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?