june 6, 2004 t.j. flynn1 multiendpoint profiling of hepatotoxicants in vitro thomas j. flynn, ph.d....

June 6, 2004 T.J. Flynn 1

Multiendpoint Profiling of Hepatotoxicants in Vitro

Thomas J. Flynn, Ph.D.

FDA, Center for Food Safety and Applied Nutrition


Disclaimer

The views presented are those of the speaker and not necessarily of the FDA/CFSAN.


Hepatotoxicity Team

Chung Kim, Ph.D. - Pharmacokinetics Paddy Wiesenfeld, Ph.D. – Apoptosis, Lipid

metabolome Saura Sahu, Ph.D. – Oxidative damage Phil Sapienza, M.S. – Research chemist Ivan Ross, M.S. – Research biologist Widmark Johnson – Technical assistance


Botanical Products Associated With Hepatotoxicity(from Willet et al., 2004)

Botanical Common Names Suspected Toxic Principle

Crotalaria spp. Rattleweed, Sunnhemp pyrrolizidine alkaloids

Heliotropium spp. White Heliotrope pyrrolizidine alkaloids

Symphytum spp. Comfrey pyrrolizidine alkaloids

Teucrium chamaedrys Germander neoclerodane diterpenes

Lycopodium serratum Jin Bu Huan levo-alkaloid

Piper methysticum Kava kava kavapyrones

Hedeoma, Mentha Pennyroyal pulegone

Larrea tridentata Chaparral nordihydroguaiaretic acid

Chelidonium majus Greater celandine ? alkaloid

Callilepsis laureola Impila atractyloside

Atractylis gummifera Gum Thistle atractyloside

Scutellaria Skullcap ?

Polygonum multiflorum He Shon Wu ?


How do you develop a relevant in vitro model for hepatotoxicity?


Mechanisms of Hepatotoxicity

Cell Death (necrosis, apoptosis) Cholestasis Steatosis Phospholipidosis Oxidative stress Mitochondrial dysfunction Modulation of CYP activities


Cell Death (Necrosis)

Total double-stranded DNA (H33258) (Rago et al., Anal. Biochem. 191: 31-34, 1990)

Resazurin reduction (“Alamar blue”) LDH, ALT, AST, ALP release Total ATP


Apoptosis

ApoStrand® Caspase-3

(Maximum sensitivity at 4 hr post-treatment)


Steatosis & Phospholipidosis

Nile red uptake (McMillian et al., In

Vitro Mol. Toxicol. 14: 177-190, 2001)


Oxidative Stress

Dichlorofluorescin diacetate oxidation (Yerushalmi et al., Hepatology 33: 616-626,

2001). Glutathione depletion DNA strand breaks Lipid peroxides (TBARS)


Mitochondrial Dysfunction

Rhodamine 123 uptake and retention (Rat et al., Cell Biol. Toxicol. 10: 329-337,

1994) (Measure 3 hr post exposure) (Also measures P-glycoprotein?)


Modulation of CYP450 Activities

EROD (CYP1A), BOROD (CYP3A) (Donato et al., Anal. Biochem. 213: 29-33, 1993)

Testosterone hydroxylation (multiple CYP)

HPLC of Hydroxylated Testosterone Metabolites

05

00

00

10

00

00

15

00

00

20

00

00

16 18 20 22 24 26 28 30 32 34 36

Time (min)

mV 6a

15b

6b15

a

7a

16a

16b

2b2a

Co

rtex

olo

ne

Tes

tost

ero

An

dro

sten

ed

Pro

ges

tero


Desirable Properties of an In Vitro Model

Display as many liver-specific functions as possible

Use post-mitotic cells (closer to “reality”) Primary cells or cell lines at confluence

Low glucose medium (closer to “reality”) “Reasonable” maximum dose (1000 mg/mL or

limit of solubility) Dose response – should not disregard usable

data (e.g., EC50)


Cell Lines Evaluated

HepG2 (human hepatocarcinoma) HepG2/C3A (human hepatocarcinoma) WRL68 (heteroploid human fetal liver) Clone-9 (normal (?) rat liver)


Compounds Used for Test System Pre-Validation

Compound Chemical Class Biological Activity Hepatotoxicity

Acetaminophen Phenolic Human drug (analgesic, antipyretic)

Yes

Androstenedione Steroid Androgen, estrogen precursor

Daidzein Isoflavone Phytoestrogen, antioxidant

Estriol Steroid Estrogenic

Fumonisin B1 Mycotoxin Inhibits sphingolipid synthesis

Yes

Genistein Isoflavone Phytoestrogen, PK inhibitor, antioxidant

Glycochenodeoxy-cholate

Steroid Detergent (bile salt) Yes


Compounds Used for Test System Pre-Validation (cont’d)

Compound Chemical Class Biological Activity Hepatotoxicity

a-Naphthoflavone Flavone (synthetic) CYP inhibitor

b-Naphthoflavone Flavone (synthetic) CYP inducer

NDGA Polyphenol Antioxidant Yes

Quercetin Flavone Antioxidant, CYP3A4 inhibitor, phospho-

diesterase inhibitor Testosterone Steroid Androgen, anabolic Yes

Valproic acid Short-chain carboxylic acid

Human drug (anticonvulsant)

Yes


96-Well Plate Template

1 2 3 4 5 6 7 8 9 10 11 12 A

PBS Resorr ufin Stds. DNA

Stds.

B 0 mg/mL

0 mg/mL

Treat-ment level 1

Treat-ment level 2

Treat-ment level 3

Treat-ment level 4

Treat-ment level 5

Treat-ment level 6

Treat-ment level 7

Treat-ment level 8

C

D

E

F

G

H DCF or R123

Stds. Stds.


Assay Protocol - 1

Plate #1 Plate #2 Plate #3 Day 1 1. Wash cells 2X with

HBSS. 2. Add DCFDA and

incubate for 30 min at 37C (no CO2).

3. Wash cells 2X with HBSS.

4. Add test agent. 5. Incubate cells at

37C.

1. Add test agent. 2. Incubate cells

at 37C.

1. Add test agent, and incubate cells 2.5 hr at 37C.

2. Add 20 mL R123 and incubate at 37C for 0.5 hr.

3. Wash cells 2X with HBSS. 4. Re-add test agent. 5. Incubate cells at 37C.

Day 2 Observe cells for cell death, crystallization of test agent, etc.

Observe cells for cell death, crystallization of test agent, etc.

Observe cells for cell death, crystallization of test agent, etc.


Assay Protocol - 2 Plate #1 Plate #2 Plate #3 Day 3 1. Remove PBS from

outer wells using suction.

2. Add DCF standards. 3. Read fluorescence

(DCFDA assay). 4. Wash cells 2X with

HBSS 5. EROD assay. 6. Wash cells 2X with

HBSS. 7. Shake plate dry,

and place in -80C freezer.

1. Wash cells 2X with HBSS.

2. Nile red assay. 3. Shake plate

dry, and place in -80C freezer.

1. Remove PBS from outer wells using suction.

2. Add R123 standards.

3. Read fluorescence (R123 assay).

4. Wash cells 2X with HBSS

5. BOROD assay. 6. Wash cells 2X

with HBSS. 7. Shake plate dry,

and place in -80C freezer.

Subsequent Day

DNA assay DNA assay DNA assay


Typical Assay Results

Effects of Valproic Acid on C3A Human Hepatocytes

01

00

20

03

00

40

0

Total DNA Nile Red R123 DCFDA EROD BOROD

% o

f C

on

tro

l

0 2.5 5 10 25 50 100 250 500Concentration (mg/mL)

*

*Significantly different from control (p < 0.05)

*

**

*

*

*

*

* *

*


Log-Log Regression

Effects of Test Compounds on Dichlorofluorescin Oxidation in C3A Cells0

100

200

0.1 1 10 100 1000

log conc. (mg/mL)

% o

f C

on

tro

l

VPA

NDG


DCFDA Assay

Dichlorofluorescin Assay for ROS

0

100

200

-10 0 10 20 30 40 50

Slope

Inte

rcep

t

VPAh

NDGh

ACAh


Rhodamine 123 Assay

Rhodamine 123

0

100

200

-10 0 10 20 30 40 50

Slope

Inte

rcep

t

TESh

ANFh

QUEh

GENh

ANDh

ACAh

DAIh


Nile Red Assay

Nile Red

0

50

100

150

-20 -15 -10 -5 0 5 10 15

Slope

Inte

rcep

t

GENh

ANFh

ANDh

ESTh

BNFh

GCDh


EROD - 1

EROD

0

1000

2000

3000

4000

-500 0 500 1000 1500 2000 2500

Slope

Inte

rcep

t

BNFh


EROD - 2

EROD

0

100

200

300

400

-50 0 50 100

Slope

Inte

rcep

t

DAIh

NDGh

VPAhFB1h

GCDh

ANFh

ESTh

ANDh

TEShQUEh


BOROD - 1

BOROD

0

100

200

300

400

500

-50 0 50 100 150 200 250

Slope

Inte

rcep

t

BNFh


BOROD - 2

BOROD

0

50

100

150

200

-20 0 20 40 60 80 100

Slope

Inte

rcep

t

GENh

ANDh

ACAh

TEShVPAh

ESTh

DAI

GCD


DNA Assay

DNA

80

90

100

110

-4 -3 -2 -1 0 1 2

Slope

Inte

rcep

t

TES

AND

ANF

QUE

EST


Species Comparisons?

C3A vs. Clone9 Cells - Comparison of Response to Test Chemicals Compound DNA Nile Red R123 DCFDA EROD BOROD Acetaminophen D S S D X X Androstenedione S S S X X X Daidzein D D S D D D Estriol S D D D D X Fumonisin B1 D D D S X X Genistein D S S D X X GCDCA S S S S X X a-Naphthoflavone D D D D D X b-Naphthoflavone S D D D S S NDGA D X D D X X Quercetin D D D S D X Testosterone S S S S X X Valproic Acid D D S D X X Different response (concentration or direction, by ANCOVA) Same response Could not compare


Conclusions

Each model compound generated a unique response pattern among the six endpoints evaluated.

The response pattern discriminated between the following pairs of closely related compounds:

Androstenedione - Testosterone a-Naphthoflavone - b-Naphthoflavone Daidzein - Genistein

The response pattern discriminated between multiple biological mechanisms of action.

For model compounds that are human drugs with known hepatotoxicity (acetaminophen and valproic acid), some endpoints responded at medium concentrations comparable to known human blood levels.

june 6, 2004 t.j. flynn1 multiendpoint profiling of hepatotoxicants in vitro thomas j. flynn, ph.d....

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