use of in vitro assays as surrogate measures of ...use of in vitro assays as surrogate measures of...

Use of In Vitro Assays as Surrogate Measures of Contaminant Bioavailability

Albert Juhasz

Future Industries Institute, University of South Australia

[email protected]

In Vivo-In Vitro Correlation –Why is this Important?

In vitro assays have the potential to overcome the

time and expense limitation of in vivo studies thereby

providing a surrogate measurement of relative

bioavailability that is quick and inexpensive compared

to animal models.

BUT …… in order to have confidence in the use of

in vitro assays as a surrogate measure of relative

bioavailability, the relationship between in vivo relative

bioavailability and in vitro bioaccessibility needs to be

established.

Pb in mine-impacted soil

RBALP and swine

(Drexler and Brattin, 2007)

Pb in soil

Rel-SBRC-I and mice

(Smith et al., 2010)

Pb in mine waste

IVG and swine

(Schroder et al. 2003)

As in soil

SBRC-G and swine / primates

(Brattin et al., 2013)

1995 2000 2010 20152005

Pb in mine waste

PBET and rats

(Ruby et al., 1996)

As in soil

SBRC-G and mice

(Bradham et al., 2011)

As, Pb in soil

UBM and swine

(Denys et al., 2012)

Pb in shooting range soils

RBALP and swine

(Bannon et al., 2009)

Pb in soil

Rel-SBRC-I and swine

(Juhasz et al., 2009).

As in soil

SBRC-G and swine

(Juhasz et al., 2007; 2009)

Can Bioaccessibility Assays Predict Contaminant Relative Bioavailability?

As in mine waste

IVG and swine

(Rodriguez et al., 1999)

As in soil

SBRC-G and swine

(Juhasz et al., 2014)

As in dust

SBRC-G and mice

(Li et al., 2014)

As in soil

SBRC-G and swine

(Juhasz et al., 2015)

As in soil

SBRC-G and mice

(Bradham et al., 2014)

Pb in dust

SBRC-G and mice

(Li et al., 2014)

As in soil

SBRC-G and MSM

(Diamond et al., 2014)

0 25 50 75 1000

25

50

75

100

As Bioaccessibility (%; SBRC-G)

As

Re

lati

ve

Bio

av

aila

bilit

y (

%)

Can Bioaccessibility Assays Predict Arsenic Relative Bioavailability?

As RBA = 0.99 * SBRC-G + 1.89 R2 = 0.92

Juhasz et al. (2009)

# of soils: 12

[As]: 42-1114 mg kg-1

[As] average: 427 mg kg-1

[As] median: 262 mg kg-1

Source: Herbicide, pesticide,

mine site, gossan

In vivo model: swine

In vitro assay: SBRC-G

# of soils – 15[As] – 233-17500 mg kg-1

Source – Mine sitesModel – Swine

As RBA (%) = 1.14*IVG-G (%) + 2.02

R2 = 0.83 (Rodriguez et al. 1999)

# of soils – 15[As] – 18-25,000 mg kg-1

Source – Mine siteModel – Swine

As RBA (%) = 1.03*UBM-G (%) – 1.51

R2 = 0.99 (Denys et al. 2012)

0

20

40

60

80

100

0 20 40 60 80 100

As Rel. Bioavailability (%)

As

Bio

accessib

ilit

y(%

)

As RBA (%) = 0.72*SBRC-G (%) + 5.64

R2 = 0.92 (Bradham et al., 2011)

# of soils – 11[As] – 173-6,899 mg kg-1

Source – Mine siteModel – Mouse


0 25 50 75 1000

25

50

75

100


As

Re

lati

ve

Bio

av

aila

bilit

y (

%)


As RBA = 0.99 * SBRC-G + 1.89 R2 = 0.92

0 25 50 75 1000

25

50

75

100


As

Re

lati

ve

Bio

av

aila

bilit

y (

%)

As RBA = 0.84 * SBRC-G + 3.56 R2 = 0.82

Juhasz et al. (2009; 2014)

Correlation

Validation

� Model bias� Model error

outlier

0

20

40

60

80

100

0 20 40 60 80 100

RB

A%

IVBA%

RBA%=0.79(IVBA%)+3.0, R2=0.87 (n=83)

Diamond et al. (2016)


� Larger number of samples

� Utilises data from different

animal models

� Incorporates variability from

bioavailability and bioaccessibility

measurement

(SBRC-G)

Correlation Between Pb Relative Bioavailability and Pb Bioaccessibility

Pb RBA (%) = 0.84*Rel-SBRC-I (%) + 10.6 R2 = 0.89

Smith et al. (2011)

# of soils: 12

[Pb]: 646-3450 mg kg-1

[Pb] average: 1618 mg kg-1

[Pb] median: 1293 mg kg-1

Source :Shooting range, Historical fill,

Incinerator waste, Mining /

smelting, Gasworks

In vivo model: Mouse

In vitro assay: Rel-SBRC-I

0 25 50 75 100 1250

25

50

75

100

125

Pb Bioaccessibility (%)

0 10 20 30 40 500

10

20

30

40

50

Pb Bioaccessibility (%)

Pb

Re

lati

ve

Bio

av

aila

bili

ty (

%)

Pb RBA (%) = 1.41*PBET (%) + 3.19

R2 = 0.93

Ruby et al. 1996 (n = 7)

Schroder et al. 2004 (n = 18)

Pb RBA (%) = 0.88*RBALP (%) – 0.028

R2 = 0.92

Denys et al. 2012 (n = 15)

Drexler and Brattin, 2007 (n = 19)

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100

y = -1,765 +1,119 * xR2 =0.78

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100

y = 2,383 +1,087 * xR2 =0.9

Pb RBA in Kidney (%)

Gastric Phase Intestinal Phase

Pb

Re

lati

ve

Bio

ac

ce

ss

ibil

ity (

%)


USEPA (2007)


Pb RBA (%) = 0.88*RBALP (%) – 0.028 R2 = 0.92

# of soils: 19

[Pb]: 1270-14200 mg kg-1

[Pb] average: 6737 mg kg-1

[Pb] median: 6940 mg kg-1

Source: Mine sites

In vivo model: Swine

In vitro assay: RBALP or SBRC-G

USEPA Method 9200

‘Non-mine site soils may not follow the observed correlation’‘Not suitable for soils with phosphate treatments’

Cd RBA (%) = 1.09*PBET-I (%) – 5.14 R2 = 0.84

Correlation Between Cd Relative Bioavailability and Cd Bioaccessibility

# of soils: 7

[Cd]: 11-267 mg kg-1

[Cd] average: 52 mg kg-1

[Cd] median: 13 mg kg-1

Source: Aircraft maintenance,

Historical fill, Mine sites,

spiked soils


In vitro assay: PBET-I

Juhasz et al. (2010)

0 25 50 75 1000

25

50

75

100

Cd Bioaccessibility (%)

Correlation Between Cd Relative Bioavailability and Cd Bioaccessibility

Schroder et al. (2003)

Gastric (stomach) phase extraction providedthe best correlation between Cd relativebioavailability and Cd bioaccessibility

# of soils: 10

[Cd]: 24-465 mg kg-1

[Cd] average: 148 mg kg-1

[Cd] median: 93 mg kg-1

Source: Mine sites

In vivo model: Swine

In vitro assay: IVG-G

Comparison of PAH Bioaccessibility and PAH Relative Bioavailability

Comparison of DDT Bioaccessibility and DDT Relative Bioavailability

# of soils: 8

[DDTr]: 578-19400 mg kg-1

[DDTr] average: 6210 mg kg-1

[DDTr] median: 4205 mg kg-1

Source :Cattle dip sites


In vitro assay: Org-PBET +

sorption sink

0 20 40 60 800

20

40

60

80

DDTr Bioaccessibility (%)

DD

Tr

Rela

tiv

e B

ioa

vaila

bilit

y (

%)

DDT RBA = 0.94 * Org-PBET(sink) + 3.49 R2 = 0.72

� Different methods give different

� relative bioavailability – bioaccessibility

� correlations.

� One method may not be suitable as

� a surrogate for the assessment of

� relative bioavailability for all metal/loids.

� Correlated methods are currently

� unavailable for organic contaminants.

Summary – Correlation Between Relative

Bioavailability and Bioaccessibility

Further Information

[email protected]

+61 418 818 121

Albert Juhasz

Future Industries Institute,

University of South Australia,

Building X1-17,

Mawson Lakes Campus,

Adelaide, 5095

use of in vitro assays as surrogate measures of ...use of in vitro assays as surrogate measures of...

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