidote is a drug, chelating substance, or a chemical that counteracts the

effects of another drug or a poison. The antidotes of cyanide poisoning include
sodium thiosulfate, amyl nitrite, sodium nitrite, 4-dimethylaminophenol,
hydroxocobalamin, dicobalt edetate, and others. Based on their mode of actions,
the antidotes can be divided into four groups, which are: the first, substances that
increase the metabolism of cyanide such as sodium thiosulphate which rely on the
endogenous cyanide metabolism pathway (Bhattacharya 2000). The second group
are the substances that bind to cyanide such as hydroxocobalamin and dicobalt
edetate. This group of substances act by formation of non toxic cyanocobalamin.
Another type of substance is methaemoglobin producer, which reacts with cyanide
to form non-toxic cyanmethemoglobin. This includes amyl nitrite, sodium nitrite,
and 4-dimethylaminophenol. Lastly, fourth substances are by reducing the
absorption of ingested cyanide such as ferrous sulphate dissolved in aqueous citric
acid and aqueous sodium carbonate (Reade et al. 2012). In this practicum, sodium
nitrate and sodium thiosulfate are discussed.

AIM

This practice aims to identify cyanide in plants and animals, and determine
the clinical symptoms of cyanide poisoning and the effect of cyanide antidote.
 MATERIALS AND TOOLS

Tools that are used in this practicum are test tube, cassava leaves, cyanide
1%, aquades, NaCN 1%, HCl 5%, NaOH, FeSO₄, FeCl3, NaCN 1%, NaNO₂ 1 %,
Na₂S₂O₃, concentrated HCl, water bath, pipette, tweezers, picrate paper, mortar,
test tube clamp, syringe, rabbit.

METHODOLOGY

Identification of cyanide present in the plants was started by put aquades

into the first test tube as a negative control, 2 or 3 ml of NaCN 1% and 5 drops of
HCl 5 % into the second test tube as positive control, and cassava leaves which
has been crushed before into the third test tube. After that the picrate papers are
put in the top of the three tubes then the tubes are closed by its cover. These tubes
are then heated on the water bath for minutes. During this time the changes of
these tubes were observed.

Next experiment is about identification of cyanide from animal samples.

The observation was started by putting NaCl 1% into a test tube, then continued
by putting NaOH 1ml, 3 drops of FeSO₄, 3 drops of FeCl3 sequentially. The tube
was then heated in the water bath for a few minutes. After that, wait for the
solution to cool down. Concentrated HCl was then put into the tube and the colour
changes were observed.

The last observation is about the clinical symptoms of cyanide and

antidote poisoning. This observation started by weighing the rabbit, then
preparing both NaNO₂ and Na₂S₂O₃ solution into two different syringes for about
2,5ml in volume. NaCN solution was taken by the dose 5-10mg/kg body weight
and put in the rabbit’s mouth. After that the clinical symptoms were observed.
Next, the antidotes solution was given to the rabbit. Lastly, NaNO₂ solution was
injected and continued by Na₂S₂O₃ injection. The rabbit’s status is being observed.

RESULT

Table 1. Identification of cyanide from plant sample

No. Test tube Color changes

1. Aquades (negative control) No changes

2. Cassava leaves Yellow to brick red

 3. NaCN 1% + HCI 5% (positive control) Yellow to brick red

Table 2. Identification of cyanide from animal sample

No. Test tube Color changes

1 NaCN 1% + 1 mL 50% NaOH + 3 drops of Brick red to prussian

FeSO₄ + 3 drops of FeCl3 and concentrated blue
HCI

Table 3. Clinical symptoms of cyanide and antidote poisoning

No Time Clinical symptoms

1. 0 minute Rapid breathing, dilation of pupils.

2. 10 minutes Hypersalivation, rapid and shallow breathing,

dilation of pupil, incoordination

3. After antidote given Pupil size, breathing and coordination back to

normal.

DISCUSSION

Cyanide is a toxic substance found in several tubers such as cassava

(Manihot esculenta), wild yam (Dioscorea hispida Dennts), some cerealia and
legumes. In the plants, it can be in the form of cyanogenic glycosides, acetone
cyanohydrin, and hydrogen cyanide (HCN) (Kuliahsari et al. 2021). Cassava
contains two different cyanogenic glucosides, linamarin and lotaustralin. Roots
and leaves contain the highest amount of linamarin. Linamarin produces the toxic
compound hydrogen cyanide (HCN) which can be hazardous to the consumer.
Linamarin is the most representative glucoside accounting for about 80% of the
total cassava glucoside (Cereda and Mattos 1996). The compartmentalization of
linamarin and lotaustralin in the vacuole and linamarase and HNL in the cell wall
prevents the formation of toxic cyanide in undamaged cells. If the cassava leaves
are crushed, sliced or chewed, causing damage to the cell walls of the tissue, the
linamarin and lotaustralin are in contact with linamarase and oxygen enzymes to
produce glucose, acetone, and cyanide acid causing chronic cyanide toxicity
among populations subsisting on cassava (Anjani et al. 2021). The breakdown of
the physical barriers between substrates and the enzymes, following tissue
damage, initiates cyanogenesis which is the potential toxicity of cassava related to
the capacity of all parts of the plant to release hydrogen cyanide from stored
cyanogenic glucosides
The alkaline picrate solution acted as a trapping agent of the liberated
HCN that evaporated. The HCN liberated slowly changed the colour of the picrate
paper strips from orange to red at 28± 20℃ (Nwokoro et al. 2010). Based on the
result of the experiment, the positive control test tube filled with NaCN 1% + HCI
5% solution showed change in color on the picrate paper and there is no change in
the negative control with aquades. This proves that traces of cyanide were found
in the cassava leaves, because the cassava leaves produce hydrogen cyanide
vapour which reacts with alkaline picrate test strips to form brick red colour on
the test strips. The colour form in the cassava leaves is less intense than positive
control due to the cyanide concentration being lower.
Cyanide is a well known potent inhibitor of haem proteins, including haem
oxygenase. Cyanide binds tightly to the ferric haem iron and inhibits the binding
of intrinsic ligands to the haem iron of a given haem protein. Generally, cyanide
coordinates to the ferric haem iron with a linear binding geometry; the Fe—C—N
angle ranges from 160 to 180°(Sugishima et al. 2007). In the experiment of
identification of cyanide from animal samples, the animal product was substituted
with NaCN. Sodium cyanide was tested against water and the test tube with
sodium cyanide turned blue upon adding HCl however the water did not. This is
because of the formation of Prussian Blue that shows the presence of ferrous
ferrocyanide (Fe⁴ (Fe(CN)6)³. The Prussian Blue colour indicates that cyanide is
tightly bound with iron.
The toxicity of cyanide is linked mainly to the cessation of aerobic cell
metabolism. Cyanide's main effect is that it inhibits oxidative phosphorylation, a
process where oxygen is utilized for the production of essential cellular energy
sources in the form of ATP. It does so by binding to the enzyme cytochrome C
oxidase and blocks the mitochondrial transport chain. After that, cellular hypoxia
and the depletion of ATP occur, leading to metabolic acidosis. The utilization of
oxygen by the tissue occurs and is followed by the impairment of vital functions
(Huzar et al. 2013).
The symptoms shown due to cyanide poisoning include dilated pupils,
weakness, and high respiratory rates. This occurs because cyanide irritates the
mucosa, both in the eyes, respiratory and digestive organs. This irritation is
mainly due to the strong alkaline strength of the hydrolysis of the sodium and
potassium cyanide salts. The toxic effect of cyanide is to block the uptake and use
of oxygen, resulting in low levels of oxygen in the tissues. Adverse effects due to
cyanide exposure can be seen prominently on the central nervous system because
of the high metabolic demand for oxygen in neurons and its control of respiratory
function. Initial stimulation of carotid and aortic bodies and effects on the central
nervous system adversely affect the function of the respiratory system.
Intravenous and inhalation of cyanide produce a more rapid onset of signs and
symptoms than exposure via the oral or transdermal route. This is due to the first
two routes providing fast diffusion into the bloodstream (Nelson 2006).
The antidote with the combination of NaNO₂ (sodium nitrite) and
Na₂S₂O₃(sodium thiosulfate) compounds was administered to treat cyanide
poisoning. Once the antidote was administered, it recovered from toxicity by
gaining back pupil size, breathing and coordination back to normal. The recovery
from toxicity is due to excretion of cyanide which is the formation of thiocyanate
(SCN-) which is excreted in the urine. Sodium nitrite oxidises the iron component
of haemoglobin, resulting in formation of methaemoglobin which cyanide binds
to preferentially over cytochrome oxidase (Braitberg 2019). This allows
resumption of cytochrome oxidase activity and the subsequent restoration of
aerobic cellular metabolism. The effectiveness of thiosulfate as a cyanide antidote
is markedly increased when combined with methemoglobin generators, and
produces a marked increase in cyanide antagonism. The addition of sodium
thiosulfate converts the cyanide to thiocyanate, which is excreted renally, while
the iron in the hemoglobin is restored to the ferrous state (Steven et al. 1992).

CONCLUSION

Cyanide is a toxic substance that affects the central nervous system and
respiratory system mainly by inhibiting oxidative phosphorylation which results
in low oxygen level in tissues. However, antidotes can be used to treat cyanide
poisoning by helping gain back pupil size, breathing and coordinating back to
normal. The antidote combination used for cyanide poisoning is sodium nitrite
and sodium thiosulfate. Sodium cyanide turns blue after adding HCl shows
presence of ferrocyanide indicates that cyanide is tightly bound with iron.
Cyanogenic glycosides content in cassava roots and leaves can lead to cyanide
poisoning.
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