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Mechanisms of Acute Toxicity
Dan Wilson, PhD, DABTScience Leader – CheminformaticsThe Dow Chemical CompanyAcute Tox WorkshopSep 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 Toxicity ranking LD50 (/kg bw) GHS Cat
Caffeine ? ? ?Arsenic ? ? ?Aspirin ? ? ?
Salt (NaCl) ? ? ?Ethanol ? ? ?Nicotine ? ? ?
Botulism toxin ? ? ?
Pop quiz: Put these in order of lethality• Which substance has lowest lethal dose? (i.e. the
most ‘toxic’)
Agent Toxicity ranking LD50 (/kg bw) GHS CatCaffeine 4 130-320 mg 3Arsenic 3 46 mg 2Aspirin 5 1000 mg 4
Salt (NaCl) 6 3000 mg 5Ethanol 7 14000 mg 5Nicotine 2 1 mg 1
Botulism toxin 1 0.02 ng 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
OrgansHeart, Liver, Kidney, Nervous System…
CellsMuscle, Cardiac, Hepatic, RBCs,
Neurons, PMNs…
OrganellesNucleus, Mitochondria, Peroxisome, Cytoplasm…
Biochemical PathwaysGluconeogenesis, 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
SO
OO
-N
O O
O
H
H
H
OH
O
O
O-
H
H
H
OH
H
H
Cholesterol
Deoxycholic acid
Taurochenodeoxycholic acid
• 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
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 energy-
rich 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
Fructose 6-F
Fructose 1,6-Bis P
Glyceraldehyde 3-P
1,3 bisPhosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate
Pyruvate
Acetyl CoA
Glucose
Dihydroxyacetone P
Glycolysis
Glycogen
Glucose 1-P
Glucose 6-P
Fructose 6-F
Fructose 1,6-Bis P
Glyceraldehyde 3-P
1,3 bisPhosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate
Pyruvate
Acetyl CoA
Citrate
Isocitrate
ɑ-Ketoglutarate
Succinyl-CoA
Succinate
Fumarate
Malate
Oxaloacetate
Kreb’s TCACycle
Lactate
Dihydroxyacetone-P
Glucose
CO2
TCA Cycle
Gluconeogenesis
Glycogen
Glucose 1-P
Glucose 6-P
Fructose 6-F
F
Gl
1,3 b
3-
NADPH
conolactone
ructose 1,6-Bis P
yceraldehyde 3-P
isPhosphoglycerate
Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate
Pyruvate
Acetyl CoA
Citrate
Isocitrate
ɑ-Ketoglutarate
Succinyl-CoA
Succinate
Fumarate
Malate
Oxaloacetate
Kreb’s TCACycle
Lactate
Dihydroxyacetone-P
Glucose
CO2
UDP Glucose
lu
NADPH
Ribose 5-P Ribulose 5-P 6-P Gluconate 6-P G
Xyulose 5-P
Sedoheptulose 7-P Erythrose 4-P
Glyceraldehyde 3-P
Pentose-Phosphate Pathway
Glycogen
Glucose 1-P
Glucose 6-P
Fructose 6-F
Fructose 1,6-Bis P
Glyceraldehyde 3-P
1,3 bisPhosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate
Pyruvate
Acetyl CoA
Citrate
Isocitrate
ɑ-Ketoglutarate
Succinyl-CoA
Succinate
Fumarate
Malate
Oxaloacetate
NADPH NADPH
Ribose 5-P Ribulose 5-P 6-P Gluconate 6-P Gluconolactone
Xyulose 5-P
Sedoheptulose 7-P Erythrose 4-P
Glyceraldehyde 3-P
Kreb’s TCACycle
Lactate
Acetoacetate
β-Hydroxybutarate
Methylmalonyl CoA
Propionyl Co A
Fatty acyl CoA (odd-number carbons)
Fructose
Fructose 1PGlyceraldehyde
Dihydroxyacetone-P
Glycerol-P
Triacylglycerol
Fatty acyl CoA
Malonyl CoA
Glycerol
Fatty acids
Glucose
CO2
UDP Glucose
Ketones
Fructose
Lipids
Glycogen
Glucose 1-P
Glucose 6-P
Fructose 6-F
Fructose 1,6-Bis P
Glyceraldehyde 3-P
1,3 bisPhosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate
Pyruvate
Acetyl CoA
Citruline
Argino-succinate
Arginine
Ornithine
Citrate
Isocitrate
ɑ-Ketoglutarate
Succinyl-CoA
Succinate
Fumarate
Malate
Oxaloacetate
NADPH NADPH
Ribose 5-P Ribulose 5-P 6-P Gluconate 6-P Gluconolactone
Xyulose 5-P
Sedoheptulose 7-P Erythrose 4-P
Glyceraldehyde 3-P
Kreb’s Urea Cycle
Kreb’s TCACycle
Asn
Asp
AlaCysGlySerThr
NH3 CO2
Carbamoyl-P
Lactate
Acetoacetate
β-Hydroxybutarate
Methylmalonyl CoA
Propionyl Co A
Fatty acyl CoA (odd-number carbons)
IleMetValThr
Homocysteine
Fructose
Fructose 1PGlyceraldehyde
Dihydroxyacetone-P
Glycerol-P
Triacylglycerol
Fatty acyl CoA
Malonyl CoA
Urea
Glycerol
Fatty acids
Glucose
LeuPheTyrTrpLys
CO2
ArgHisPro
Gln
GluPheTyr
Ile
Fumarate
UDP Glucose
Amino Acids
Glycogen
Glucose 1-P
Glucose 6-P
Fructose 6-F
Fructose 1,6-Bis P
Glyceraldehyde 3-P
1,3 bisPhosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate
Pyruvate
Acetyl CoA
Citruline
Argino-succinate
Arginine
Ornithine
Citrate
Isocitrate
ɑ-Ketoglutarate
Succinyl-CoA
Succinate
Fumarate
Malate
Oxaloacetate
NADPH NADPH
Ribose 5-P Ribulose 5-P 6-P Gluconate 6-P Gluconolactone
Xyulose 5-P
Sedoheptulose 7-P Erythrose 4-P
Glyceraldehyde 3-P
Kreb’s Urea Cycle
Asn
Asp
AlaCysGlySerThr
NH3 CO2
Carbamoyl-P
Lactate
Acetoacetate
β-Hydroxybutarate
Methylmalonyl CoA
Propionyl Co A
Fatty acyl CoA (odd-number carbons)
IleMetValThr
Homocysteine
Fructose
Fructose 1PGlyceraldehyde
Dihydroxyacetone-P
Glycerol-P
Triacylglycerol
Fatty acyl CoA
Malonyl CoA
Urea
Glycerol
Fatty acids
Glucose
LeuPheTyrTrpLys
CO2
ArgHisPro
Gln
GluPheTyr
Ile
Fumarate
UDP GlucoseNiacin
Thiamin
Niacin
Niacin
Niacin
Niacin
RiboflavinNiacinVit B12
Pyridoxine
Biotin
Niacin
Riboflavin
Pyridoxine Niacin
RiboflavinNiacin
ThiaminNiacinLipoic Acid
ThiaminNiacinLipoic AcidRiboflavin
Biotin
Vit B12
Pyridoxine
Biotin
Vitamins
Glycogen
Glucose 1-P
Glucose 6-P
Fructose 6-F
Fructose 1,6-Bis P
Glyceraldehyde 3-P
1,3 bisPhosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate
Phosphoenolpyruvate
Pyruvate
Acetyl CoA
Citruline
Argino-succinate
Arginine
Ornithine
NADPH NADPH
Ribose 5-P Ribulose 5-P 6-P Gluconate 6-P Gluconolactone
Xyulose 5-P
Sedoheptulose 7-P Erythrose 4-P
Glyceraldehyde 3-P
Kreb’s Urea Cycle
Asn
Asp
AlaCysGlySerThr
NH3 CO2
Carbamoyl-P
Lactate
Acetoacetate
β-Hydroxybutarate
Methylmalonyl CoA
Propionyl Co A
Fatty acyl CoA (odd-number carbons)
IleMetValThr
Homocysteine
Fructose
Fructose 1PGlyceraldehyde
Dihydroxyacetone-P
Glycerol-P
Triacylglycerol
Fatty acyl CoA
Malonyl CoA
Urea
Glycerol
Fatty acids
Glucose
LeuPheTyrTrpLys
CO2
ArgHisPro
Gln
GluPheTyr
Ile
Fumarate
UDP GlucoseNiacin
Thiamin
Niacin
Niacin
Niacin
Niacin
RiboflavinNiacinVit B12
Pyridoxine
Biotin
Niacin
Riboflavin
Pyridoxine Niacin
RiboflavinNiacin
ThiaminNiacinLipoic Acid
ThiaminNiacinLipoic AcidRiboflavin
Biotin
Vit B12
Pyridoxine
Biotin
MitochondrialElectron Transport
Citrate
Isocitrate
ɑ-Ketoglutarate
Succinyl-CoA
Succinate
Fumarate
Malate
Oxaloacetate
O
O
OH
CH3S
O
Thiamin coenzyme
Lipoic acid coenzyme
Coenzyme A-SH
Flavin coenzyme
NAD+
CO2
Thiamin coenzyme
Lipoic acid coenzyme
Flavin coenzyme
NADH + H+
CoA
Pyruvate Dehydrogenase Complex
Acetyl CoAPyruvate
Mitochondria
Arsenicinhibits
Pyruvate Dehydrogenase Complex
Lipoic Acid: Cofactor for 2nd Enzyme
NN
NN
NH2
OOH
OPOOH
OHO
PO
O
OHP
OO
OH OH
O
NH NH
O
S
O
NN
NN
NH2
OOH
OPOOH
OHO
PO
O
OHP
OO
OH OH
O
NH NH
O
SH
+
N+
R
SR
OH
Hydroxyethyl derivative bound to reactive carbon of TPP
TPP
SS
O
OHH
Lipoic acid
O
SHS
O
OH
H
O
SHS
O
OH
H
SHSH
O
OH
H
Coenzyme A-SH
Acetyl CoA Reduced Lipoic acid
+
+
Arsenicinhibits
Dihydrolipoyl transacetylase
Niacin – Vitamin B3OH
O
N
NH2
O
N
N
N
N
N
NH2
O
OHOH
O OP
O
O
O-
P
O
OH
O
OH OH
NH2
O
N+
N
N
N
N
NH2
O
OHOH
O OP
O
O
O-
P
O
OH
O
OH O
NH2
O
N+
PO
OHOH
NH
O
NH2
OH
Nicotinic acid
Nicotinamide
Tryptophan
NAD+ NADP+
R
NH2
O
N+
HH
R
NH2
O
N
NAD+
NADH
:H- Hydride ion
Riboflavin – Vit B2
FMN and FAD tightly bound to enzymes that catalyze oxidation or reduction
N N
NNH
R
O
OCH3
CH3
H
H
N N
NNH
R
O
OCH3
CH32H+, 2e-
FAD (oxidized quinone form) FADH2 (reduced hydroquinone form)
Fish acute toxicity vs. ToxCast HTS
Mitochondrial Electron Transport
III
III
Intermembrane space
Matrix
ATP Synthase
H+TCA
Cycle
SuccinateFADH2
Q
H+ H+
IIIIV
H2O O2
ADP ATP
H+
NADH
H+ H+ H+
Mitochondria membrane potential assay
Sakamuru S et al. Physiol. Genomics 2012;44:495-503
Mitochondrial tox predicts upper boundary to Daphnia toxicity
AC50Inhibition of Mitochondrial Membrane Potential (
Dap
hnia
acu
te L
C50
(mg/
l)
AC50 Inhibition of Mitochondrial Membrane Potential (uM)
AC50 Inhibition of Mitochondrial Membrane Potential (
Mitochondrial toxicity predicts upper bound to acute intravenous toxicity
AC50 Inhibition of Mitochondrial Membrane Potential (uM)
Rat
Intr
aven
ous
LD50
(mg/
kg)
AC50Inhibition of Mitochondrial Membrane Potential (
AC50 Inhibition of Mitochondrial Membrane Potential (
Mitochondrial toxicity doesn’t predict upper bound to oral rat toxicity
AC50 Inhibition of Mitochondrial Membrane Potential (uM)
Rat
Ora
l LD
50(m
g/kg
)
AC50Inhibition of Mitochondrial Membrane Potential (
AC50 Inhibition of Mitochondrial Membrane Potential (
LD50 values adjusted downward by % bioavailable
ester group
AC50 Inhibition of Mitochondrial Membrane Potential (uM)
Rat
Ora
l LD
50(m
g/kg
)
AC50Inhibition of Mitochondrial Membrane Potential (
AC50 Inhibition of Mitochondrial Membrane Potential (
Antimetabolites
HHH
NH
O
O
NH10
O
OHOH
N
NH
N5
NH
O
NH2
HHH
O N10
NH
N5
HHH
N+
10
NH
N5 H
H
HHH
N10
NH
N5OH2
HHH
HHH
NH10
NH
N5
Tetrahydrofolate (THF)
N10-Formyl-THF N5N10-Methenyl-THF N5N10-Methylene-THF N5-Methyl-THF
NADPH + H+
NADP+ NADH + H+
NAD+
Methionine
TMP
Purines
Dihydrofolate reductase2-steps requiring 2 NADPH Methotrexate
LD50 135 mg/kg
Purine synthesisGln
Gla
Asp
OO-
O-
OP H
O
OHOH
O
O- OP
O-O-
O
P
O
O
NH2
OH
NH2
OH
OO
O-
NH2Gln Gla
CO2
OO-
O-
OP NH2
O
OHOH
O
OHNH2
O
NH2O
O-
O-
OP NH
O
OHOH
ATP ADP + Pi+
N10 Formyl-Tetrahydrofolate
THF
O
O
NH2
OH
NH2
OH
O O
O-
NH2NH2
NO
O-
O-
OP NO
OHOH
O
NHNH
OO- O-
OP
NHO
OHOH
O
ONH
OO-
O-
OP NHO
OHOH
OOHNH2
NO
O-
O-
OP NO
OHOH
O
O
NH2
OH
OH
ATP+
ADP + Pi
O
O
NH
OH
OH
ONH2
NO
O-
O-
OP NO
OHOH
NH2
ONH2
NO
O-
O-
OP NO
OHOH
N10 Formyl-Tetrahydrofolate
THFO
NH2
ONH
NO
O-
O-
OP NO
OHOH
NN
NNH
O
O
OHOH
OOHP
O
OH
PRPP
GlnATPADPATPADP
H2O
O
O
O-
O-
H
H
FumarateH2O
Inosine 5'-Monophosphate (IMP)
FOLIC ACID ANALOGSMethotrexate etc inhibit reduction of dihydrofolate to THF; ↓ DNA replication in cancer and normal cells
Purines built on existing ribose sugar supplied by Pentose Phosphate Path
Pyrimidine Biosynthesis
O
NH2O
-
O-
OP
O
O
NH2
OH
OH
O
O
NH
OH
OH
O
NH2
H
NHNHO
OO
OH
NHNHO
O
O
OH
N
NH
OP OO
O
O
OH
OH
OH
OH
OH
ON
NH
OP OO
O
O
OH
OH
OH
OH
O
O
NH2
OH
NH2
OH
OO
O-
NH2Gln Gla Carbamyl Phosphate
AspDihydro Orotate
ATP ADP
CoQ
CoQH2
FMN
FAD
Orotate
PRPPPPCO2
CO2
UMP
H2O
dUMPdTMP
N5N10 Methylene Tetrahydrofolate
THF
Base synthesized then added to preformed ribose
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
O
OH
NH2
OH O
OH
O
O
OH
NH2OH
Glutamate CarboxyglutamateO
O
CH3
CH3
CH3CH3
CH3CH3
Vitamin K
Warfarin is structural analog of Vit KLD50 8.7 mg/kg
Chelators
104mg/kg rat iv280 ug/kg rat iv
Signal transduction
Tetrodotoxin
Inhibits voltage-gated sodium channelsOral 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
AcroleinLD50 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?
