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. 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. 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. 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. 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. 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. 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
O
R O
present in
chrysanthemums

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. 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. 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. Hydrolysis of Pyrethyroids (HPLC)
impurity from
intestinal mics
o-Br2CA 3-PBCOOH
t-Cl2CA 3-PBAlc

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. 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. 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. Hydrolysis by Individual HLCs
• 2 substrates
• 10X variability
• Correlated well
• Same CE enzymes
catalyze these
reactions

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. 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. 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. 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. 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. 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. 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. 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