In vitro hepatic and gastrointestinal biotransformation of

In vitro hepatic and gastrointestinal biotransformation of

hydrophobic chemicals in fish: Consideration of gastrointestinal biotransformation

in in vitro to in vivo extrapolation models

Leslie J. Saunders1, Patrick N. Fitzsimmons2, John W. Nichols2, Frank A.P.C. Gobas1,3

1. Department of Biological Sciences, Simon Fraser University, Burnaby, BC2. U.S. Environmental Protection Agency, National Health and Environmental Effects

Research Laboratory , Mid-Continent Ecology Division, Duluth, MN3. School of Resource and Environmental Management, Simon Fraser University,

Burnaby, BC

+

Biotransformation

• Enzymatic alteration of a chemical within an organism

• Conversion of a chemical to different chemical forms

• Relevant for chemical bioaccumulation assessment

If chemicals are readily metabolized their potential to bioaccumulate is reduced

Chemicals may be misidentified as bioaccumulative if biotransformation is not considered

Dietary uptake (kD)

Gill uptake (k1)

Gill elimination (k2)

Biotransformation (kMET)

Growth dilution (kG)

Fecal egestion (kE)

• Biotransformation can be an important determinant of a chemical’s bioaccumulation potential

Arnot & Gobas. 2004. Environ Toxicol Chem 23(10): 2343–2355

Arnot & Gobas Bioaccumulation Model

Estimating Biotransformation Potential

• In vivo biotransformation rates may be estimated by in vitro biotransformation data

BCF > 5,000 ?

in vitro liver

systemWhole liver Whole organism

(in vivo)

Bioaccumulation Model

• Major assumption: hepatic biotransformation represents whole body chemical biotransformation in fish

• Biotransformation in other tissues may significantly contribute to chemical elimination in fish

• The gastrointestinal tract (GIT) may contribute substantially to chemical elimination in fish

Gastrointestinal Biotransformation

• Diet often the primary exposure route for bioaccumlative contaminants

• Influence of GIT metabolism is generally overlooked Standardized methods for measuring GIT

biotransformation in fish do not currently exist

G.I. TractDietary

Uptake

Hepatic

portal

vein

Liver

1. To develop and optimize a method for preparing a trout GIT in vitro system (S9)

2. Compare the ability of the GIT to metabolize hydrophobic compounds to that of the liver

1.

3. Measure GIT scaling factors to predict in vivobiotransformation impacts

Research Objectives

Hepatic and Intestinal in vitro systems (S9 fractions)

3 pools of 3 fish per pool (n=3)

S9

9000 g

S9

9000 g

Whole livers were homogenized

Mucosal epithelial cells of intestines were

homogenized

Centrifugation at x 9000g produces

S9 fractions

ContainPhase I and II

biotransformation enzymes involved

in xenobiotic metabolism

6 cm

Incubation period

(1-60 min)

Lo

g (

Co

nc)

Time

Test

Control

Incubation system

buffer & cofactors

Test chemicalLiver orGIT S9

Extraction & GC/MS Analysis

in vitro biotransformation assays

y=mx+b, where m = in vitro biotransformation rate constant

Incubation period

(1-60 min)

Lo

g (

Co

nc)

Time

Test

Control

Incubation system

buffer & cofactors

Test chemicalLiver orGIT S9

Extraction & GC/MS Analysis

in vitro biotransformation assays

0.00

0.02

0.04

0.06

A B C

In v

itro

bio

tran

sfo

rma

tio

n

rate

c

on

sta

nt

(min

-1)

Test Chemical

Liver GIT

y=mx+b, where m = in vitro biotransformation rate constant

Selected Test Chemicals

Octocrylene

logKOW: 6.88

2-Ethylhexyl-4-

trimethoxycinnamate (EHMC)

logKOW: 5.80Benzo(a)pyrene

Pyrene

log KOW = 4.88

log KOW = 6.04

Polycyclic Aromatic

Hydrocarbons (PAHs)

Organic Sunscreen

Agents (UVFs)

In vitro biotransformation assays

0

0.1

0.2

0.3

0.4

�Pyrene

in v

itro

bio

tra

nsfo

rma

tio

n r

ate

co

nsta

nt

(min

-1)

Pyrene

Liver

GIT

0

0.2

0.4

0.6

0.8

1

�BaP

in v

itro

bio

tra

nsfo

rma

tio

n r

ate

co

nsta

nt

(min

-1)

Benzo(a)pyrene

Liver

GIT

• in vitro biotransformation rate constants in the liver are ~ 2 to 5 fold higher than those measured in GIT

0

0.02

0.04

0.06

0.08

0.1

�EHMC

in v

itro

bio

tra

nsfo

rma

tio

n r

ate

co

nsta

nt

(min

-1)

EHMC

Liver

GIT

Data presented are the mean ± standard error (n=3)

• Octocrylene: Biotransformation rates ~2x higher in the GIT vs the liver

• OC may be metabolized by an enzyme that has higher activities in the GIT vs liver

• For some chemicals, intestinal metabolism may greatly contribute to biotransformation in fish

0

0.01

0.02

0.03

0.04

�Octocrylene

In v

itro

bio

tra

ns

form

ati

on

ra

te c

on

sta

nt

(min

-1)

Octocrylene

Liver

GIT

In vitro biotransformation assays

Data presented are the mean ± standard error (n=3)

in vitro to in vivo Extrapolation Factors

• Physiological Measurements Dissections to determine

proportional tissue weightsof GIT and liver

Histology work to determine the % epithelial cells in the GIT

• Biochemical Measurements Total CYP characterization Protein contents of S9 and tissues

• in vitro hepatic and intestinal biotransformation data normalized to CYP protein and tissue weights for extrapolation

• Multiple data collected to extrapolate in vitrobiotransformation data to the in vivo level

Images provided by C. Blanksma, Histotechnologist, US EPA

Pyloric cecum Anterior GIT

0

0.1

0.2

0.3

0.4

0.5

1

in v

ivo

bio

tra

ns

form

ati

on

ra

te c

on

sta

nt

(da

y-1

) EHMC

in vivo predicted- Liver only

in vivo predicted - Liver & GIT

0.0

1.0

2.0

3.0

1

in v

ivo

bio

tra

ns

form

ati

on

ra

te c

on

sta

nt

(da

y-1

) Pyrene



0.0

0.5

1.0

1.5

1

in v

ivo

bio

tra

ns

form

ati

on

ra

te c

on

sta

nt

(da

y-1

) BaP



in vivo biotransformation rate constants

0

0.1

0.2

0.3

0.4

0.5

1

in v

ivo

bio

tra

ns

form

ati

on

ra

te c

on

sta

nt

(da

y-1

) EHMC



in vivo measured

0.0

1.0

2.0

3.0

1

in v

ivo

bio

tra

ns

form

ati

on

ra

te c

on

sta

nt

(da

y-1

) Pyrene

in vivo predicted- Liver onlyin vivo predicted - Liver & GITin vivo measured

0.0

0.5

1.0

1.5

1

in v

ivo

bio

tra

ns

form

ati

on

ra

te c

on

sta

nt

(da

y-1

) BaP

in vivo predicted- Liver onlyin vivo predicted - Liver & GITin vivo measured


In vivo biotransformation rate constants measured in rainbow trout. Pyrene and BaP in vivo data from Arnot et al., 2008. Environ. Tox. Chem. 27 (2): 341-351; EHMC in vivo data collected by L. Saunders (In Preparation)


0.00

0.05

0.10

0.15

1

in v

ivo

bio

tran

sfo

rmati

on

rate

co

nsta

nt

(day

-1)

Octocrylene




0.00

0.05

0.10

0.15

1

in v

ivo

bio

tran

sfo

rmati

on

rate

co

nsta

nt

(day

-1)

Octocrylene



in vivo measured

Octocrylene in vivo biotransformation rate constant measured in rainbow trout. Collected by L. Saunders (In Preparation)

Conclusion

• GIT biotransformation can contribute significantly to the overall biotransformation in fish

• Using only hepatic tests may underestimate in vivobiotransformation rates for some contaminants

Potential overestimates respective chemical bioaccumulation potential in fish

• Need to consider GIT biotransformation rates in in vitro to in vivo extrapolation approaches

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• Funding and Scholarships Unilever Research and Development

Natural Sciences and Engineering Research Council of Canada (NSERC)

Simon Fraser University

• Prof. Frank Gobas, SFU

• School of Resource and Environmental Management Toxicology Lab, Simon Fraser University

• Dr. John Nichols, US EPA

• Toxicokinetics Research Group

Alex Hoffman, Patrick Fitzsimmons, Melanie Ladd

Acknowledgements

Liver and GIT Enzyme Activities

Tissue

Cytochrome P450

(CYP) content

Ethoxyresorufin-O-deethylase

(EROD)

UDP-glucuronosyltransferase

(UGT)

Glutathione-S-transferase

(GST)

nmole/g tissuepmol/min/mg

proteinpmol/min/mg protein

nmol/min/mg protein

Liver S9 10.7 ± 2.1 3.9 ± 0.21014 ±

231725 ± 64

GIT S9 11.0 ± 1.9 1.7 ± 0.3 899 ± 136 381 ± 46

• Enzyme characterization assays to characterize Phase I and Phase II biotransformation enzymes

Data Collected by P. Fitzsimmons and M. Ladd, US EPA

• GIT metabolism assumed to operate in series with hepatic metabolism

• Independently calculated liver and GIT clearance terms, then combine to calculate total clearance (CLTOT; L/d/kg fish):

CLTOT = CLGIT + CLH ((QH-CLGIT)/QH)

• Scaling factors used to estimate the in vivo biotransformation rate constant from total clearance

QH = total liver blood flow, CLH = hepatic clearance, CLGIT = clearance by tissues of the GIT

in vitro to in vivo extrapolation of liver and GIT biotransformation

Fish

QH CLTOTCLH and CLGIT kMET

Documents

In vitro hepatic and gastrointestinal biotransformation of