hydrolysis of pyrethroids by human and rat tissues
TRANSCRIPT
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)
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
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
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
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
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
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)
a-cyano group
O
O
O
R
Pyrethrins
present in chrysanthemums
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
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
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
Hydrolysis of Pyrethyroids (HPLC)
3-PBCOOH
3-PBAlct-Cl2CA
o-Br2CA
impurity from intestinal mics
Pyrethroid Metabolism by Intestinal Mics
• Metabolism by human intestinal mics is similar to hCE-2 profileKm = 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)
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
trans-Permethrin Metabolism by HLM and HLC
50 µM trans-permethrinHLM are 3X more active than HLC
HLM: Km = 21 µM, Vmax = 1120 pmol/min/mgHLC: Km = 3 µM, Vmax = 469 pmol/min/mghCE-1: Km = 24 µM, kcat = 3.4 min-1
Hydrolysis by Individual HLCs
• 2 substrates
• 10X variability
• Correlated well
• Same CE enzymes catalyze these reactions
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
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
trans-Permethrin: Human (pooled, 25) vs Rat Liver
• 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
Rat hydrolase A 7 2.2 min-1Rat hydrolase B 10 1.5hCE-1 24 3.4
Whole Rat Serum
• 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
Type 2
Type 1
50 µM pyrethroid + Rat Serum
Purified Rat Serum CE
• Stained with
4-MUA
• Purified
rat serum CE
• CPO (5 µM) inhibits rat serum CE but not rat albumin esterase activity
Purified Rat Serum CE
• 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
Type 2
Type 1
50 µM pyrethroid + Rat Serum
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
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