Joachim 1 - biomarkers

Biomarker def

biological (often biochemical) response indicating exposure to and often effect of toxic substance(s)

Example of biomarkers:

– Induction of detoxification enzymes (e.g. EROD)

– Bile PAH

– Induced antioxidant defence system

– Induction of yolk protein in male fish

– DNA adducts

Effects on organisim level biomarkers

Retarded

Spinal deformities

Malformed digestive tract

Abnormal heads

Tissue/ organ level biomarkers

Liver histology

Intersex

Fish disease and histopathology

• Organ and cellular pathology gives a biological endpoint of exposure history.

• Pathological effects can be associated with exposure to pathogens, xenobiotics and other environmental stressors.

• A range of lesions associated with carcinogenic, toxic and endocrine disruptive insult can be detected. Gives the widest appreciation of ‘health status’ in animals chosen for our studies.

• Wide species range.

• Material collected for pathology can be applied to other studies (e.g. immunohistochemistry, proteomics, genomics etc)

Cellular level biomarkers

Lysosomal stability (Neutral red retention)

Nutral red retention

unhealthy cell with enlarged lysosome and dye leakage

Biochemical / molecular level biomarker

Neuro transmitters - Cholinergic

EROD

VTG

AChE inhibition

Increased stimuli = increased contraction

AChE def

AcetylCholineEsterase

Substances with cholinergic activity due to due to AChE inhibition

• Insecticides

– Organic phosphates

– Carbamates

• Mustard gas (sarin)

How does AChE inhibitors (e.g. Sarin) affect the individual?

• Acute

– Stopped breathing

– Epilepsy

– Shaking / spasms

• Long term (ex.Gulf War syndrome)

– Apathy

– Difficulties with concentration

– Memory problems

– Irritation

delta-aminolevulinic acid dehydratase (ALA-D)

The heme group is the oxygen binding molecule in hemoglobin and is synthesized in a multiple step process.

ALA-D catalyzes the second step

ALA-D is strongly inhibited by lead

– The consequence is decreased oxygen uptake

How can you measure oestrogen in effluents?

• Chemistry

• Biological methods

• Transformed MCF-7 cells. MCF-7 is a human cell line that requires oestrogen to grow.

• Manipulated yeast cells

• Stimulation of yolk protein (vitellogenin) synthesis in fish cells.

Genotoxicity biomarkers

• Comet assay

• Micronuclei

• DNA adducts

Comet assay overview

1. Living cells from culture media, blood, or tissue

2. DNA damadge (chemical, UV or radiation)

3. Cells with damadged DNA having single-stranded /double strand braks

4. Singel cells are embeded on agarouse coated slide and lysed

5. After electrophoresis and flourecenct staining the damadged DNA is seperated from the intact DNA (the heas) and generates a tail

How to select biomarker

• Biological level?

• Relevance?

• Feasibility?

• Cost?

Biochemical parameters of interest

What more do we know about PAH and effects in the environment?

• Slowly degradable, especially larger PAH

• Bioaccumulate

• Toxic/ecotoxic

– Genotoxic (mutagenic)

– Cause cancer

– Disturb reproduction

• Transported to the environment

• Effects in the environment