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Hydrolysis of Pyrethroids by Human         and Rat Tissues          Crow, Borazjani, Potter, Ross        Mississippi State...
Human Carboxylesterases (hCEs)• hCE-1 and hCE-2  – 48% sequence homology  – Large quantities in various tissues, but rathe...
Previous Work• Human hepatic CEs are involved in pyrethroid  metabolism• Purified CEs and pyrethroids   – hCE-1, hCE-2, ra...
Objectives of this Study• Expression and activity of CEs in:   – Human      • Intestinal mics      • Hepatic mics and cyto...
Materials• Pyrethroids, metabolites and inhibitors were purchased• hCE-1 and hCE-2 were expressed• Rat serum was purified•...
Tissue Preparations• Pooled human intestinal microsomes (5 individuals)   – Individual mics and cytosol are unavailable• P...
Pyrethroid Insecticides    •    Used extensively in agriculture and public health          –   Sodium channel toxin  seiz...
Microsomal, Cytosolic, and Serum Incubations• Pyrethroid substrate (5-100 µM or 50 µM)• 50 mM Tris buffer (pH 7.4)• Total ...
Pure CE and Lipase Incubations• Pyrethroid substrate (5-100 µM)• 50 mM Tris buffer (pH 7.4)•  Deoxycholic or cholic acid (...
Native PAGE Analysis• 100 ng purified protein or• 40 µg homogenate-supernatant•   100 µM 4-MUA•   100 mM KPO4 (pH 6.5)•   ...
Hydrolysis of Pyrethyroids (HPLC)        impurity from        intestinal mics                           o-Br2CA    3-PBCOO...
Pyrethroid Metabolism by Intestinal Mics                      •   Metabolism by human intestinal                          ...
Native PAGE analysis         •   hCE-1 and hCE-2 are present in HLC             and HLM         •   Trans-permethrin:     ...
trans-Permethrin Metabolism by HLM and HLC                50 µM trans-permethrin                HLM are 3X more active tha...
Hydrolysis by Individual HLCs                       • 2 substrates                       • 10X variability                ...
hCE-1 Protein Levels in HLC•   Variable amounts (CV = 56%, unlike HLM levels where CV = 9%) that correlated well    with h...
4-MUA Staining of HLC              • hCE-1 trimers and                monomers              • Esterase D              • CP...
trans-Permethrin: Human (pooled, 25) vs Rat Liver    Rat hydrolase A              7                   2.2 min-1    Rat hyd...
Whole Rat Serum    50 µM pyrethroid + Rat Serum                              Type 1           Type 2•   Rat:     –     Typ...
Purified Rat Serum CE                              • CPO (5 µM) inhibits                                rat serum CE but n...
Purified Rat Serum CE     50 µM pyrethroid + Rat Serum                       Type 1         Type 2•   Same order of substr...
Conclusions• hCE-2 plays a significant role in the metabolism of trans-permethrin    –   But not other Type 1 or Type 2 py...
Summary (cont’d)•   Should use whole tissue homogenates when assessing overall esterase    activity•   Variability in live...
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Hydrolysis of pyrethroids by human and rat tissues

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Hydrolysis of pyrethroids by human and rat tissues

  1. 1. Hydrolysis of Pyrethroids by Human and Rat Tissues Crow, Borazjani, Potter, Ross Mississippi State Univ, St. Jude’s Toxicol. Applied Pharmacol. 221, 1-12 (2007)
  2. 2. Human Carboxylesterases (hCEs)• hCE-1 and hCE-2 – 48% sequence homology – Large quantities in various tissues, but rather inefficient as enzymes • hCE-1 in liver • hCE-2 in intestine  reduced bioavailability – Rats and mice have CEs in their plasma, but humans do not – Rats and mice have >two CEs in their livers • Rat hydrolase A and B are 70-80% identical to hCE-1 and <50% to hCE-2 – Human and rat adipose tissue contain lipases • Pancreatic lipases are secreted into the small intestine and stimulated by bile salts – Exhibit hydrolytic activity toward: • Drugs • Lipids • Other xenobiotics – Pyrethroid insecticides
  3. 3. Previous Work• Human hepatic CEs are involved in pyrethroid metabolism• Purified CEs and pyrethroids – hCE-1, hCE-2, rabbit CE, 2 rat CEs – Km, Vmax
  4. 4. Objectives of this Study• Expression and activity of CEs in: – Human • Intestinal mics • Hepatic mics and cytosol • Serum – Rat • Intestinal mics and cytosol • Serum• Kinetic properties and substrate specificity – Purified rat serum CE and lipases
  5. 5. Materials• Pyrethroids, metabolites and inhibitors were purchased• hCE-1 and hCE-2 were expressed• Rat serum was purified• Lipases were purchased• Antibodies were obtained through collaboration
  6. 6. Tissue Preparations• Pooled human intestinal microsomes (5 individuals) – Individual mics and cytosol are unavailable• Pooled human liver microsomes (18 individuals)• Individual human liver cytosol preps (11 individials)• Pooled human liver cytosol preps (20 individuals)• Pooled rat blood (5 individuals) – Stand 1 hr to clot and then centrifuge at 2000 x g for 20 min  serum• Rat liver and intestinal microsomes and cytosol
  7. 7. Pyrethroid Insecticides • Used extensively in agriculture and public health – Sodium channel toxin  seizures – 500,000 lbs used in CA in 1999 (17% of global market in 2002) • Replacing more acutely toxic OP insecticides (considerably less toxic to animals) – Lowest lethal dose in adults is 1 g/kg (pyrethrum) – Cis more toxic than trans (slower metabolism) -cyano group Pyrethrins O OR O present in chrysanthemums
  8. 8. Microsomal, Cytosolic, and Serum Incubations• Pyrethroid substrate (5-100 µM or 50 µM)• 50 mM Tris buffer (pH 7.4)• Total volume = 250 µL• 5 min preincubation• 0.5 mg/mL tissue fraction or 25-50 uL pooled serum initiates reaction• 15 or 30 min incubation• Quenched by addition of 250 µL ice-cold ACN• IS = 3-(4-methoxy)-phenoxybenzaldehyde (10 µM)• 5 min centrifugation, 16100 x g• HPLC analysis
  9. 9. Pure CE and Lipase Incubations• Pyrethroid substrate (5-100 µM)• 50 mM Tris buffer (pH 7.4)• Deoxycholic or cholic acid (5 mM) for lipase reactions• Total volume = 100 µL• 5 min preincubation• 2.5 µg pure CE or lipase initiates reaction• 30 min incubation• Quenched by addition of 100 µL ice-cold ACN• IS = 3-(4-methoxy)-phenoxybenzaldehyde (10 µM)• 5 min centrifugation, 16100 x g• HPLC analysis
  10. 10. Native PAGE Analysis• 100 ng purified protein or• 40 µg homogenate-supernatant• 100 µM 4-MUA• 100 mM KPO4 (pH 6.5)• Rocked for 15 min• Visualize with UV transilluminator plate• Quantitate by densitometry
  11. 11. Hydrolysis of Pyrethyroids (HPLC) impurity from intestinal mics o-Br2CA 3-PBCOOH t-Cl2CA 3-PBAlc
  12. 12. Pyrethroid Metabolism by Intestinal Mics • Metabolism by human intestinal mics is similar to hCE-2 profile Km = 9 µM, kcat =1.7 min-1 • No hCE-1 like-protein in rat or human intestinal mics • Selective hCE-2 inhibitor (Ki = 9 vs 3300 nM) inhibits trans- permethrin metabolism (1.1 µM  50% decrease in hCE-2 activity) • trans-permethrin: Human intestinal mics 4-5X more active than rat (~ 2.5% of total rat hydrolysis)
  13. 13. Native PAGE analysis • hCE-1 and hCE-2 are present in HLC and HLM • Trans-permethrin: hCE-1: Km = 24 µM, kcat = 3.4 min-1 hCE-2: Km = 9 µM, kcat =1.7 min-1 • hCE-1 is not present in HIM • hCE-1: HLM >> HLC
  14. 14. trans-Permethrin Metabolism by HLM and HLC 50 µM trans-permethrin HLM are 3X more active than HLC HLM: Km = 21 µM, Vmax = 1120 pmol/min/mg HLC: Km = 3 µM, Vmax = 469 pmol/min/mg hCE-1: Km = 24 µM, kcat = 3.4 min-1
  15. 15. Hydrolysis by Individual HLCs • 2 substrates • 10X variability • Correlated well • Same CE enzymes catalyze these reactions
  16. 16. hCE-1 Protein Levels in HLC• Variable amounts (CV = 56%, unlike HLM levels where CV = 9%) that correlated well with hCE-1 activities – Variation ~ 6X – pNPVa, trans-permethrin, and bioresmethrin activity – Indicate a role for hCE-1
  17. 17. 4-MUA Staining of HLC • hCE-1 trimers and monomers • Esterase D • CPO (1 µM) inhibits hCE-1 and hCE-2 but not Esterase D
  18. 18. trans-Permethrin: Human (pooled, 25) vs Rat Liver Rat hydrolase A 7 2.2 min-1 Rat hydrolase B 10 1.5 hCE-1 24 3.4• HLM Vmaxs vary 6X while hCE-1 protein levels do not vary – Other esterases involved that are probably not in the HLC fraction• Rat appears to be a reasonable model for human hepatic metabolism of trans-permethrin
  19. 19. Whole Rat Serum 50 µM pyrethroid + Rat Serum Type 1 Type 2• Rat: – Type 1 exhibited Michaelis-Menten kinetics – Type 2 did not exhibit hyperbolic kinetics – Estimate that rat serum possesses ~ 4% of the total hydrolytic capacity for pyrethroids• Human serum did not catalyze hydrolysis of Type 1 or Type 2 pyrethroids• Purified human AChE and BuChE did not hydrolyze Type 1 or Type 2 pyrethroids
  20. 20. Purified Rat Serum CE • CPO (5 µM) inhibits rat serum CE but not• Stained with • Purified rat albumin esterase 4-MUA rat serum activity CE
  21. 21. Purified Rat Serum CE 50 µM pyrethroid + Rat Serum Type 1 Type 2• Same order of substrate hydrolysis as whole rat serum• Bioresmethrin: Km = 16 µM and kcat = 1.65 min-1• Trans-permethrin: Km = 24 µM and kcat = 1.30 min-1• Lipases were not able to hydrolyze the pyrethroids
  22. 22. Conclusions• hCE-2 plays a significant role in the metabolism of trans-permethrin – But not other Type 1 or Type 2 pyrethroids – Metabolism of pyrethroids in the intestine depends on the structure – Rat intestine was 4-5X less active than human – hCE-1 and hCE-2 in the liver have similar kinetic properties with trans- permethrin  therefore probably both involved in its metabolism• There are differences in the CEs expressed in rat and human intestine – rCE-1 and two rCE-2 like enzymes vs. just hCE-2 – No hCE-1 in human intestine• Rat metabolism of trans-permethrin: – 4% by serum, 2.5% by intestine, 40% by liver cytosol, 50% by liver microsomes• Human metabolism of trans-permethrin: – 0% by serum, 10-12% by intestine, 20-60% by liver cytosol (average = 40), 30- 70% by liver microsomes
  23. 23. Summary (cont’d)• Should use whole tissue homogenates when assessing overall esterase activity• Variability in liver cytosolic hCE-1 might be due to: – Only partial solubiliization in the purification protocol – Cytosolic CE lacks the N-terminal signal sequence – Some unknown mechanism directs the CE to the cytosol• No detectable pyrethroid metabolism in human blood – Lack of CEs – Rat may not be a good model when a compound is metabolized to a significant extent in rat blood – May need a transgenic rat to predict PK for these compounds – Rat and mouse may not be good models to use for risk assessment

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