the role of cell transport systems in medicating the toxic effects of drugs and chemicals....
TRANSCRIPT
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%