The role of cell transport systems in medicating the toxic effects of drugs

and chemicals. Mechanisms of paraquat poisoning.

Lewis L Smith

Syngenta

Toxicology

• Identification of Hazard

• Characterisation of Hazard

• Relevance to MAN

• RISK Assessment

• RISK Management

Descriptive R

esearch

Mechanisms of Toxicity

Dose & Route of Exposure

Distribution & Metabolism

Primary Biochemical effect

Effect on Organism

Relevant to Man?

Similar to Man?

Qualitatively or quantitatively similar to Man?

Inhibition of putrescine (1µM) accumulation into rat lung slices by selected amino acids and their decarboxylated derivatives

Compound Structure at pH 7.4 I50 (µM)L-ornithine H3

+N(CH2)3 CHN+H3

I COO-

>1000

L-lysine H3+N(CH2)4CHN+H3

I COO-

>1000

Cadaverine(1,5-diaminopentane)

H3+N(CH2)5N+H3

25.6±7.8

L-arginine H2N-C-NH(CH2)3CHN+H3

II I NH2 COO-

+

>1000

Agmatine H2N-C-NH(CH2)4N+H3

II NH2 +

4.9 ± 0.7

Slices of rat lung were incubated in KRP (pH 7.4) containing 1 µM [14C)-putrescine in the presence of various concentrations of the compounds indicated. I50 values were determined by the method of Ross and Krieger, each animal acting as its own control. Results are expressed as the mean ± SEM, at least 3 animals being employed per compound studied.

The inhibitory potencies of various triamines against putrescine (1µM) accumulation by rat lung slices

Compound Structure

H2N(CH2)3NH(CH2)xNH2

I50 (µM)

N-[3 aminopropyl]-1,3-diaminopropane x=3 10.0±1.9

N-[3-aminopropyl]-1,4-diaminobutane x=4 10.9±4.5

N-[3-aminopropyl]-1,5-diaminopentane x-=5 12.4±4.5

N-[3-aminopropyl]-1,6-diaminohexane x=6 9.9±3.9

N-[3-aminopropyl]-1,7-diaminoheptane x=7 7.7±3.6

Slices of rat lung were incubated in KRP medium containing putrescine (1µM) in the presence of the triamine indicated. I50 values expressed as the mean ± SEM (n>3) derived by the method of Ross and Krieger.

The inhibitory potencies of methylglyoxal-bis (guanylhydrazone) against the accumulation of putrescine (1 (µM) by rat lung slices

Compound Structure I50 (µM)

Methylglyoxal-bis (guanylhydrazone)

H2N H CH3 NH2

C-NH-N=C-C=N-NH-C

HN NH

1.0±0.2

Eadie-Hofstee plot for the derivation of Km and Vmax of the accumulation process for MGBG into rat lung slices 100-

90-

80-

70-

60-

50-

40-

30-

20-

10-

0- 0 10 20

.. . .

.V (nmoles/g.wet wt.lung/hr)S (medium substrate conc. (µM)

Rat lung slices were incubated at 37o in the presence of various concentrations of [14C]-MGBG. Accumulation of [14C]-MGBG(V) was determined after 30,15 and 5 min for substrate medium concentrations <1µM and after 60,30 and 15 min for substrate medium concentrations >1µM. Triplicates were determined at each time point and all slices taken from the same rat. Rates of accumulation were determined by linear regression analysis. The results shown are from a single experiment typical of 3 and by linear regression analysis the estimated kinetic parameters from this experiment were: Km=4.6µM. Vmax=83.5nmols/g wet wt. Lung/hr (correlation coefficient r= -0.93).

NH2

N+

NH2

C2H5Br

O

NH2

NH

O

NH2

NHOH

S OH

O

O

.2 .2

N O

NHNH2

OH P OH

OH

O

.2

NCl

NH

CH3

N CH3

CH3

OH P OH

OH

O

.2

Ethidium 139.9 (±50.1)

Pentamidine17.9±3.8

Primaquine 51.8 (±23.9)

Chloroquine 20.1 (±11.3)

Compounds Structure I50 (µM)

30

60

90

120

150

180

00 100 200 300 400 500

s/v

(S) (uM)

..

.

....

KM = 503uM

KM = 12uM

Hanes – Woolf Plot of Cystamine Uptake

VMAX = 1/Slope KM = Y-Intercept x VMAX

Cystamine Cystaldimine

ThiocysteamineCysteamine

Hypotaurine TaurineNAD+ NADH

2GSH

GSSCys

1 3

4

5

6

H3N(CH2)2S-S(CH2)2NH3

++

H3N(CH2)2SH+

H3N-CH2-CH2-S-SH+

H3N-(CH2)2-SO3

+ -H3N-(CH2)2-SO2

+ -

CH2 CH

N

CH2 CH2

S

S

1 Postulated thiol-disulphide exchange2 Amine:oxygen oxidoreductase (deaminating)3 Spontaneous4 Uncharacterised5 Cysteamine dioxygenase6 Hypotaurine dehydrogenase

Possible Routes of Cystamine Metabolism

External Cystamine

Conc

Cystamine/

Cysteamine

Taurine M2 Protein

Association

5µM 15% 75% N.D. 5-10%

100µM 50% 25% 10-15% 5-10%

State of 14C-Label after 30’ Incubation

Aims of Current Studies

• Holistic identification of molecular pathways involved in PQ-induced lung fibrosis using Toxicogenomics

• Develop testable hypotheses for therapeutic intervention

Overview of molecular functions and pathwaysassociated with PQ-responsive genes

543 Paraquat-responsive genes

Gene Ontology terms(biological functions)

165 genes had no annotation

No annotation43.3%

Others25.5%

Collagen synthesis0.8%

TGFbeta pathway1.6%

Redox/Oxidative stress2.4%

G-protein related3.9%

Extracellular and matrix component

4.5%

Metalloprotease related1.3%

Lung development0.8%

cell maintenance: replication,

transcription, splicing and apoptosis

5.8%

Other growth factor/cytokine/signall

ing/Inflammation5.2% Transport,

endocytosis, exocytosis

5.0%