Studies on the in vitro Metabolism of 

Deltamethrin by Rat and Human Tissue 

Preparations

Brian G. Lake

LFR Molecular Sciences and Centre 

for Toxicology, University of Surrey

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Outline of Presentation

• Purpose

• Study design

• Results

• Follow-up -hepatocyte and liver 

microsome/cytosol studies

• Implications of Results

Acknowledgements. Miyoung Yoon (Hamner Institutes) 

and Brian Houston (University of Manchester) 

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Purpose

• PBPK modelling needs intrinsic clearance 

(CLint) values for human tissues and Km 

and Vmax values for rat tissues

• Compare current findings with literature 

reports

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Determination of Rates of DLM Metabolism 

by Rat and Human Liver Preparations

• Phase I metabolism of DLM and other pyrethroids 

is by cytochrome P450 (CYP) and/or 

carboxylesterase (CES) enzymes

● Species studied

– Human

• Liver microsomes (CYP and CES)

• Cytosol (CES)

– Rat 

• Liver microsomes, cytosol and plasma (CES 

enzymes). 

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Determination of Rates of DLM Metabolism 

by Rat and Human Liver Preparations

• DLM metabolism by substrate depletion

– Monitoring loss of parent pyrethroid from tissue incubations 

at various times. Determine intrinsic clearance (CLint) values 

at low (e.g.1 μM) substrate concentration.

– Dissolve substrate in 50% acetonitrile (1% solvent).

– Perform incubations in glass vials to avoid binding to 

plastics. Stop with acetonitrile containing bifenthrin internal 

standard. 

– Analyse pyrethroids by Waters Acquity UPLC linked to 

Waters Xevo TQ mass spectrometer (UPLC-MS-MS). 

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DLM Metabolism with Rat and Human Liver 

Microsomes and Cytosol - Results

• Species differences confirmed

– Human liver - mainly by CES 

– Rat liver –both CYP and CES 

• CES are present in liver microsomes and cytosol

– Determination of rates of metabolism with microsomes 

alone will underestimate actual hepatic clearance. 

• Nonspecific binding to liver microsomes and cytosol. 

– Needs to be taken into account to obtain correct kinetic 

data – need to determine fraction unbound (fu) 

– Allowing for nonspecific binding results in higher CLint 

values and different Km values compared to previous data. 

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Comparison of EPA and LFR Data on DLM 

Metabolism in Rat and Human Liver Microsomes

• Godin et al. (2006) utilised rat and human liver 

microsomal protein concentrations of 1 mg protein/ml 

incubation mixture.

• Reported scaled CLint values expressed as ml/min/kg 

body weight converted to ml/min/mg protein using 

scaling factors (liver microsomal protein content and liver 

weight per kg body weight) reported by Godin et al. 

(2006).

• LFR studies performed with pooled male SpragueDawley rat and commercially obtained human (150 

donor pool) liver microsomes. 

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DLM Metabolism in Rat and Human Liver 

Microsomes: Comparison of EPA and LFR Data

Liver 

microsomes 

Apparent CLint (ml/min/mg 

protein

EPA LFR

Rat 0.077 0.040 (52%)

Human 0.077 0.174 (226%)

Determined at a microsomal protein concentration of 1.0 mg/ml in the 

presence of NADPH (i.e. CYP and CES metabolism) with a DLM 

substrate concentration of 1 μM. Note: EPA data of Godin et al. (2006) 

obtained with Long-Evans and not with Sprague-Dawley rats and with 

different human liver microsomal preparations.

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Issues Concerning Kinetics of DLM 

Metabolism in Rat Liver

• Corrected CLint values for DLM metabolism by 

rat liver microsomes and cytosol are higher than 

those previously reported values predicted by the 

current Hamner DLM PBPK models – need to 

reconcile

• Hypothesis: Rate limiting step in the clearance 

by metabolism of DLM in vivo may be the rate of 

uptake into hepatocytes.

– Example: Saquinavir the rate of metabolism in 

microsomes at low substrate concentrations is greater 

than the rate of metabolism in hepatocytes 

(Parker and Houston (2008) Drug Metab Dispos 36, 1375-1384). Exclusive Property of the Council for the Advancement 

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Studies to Resolve Issues Concerning Kinetics 

of DLM Metabolism in Rat Liver

• Obtained cryopreserved adult (3 month old) male 

Sprague-Dawley rat hepatocytes from Triangle Research 

Labs (USA). 

• Compared rates of DLM metabolism in hepatocytes with 

two preparations of 90 day old male SD rat liver 

microsome and cytosol fractions. Rat livers supplied by 

Charles River from both USA (designated CRL) and 

previously from UK (designated LFR). 

• Performed hepatocyte incubations in glass vials using an 

Eppendorf ThermoMixer C shaker/incubator.

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Studies to Resolve Issues Concerning Kinetics 

of DLM Metabolism in Rat Liver

• Studies conducted at a substrate concentration 

of 1 μM over a range of rat hepatocyte 

concentrations. 

– Assumption: only viable cells (based on Trypan blue 

exclusion) can metabolise DLM, but nonspecific 

binding of DLM will occur with both viable and non 

viable hepatocytes. 

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DLM Metabolism in Rat Hepatocytes: 

Effect of Cell Concentration

1 μM DLM. Adult male SD rat hepatocytes 0.018-

0.242 million total cells/ml incubation.

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Kinetics of DLM Metabolism in Rat 

Hepatocytes, Liver Microsomes and Cytosol

• Obtained apparent CLint values for DLM metabolism in rat 

hepatocytes and liver microsomes and cytosol over a range 

of protein concentrations.

• Calculated unbound CLint and affinity constant (Ka) with the 

equation:

CLint (apparent) = CLint

1 + Ka.P

Where P is incubation cell or protein concentration

• Calculated fraction unbound (fu) values with the equation: 

fu = 1

1 + Ka.P

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Rates of DLM Metabolism in Rat Hepatocytes 

and Liver Microsomes and Cytosol

• Compared rates of metabolism of 0.05, 0.1, 0.3 

and 1 μM DLM in hepatocytes, microsomes 

(with NADPH) and cytosol.

• Experimentally observed CLint values were 

corrected by allowing for the fraction unbound 

(fu), by dividing apparent CLint by the fu value. 

– The corrected (unbound) CLint values were scaled to 

per gram of liver

– The data for liver microsomes and cytosol were 

summed to compare with hepatocytes. 

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Determination of Rates of DLM Metabolism in 

Rat Hepatocytes and Liver Microsomes and 

Cytosol

Tissue 

preparation

Incubation 

conditions 

Affinity 

constant 

(Ka)

Fraction 

unbound 

(fu)

Scaling 

factor per 

gram of 

liver*

Hepatocytes 0.1 x 106

viable 

cells/ml

3.8583 0.722 120 million

Microsomes 0.25 mg 

protein/ml

7.7635 0.340 45 mg 

protein

Cytosol 0.4 mg 

protein/ml

7.6466 0.246 90 mg 

protein

*Data from Houston and Galetin (2008) Curr Drug Metab 9, 

940-951.

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Effect of Substrate Concentration on Rates of 

DLM Metabolism in Rat Hepatocytes

Parameter DLM substrate (μM)

0.05 0.1 0.3 1

Apparent CLint* 0.328 0.291 0.234 0.172

Unbound CLint* 0.454 0.404 0.324 0.239

Scaled CLint** 54.48 48.44 38.93 28.64

Results are mean values (n=3 experiments)

*Units ml/min/1 x 106 viable cells.

**Units ml/min/gram liver.

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Comparison of Scaled Rates of DLM 

Metabolism in Rat Hepatocytes with Liver 

Microsomes and Cytosol

Tissue fraction DLM substrate (μM)*

0.05 0.1 0.3 1

Hepatocytes 54.48 48.44 38.93 28.64

CRL microsomes 57.47 52.91 49.12 40.04

CRL cytosol 4.64 8.98 11.25 7.98

CRL sum 62.11 61.89 60.37 48.02

LFR microsomes 59.72 47.78 36.22 27.88

LFR cytosol 10.63 10.05 13.22 7.75

LFR sum 70.34 57.83 49.44 35.63

*Units ml/min/gram liver.

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Comparison of Scaled Rates of DLM 

Metabolism in Rat Hepatocytes with Liver 

Microsomes and Cytosol

Tissue fractions DLM substrate (μM)

0.05 0.1 0.3 1

Hepatocytes* 54.48 48.44 38.93 28.64

CRL sum** 1.14 1.28 1.55 1.68

LFR sum** 1.29 1.19 1.27 1.24

*Units ml/min/gram liver.

**Fold hepatocytes (i.e. sum of rates liver microsomes and 

cytosol divided by rate in hepatocytes). 

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Comparison of Scaled Rates of DLM 

Metabolism in Rat Hepatocytes with Liver 

Microsomes and Cytosol

• No marked difference between rates of DLM 

metabolism in rat hepatocytes compared to liver 

microsomes and cytosol 

– Thus rates of DLM metabolism can be estimated from 

CYP- and CES-dependent activities in liver microsomes 

and cytosol with no further contribution from other 

enzymes (e.g. phase II conjugative enzymes) present in 

intact hepatocytes. 

• Limitation of uptake into hepatocytes does not 

provide an explanation for the higher rates of DLM 

metabolism observed in LFR studies in rat tissue

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Implications of Results

• Reconciliation of differences in metabolic 

clearance necessitates other explanation 

and accommodation in the PBPK model

– To be discussed by Dr. Yoon 

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