ANALYTICAL CHARACTERISTICS OF THE H1L6.1 AND THE H1L7.5 MOUSE
HEPATOMA CELL LINES
Van Langenhove K.1, Croes K.1, Denison M. S.2, Baston D. S.2, Elskens M.1, Baeyens W.1
1

Vrije Universiteit Brussel (Free University of Brussels), Department of Analytical and Environmental Chemistry(ANCH), Pleinlaan 2, 1050 Brussels, Belgium.
2
Department of Environmental Toxicology, Meyer Hall, University of California, Davis, CA 95616, USA.
Introduction
Polychlorinated dibenzo-p-dioxins, -furans and biphenyls form a group of ubiquitous environmental contaminants
that persist in time, accumulate in a non-polar matrix as well as biomagnifie in the trophic chain1. Due to thepotency of the most toxic congener, the 2,3,7,8-TCDD, only very small amounts are needed to exert health effects. It
is therefore needed to have sensitive tools that allow quantification of these low amounts of toxic compounds inbiological and environmental samples.
One of the most utilized cell lines at this point is the H1L6.1 cell line2,3, a wild type Hepa1c1c7 cell that has beenstably transfected with a luciferase gene under control of the Aryl hydrocarbon receptor, allowing induction ofluciferase by dioxin and dioxin-like compounds in a time-, dose-, ligand- and AhR-dependent manner.
The more recently developed H1L7.5 cell line4 differs from the previous one because of the number of inserted
DRE’s (Dioxin Responsive Elements). These increased from 4 (H1L6.1) to 20 (H1L7.5), where 4 DRE’s constituteone DRD (Dioxin Response Domain). Increasing the number of DRE’s under control of the AhR augments the light
output for each molecule of TCDD that binds to the AhR. Hence, with these third generation (G3) luciferase
bioassays, lower amounts of TCDD and other related compounds can be distinguishable from basal levels, while
they were previously undetected by the commonly used H1L6.1 cell line.
This newly developed cell line could be particularly of interest in analysis of blood samples4 and low detectablecontamination-level samples such as breast milk and deposition samples. In this article, the focus has been put
primarily on these two cell lines with respect to their induction capabilities, background values and rough EC50values as depicted on the graphs. A descriptive comparison of these values (as established in our lab) with literature
values, (when available) will allow to assess differences between different cell lines expressing the same vector(pGudLuc6.1 or -7.5) in terms of EC50 and fold induction5.
Materials and methods
Both the H1L6.1 and the H1L7.5 cell lines were investigated for their performance in relationship to 2,3,7,8-tetrachlorodibenzo-p-dioxin. The same cell protocol was used when growing, seeding and dosing the plates for theH1L7.5 cell line as was previously done for the H1L6.1 line.
ProtocolBoth mouse hepatoma cells were grown in α-MEM (Gibco, UK) supplemented with 10% FBS (South American;Gibco, UK) to near confluence (roughly 80 to 85% coverage of the tissue culture plate). Cells were seeded into clear
bottom 96-well plates (Perkin Elmer, USA) by adding 100 µl of a homogenous cell suspension at a cell density of
7.5 105 cell/ml using a 12-channel multipipettor (Brand).
Cells were incubated for 20-24 hours at 37°C in 5% CO2 and 80% relative humidity. These were afterwards dosedwith a 100-fold dilution (4 µl in 396 µl of media) of TCDD (Accustandard) standard solutions as mentioned in

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Figures 2 and 3. Dosing of the cells was accomplished by adding 100 µl to the wells in triplicate with all 96 wells
used for 10 TCDD standards, a DMSO and media blank as well as 20 remaining positions for serial dilutions ofsamples.
Finally, cells were incubated for another 23-24 hours, the wells were rinsed with 50 µl of 1X PBS (Gibco, UK),
checked for viability and any altered morphology, followed by addition of 50 µl of 1X lysis buffer (Promega, USA).
Afterwards, wells were read using a Glomax 96-well plate luminometer (Promega, USA) with a 5.6 second
incubation time for both cell lines, after injection of 50 µl of the reconstituted luciferase assay buffer. The H1L6.1
cell line had a 5 second integration time, whereas only 3 seconds were used in the case of the H1L7.5 cell line.
For both cell lines, values are reported as the percentage of maximum RLU induced by 2,3,7,8-TCDD. The protocol
described above uses a molar-derived representation for the percentage induction, where both the 1 nM and 10 nM
TCDD treatment solutions are present in the data set for the H1L6.1 cell line, the 1 nM is also present in the new
H1L7.5 cell line treatment set.
Results and discussion
For both the 6.1 and 7.5 cell lines, a total of 9 TCDD dose curves on individual plates were analyzed over the courseof 3 different days. Results are reported on a molar basis. Graphical representations of both cell lines with full dose
TCDD standard-curves are presented in Figures 2 and 3. The absolute, raw values (Figure 1) for the H1L6.1 andH1L7.5 cell lines can be checked and show a tremendously increased output in number of relative light units for thelatter cells. Average (triplicate measurement) maximum values are labeled in this bar chart for reasons ofcomparison.
Experimental backgrounds were determined for DMSO (1% v/v) and for media. On average, a 2.3-fold increase of
the blank in DMSO relative to media was observed for the H1L6.1 cell line and a 1.7-fold increase in the case of theH1L7.5 cell line (Table 1).
Table 1: Average values for the H1L6.1 and H1L7.5 cell line over a total of 9 plates.
RLU media blank

RLU DMSO blank

Maximum RLU

8,000 ± 2,000
1,750,000 ± 200,000

18,000 ± 3,000
3,000,000 ± 200,000

280,000 ± 27,000
32,500,000 ± 3,000,000

Media (% Max RLU)DMSO Blank (% Max RLU)

H1L6.1
H1L7.52.7 ± 0.5
5.4 ± 0.4

6.4 ± 0.6
9.5 ± 1.1

EC50 value (pM)

LOD (% Max RLU)

LOQ (% Max RLU)

H1L6.1H1L7.5

Half of maximum TCDD response~ 18-27~5.5-7.0

DMSO Blank + 3,29 stdev
8.2
13.2

DMSO Blank + 10 stdev
12.0
20.8

H1L6.1
H1L7.5The blanks, either from the media or the DMSO, represent between 2.7% and 9.5% of the maximum induction of thecell lines. The fold induction, which is defined as the maximum induction divided by that of the DMSO blank,
differs between the two cell lines. A low fold induction is obtained for the 6.1 line (16) and an even lower one for
the G3 cell line (11). This is in contrast to values found in the literature5 which specify factors of 22 and 24 forrespectively the 6.1 and a G3 cell line5.

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Comparing the DMSO blanks for both cell lines reveals that a higher background value for the 7.5 cell line (9.5%) ispresent in contrary to the 6.1 (6.4%). Summation of these values (6.4% and 9.5%) and their standard deviation
multiplied with an appropriate coverage factor, provide the Limit of Detection (LOD) and of Quantification (LOQ);
all are mentioned in Table 1. Contradictory to what perception might suggest, the higher LOD for the 7.5 cell line
compared to the 6.1 is based on a percentage maximum RLU and as such does not mean a higher LOD in terms ofconcentration TCDD; the 7.5 cell line is by far more sensitive.
The largest difference between both cell lines is noticeable in the raw RLU value. This is reflected in the output
generated by the luminometer with values mounting to almost 40,000,000 RLUs for the 7.5 line. A comparison wasmade in Figure 1, where values are barely visible for the 6.1 line. These values, however, relate to the type ofequipment and conditions used in our lab and by no means indicate an absolute, maximum value for this type of cellline.
Figure 1: Comparison of Raw RLU response values for the H1L6.1 and H1L7.5 cell lines.
Comparison cell lines - Maximum Response in Raw RLU
4,000,000

40,000,000
H1L7.5 - M aximum Raw RLU

37,285,724

H1L6.1 - M aximum Raw RLU

36,647,405

34,103,186

35,000,000

33,468,355

3,500,000

31,869,863

RLU values H1L7.5

30,085,395

29,964,566

28,901,829

30,000,000

3,000,000

25,000,000

2,500,000

20,000,000

2,000,000

15,000,000

1,500,000

10,000,000

1,000,000

5,000,000

300,313

303,441

256,333

262,502239,759

275,380

317,784

303,640

304,402

12

3

4

5

6

78

9

RLU Values H1L6.1

30,398,329

500,000

00

Plate number

Regarding EC50 values for the 6.1 cell line, they are ranging between 18 and 27 pM (Figure 2) and are in goodcomparison to values reported in literature: 30 pM3. Looking to Figure 3, a value of 5.5 to 7 pM can be extrapolated
for the G3 cells.
Repeatability and reproducibility experiments were performed by having multiple plates over a period of severaldays. The average ± stdev (n=9) of a TCDD treatment solution of 4.95E-13 M amounted to (2.63 ± 0.54) % max
RLU and (8.10 ± 1.16) % max RLU, with the former referring to the H1L6.1 cell line, the latter to the 7.5. For atreatment solution of 9.83E-10 M, these values (n=9) resulted in (97.81 ± 5.89) % max RLU for the 6.1 and (99.97 ±4.91) % max RLU in the case of the 7.5. For both cell lines, these data points are, respectively, situated in the lower
and upper plateau. Concentrations within this range showed, in general, lower absolute standard deviations and weresimilar to each other (data not shown). RSD values calculated (n=9) did not exceed 20%, except for the lowest
standard in the H1L6.1 protocol. This does not occur with the H1L7.5 cell line since DMSO blanks and low

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concentration TCDD standards yield much higher responses for a similar standard deviation, hence resulting inlower RSD values.
Future considerations include expanding the data set to have more statistical power concerning DMSO blank levels
and EC50 values. As a next step, sample addition, originating from different matrices, can be investigated. This willallow assessment of BEQ-values for the same sample with the different cell lines.
Acknowledgements
The cell lines were kindly provided by Prof. M. Denison, from the Department of Environmental Toxicology, Meyer
Hall, University of California, Davis, CA 95616, USA.
References:
1.
2.
3.
4.
5.
6.

Van Larebeke N, Covaci A, Schepens P, Hens L (2002) Journal of Epidemiology & Community Health 56:828-830.
Dalho Han, Scott R. Nagy and Michael S. Denison (2002) Organohalogen Compounds 58: 421-424.
Michael S. Denison, Bin Zhao, David S. Baston, George C. Clark, Hiroshi Murata, Dalho Han (2004)
Talanta 63: 1123-1133.
Guochun He, David S. Baston, Michael S. Denison and Tomoaki Tsutsumi (2008) Organohalogen
Compounds 70: 000772-000775.
Guochun He, David S. Baston, Michael S. Denison and Tomoaki Tsutsumi (2009) Organohalogen
Compounds 71: 002399-002403.
Dalho Han, Scott R. Nagy and Michael S. Denison (2004) BioFactors 20: 11-22.

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Figure 2: Standard curves for the H1L6.1 cell line - Percentage max. RLU vs. TCDD treatment solution.
H1L6.1 Cell line - TCDD Standard Curves
110.00100.00Percentage Maximum RLU TCDD-induced (%)

90.00
80.00
70.00

60.00
50.00

Standardsolution (M)8.23E-12
4.95E-11
2.98E-10
6.44E-10
1.19E-09
2.18E-09
3.47E-09
5.95E-09
9.83E-08
9.90E-07

40.00
30.00
20.00

10.00
0.00
1.00E-14

1.00E-13

1.00E-12

1.00E-11

1.00E-10

1.00E-09

1.00E-08

1.00E-07

TCDD Treatment solution (M)Plate 1

Plate 2

Plate 3

Plate 4

Plate 5

Plate 6

Plate 7

Plate 8

Plate 9Figure 3: Standard curves for the H1L7.5 cell line - Percentage max. RLU vs. TCDD treatment solution.
H1L7.5 Cell line - TCDD Standard Curves
110.00100.00Percentage Maximum RLU TCDD-induced (%)

90.00
80.00
70.00

60.00
50.00

Standardsolution (M)3.29E-12
2.23E-11
4.95E-11
1.49E-10
2.49E-10
4.96E-10
8.93E-10
1.98E-09
2.47E-08
9.83E-08

40.00
30.00
20.00

10.00
0.00
1.00E-14

1.00E-13

1.00E-12

1.00E-11

1.00E-10

1.00E-09

1.00E-08

1.00E-07

TCDD Treatment solution (M)Plate 1

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Plate 2

Plate 3

Plate 4

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Plate 6

Plate 7

Plate 8

Plate 91096