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Hydrolysis of pyrethroids by human and rat tissues
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Transcript of Hydrolysis of pyrethroids by human and rat tissues
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