Toxicology

Lecture 2

Biochemical and Molecular Methods in

Toxicology
This lecture summarizes different methods for the
investigation of xenobiotic metabolism
CELL CULTURE TECHNIQUES:
Replace whole organism for toxicity testing

Loss of differentiated cell function is a common problem

Media constituents shows unwanted and undefined effects

Two types of cell culture: Cell Suspension & Monolayer Cell Culture

MOLECULAR TECHNIQUES
Responses to toxicants involves gene expression analyzed by
microarray
Studies of polymorphisms in xenobiotic-metabolizing and other
enzymes
Analysis of chemically induced mutations
Development of knockout animals lacking a particular gene
Transgenic animals with an additional transgene
Toxic effects on transcription, mRNA processing and translation
and protein synthesis

MOLECULAR TECHNIQUES
1. Molecular Cloning (Insertion of a DNA segment into a suitable
vector)
2. cDNA and Genomic libraries (used in many screening
procedures and many transgenic proteins)
3.

Northern and Southern Blot Analyses

Northern analysis is used to identify and quantitate specific mRNAs in

a sample

Southern to determine presence and copy number of gene of

interests

Restriction fragment length polymorphism (RLFP)

4.Polymerase Chain Reaction (PCR)

Amplification of small quantity of
DNA in to large
Polymorphisms in xenobioticmetabolizing enzymes
Isolating genes from cDNA and
genomic libraries and for
mutational analysis

IMMUNOCHEMICAL TECHNIQUES
Immunolocalization:
is a technique for identifying the presence of a
protein within the cell, its relative abundance
and its subcellular localization

Immunoaffinity Purification
Involves the use of antibodies, bound to
an insoluble matrix, for chromatography.

Immunoprecipitation
immunoaffinity
means

to

is

purification

remove

variant
and

protein

of

is

from

complex mixture in a highly specific

Western Blotting:
is a widely used technique in which antibodies
are used to detect proteins following
electrophoresis