NEUROTOXICITY OF CYCASIN IN CHICK EMBRYO

NEUROTOXICITY OF CYCASIN

IN

CHICK EMBRYO

ROBERT BROWN FOR THE FIRST TIME POINTED OUT THAT…..

WHAT HAD TILL BEEN REGARDED AS THE FEMALE FLOWER OF THE CYCADS WAS ACTUALLY

A NAKED OVULE

Robert Brown (1773–1858)

AS A RESULT OF THIS REMARKABLE DISCOVERY…………

CYCAS WAS EXCLUDED FROM DICOTYLEDONS &………

CAME TO RECOGNISED AS ..

GYMNOSPERM

……….However the first use of the Greek word gymnosperm have been already made as early as 300 b.c by the Greek philospher ……

THEOPHRASTUS in his “ENQUIRY INTO PLANTS”

GYMNOSPERM…..

An ancient group of plants dating back to the upper Devonian , roughly about 375 million year ago……..

A group of vascular plants whose seeds are not enclosed by a ripened ovary (fruit).

A group of plant which lack double fertilisation in the formation of endosperm

They are true land plants………..

Because they show……….

•Protection of gametophyte and young developing sporophyte from dessication and injury .

•Provision of nourishment for their proper development.

•Freedom from external water for the transference of the gametes

Gymnosperm achieved all these conditions by mustering 3 succesive generation in a single organ…….

SEED……..

•The parent sporophyte as …integuments & nucellus

•The gametophyte as ….endosperm

•The daughter sporophyte as……. embryo

integuments

nucellus

endosperm

embryo

gymnosperm

Cycadophyta (cycads)

Ginkophyta

(ginko)

Coniferophyta (conifers)

Gnetophyta (gnetae)

CYCAS

• SCIENTIFIC CLASSIFICATION

• Kingdom: PLANTAE Division: CYCADOPHYTAClass: CYCADOPSIDAOrder: CYCADALESFamily: CycadaceaePersoonGenus: Cycas

– They are evergreen, gymnospermous and dioceious plants having large pinnately compound leaves.

CYCAS…….

About 95 species are currently accepted. The best-known species is Cycas revoluta widely cultivated under the name "Sago Palm" or "King Sago Palm" due to its palm-like appearance although it is not a true palm

Approximate world distribution of Cycadales

EXPERIMENT……..

COMMENCMENT : 10 FEB 2008

FINISHED : 11 APRIL 2008

DURATION : ALMOST 2 MONTH

AIM…

TO STUDY THE EFFECT OF NEUROTOXIN PRESENT IN CYCAS SEEDS IN GROWING CHICK EMBRYO

MATERIAL REQUIRED….

1.PHYICAL APPARATUS

.CHINA DISH

.PISTAL & MORTAR

.BEAKER

.CUTTER

.PETRIDISH

.EGG STAND

.WATCH GLASS

2.CHEMICAL

.ETHANOL

.DISTILLED WATER

.FORMALDEHYDE

.SODIUM CHLORIDE(NaCl): 8g

.CALCIUM CHLORIDE(CaCl2):0.24g

.POTTASIUM CHLORIDE(KCl) :0.42g

.SODIUM BICARBONATE(NaHCO3):0.20g

3.BIOLOGICAL MATERIAL

.Cycas pectinata

.fertilised egg of hen

2 days old

4 days old

9 days old

15 days old

INSTRUMENTS……

.AN OVEN (set at 52 degree celcius )

this temperature depends on environmental condition.

.AN INCUBATOR (set at 37 degree celsius)

we used orbital shaker

.A DIGITAL CAMERA(for the pics)

Theory ……

Cycads produce several secondry compounds that arecarcinogenic and neurotoxic. Because of their uniqueplacement in terrestrial plant evolution, molecularstudies should help to define the origins of structuresthat led to the rise of seed plants and the role of neurotoxiccompounds that are found in cycads.

The subsequent search for a neurotoxiccompound in cycads lead to the identification of a

•Novel non-protein amino acid called BMAA (beta-methylamino L-alanine)

•methylazoxymethanol(MAM)-glycocides( cycasin macrozamin and neocycasin )

•sterol beta-D glucoside

STRUCTURE……

CYCASIN N MACROZAMIN

The toxic azoxyglycosides from the seeds of several species of Cycas.

Carcinogenic in mice, rats, hamsters, guinea-pigs, rabbits and fish.

Azoxyglycosides may have played an important ecological role as antiherbivore defenses.

ELECTRICAL

WEIGHING MACHINEWEIGHING LIMIT:

FOR MICROGRAM AND GRAM

Cycasin

1.3. Molecular formula, weight and structure:

2.C8H16N2O7; 253.233.4. Density: No data.4.5. Absorption spectroscopy: UV

absorption spectra have been reported (Riggs, 1956); λ (log ε)= 2187.

5.Solubility: Very soluble in water and dilute ethanol; less soluble in absolute ethanol; insoluble in

6.organic solvents.7.8. Description, appearance: Colorless, long,

needle-like crystals.

METABOLISM OF CYCASIN AND MACROZAMIN

The active component of all theses compounds is MAM (methylazoxyglycoside),

which is metabolised to a toxic intermediate by the p450 enzyme present in the liver and in the olfactory epithelium in the nose and inner membrane of mitochondria

TOXIC PRINCIPLEMETABOLISED

BY P450 ENZYME

NON TOXIC FORM

P450 ENZYME

Cytochrome P450 (abbreviated CYP, P450, infrequently CYP450) is a very large and diverse superfamily of hemoproteins found in all domains of life…

ncluding mammals,birds, fish, insects, worms, sea squirts, sea urchins, plants, fungi slime molds, bacteria and archaea

the most common reaction catalysed by cytochrome P450 is a monooxygenase reaction :RH + O2 + 2H+ + 2e– → ROH + H2O

PROCEDURE….

our first aim to isolate cycasin from cycas seeds..!

steps :

1 we were provided with 7 seeds of cycas pectinata

2 seed coat of cycas seed was quite hard so we first isolated seed form seed coat with the help of cutter.

3 we placed the naked cycas seeds in petridish

CYCAS SEED COVERED WITH HARD SEED COAT

MAIN CYCAS SEEDNAKED SEED

4 since before extraction of cycasin we need to quantitise the cycasin in cycas seeds, therefore we measured them in electrical weighing machine one by one and recorded them.RECORD : SEEDS WET WEIGHT(grams)

1 12.41

2 12.08

3 10.16

4 9.84

5 10.42

6 8.32

7 7.31

WET CYCAS SEEDS

PARTION BETWEEN THE SEED

5 since seeds were wet so we kept them in oven at 54 degree celcious to dry them.

6 our next step will be the crushing of dried seeds and soaking in water for extraction of cycasin.........

RECORD EARLIER WEIGHT : DRY WEIGHT1 12.41 : 6.50

2 12.08 : 4.71

3 10.16 : 4.922

4 9.84 : 5.884

5 10.42 : 4.69

6 8.32 : 4.87

7 7.31 : 3.10

After drying cycas seeds for 4 days in oven at 54degree celcius,

it was crushed in mortar and pistil untill it become fined powedered.

the powdered form of seeds were again weighed....,and it was found that their dry weight were almost 50% less than there originall weight..!

CYCAS FLOUR

CYCASIN SOLUTION

SOL A

CONC: 0.2 g/ml

Wash : 1st

Wash : 2nd

SOL A

CONC: 0.2 g/mlWash : 1stWash : 2nd

SOL B

CONC: 0.1 g/mlWash : 1stWash : 2nd

SOL C

CONC: 0.05 g/mlWash : 1stWash : 2nd

CYCAS SOLUTION

Solute: cycas flour

Solvent: distill water

Concentration :0.1 g /ml

DEALING WITH FERTILISED EGG

steps:

1 firstly we fixed the embryo in appropriate size beaker in such a way that narrow portion of head facing downward and broder portion facing ourself.

2 then with the help of needle a hole was made in the broder portion of egg.

3 then widened this hole with the help of scissor , this should be done very carefully without disturbing the vitelline membrane.

NORMAL EGG (CONTROL): WITHOUT CYCAS SOLUTION

EXPERIMENTAL EGG WITH CYCAS SOLUTION

INVITRO FERTILISED EGG

HOLES ARE MADE WITH NEEDLES WITHOUT DISTURBING THE VITELLINE MEMBRANE

HOLES ARE ALSO MADE IN NORMAL EGG FOR EQUIVALENT PARAMETERES

EGGS WERE EXAMINED AFTER 6 HOURS AND 24 HOURS

FERTILISED EGG YOLK

ALBUMIN TURNED GREENISH

YOLK

2 DAYS OLD EGG TREATED WITH CYCAS SOLUTION OBSERVED AFTER 24 HOURS

MORE TRANSPARENT

CONTROL EGG 2 DAYS OLD WITHOUT CYCAS SOLUTION

THIK YOLK

ALBUMEN

AFTER 24 HOURS (EXPERIMENTAL EGG 4 DAYS OLD)

A NORMAL (CONTROL) 4 DAY OLD EGG

RED SPOT INDICATE THE NERVE NETWORK

RED PORTION DENOTES SOMATOCYTES

NORMAL EGG 4 DAYS OLD

CYCAS SOLUTION TREATED 4 DAYS OLD EGG

NORMAL EGG 4 DAYS OLD

NERVE STRANDEXPERIMENTAL EGG

NERVE STRAND IN CONTROL EGG

A 9 DAY OLD CHICK EMBRYO

TREATED WITH CYCAS SOLUTION ..

REMAINED ALIVE AFTER 6 HOURS WITH ALL THE CONCENTRATIONS HOWEVER DIED AFTER 24 HOURS WHEN TREATED WITH SOLUTION A

A 9 DAYS OLD CHICK EMBRYO

DIED AFTER 24 HOURS WHEN TREATED WITH SOL A(HIGHLY CONCENTRATED ,1ST WASH)

A 9 DAYS OLD CHICK EMBRYO( ALIVE) NORMAL I.E NOT TREATED WITH CYCAS SOLUTION

A 18 DAYS OLD CHICK EMBRYO

SURVIVE WITH ALL THE CONCENTRATION AND WASHS

EGG EXPERIMENT OBSERVATION INFERENCE

2 DAYS

OLD

SOL A

WASH 1

WASH 2

ALBUMEN TURNED GREENISH IN BOTH CASE

MAY BE BECAUSE OH HIGH CONCENTRATION TOXIC IN CYCAS SOLUTION

SOL B

WASH 1

WASH 2

ALBUMEN TURNED YELLOWNO CHANGE

FIRST WASH CONTAIN RELATIVELY HIGH AMOUNT OF TOXIN THAN 2ND WASH

SOL C

WASH 1

WASH 2

NO CHANGENO CHANGE

EXTREMLY SMALL QUANTITY OF TOXIN IN BOTH WASH

OBSERVATION &DESCUSSION

EGG EXPERIMENT OBSERVATION INFERENCE

4 DAYS OLD

SOL A

WASH 1 –6HOURS

24 HOURS

WASH 2-6 HOURS

24 HOURS

NERVE NETWORK DEVELOPMENT RETARTED IN E.E IN COMPARISON TO N.EEGG SPOILED AND A VERY FOUL SMELL CAME & NERVE DEVELOPMENT WAS VERY LITTLE IN COMPARISON 2 N.ENERVE NETWORK REMAINS ALMOST SAME IN BOTHNERVE RETARTED BUT FOUL SMELL DIDN’T COME

HIGH CONCENTRATION OF TOXIN MIGHT KILLED SOME OF THE SOMATOCYTES HIGH CONCENTRATION MIGHT KILLED ALL THE SOMATOCYTES IN E.E & DESTROYED THE EGG IN SUCH A LONG TIME

COMPARETIVELY LOW CON. AND VERY LESS TIMELOW CON. AND LONG ACTION TIME

EGG EXPERIMENT OBSERVATION INFERENCE

4 DAYS

OLD

SOL B

WASH 1 –6HOURS

24 HOURS

WASH 2-6 HOURS

24 HOURS

NOT DISHTINGUISHABLE OBSERVATION

NERVE NETWORK SHOW RETARDATION IN DEVELOPMENT

NO CHANGE ALMOST SAME

NO CHANGE ,E.E DIDN’T SPOIL

LOW CONCENTRATION &LESS ACTION TIME

LOW CONCENTRATION &BUT RELATIVELY LONG ACTION TIME

EXTREMLY LOW CONCENTRATION

EGG EXPERIMENT OBSERVATION INFERENCE

4 DAYS

OLD

SOL C

WASH 1 –6HOURS

24 HOURS

WASH 2-6 HOURS

24 HOURS

NO CHANGE

NO CHANGE

NO CHANGE

NO CHANGE

MIGHT BE DUE TO EXTREMLY LOW OR ALMOST NEGLIGIBLE CONCENTRATION OF TOXIN

EGG EXPERIMENT OBSERVATION INFERENCE

18 DAYS

OLD

SOL A

WASH 1 –6HOURS

24 HOURS

WASH 2-6 HOURS

24 HOURS

NO EXTERNAL OBSERVATION

CHICK REMAINED ALIVE IN ALL THE CONDITIONS

HIGH RESISTANCE TO TOXIN DUE TO WELL DEVELOP IMMUNE SYSTEM

MIGHT BE THERE WERE CHANGES IN NERVOUS SYSTEM ACCORDING TO STUDIES BUT WE WERE UNABLE TO FIND THAT

THANX……..

DR.RITA MAM

2. JIBAN SIR

3. B.T DEPARTMENT

4.SAKSHI MAM

5.SWATI MAM

6.INTERNET SOURCES,

7. INTRODUCTION TO GYMNOSPERM BY DIVYA DARSHAN PANT.

8 PLANT ENCYCLOPEADIA

9CHICK EMBRYOLOGY..

ROMEIO N JULLIET