1. The role of cell transport systems in medicating the toxic effects of drugs and chemicals. Mechanisms of paraquat poisoning. Lewis L Smith Syngenta

2. Toxicology Identification of Hazard Characterisation of Hazard Relevance to MAN RISK Assessment RISK Management Descriptive Research

3. 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?

17. Inhibition of putrescine (1µM) accumulation into rat lung slices by selected amino acids and their decarboxylated derivatives CompoundStructure at pH 7.4I50 (µM) L-ornithine H 3 + N(CH 2 ) 3 CHN + H 3 I COO - >1000 L-lysine H 3 + N(CH 2 ) 4 CHN + H 3 I COO - >1000 Cadaverine (1,5-diaminopentane) H 3 + N(CH 2 ) 5 N + H 3 25.6±7.8 L-arginine H 2 N-C-NH(CH 2 ) 3 CHN + H 3 II I NH 2 COO - + >1000 AgmatineH 2 N-C-NH(CH 2 ) 4 N + H 3 II NH 2 + 4.9 ± 0.7 Slices of rat lung were incubated in KRP (pH 7.4) containing 1 µM [ 14 C)-putrescine in the presence of various concentrations of the compounds indicated. I 50 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.

18. The inhibitory potencies of various triamines against putrescine (1µM) accumulation by rat lung slices CompoundStructure H 2 N(CH 2 ) 3 NH(CH 2 ) x NH 2 I 50 (µM) N-[3 aminopropyl]-1,3-diaminopropanex=310.0±1.9 N-[3-aminopropyl]-1,4-diaminobutanex=410.9±4.5 N-[3-aminopropyl]-1,5-diaminopentanex-=512.4±4.5 N-[3-aminopropyl]-1,6-diaminohexanex=69.9±3.9 N-[3-aminopropyl]-1,7-diaminoheptanex=77.7±3.6 Slices of rat lung were incubated in KRP medium containing putrescine (1µM) in the presence of the triamine indicated. I 50 values expressed as the mean ± SEM (n>3) derived by the method of Ross and Krieger.

19. The inhibitory potencies of methylglyoxal-bis (guanylhydrazone) against the accumulation of putrescine (1 (µM) by rat lung slices CompoundStructure I 50 ( µM) Methylglyoxal-bis (guanylhydrazone) H2N H CH3 NH2 C-NH-N=C-C=N-NH-C HN NH 1.0±0.2

20. 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 37 o in the presence of various concentrations of [ 14 C]-MGBG. Accumulation of [ 14 C]-MGBG(V) was determined after 30,15 and 5 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=4.6 µM. Vmax=83.5nmols/g wet wt. Lung/hr (correlation coefficient r= -0.93).

21. Ethidium 139.9 (±50.1) Pentamidine 17.9±3.8 Primaquine 51.8 (±23.9) Chloroquine 20.1 (±11.3) CompoundsStructureI 50 (µM)

22. 30 60 90 120 150 180 0 0100200300400500 s/v (S) (uM)....... KM = 503uM KM = 12uM Hanes – Woolf Plot of Cystamine Uptake VMAX = 1/Slope KM = Y-Intercept x VMAX

23. CystamineCystaldimine ThiocysteamineCysteamine HypotaurineTaurine NAD + NADH 2 GSH GSSCys 1 3 4 5 6 H 3 N(CH 2 ) 2 S-S(CH 2 ) 2 NH 3 + + H 3 N(CH 2 ) 2 SH + H 3 N-CH 2 -CH 2 -S-SH + H 3 N-(CH 2 ) 2 -SO 3 + - H 3 N-(CH 2 ) 2 -SO 2 + - CH 2 CH N CH 2 S S 1 Postulated thiol-disulphide exchange 2 Amine:oxygen oxidoreductase (deaminating) 3 Spontaneous 4 Uncharacterised 5 Cysteamine dioxygenase 6 Hypotaurine dehydrogenase Possible Routes of Cystamine Metabolism

24. External Cystamine Conc Cystamine/ Cysteamine TaurineM2Protein Association 5 µM 15%75%N.D.5-10% 100 µM 50%25%10-15%5-10% State of 14 C-Label after 30’ Incubation

28. Aims of Current Studies Holistic identification of molecular pathways involved in PQ-induced lung fibrosis using Toxicogenomics Develop testable hypotheses for therapeutic intervention

29. Overview of molecular functions and pathways associated with PQ-responsive genes 543 Paraquat-responsive genes Gene Ontology terms (biological functions) 165 genes had no annotation