Mechanisms of Acute Toxicity

Mechanisms of Acute
Toxicity
Dan Wilson, PhD, DABT
Science Leader – Cheminformatics
The Dow Chemical Company
Acute Tox Workshop
Sep 24, 2015
Acknowledgements
•
•
•
•
Barun Bhhatarai
Ed Carney
Amanda Parks
Paul Price
Pop quiz: Put these in order of lethality
• Which substance has lowest lethal dose? (i.e. the
most ‘toxic’)
Agent
Caffeine
Arsenic
Aspirin
Salt (NaCl)
Ethanol
Nicotine
Botulism toxin
Toxicity ranking
?
?
?
?
?
?
?
LD50 (/kg bw)
?
?
?
?
?
?
?
GHS Cat
?
?
?
?
?
?
?
Pop quiz: Put these in order of lethality
• Which substance has lowest lethal dose? (i.e. the
most ‘toxic’)
Agent
Caffeine
Arsenic
Aspirin
Salt (NaCl)
Ethanol
Nicotine
Botulism toxin
Toxicity ranking
4
3
5
6
7
2
1
LD50 (/kg bw)
130-320 mg
46 mg
1000 mg
3000 mg
14000 mg
1 mg
0.02 ng
GHS Cat
3
2
4
5
5
1
1
Acute classification categories
Requirement is to classify by LD/LC50
• This can be done using guideline animal studies
• Do in vitro alternative methods offer a replacement?
• Do in silico alternative methods offer a replacement?
In silico approaches - QSAR
Why study acute mechanisms?
•
•
•
•
•
•
•
•
•
In vitro and in silico approaches not yet a total replacement
May direct in vitro HTS assays and enable building of QSARs
The ‘future’ is mechanism- (AOP-) based
Better enable read-across
Focus on identify compounds of high inherent toxicity
Important in poisoning cases
Acute mechanism in scope for repeat-dose studies
Understand if animal data relevant to humans
Understanding mechanisms makes us better toxicologists and
better able to interpret and troubleshoot studies
Challenges of identifying acute MOAs
•
•
•
•
•
•
•
•
•
A workshop like THIS has never been held…
Not a guideline study requirement
Study doesn’t include organ weights, histopath or clin path
DBs of LD/LC50 values don’t contain other mechanistic info
Studies often conducted at CROs blinded to TM identity
Specific mechanisms rarely examined
Relationship of mechanistic effect to apical effect not clear
Risk assessors didn’t consider acute toxicity ‘sexy’ – rare focus
Mechanistic in vitro HTS assays may only look above
cytotoxicity noise level yet the MOA may drive cytotoxicity
Facts about acute data
• Distribution of GHS classification not evenly distributed for oral
route - most compounds are GHS 4-5
• Provides information on inherent toxicity
• IV Data
– Compounds average 40x more toxic by iv than oral route
– Sometimes it’s the only data you have, especially for highly insoluble
compounds
– Compounds that pass limit dose orally may cause lethality in seconds
intravenously
– Directly applicable for medical devices
Ways to identify potential mechanisms
•
•
•
•
•
•
•
•
•
•
•
Determine whether ‘reactive’ or ‘pharmacologic’
3-D crystalline protein structure mapping
HT Gene expression data-mining
Identify protein targets using wet-lab binding interactions
Examine pathology and clinical pathology data
Consider Time-to-death
Examine relationship of acute toxicity to HTS data results
Similarity to compounds with known mechanisms
Years of experience resulting in a logical ‘hunch’
Use systems biology approach
Focus on critical targets for high acute toxicity
Chemical reactivity
•
•
•
•
•
•
•
•
•
Electrophilicity
Hardness (HOMO/LUMO)
Acylation
Schiff base formation
Michael addition reaction
SN1 mechanism
SN2 mechanism
SNAr mechanism
Polarizability
•
•
•
•
•
•
Molecular wt
Protein/DNA binding
Substructures
Solubility
pKa
Log KoW
Systems biology approach
Organism
Organs
Heart, Liver, Kidney,
Nervous System…
Cells
Muscle, Cardiac, Hepatic, RBCs,
Neurons, PMNs…
Organelles
Nucleus, Mitochondria, Peroxisome, Cytoplasm…
Biochemical Pathways
Gluconeogenesis, Pentose phosphate pathway, Kreb’s cycle,
Electron transport, Fatty acid metabolism, Lipolysis, Lipogenesis,
Pyrimidine/Purine biosynthesis, Urea cycle, Glycolysis, Vitamins…
Some mechanisms of acute toxicity
•
•
•
•
•
•
•
•
•
•
Inhibit energy production
Antimetabolites
Anticoagulants
Chelants
Inhibit signal transduction
Ion channel blockers
Inhibit Na+/K+ ATPase
Protein synthesis inhibitors
Non-specific high chemical reactivity
Physico-chemical properties
– Acids, Bases
– Surfactants
– Accept protons and uncouple mitochondrial during diffusion
Metabolism - Bioavailability
• Physical
–
–
–
–
–
Mucous
Chewing
Mixing/churning
Acid
Emulsification
• Hormones
• Enzymes
Protein Digestion
• Stomach
– HCl denatures
– Pepsinogen → pepsin
• Small Intestine
– Hormones
• Cholecystokinin
• Secretin
– Pancreatic enzymes
• Trypsin, peptidases, elastase
• Amino acids ↑ insulin, ↓ glucagon
• No storage form for protein
– amino acids → protein; carbons → carbohydrate/lipid; amino “N” as urea
Carbohydrate Digestion
• Starch: glucose polymer α(1→4) glycosidic bonds
– Amylose
• linear, 100’s glucoses
– Amylopectin
• branched, 1000’s units
• linear α(1→4)
• branch α(1→6) each 24-30 units
– Glycogen
• branch each 8-12 units
• Pancreatic amylase breaks α-1,4-bonds
Lipid Digestion
• Stomach
– Lingual and Gastric Lipase
• Small intestine
– Cystokinin → gallbladder
• ↓ gastric motility
– Secretin → pancreas
• bicarbonate neutralizes pH
–
–
–
–
–
–
Emulsification → Bile saltss
Pancreatic Lipase → FA at C1 and C3
Colipase → stabilizes Lipase
Cholesteryl ester hydrolase
Phospholipase A2 → FA at C2
Lysophospholipase → C2
Cholesterol
O
H
H
-
O
O
H
O
Deoxycholic acid
H
H
O
H
O
H
N
Taurochenodeoxycholic acid
H
H
O
O SO
O
H
O
Potentially labile subfragments
Modeling Systemic Bioavailability
Simulations Plus
Energy production
• Adenosine Triphosphate (ATP) used for most energy requiring reactions
(e.g., active transport). Isn’t stored, consumption closely follows synthesis
• 1 kg created-recycled/hr. A cell uses 10 million ATP molecules/sec and
recycles all of its ATP every 20-30 sec
• Guanosine Triphosphate (GTP) equivalent to ATP in energy content and
preferentially used in some cellular reactions
• Flavin Adenine Dinucleotide (FAD) a cofactor reduced to FADH2, an energyrich molecule
• Nicotinamide Adenine Dinucleotide (NADH): a cofactor; reducing potential
converted to ATP through the electron transport chain
• Nicotinamide adenine dinucleotide phosphate (NADPH) is used in fatty acid
and nucleic acid synthesis
• Phosphocreatine is used to replenish ATP from creatine and ADP
Glycogen
Glucose 1-P
Glucose 6-P
Glucose
Fructose 6-F
Fructose 1,6-Bis P
Glyceraldehyde 3-P
1,3 bisPhosphoglycerate
Glycolysis 3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate
Pyruvate
Acetyl CoA
Dihydroxyacetone P
Glycogen
Glucose 1-P
Glucose 6-P
Glucose
Fructose 6-F
Fructose 1,6-Bis P
Glyceraldehyde 3-P
Dihydroxyacetone-P
1,3 bisPhosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate
Lactate
Gluconeogenesis
Pyruvate
CO2
Acetyl CoA
Citrate
Oxaloacetate
Malate
Fumarate
Isocitrate
Kreb’s TCA
Cycle
ɑ-Ketoglutarate
Succinyl-CoA
Succinate
TCA Cycle
Glycogen
NADPH
Glucose 1-P
6-P Gluconolactone
Glucose 6-P
NADPH
Ribose 5-P
Ribulose 5-P
6-P Gluconate
Glucose
Fructose 6-F
Xyulose 5-P
Sedoheptulose 7-P
UDP Glucose
Fructose 1,6-Bis P
Glyceraldehyde 3-P
Erythrose 4-P
Dihydroxyacetone-P
1,3 bisPhosphoglycerate
3-Phosphoglycerate
Glyceraldehyde 3-P
2-Phosphoglycerate
Pentose-Phosphate Pathway
Phosphoenolpyruvate
Lactate
Pyruvate
CO2
Acetyl CoA
Citrate
Oxaloacetate
Malate
Fumarate
Isocitrate
Kreb’s TCA
Cycle
ɑ-Ketoglutarate
Succinyl-CoA
Succinate
Glycogen
NADPH
Ribose 5-P
Ribulose 5-P
6-P Gluconate
NADPH
Glucose 1-P
6-P Gluconolactone
Glucose 6-P
Fructose
Glucose
Fructose
Fructose 6-F
Xyulose 5-P
Sedoheptulose 7-P
UDP Glucose
Erythrose 4-P
Fructose 1,6-Bis P
Glyceraldehyde
Glyceraldehyde 3-P
Dihydroxyacetone-P
1,3 bisPhosphoglycerate
Glycerol-P
3-Phosphoglycerate
Glyceraldehyde 3-P
Phosphoenolpyruvate
Fatty acyl CoA
Pyruvate
Malonyl CoA
CO2
Lipids
Fatty acids
Acetyl CoA
Acetoacetate
Citrate
β-Hydroxybutarate
Oxaloacetate
Malate
Glycerol
Triacylglycerol
2-Phosphoglycerate
Lactate
Fructose 1P
Isocitrate
Kreb’s TCA
Cycle
Ketones
ɑ-Ketoglutarate
Methylmalonyl CoA
Fumarate
Succinyl-CoA
Succinate
Propionyl Co A
Fatty acyl CoA
(odd-number carbons)
Glycogen
NADPH
Glucose 1-P
6-P Gluconolactone
Glucose 6-P
NADPH
Ribose 5-P
Ribulose 5-P
6-P Gluconate
UDP Glucose
Glucose
Fructose
Fructose 6-F
Xyulose 5-P
Sedoheptulose 7-P
Erythrose 4-P
Fructose 1,6-Bis P
Glyceraldehyde
Glyceraldehyde 3-P
Dihydroxyacetone-P
1,3 bisPhosphoglycerate
3-Phosphoglycerate
Glyceraldehyde 3-P
Ala
Cys
Gly
Ser
Thr
Amino Acids
NH3
Glycerol-P
CO2
Carbamoyl-P
Urea
Fatty acyl CoA
Pyruvate
Malonyl CoA
Citruline
Kreb’s
Urea
Cycle
Arginine
Acetyl CoA
Arginosuccinate
Fumarate
Homocysteine
Phe
Tyr
Ile
Met
Val
Thr
Oxaloacetate
Fatty acids
Acetoacetate
Ile
Citrate
Asn
Asp
Ornithine
Phosphoenolpyruvate
CO2
Glycerol
Triacylglycerol
2-Phosphoglycerate
Lactate
Fructose 1P
β-Hydroxybutarate
Leu
Phe
Tyr
Trp
Lys
Isocitrate
Gln
Malate
Kreb’s TCA
Cycle
ɑ-Ketoglutarate
Glu
Methylmalonyl CoA
Fumarate
Succinyl-CoA
Succinate
Propionyl Co A
Fatty acyl CoA
(odd-number carbons)
Arg
His
Pro
Glycogen
Ribulose 5-P
6-P Gluconolactone
Glucose 6-P
Niacin
NADPH
Ribose 5-P
NADPH
Glucose 1-P
6-P Gluconate
Ala
Cys
Gly
Ser
Thr
Thiamin
Niacin
Lipoic Acid
Riboflavin
Carbamoyl-P
Niacin
Pyridoxine
Urea
Arginine
CO2
Biotin
Fumarate
Homocysteine
Vit B12
Phe
Tyr
Ile
Met
Val
Thr
Pyridoxine
Fatty acids
Niacin
Leu
Malonyl CoA
Phe
Biotin Acetoacetate
Tyr
Niacin
Trp
β-Hydroxybutarate Lys
Acetyl CoA
Citrate
Oxaloacetate
Ile
Isocitrate
Niacin
Arginosuccinate
Fructose 1P
Fatty acyl CoA
Pyruvate
Asn
Citruline
Kreb’s
Urea
Cycle
Phosphoenolpyruvate
Lactate
Asp
Ornithine
Glyceraldehyde
Glyceraldehyde 3-P Dihydroxyacetone-P
Niacin
1,3 bisPhosphoglycerate
Glycerol-P
Glycerol
3-Phosphoglycerate
Riboflavin Triacylglycerol
2-PhosphoglycerateNiacin
Erythrose 4-P
Glyceraldehyde 3-P
CO2
Fructose
Fructose 1,6-Bis P
Thiamin
Sedoheptulose 7-P
NH3
Glucose
Fructose 6-F
Xyulose 5-P
Vitamins
UDP Glucose
Pyridoxine
Niacin
Gln
Arg
His
Pro
Riboflavin ɑ-Ketoglutarate
Glu
Thiamin
Vit B12
Niacin
Methylmalonyl CoA
Lipoic Acid
Biotin
Fumarate
Succinyl-CoA
Propionyl Co A
Malate
Succinate
Riboflavin
Fatty acyl CoA
Niacin
(odd-number carbons)
Glycogen
Ribulose 5-P
6-P Gluconolactone
Glucose 6-P
Niacin
NADPH
Ribose 5-P
NADPH
Glucose 1-P
6-P Gluconate
Glyceraldehyde
Ala
Cys
Gly
Ser
Thr
Thiamin
Niacin
Lipoic Acid
Riboflavin
Niacin
CO2
Biotin
Asp
Kreb’s
Urea
Cycle
ArginoMitochondrial
Ornithine
succinate
Urea
Electron Transport Phe
Tyr
Fumarate
Homocysteine
Vit B12
Ile
Met
Val
Thr
Pyridoxine
Fatty acyl CoA
Fatty acids
Niacin
Leu
Malonyl CoA
Phe
Biotin Acetoacetate
Tyr
Niacin
Trp
β-Hydroxybutarate Lys
Pyruvate
Acetyl CoA
Citrate
Asn
Citruline
Arginine
Phosphoenolpyruvate
Lactate
Pyridoxine
Fructose 1P
Glyceraldehyde 3-P Dihydroxyacetone-P
Niacin
1,3 bisPhosphoglycerate
Glycerol-P
Glycerol
3-Phosphoglycerate
Riboflavin Triacylglycerol
2-PhosphoglycerateNiacin
Erythrose 4-P
Glyceraldehyde 3-P
Carbamoyl-P
Fructose
Fructose 1,6-Bis P
Thiamin
Sedoheptulose 7-P
CO2
Glucose
Fructose 6-F
Xyulose 5-P
NH3
UDP Glucose
Oxaloacetate
Ile
Isocitrate
Niacin
Pyridoxine
Niacin
Gln
Arg
His
Pro
Riboflavin ɑ-Ketoglutarate
Glu
Thiamin
Vit B12
Niacin
Methylmalonyl CoA
Lipoic Acid
Biotin
Fumarate
Succinyl-CoA
Propionyl Co A
Malate
Succinate
Riboflavin
Fatty acyl CoA
Niacin
(odd-number carbons)
Pyruvate Dehydrogenase Complex
CH3
Thiamin coenzyme
CO2
Lipoic acid coenzyme
Thiamin coenzyme
Coenzyme A-SH
Lipoic acid coenzyme
Flavin coenzyme
Flavin coenzyme
NAD+
NADH + H+
Arsenic
inhibits
S
O
OH Pyruvate Dehydrogenase Complex
CoA
O
O
Pyruvate
Acetyl CoA
Mitochondria
Lipoic Acid: Cofactor for 2nd Enzyme
HO
O
S
S
R
+
N
S
R
H
Hydroxyethyl derivative bound to
reactive carbon of TPP
OH
TPP
O
H
Lipoic acid
Arsenic
inhibits
NH2
N
N
S HS
OH
+
O
N
N
O
HO
HO
P
O
O
O
O
O
O O
P
HO
O
P
O
NH
OH
O
NH
S HS
SH
OH
+
OH
OH
H
Coenzyme A-SH
O
NH2
N
N
N
N
P
O
O
O
O
HO
O
SHHS
O
HO
HO
O O
P
O
P
O
NH
OH
OH
NH
OH
+
S
O
H
OH
Acetyl CoA
Reduced Lipoic acid
Dihydrolipoyl transacetylase
O
Niacin – Vitamin B3
O
OH
N
NH2
NH2
Nicotinic acid
N
N
O
N
NH2
O
N
O
O
N
O
Nicotinamide
HO
NH2
N
H
HO
P
NH2
O
O
OH
P
O
N
N
N
N
O
N
O
O
+
O
O
O
OH
HO
OH
HO
P
NH2
O
O
OH
P
O
OH
+
NAD
N
+
O
O
HO
NADP
+
O
O
HO P OH
O
O
Tryptophan
NH2
NAD+
+
N
R
:H- Hydride ion
H H
O
NH2
NADH
N
R
Riboflavin – Vit B2
O
H3C
N
H3C
N
H
NH
N
R
FAD (oxidized quinone form)
O
2H+, 2e-
O
H3C
N
H3C
N
N
R
H
NH
O
FADH2 (reduced hydroquinone form)
FMN and FAD tightly bound to enzymes that catalyze oxidation or reduction
Fish acute toxicity vs. ToxCast HTS
Mitochondrial Electron Transport
Intermembrane space
H+
H+
H+
ATP
Synthase
Matrix
III H O
ADP
H+
TCA
Cycle
ATP
O2
2
Succinate
NADH
FADH2
Q
IV
H+
III
H+
II
I
H+
Mitochondria membrane potential assay
Sakamuru S et al. Physiol. Genomics 2012;44:495-503
Mitochondrial tox predicts upper boundary to
Daphnia toxicity
Daphnia acute LC50 (mg/l)
AC50 Inhibition of
Mitochondrial Membrane
Potential (
AC50 Inhibition of Mitochondrial Membrane Potential (uM)
AC50
Inhibition of
Mitochondri
al Membrane
Potential (
Mitochondrial toxicity predicts upper bound to
acute intravenous toxicity
AC50
Inhibition of
Mitochondri
al Membrane
Potential (
Rat Intravenous LD50 (mg/kg)
AC50 Inhibition of
Mitochondrial Membrane
Potential (
AC50 Inhibition of Mitochondrial Membrane Potential (uM)
Mitochondrial toxicity doesn’t predict upper bound
to oral rat toxicity
AC50
Inhibition of
Mitochondri
al Membrane
Potential (
Rat Oral LD50 (mg/kg)
AC50 Inhibition of
Mitochondrial Membrane
Potential (
AC50 Inhibition of Mitochondrial Membrane Potential (uM)
LD50 values adjusted downward by %
bioavailable
AC50
Inhibition of
Mitochondri
al Membrane
Potential (
Rat Oral LD50 (mg/kg)
AC50 Inhibition of
Mitochondrial Membrane
Potential (
ester group
AC50 Inhibition of Mitochondrial Membrane Potential (uM)
Antimetabolites
H
N
H2 N
Dihydrofolate reductase
2-steps requiring 2 NADPH
H
N
Tetrahydrofolate (THF)
H
H
H
N
N
5
O
Methotrexate
LD50 135 mg/kg
O
HN
10
HN
O
Purines
HO
H
N
H
H
N
H
H
N
H
H2O
5
O
Methionine
O
N
10
N10-Formyl-THF
HO
H
N
H
H
N
5
N
+
TMP
H
NADPH + H+
NADP+
10
N5N10-Methenyl-THF
H
N
H
H
H
N
5
H
N
N
NADH + H+
NAD+
10
H
N5N10-Methylene-THF
H
H
5
H
HN
H H
10
N5-Methyl-THF
Purine synthesis
O
O
O
O
O
P
O
P
H
O
-
O
O
O
NH2
O P O-O
-
-
-
O
OH HO
Gln
O
O
Gla
NH2
+
-
NH2
P
-
OH
ATP
O
-
O
ADP + Pi
O
NH2
O
OH
HO
O
O
-
O
O
O
HO
Gln
H 2N
P
-
O
NH2
NH
O
O
HO
OH
OH
PRPP
Purines built on existing ribose sugar
supplied by Pentose Phosphate Path
O
-
O
P
O
ADP
N
O P
ATP
NH
NH2 Gla
ADP
O
O
ATP
O
O
P
-
NH
O
O
-
NH
O
O
NH
O
O
HO
OH
OH
Asp
P
-
O
N
HO
O
O
OH
HO
+
ATP
O
-
HO
H2N
ADP + Pi
N
O
O
-
O
P
-
O
O
O
O
NH
N
N
-
O
O
H2O
OH
O
-
H2N
H
Fumarate
O
-
O
O
P
OH
O
-
O
N
NH2
N
O
O
O
HO
OH
O
H
NH2
O
H2N
OH
HO
O
CO2
O
THF
NH2 Gln
O
-
N10 Formyl-Tetrahydrofolate
O
NH2
OH HO
O
HN
O
N
O
O
-
-
O
-
H2N
O
O
HO
HO
OH
OH
N10 Formyl-Tetrahydrofolate
THF
O
FOLIC ACID ANALOGS
Methotrexate etc inhibit reduction of
dihydrofolate to THF; ↓ DNA
replication in cancer and normal cells
HO
HO
P
O
N
N
O
O
N
HN
HN
H2O
O
O
-
O
O
P
-
O
N
O
O
HO
OH
Inosine 5'-Monophosphate
(IMP)
HO
OH
NH2
N
Pyrimidine Biosynthesis
O
NH2
O
O
O
OH HO
Gln
NH2
O
-
NH2
Gla
O
CO2
O
P
H2 N
ATP
-
O
ADP
Carbamyl
Phosphate
O
-
OH
O
O
OH
O
NH
NH2
O
NH2
HO
HN
THF
dUMP
HO
N5N10 Methylene
Tetrahydrofolate
P O
OH
N
O
FMN
CoQH2
FAD
CO2
HO
P
O
O
O
N
O
O
OH
PP
PRPP
O
O
HO
Dihydro Orotate
CoQ
HN
OH
dTMP
OH
O
O
O
N
H
O
HO
O
O
HN
O
Asp
H
H2O
OH
H
N
O
HN
OH
HO
OH
UMP
Base synthesized then
added to preformed ribose
O
Orotate
Anticoagulants
• Cofactor of enzyme that carboxylates γ-glutamyls in
Prothrombin and Factors VII, IX and X
• Without carboxylation, don’t bind membrane phospholipids
• Deficiency in infants - hemorrhagic disease of the newborn
• Natural K vitamins free of toxic side effects
O
CH3
O
OH
CH3
CH3
OH
O
NH 2
HO
NH2
CH3
O
O
HO
Vitamin K
O
Glutamate
H3C
CH3
Warfarin is structural analog of Vit K
LD50 8.7 mg/kg
OH
O
Carboxyglutamate
Chelators
104mg/kg rat iv
280 ug/kg rat iv
Signal transduction
Tetrodotoxin
Inhibits voltage-gated sodium channels
Oral LD50 334ug/kg
Cardiac glycosides (Inh Na+/K+ ATPase)
28.3 mg/kg rat LD50 oral
10.8 mg/kg rat LD50 iv
Michael acceptors
Acrolein
LD50 26 mg/kg
Acids
• Trifluoromethanesulfonic acid
• pH of 10% solution = 0.1
• Acute oral LD50: 1605.3 mg/kg bw
– GHS Cat 4; H302: Harmful if swallowed
• Acute dermal LD50: > 50 mg/kg bw
– test results inconclusive because of severe local
effects on skin at 2000 mg/kg bw
• Acute inhalation LC50: ????
– study scientifically unjustified
Future mechanistic approaches…
Questions?