THE APPLICATION OF LOWERED INCUBATION TERMERATURE TO
ENHANCE CALUX AND CAFLUX CELL BIOASSAY RESPONSES INENVIRONMENTAL ANALYSIS
Bin Zhao, David S. Baston and Michael S. Denison
Department of Environmental Toxicology, University of California, Davis, CA, USA
Introduction
Reporter genes produce a protein product in transfected cells that can be easily measured in intact or lysed cells
and they have been extensively used in numerous basic and applied research applications.1

Over the past 10years, reporter gene assays have become widely accepted and used in epidemiological, risk assessment andexposure analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related dioxin-like halogenatedaromatic hydrocarbons (HAHs) in the species of interest and in various exposure matrices (i.e. biological,environmental, food and feed) given that high-resolution instrumental analysis techniques are impractical forlarge scale screening analysis.2

The majority of these bioanalytical systems take advantage of the ability ofthese chemicals to activate one or more aspects of the AhR-dependent mechanism of action.

The most sensitivecell-based bioassay systems developed to date are CALUX (Chemically Activated Luciferase Expression) andCAFLUX (Chemically Activated Fluorescent Expression) bioassays, which utilize recombinant cell lines
containing stably transfected dioxin (AhR)-responsive firefly luciferase or enhanced green fluorescent protein(EGFP) reporter genes, respectively.2

Treatment of these cells with TCDD and related HAHs, other AhR

agonists and extracts containing AhR agonists, results in induction of these reporter gene expression in a time-,
dose-, AhR-, and chemical-specific manner and the level of reporter gene expression correlates with the total
concentration of TCDD-like AhR inducers (agonists) in the sample.2,3,4

While the current CALUX andCAFLUX bioassays are very sensitive, increasing their lower limit of sensitivity as well as magnitude ofresponse and dynamic range for chemical detection would greatly increase their utility, particularly for thosesamples that contain low levels of dioxin-like HAHs (i.e. serum).

The previous studies showed thattemperature appears to be important on the activity of these two reporter genes, but very few analyzed the effectof cell growth temperatures on the overall activity of these reporter gene products in transfected mammaliancells.

Here we have examined the effect of temperature on overall responsiveness of the reporter gene inCALUX and CAFLUX cells respond to HAHS and HAH containing extracts of environmental samples.
Materials and Methods
Chemicals: TCDD was generously provided by Dr. Steven Safe (Texas A&M University, College Station, TX).
All solvents used for preparation of sediment sample were purchased from Fisher Scientific (Pittsburgh, PA).
Cell culture reagents and media were purchased from Gibco/BRL (Grand Island, NY) and G418 was fromGemini Bio-Products (Woodland, CA).

Organohalogen Compounds, Volume 70 (2008) page 001124

Preparation of Environmental Sample and CALUX and CAFLUX Assays: Environmental sediment samples
were extracted and cleaned up through a standard procedure to allow isolation of halogenated dioxins, furans andbiphenyls.5

Mouse hepatoma H1L6.1c2 and H1G1.1c3 were prepared and grown as previously described.3,6The stably transfected H1L6.1c2 cells, that contained the firefly luciferase gene under the control of 4 DREs, andthe stably transfected H1G1.1c3 cells, that contained the EGFP (enhanced green fluorescent protein) gene under
the control of 4 DREs.3,7Cells were incubated with chemicals or extracts of environmental sample for 24 hours

at indicated temperature and assayed as previously described.3,5,6Condition for two incubators was set exactly

the same except for the temperature, which was 33ºC and 37ºC.
Results and Discussion
In temperature range finding experiments, we previously found that 33ºC was the most optimal incubation
temperature for TCDD to induce reporter gene expression in either H1L6.1c2 (luciferase) or H1G1.1c3 (GFP)
cells.

Here, we extended these studies to investigate whether reduced temperature (33ºC) would also improveCALUX and CAFLUX analysis when applied to environmental sample extracts. Sediment samples werecollected and cleaned up in the lab followed the standard procedures to remove PAHs and other AhR agonists
from the desired HAHs (dioxin like PCDD, PCDF and PCBs). The TCDD standard was run in parallel withenvironmental sample as a positive control and for comparative purpose. The results of TCDD dose response
studies for luciferase (CALUX assay) at 37ºC and 33ºC are shown in Figure 1A and consistent with previousresults, cells incubated at 33ºC exhibited significantly more luciferase activity than that at 37ºC. Not only was
the luciferase activity at 33ºC, 5-15 fold greater than that observed in cells incubated at 37ºC at all TCDD
concentrations, but it increased the amount of luciferase activity at the minimal detection limit of 0.45 pg
TCDD/ml (compare 46.0 ± 19.2 relative light units (RLUs) at 33ºC to 1.3 ± 1.6 RLUs at 37ºC).

Notsurprisingly, the luciferase activity at 33ºC was similarly 5-15 fold greater than that observed in cells incubatedat 37ºC when incubated with HAH containing sediment extracts (Figure 1B). Most significantly, the amount ofdioxin or dioxin-like compounds extracted from less than 1 μg of sediment was essentially not detectable(produced less than 10 RLU over the solvent control) in the standard CALUX (37ºC) while they became easilydetectable when incubated at 33ºC since they induced significantly higher luciferase activity (up to 300 RLU)
over the solvent control. Interestingly, this small temperature change (from 37ºC to 33ºC) could significantly
increase our bioassay’s detection of TCDD-like compounds in environmental samples by up to 2 orders ofmagnitude.

Similar results were obtained using the CAFLUX assay, where about a 2 fold greater amount offluorescence activity from GFP was obtained from cells that had been incubated at 33ºC compared to thoseincubated at 37ºC with either TCDD standard or environment extract (Figure 1C & 1D). Not only was GFP
fluorescence activity in cells treated with 0.45 pg TCDD/ml for 24 hours at 33ºC significantly higher than thatobserved from cells incubated at 37ºC, but the overall activity at 33ºC was significantly above background, while
GFP activity at 37ºC was indistinguishable from background (Figure 1C). The GFP fluorescence activity
induced by TCDD at 33ºC was ~2 fold greater than that observed in cells incubated at 37ºC at all concentrationsof sediment extract (Figure 1D). We observed similar temperature-dependent differences in reporter geneactivity (with optimum at 33ºC) using rat and human liver and guinea pig intestinal cell lines containing a stably

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transfected AhR-responsive luciferase or EGFP reporter gene (data not shown), and in human ovarian and breast
carcinoma cells containing an estrogen- or androgen-responsive luciferase reporter gene (data not shown).
These results demonstrate that our observed temperature-dependence of reporter gene activity is due to an effecton the reporter gene itself, rather than an effect on single species or on the AhR signaling pathway itself. Here
we have demonstrated that typical EGFP and firefly luciferase gene expression protocols in transfectedmammalian cells for environmental analysis are not carried out at suboptimal temperatures necessary for
production of maximal reporter gene activity.

In those situations where optimal reporter gene activity intransfected mammalian cells is desired, cells should be incubated at lower temperatures (33ºC or optimal for thetarget cell line) during the gene expression period.

This simple modification of gene induction protocols canlead to substantial increases in measurable reporter gene activity and therefore significantly increases thedetectable limit and range of bioassay for dioxin and dioxin-like compounds in extract from environmentalsamples.
Acknowledgements
This work was supported by grants from the National Institutes of Environmental Health Sciences (ES12489 anda Superfund Basic Research Grant (ES04699)) and the California Agriculture Experiment Station.
References
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Alam J. and Cook J. L. Anal Biochem 1990; 188: 245.

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Denison M., Zhao B., Baston D., Clark G. C., Murata H. and Han D. Talanta 2004; 63: 1123.

3.

Nagy S. R., Sanborn J. R., Hammock B. D. and Denison M. S. Toxicol Sci 2002; 65: 200.

4.

Ziccardi M. H., Gardner I. A. and Denison M. S. Toxicol Sci 2000; 54: 183.

5.

Brown D. J., Orelien J., Gordon J. D., Chu A. C., Chu M. D., Nakamura M., Handa H., Kayama F.,
Denison M. S. and Clark G. C. Environ Sci Technol 2007; 41: 4354.

6.

Han D., Nagy S. R. and Denison M. S. Biofactors 2004; 20: 11.

7.

Rushing S. R. and Denison M. S. Arch Biochem Biophys 2002; 403: 189.

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CAFLUXCALUXAC

3000

1400
oo

37 Co
33 C37 Co
33 C

1200

2500

1000

RFU

RLU

2000

1500

800

600

400

1000

200
500
00
1e-5

1e-4

1e-3

1e-2

1e-1

1e+0

1e+11e-5

TCDD Standard (ng/ml)

1e-2

1e-1

1e+0

1e+1D
1600
o

37 Co
33 Co

1400

1200

1200

1000

1000

RFU

RLU

1e-3

TCDD Standard (ng/ml)

B
1600
1400

1e-4

800
600

37 Co
33 C

800

600

400

400

200

200

01e-7 1e-6 1e-5 1e-4 1e-3 1e-2 1e-1 1e+0 1e+11e-7 1e-6 1e-5 1e-4 1e-3 1e-2 1e-1 1e+0 1e+1Sediment Mass (mg)

Sediment Mass (mg)Figure 1. Temperature-dependent reporter gene expression in mouse CALUX (H1L6.1c2)
and CAFLUX (H1G1.1c3) cell lines. H1L6.1c2 and H1G1.1c3 cells were incubated at theindicated temperature with either increasing concentrations of TCDD standard (A and C) orextract of environmental sediment sample (B and D) for 24 hours and luciferase and EGFP
activity were determined as described. Values are expressed as relative light or fluorescence
units (RLU and RFU) and represent the mean ± SD of triplicate determinations.

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