METABOLISM

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The McKim Conference on the Use QSARs and Aquatic
Toxicology in Risk Assessment June 27-29, 2006
METABOLISM in-silico simulation
Sabcho Dimitrov Ovanes Mekenyan
Laboratory of Mathematical Chemistry University
Prof. As. Zlatarov, Bourgas, Bulgaria
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Research Partners from Regulation Agencies
US EPA, Athens, GA 5 yrs coop US EPA, Duluth,
MN Environment Canada NITE Japan
Research Partners from Industry
PG ExxonMobil Unilever BASF
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Overview

• (Q)SARs
• Metabolism of Prokaryotes
• Metabolism of Eukaryotes
• Simulation of metabolism

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(Q)SARs
Biodegradation Bioaccumulation Acute
Toxicity Chronic Toxicity Hormone Toxicity Skin
sensitization Mutagenicity
Metabolism
(Q)SARs
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The Five Kingdoms
Hormone Toxicity Skin sensitization Mutagenicity
BCF
Acute Chronic toxicity
Biodegradation
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Metabolism
Energy-generating component Catabolism Produce
energy (as ATP) and simple oxidized compounds
Energy-consuming component Anabolism Build
cell material
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Intermediary metabolism
Set of reactions involving metabolites that are
intermediates in the degradation or biosyntheses
of biopolymers
Common energy metabolism tricarboxylic acids
cycle, glycolysis Common biosynthetic
pathways synthesis of amino acids, lipids, and
carbohydrates
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Anabolism
Catabolism
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Prokaryotes Kingdom Monera
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The Kingdom Monera (Prokaryotes)
Constitutive enzymes always produced by
cells The enzymes that operate during glycolysis
and the tricarboxylic acid cycle Inducible
enzymes produced ("turned on") in cells in
response to a particular substrate they are
produced only when needed
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The Kingdom Monera (Prokaryotes)
Operon - grouping of genes in bacteria under the
control of the same regulatory system. Bacteria
tend to group genes that are functionally related
together on the chromosome.
Lactose operon contains genes that encode
enzymes responsible for lactose metabolism
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The Kingdom Monera (Prokaryotes)
Operon
Transposons - segments of DNA that can move from
one site in a DNA molecule to other target sites
in the same or a different DNA molecule within or
among organisms.
DNA
DNA
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The Kingdom Monera (Prokaryotes)
Operon
Transposons

• Plasmids - extrachromosomal genetic elements.
• Contain genes that help the host organism to
  survive under specific conditions or they contain
  genes for catabolism of non-intermediary organic
  compounds
• Replicate independently of the chromosome
• Can be transfered from the donor bacterium to
  other recipient bacteria

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Eukaryotes Kingdoms Protista Planta Fungi Animal
ia
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Eukaryotes
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Multienzyme complex
In a number of cases, enzymes that catalyze
sequential reactions in the same metabolic
pathway have been found to be physically
associated

• Active sites are found on a single,
  multifunctional peptide chain
• Several individual enzymes are non-covalently
  associated
• Attachment to membranes

The formed enzyme complexes allow channeling of
reactants between active sites the product of
one reaction can be transferred to the next
active site.

• Speed up the reactions of the pathway
• Protect unstable intermediates

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Summary
1. The application of metabolic transformations
is strongly organized.
2. This is a premise for development of
metabolic simulators.
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Simulation of molecular transformations
Half-lives, 250C, pH 7 2 years 7 days 4
minutes
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Simulation of molecular transformations
X F, Cl, Br
not CX2 X F, Cl, Br
not Csp2 and CX2 X F, Cl, Br
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Simulators of metabolism
Rule based systems Predictions single best or
multiple alternative metabolic pathways
BESS (PG and Michigan State University) META
(MultiCASE Inc) METEOR (Lhasa Ltd) CATABOL (PG
and LMC, Bourgas As. Zlatarov University) TIMES
(LMC, Bourgas As. Zlatarov University) PPS
(UM-BBD, http//umbbd.msi.umn.edu/) MEPPS (under
development, Lhasa Ltd)
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Simulators of metabolism
Rule based systems Predictions single best or
multiple alternative metabolic pathways
BESS (PG and Michigan State University) META
(MultiCASE Inc) METEOR (Lhasa Ltd) CATABOL (PG
and LMC, Bourgas As. Zlatarov University) TIMES
(LMC, Bourgas As. Zlatarov University) PPS
(UM-BBD, http//umbbd.msi.umn.edu/) MEPPS (under
development, Lhasa Ltd)
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Simulators of metabolism
Rule based systems Predictions single best or
multiple alternative metabolic pathways
METEOR (Lhasa Ltd) PPS (UM-BBD)
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Simulators of metabolism
Rule based systems Predictions single best or
multiple alternative metabolic pathways
CATABOL (PG and LMC, Bourgas As. Zlatarov
University) TIMES (LMC, Bourgas As. Zlatarov
University)
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CATABOL Simulation of catabolism
O
H
C
O
C
O
H
O
H
O
H
O
O
O
O
O
H
O
O

O
H
O
O
O
C
C
N
H
O
2
C
C
C
H
3
O
H
C
N
C
N
H
H
N
C

2
2
N
C
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P 1.00
O
H
C
O
C
O
H
P 0.99
O
H
O
H
O
O
O
O
P 0.95
O
H
P 0.90
O
O

O
H
O
O
O
P 0.75
C
C
N
H
O
2
C
C
P 0.40
C
H
3
O
H
P 0.001
C
N
C
N
H
H
N
C

2
2
N
C
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P 1.00
O
H
Match?
- No!
C
O
C
O
H
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P 0.99
O
H
O
H
Match?
- No!
O
O
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Match?
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RESULT
Match?
- Yes!
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The most plausible biotransformation pathway
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Comparison of metabolic pathways
Pathway A
Pathway B
Complement of A in B B\A or B-A
Complement of B in A A\B or A-B
Intersection AnB
Accounting (or not) for pathway structures
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Trivial measures of similarity/dissimilarity pathw
ay structure is not accounted for
Measures of similarity/dissimilarity Tanimoto, D
ice, etc.
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Non-trivial measures of similarity/dissimilarity p
athway structure is accounted for
A in B Card(AnB)/Card(A) B in A
Card(AnB)/Card(B) A out of B Card(A\B)/Card(A) B
out of A Card(B\A)/Card(B)
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Observed versus simulated pathways
Observed and predicted catabolism
Union of pathways


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Union of pathways
Probability that the metabolite is
observed, given that the metabolite is predicted
(predictability)
Probability that the metabolite is
observed, given that the metabolite is truly
observed (sensitivity)
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Probability that the metabolite is not predicted
given that the metabolite is truly observed
(false negatives)
Union of pathways
Probability that the metabolite is predicted
given that the metabolite is truly not observed
(false positives)
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Reliability of generated metabolic pathway
- the numbers of successful applications of the
transformation - the numbers of unsuccessful
applications of the transformation
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CATABOL, mathematical formalism
BOD or CO2 production
Biodegradability - of theoretical
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CATABOL, mathematical formalism
BOD or CO2 production
Lyons, C. D., S. Katz, R. Bartha, Appl Environ
Microbiol, 1984, 48, N 3, pp. 491-496
Simulated catabolism
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CATABOL, mathematical formalism
Quantities of metabolites
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CATABOL, mathematical formalism
First order kinetics
Ultimate half-life
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CATABOL, mathematical formalism
First order kinetics
Ultimate half-life
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CATABOLTD time dependent model
Training data
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CATABOLTD time dependent model
Training data
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CATABOLTD time dependent model
Training data
BOD within 10-d window
10-d window
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CATABOLTD time dependent model
Probabilistic approach First order kinetics
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CATABOLTD time dependent model
The model is able to predict

• Primary half-life half-life of parent chemical
• Ultimate half-life half-life by BOD
• Biodegradation as a function of time
• Metabolites quantity as a function of time
• Biodegradation within 10 days window

Metabolite
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Biodegradation
Metabolite BOD0.016 LC500.34 mg/l
Parent CAS 31570-04-4 Phenol 2,4-bis(1,1-dimethyl
ethyl), phosphite (31) BOD0.038 LC50145835248
mg/l
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Biodegradation
Metabolite BOD0.39 LC501.2.106 mg/l
Parent CAS 124-28-7 N-n-Octadecyl-N, N-dimethyl
amine BOD0.83 LC500.94 mg/l
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CATABOLTD time dependent model
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TIMES Skin Sensitization Model
Phase II
Reactive species
Metabolism
No sensitization
Parent
Reactive species
Phase II
Reactive species
Dimitrov et al, 2005. Skin sensitization
Modeling based on skin metabolism simulation and
formation of protein conjugates. International
Journal of Toxicology 24, 189-204.
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Fish liver simulator
Bioccumulation
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TIMES Skin Sensitization Model
Phase II
Reactive species
Metabolism
No sensitization
Parent
Reactive species
Phase II
Reactive species
Dimitrov et al, 2005. Skin sensitization
Modeling based on skin metabolism simulation and
formation of protein conjugates. International
Journal of Toxicology 24, 189-204.
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Skin sensitization
Predicted metabolism of isoeugenol
Sulphate conjugation
Dealkylation
Formation of semiquinone free radicals
Glucoronidation
Michael addition on quinones
Free radical reaction on proteins
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Mutagenicity
N-Nitrosoamine Aliphatic C-Oxidation
N-Nitrosoamine Oxidative N-Dealkylation
N-Nitrosoamine Oxidative N-Dealkylation
Electrophilic Species Generation
Aliphatic C-Oxidation
Reacting with DNA
O-Glucuronidation
Amino Acid Conjugation
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Next Major Activity
Laboratory conditions
Intact Organism
Intact Cells, Tissue, Organism
Sub-cellular fractions
cDNA-expressed Individual CYPs
Microsomes Cytosol
In vivo
Primary hepatocytes
Rat liver homogenate (S9 fraction)
Liver slices
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Research Partners from Regulation Agencies
US EPA, Athens, GA US EPA, Duluth, MN Environment
Canada NITE Japan
Research Partners from Industry
PG ExxonMobil Unilever BASF
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Metabolism logic

• References
• Villee C.A., Dethier V.G., Biological Principles
  and Processes, W. B. Saunders Company, London,
  1971.
• Hames B.D., Hooper N.M., Houghton J.D., Instant
  Notes in Biochemistry, Springer-Verlag New York
  Inc., NY, 1997.
• Horton H.R., Moran L.A., Ochs R.S., Rawn J.D.,
  Scrimgeour K.G., Principles of Biochemistry, Neil
  Patterson Publishers, Prentice Hall, Inc, NJ,
  1992.
• Wackett L.P., Hershberger C.D., Biocatalysis and
  Biodegradation Microbial transformation of
  Organic Compounds, ASM Press, Washington, 2001.

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Metabolism logic
Supraorganism Enzymes Metabolism Prokaryotes Eukar
yotes Conclusions
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Supraorganism concept
Organization of biodegradation information Indivi
dual Bacteria Individual Transformations
Individual DNA
Virtual Supraorganism
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Endoplasmic reticulum
The main function of the ER is to transport
materials through the cell and to serves as the
site of biochemical reactions
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Rough Endoplasmic Reticulum
Dotted with ribosomes Provides a large surface
area for the organization of chemical reactions
and synthesis Can synthesize itself including
both protein and lipid parts Some of the
synthesized membrane replace nuclear membrane,
form the Golgi complex, lysosomes or plasma
membrane
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Smooth Endoplasmic Reticulum
Membraned matrix proteins having enzyme
activity A great deal of non-protein synthesis,
including lipids and carbohydrates Dedetoxication
functions, particularly in the smooth ER found in
liver cells
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Lysosmes
Membrane-enclosed spheres Contain powerful
digestive enzymes Breakdown of cellular
molecules, ingested bacteria and old organelles
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Peroxisome
Metabolism of free oxygen radicals Synthesis of
cholesterol and ether lipids Bile acid
formation Catabolism of long chain fatty
acids Catabolism of purines, prostaglandins,
leucotriens Alcohol detoxification in liver
Metabolism of estradiol
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Mitochondrion
The Matrix contains many of the enzymes involved
in aerobic energy metabolism Carbohydrates, fatty
acids, and amino acids are oxidized to CO2 and
water The released energy is conserved in
energy-rich molecule ATP
Divides using their own circular strand of DNA
Releases a chemical that trigger programmed cell
death (apoptosis) Exercises a veto, which eggs in
a woman should be released during ovulation and
which should be destroyed (atresia) In liver
cells contains enzymes that allow them to
detoxify ammonia