ALTERNATIVE METHODS

IN

PESTICIDES TOXICOLOGY

Dr. Alaa Eldin Bayoumi Abd-ElKhalek Moustafa

X: Xenobiotic, Pesticide

EVALUATION OF TOXICITY IN VIVO

Prediction of the Toxicity on

the human and the Environment.

Laboratory Animal and/or

Aquatic system

Acute, subchronic, chronic and long-term

toxicity studies

High

Economic

Coast

500.000

to

1.500.000 $

Long time

2-3 years

1500 Animal

mouse 65 %

rat 22 %

other rodents 3.19 %

rabbit 1.62 %

reptiles and

amphibians 0.57 %

fish 4.80 %

ungulates 2.01 %

carnivores 0.55 %

pigs 0.46 %

monkeys 0.16 %

DISTRIBUTION OF SUFFERING BETWEEN ANIMAL SPECIES

HHISTORY OF THE ALTERNATIVE METHODS IN VITRO

1798, Jeremy Bentham : The question is not, can they reason, nor can they talk, but can they suffer?

Century XIX, Marchal Hall: (avoidance the duplication of experiments

and the inneseccary suffering of animals).

1959, Three R´s Principles by Russell and Burch: Principles of Human Experimental Technique.

(Progressive move to reduce, refine and replace the use of animals in experiments)

1967, UAA (The united action for Animals).

1969, FRAME (Fund for Replacement of Animals in Medical Experiments).

1971, European Center of Decumentation and information of the Alternative Methods.

1986, ECVAM (European Center for Validation of Alternative Methods).

1995, GTEMA, (Grupo de Trabajo Especializado en Métodos Alternativos).

Decrease

of Animals Number

PRINCIPLE OF THREE R´s

William Russell and Rex Burch (1959)

Progressive move to

reduce,

refine and

replace

the use of animals in experiments

Alternatives: methods which reduce the number of animals necessary for an

experiment, refine existing tests by minimizing animal distress, or replace

whole-animal use with in vitro other tests.

3Rs principles

Reduction: Cutting the number of animals involved in a scientific experiment to the bare

minimum that will give results that are

scientifically valid.

Refinement: Ensuring that any animals used in experiments experience the minimum of

suffering, and that their welfare is safe-

guarded in every possible way.

Replacement: The use of experimental techniques that do not involve living animals, such as

human volunteers, cell and tissue

culture, and computer modelling.

3Rs PRINCIPLES

Any strategy that will result in less animals being used to

obtain the same amount of information, limiting or avoiding

the subsequent use of additional animals.

Appropriate experimental design and appropriate

analysis of the results based on statistical principles.

1.- Reduction

1.1.- Environmental enrichment of animal housing.

1.2.- Laboratory staff must be well trained and competent in the handling

of the species.

1.3.- Anaesthesia and analgesia should be used whenever appropriate and

possible.

1.4.- Use techniques and parameters which are least invasive and which can

be assessed as early as possible, in order to minimise both the distress

caused to the animals and the duration of the study.

1.5.- At the end of the experiment, the most humane method of euthanasia

should be chosen.

2.- Refinement The term refinement signifies the modification of any procedures that

operate from the time a laboratory animal is born until its death, so

as to minimise the pain and distress experienced by the animal, and to

enhance its well-being.

Any experimental system which does not entail the

use of a whole, living animal Is Considered to be a

replacement alternative.

3.- Replacement

3.1.- information

3.2.- computer-based systems

3.3.- physico-chemical techniques

3.4.- the use of lower organisms and embryo stages

3.5.- cell, tissue and organ cultures

SOURCES OF ALTERNATIVE METHODS IN VITRO

Vertibrates , Animals

Microorganisms Bacteria : Vibrio fischeri(Microtox ), Pseudomonas, Fungi and Algae. Invertibrates, insects

*- Cultures of animal embryos

*- Organs Bath

*- Organs Perfusion

*- Explants and Tissue Culture

*-Cell Cultures Primary Culture or Established Cell lines

*- Subcellular fractions mitochondria, DNA, enzymes ...etc.

Embryo Fecunded egg

Kidney Pancreas

Liver Brain

Se

lecte

d A

nim

al

Liver

Data: Information, computer-based systems and physico-chemical techniques

CLASSIFICATION OF ALTERNATIVE METHODS

USING LIVING ORGANISMS

I- Alternative Methods at Biological Levels: Basal Cytotoxicity Tests: Irritation assay system

Microtox test system Ames Test Neutral Red Incorporation (NRI) assay Total Cellular Protein content LDH leakage test

Energy Metabolism Tests: MTT assay Cellular ATP content

Special Cytotoxicity Tests: Chromosome aberrations Sister Chromatid Exchange Reconstructed Human Skin Mechanism of Cellular Death (apoptosis).

II- Alternative Methods at Biochemical Levels: Measurement of Lipid peroxidation. Glutathione Redox balance, i.e. cellular glutathione content, (total, reduced or oxidized), cellular glutathione related enzymes (GST, Grd or Gpx).

I- Alternative Methods at Biological Levels:

THE IRRITECTION ASSAY SYSTEM (IAS )

IAS is a quantitative in vitro test method employed to detect, the ocular and dermal irritation potential of cosmetics, consumer products,

pharmaceuticals, pesticides and chemical raw materials.

Irritection data may be obtained in 5 h, vs.

the 2-3 w required for in vivo studies.

IAS are highly reproducible, allowing com-

Parative ranking of samples.

Studies have demonstrated that IAS results

are highly correlated with standard Draize

tests.

Vibrio Fischeri, under optimum growth conditions, it is a very

brightly glowing species.

THE MICROTOX ASSAY FOR TOXIC ASSESSMENT

The Microtox toxicity test system exposes natural luminescent bacteria

(Vibrio fischeri) to increasingly concentrated solutions of test sample. Reductions in the light output of the microorganisms are monitored

in a temperature controlled photometer, reflecting the toxicity of the sample.

Test durations range from 5 to 30 minutes permitting a rapid turnaround of

results. Specialized computer software reduces the data generated to

estimate a median effective concentration.

temperature controlled

photometer

The assay is based upon the reversion of mutations in the histidine

(his) operon in the bacterium Salmonella typhimurium. Strains with mutations in the his operon are unable to grow without added

histidine. Revertants will grow on minimal medium plates without his.

This provides a simple, sensitive selection for revertants of his mutants.

TA1575 is a substitution his mutant

TA1578 is a frameshift his mutant

rfa mutation makes the outer membrane more permeable to large

molecules.

uvrB mutation eliminates excision repair gene.

The plasmid pKM101 increases error-prone repair of DNA damage

The AMES test for genotoxicity

The AMES test for genotoxicity (Cont.)

Plating in His-

medium

Growing in His+ liquid medium

Growth in His- indicates

genotoxicity

SOME SPECIES USED IN PESTICIDE TOXICITY ASSESSMENT

Some lower species of invertebrates are used as alternatives of re-

placement in the assessment of environment contamination.

Daphnia magna

CELLULAR TARGETS FOR ASSESSMENT

Protein synthesis

Total protein content

cell viability

Neutral red uptake (NRU)

DNA synthesis

RNA synthesis proliferation

Mutagenesis

ATP content

MTT Energetic metabolism

ATPase

Cell membrane

Leakage (LDH)

DIFFERENT IN VITRO CELLULAR MODELS

Type In vitro/in vivo Type of Standar. Most commonly

Characteristics culture used cell lines

Freshly isola- - good - few hours - poor - hepatocytes

ted cells - karyotipe - suspension - endothelial

normal - intestinal

Primary - good - few days, - poor - fibroblasts

cultures - karyotipe - monolayer - epithelial

normal

Finite cell - variable - month/years - good - fibroblasts

lines - diploid - monolayer - epithelial

Continuous - poor - years - very - epithelial - -

monolayer -aneuploid good - transformed

(established) or tumoral

CONTINUOUS CELL LINES USED IN TOXICOLOGY

Name Origin Characteristics Replication

CHO Chinese hamster - epithelial-like 15-20 x (7 d)

Ovary cells - polyploid

KB Human epidermal - epithelial-like 10-15 x (7 d)

oral carcinoma - polyploid

BHK Syrian hamster - fibroblast-like 2 x (4 d)

kidney - polyploid

HeLa Human cervix - epithelial-like 15 x (7 d)

carcinoma - aneuploid

3T3 Mouse embryos - fibroblastoid 4-6 x (6 d)

- aneuploid

V79 Chinese hamster - fibroblastoid 15-20 x (6 d)

lung - aneuploid

Endpoint

Cell Morphology Cell size and shape

Cell to cell contacts

Nuclear size, shape and inclusions

Nucleolar vacuole formation

Cytoplasmic vacuole formation

Cell adhesion Attachment to culture surface

Dettachment from culture surface

Cell to cell adhesion

BIOLOGICAL ENDPOINTS FOR CYTOTOXICITY (1)

BIOLOGICAL ENDPOINTS FOR CYTOTOXICITY (2)

Endpoint

Cell viability Vital dye uptake

Trypan blue exclusion

Cell number

Replating efficiency

Cell proliferation Increase in total DNA

Increase in total RNA

Increase in total protein

Colony formation

Endpoint

Membrane damage Loss of enzymes (LDH)

Loss of co-factors (NADPH)

Leakage of preloaded cells

Radioactive precursors Tritium thymidine to DNA

Tritium uridine to RNA

Tritium or sulphur amino

acids to proteins

BIOLOGICAL ENDPOINTS FOR CYTOTOXICITY (3)

The FRAME modified NRI cytotoxicity test

Balb 3T3-L1 cells from mice

The basis of this test is that a cytotoxic chemical (regardless of site or

mechanism of action) will interfere with the cellular lysosomes, hence

The lysosomes of the affected cells can not accumulate the neutral red

dye (NR).

Neutral Red

dye accumulation

RN

lysosomes

Basal Cytotoxicity Tests:

The LDH Leakage test

u u u u u u u u u

u u u u

u u u u u

u u u u

u u

Transfer of

supernatant

ELISA-reader

Incubation

With the tested

compound

(2-48 h)

Incubation with

reaction mixture

Lactate dehydrogenase (LDH) is a

stable cytoplasmic enzyme present

in all cells. It is rapidly released in-

to cell culture when the plasma

membrane is damaged.

The use of microplate reader permits the measurement of multiple samples.

Release

of LDH

RN

mitochondria

Reduction by SDH

Methyl tetrazolium salt

Formazan

INTERFERENCE WITH THE METABOLIC PROCESS,

The basis of this test is that a cytotoxic chemical (regardless of site or

mechanism of action) will interfere with the cellular mitochondria,

hence the mitochondria of the affected cells can not transform the

tetrazolium salt to formazan by its Succinate dehydrogenase (SDH).

Energy Metabolism Tests: MTT ASSAYS

X

Selection oa animal and tissue Using an established cell line

(ATCC)

Cuturing

Isolation of tissue and cells

Adding the tested

compound

Observation

Morphological changes Cell counting

Sellected Cell line

Culture and

Subculture

Subcultur a 65%

confluence

Adding the desired

Concentration of the

tested pesticide

Measurement of the absorbance by ELISA

0 1 10 100

4,2

3,0

5,0

6,0

7,0

Data analysis

Sigma

plot Estimation of toxicity lines

and calculation of

cytotoxicity values

Following the procedure of

The sellected assay

1 10 100

3

4

5

6

7

Pro

bit

LC75 LC50 Comparative Cytotoxicity

Comparative Biochemistry

LC25

LC12,5

LC6,25

RELATION BETWEEN

CYTOTOXIXITY VALUES

AND BIOCHEMICAL

PARAMETERS

Diclorodiphenylethanes

HCH derivatives

Cyclodienes

Herbicides

More

toxic

Less

toxic

PESTICIDE CYTOTOXICITY IN SEVERAL CELL LINES USING

FOUR ALTERNATIVE METHODS

Bayoumi A.E. et al. (1999) Rev.Toxicol.

BF - 2 RTG - 2 CHO - K1

RN Proteíns RN Proteins RN Proteins MTT ATPase

TDE TDE TDE TDE TDE TDE TDE TDE

DDT DDT DDT DDT DDT DDT DDT DDT

DDE DDE DDE DDE DDE Metoxi. Metoxi DDE

Metoxi Metoxi Metoxi Metoxi Metoxi DDE DDE Metoxi

HCH HCH HCH HCH HCH H CH Lind. HCH

Lind . Lind . Lind . Lind . Lind . Lind . HCH Lind .

Clord . Clord Endos. Aldrín Clord Clord Aldrín Aldrín

Endos Hept. Clord Dield. Hept. Hept. Dield. Dield.

Aldrín Endos Hept Clord. Aldrín Aldrín Endos Clord

Hept Aldrín Aldrín Hept Dield. E ndos Clord Endos

Toxaf . Dield. Dield. Endos Endos Toxaf. Hept Hept

Dield. Toxaf. Toxaf. Toxaf. Toxaf. Dield. Toxaf. Toxaf.

Atraz. Atraz Atraz Atraz Atraz Atraz Atraz N.D.

N.D. Paraq. Paraq. Paraq. Paraq. Paraq. Paraq. N.D.

Compounds

Reconstructed skin is a model of human skin, created from human

cells which are grown in vitro. The culture medium feeds the system and enables the cells to multiply.

At the final stage of growth, the culture

is exposed to the open air to foster the

formation of a corneous layer.

Epidermis which include melanocytes

exposed to UV rays actually tan.

RECONSTRUCTED HUMAN SKIN: AN EXAMPLE OF REPLACEMENT

USE OF ARTIFICIAL SKIN FOR TOXICITY ASSAYS

Reconstructed skin

contents all cellular

elements and layers

appearing in normal

skin.

Cytogenetic test using CHO cultures

The cytogenetic analysis of in vitro cultured CHO cells can be used as a mutagenicity test for chromosome abberations and Sister Chromatide Exchanges

(SCE). After completion of the cell cycle stained metaphase preparations are

examined with the microscope.

Chormosome Abberations Sister Chromatid

Exchanges (SCE)

The sister chromatid exchange

(SCE) assay is used to evaluate

whether cytogenetic damage has

been caused by chemical or

physical agents. Chemical

mutagens often induce SCEs at

concentrations which are lower

than those required to produce

significant yields of chromosomal

aberrations. When cells are

treated with compounds that

cause lesions that persist through

cell division, exchanges will

occur between the daughter

chromatids.

Special Cytotoxicity Tests:

MECHANISMS OF CELLUAL DEATH (APOPTOSIS)

Necrosis

Normal reversible Swelling

irreversible

Swelling Disintegration

Conserved chromatin

Mitochondrial changes

Apoptosis

Normal Condensation

(Cell blebbing)

DNA

Fragments

Mitochondrial preserved

Fragmentation

Intact membranes

apoptic bodies

Formation of

apoptic bodies

Nuclear changes

Cytotoxic values of chlordane and toxafene that induced

apoptosis at Levels of NR25 and NR50 in CHO cell line.

Absorb

ance (4

90

nm

)

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0 2 4 6 8 10

chlordane-NR25

toxafene-NR25

A

0 2 4 6 8 10

chlordane -NR50

toxafene-NR50

B

*** ***

*** *** ***

***

**

*** *** ***

***

**

***

*** ***

***

***

**

***

**

0

Time (h)

Kinetics of p,p´-DDT, endosulfan and dieldrin which induced apoptosis at levels of NR25 (A) and NR50 (B) in CHO-K1 cells.

Time (h)

Absorb

ance (

49

0 n

m)

0 2 4 6 8 10

p,p´-DDT endosulfan

dieldrin

***

***

*** ***

***

** * **

**

**

** *

0,0

0,1

0,2

0,3

0,4

0,5

0 2 4 6 8 10

p,p´-DDT endosulfan

dieldrin

**

**

**

** ***

** **

0,6

A B

APPLICATIONS OF THE ALTERNATIVE METHODS

Animal Test

Draize test in

Rabbit´s eye

Corrosivity testing

On rabbit skin

Neurotoxicity test

of organopho-

sphorus

compounds

in Chicken

In vitro Method

Eye irritation

Skin corrosion

Human skin cultures

NTE esterases

determination

in neuroblastoma

cells

Degree of Replacement

Complete replacemnent

Complete replacement

Partial replacement for

Esterase inhibitors

Regulatory authorities

acceptance

EU

US, Canada

Worldwide

GENERAL SCHEME FOR USING ALTERNATIVE METHODS

FOR ACUTE TOXICITY RTESTING,

1- Examination of physicalchemical properties, literature review and QSAR.

2- Integrate data.

3- Is information suffiecient for hazard characterization ?

4- Conduct in vitro biological tests

5- Integrate all data

6- Is information suffiecient for hazard characterization ?

7- In cases where human data are necessary,

Is information suffiecient to aalow controlled human exposure ?

8- Conduct animal studies Conduct human trial

after ethical review

Integrate all data

Is information suffiecient for hazard characterization ?

Yes

Yes

No

No

No Yes

No Yes

Evaluate hazard and use

Results in safety assessment

Spielmann

and Goldberg,

(1999)

II. ALTERNATIVE METHODS AT BIOCHEMICAL LEVELS

NADPH + H +

NADP +

PENTOSES PHOSPHATE CYCLE

XENOBIOTIC

MERCAPTURIC ACIDS

GSSG

H2O2

catalase

O2

O 2

- SOD

O2

2 GSH

Gpx

Gpx: glutathione peroxidase

Grd: glutathione reductase

GST: glutathione S-transferase

SOD: Superoxide dismutase

Grd

GST

H2O

According to the fact that the majority of pesticides are producing Free

radicals such as ROS which in turn causing oxidative stress which May lead to

lipid peroxidation, so that, it is well documented that the glutathione redox

balance is considered as the most important biomarker That can be used to

detect the cell or tissue injury caused by free radical.

Glutathione Redox Balance

Glutationasa

ADP + Pi

ATP

Glutamic acid

-Glutamylcisteina sinthitase

-Glutamyl cisteine

Glutathione synthitase

X

NADPH + H+

NADP+

O2

H2O

H2O2 O2

O2 -

Superoxid distmutase

GSSG

Glutathione Glutathione

reductase peroxidase

2 GSH

Glutathione

S-transferase

Catalase

-GLU-CYS-GLY

X

Dipeptidase

Glutamyltranspeptidase

Mercapturic acid

PENTOSES PHOSPHATE CYCLE

HCH and LINDANE on Gpx and Grd

(nm

ole

s/m

in/m

g p

rote

inas)

100

200

300

400

500

600

700

NR Units

0 6,25 12,5 25

Lindano A HCH ***

***

**

***

***

Glutathione peroxidase

NR50 HCH = 55,70 mg/ml NR50 lindane = 144,06 mg/ml

(nm

ole

s/m

in/m

g p

rote

inas)

0

40

80

120

160

NR Units

0 6,25 12,5 25

HCH Lindano

B

***

***

**

Glutathione reductase

Bayoumi A.E. et al., (2002) Xenobiotica.

CYCLODIENE INSECTICIDES ON Gpx AND Grd

100

200

0

NR Unit

0 6,25 12,5 25

chlordane

heptachlor

**

**

**

(nm

ole

s/m

in/m

g p

rote

inas)

Glutathione peroxidase

NR50 chlordane = 22,16 mg/ml NR50 heptachlor = 23,90 mg/ml

90

30

60

0

NR Unit

0 6,25 12,5 25

(nm

ole

s/m

in/m

g p

rote

inas)

chlordane

heptachlor

**

**

*

Glutathione reductase

Bayoumi A.E. et al., (2001) Comp Biochem Physiol C Toxicol Pharmacol.

Glutathione peroxidase

400

NR Unit

0 6,25 12,5 25

0

100

200

300

(nm

ole

s/m

in/m

g p

rote

inas)

Aldrín

Dieldrín

**

***

**

***

**

NR50 aldrín = 56,63 mg/ml NR50 dieldrín = 65,96 mg/ml

(nm

ole

s/m

in/m

g p

rote

inas)

60

20

30

40

50

NR Unit

0 6,25 12,5 25

Aldrín

Dieldrín

***

***

**

*

Glutathione reductase

CYCLODIENE INSECTICIDES ON Gpx AND Grd

Bayoumi A.E. et al., (2002) Comp Biochem Physiol C Toxicol Pharmacol.

Pesticides -Glutathione S-transferase

Ac

tivit

y g

luta

thio

ne

S

-tra

ns

fera

se

(nm

ol/

min

/mg

pro

tein

)

Cytotoxicity (NR25 mg/ml) 10 100 1000

0

10

20

30

40

50

60

r =- 0.831

p,p´-DDT clordano heptachloro

aldrin

methoxychlor

HCH

endosulfan

lindane

atrazina

paraquat

p,p´-TDE

Cytotoxicity (RN25 mg/ml)

Activity o

f glu

tath

ione p

ero

pxid

ase

(nm

ol/m

in/m

g p

rote

in)

10 100 1000

0

100

200

300

400

500

600

700

r = 0,725

p,p´-TDE

chlordane

heptachlore

p,p´-DDT

aldrin

methoxychlore

HCH

lindane

atrazina

paraquat

Glutation peroxidasa-Plaguicidas

Cytotoxicity (NR25 mg/ml)

Pesticide- Total Glutathione

tota

l G

luta

thio

ne

(nm

ol/m

g p

rote

in)

10

20

30

40

50

60

70

10 100 1000 0

p,p´-TDE

chlordane

heptachlore

aldrin

methoxychlore

HCH p,p´-DDT

lindane atrazina

paraquat r = 0,847

ADVANTAGES AND LIMITATIONS OF IN VITRO ALTERNATIVE METHODS

ADVANTAGES

Testing is fast and cheap. Controlled testing conditions. Reduction of systemic effects. Reduction of testing in animals. Presenting high data reproducibility. Human cells and tissues can be used. Reduction variability between experiments. Very small amount of test material is required. Utilizing a biological material highly homogenic. Trangenic cells carrying human genes can be used. Time-dependent studies can be performed and samples taken. Permit and facilitate the study of mechanism of action at the cellular level. Same dose range can be tested in a variety of test systems (cells and tissues). LIMITATIONS

General side and systemic effects (behaviour) cannot be assessed. Chronic effects cannot be tested.

EXAMPLES OF PESTICIDES THAT COULD BE

TESTED USING ALTERNATIVE METHODS

All the organochlorine compounds and their metabolites, i.e. DDT, TDE, DDE, methoxychlor,

aldrin, dieldrin, endrin, chlordane, heptachlor, endosulphan,......etc

All the organophosphorus compounds and their major metabolites, i.e. Malathion, parathion,

fenthione, diazinon, chlorpyriphos, dichlorvos, methidathion, Acephate, dimethoat, ............etc

All the carbamate compounds, i.e. methomyl, carbaryl, .........etc

All the dithiocarbamate compounds which acting as fungicides

and their metabolites, i.e. maneb, zineb, mancozeb.......etc

The majority of herbicides, 2,4-D, paraquat, triazines compounds.

The majority of pyrethroids compounds, i.e. fenvalerate, deltamethrin,

permethrin, Tetramethrin, .......etc

Some of antimolting agents, i.e. diflubenzuron, hexaflumurun, pyriproxyfen.

Some of synthetized insect sex pheromones, i.e. Pectinophora gossypiella and Heliothis armigera