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