Abstract2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most widely studied ligand of the aryl hydrocarbon receptor (AHR). The AHR-dependent TCDD-induced mitochondrial hyperpolarization (Tappenden et al., 2011) [1] and reduced oxygen consumption rate of intact mouse hepatoma cells (Huang et al., in press) [2] in the previous studies suggest that these alterations can be related to enzymatic activities of the electron transport chain (ETC) and ATP synthase in oxidative phosphorylation (OXPHOS) system. Here, we evaluated the activity of each complex in the OXPHOS system using in vitro enzymatic assays. The calculated enzymatic activity of each complex was normalized against the activity of citrate synthase. To combine each value from an independent experiment, normalized enzyme activities from cells exposed to TCDD were converted to fold changes via comparison to the activity relative to time-matched vehicle control. The averaged fold change for each treatment suggests more replicates are needed in order to clearly evaluate a difference between treatments

Dunivin, Taylor K.

Hwang, Hye Jin

LaPres, John J.

Rizzo, Mike

Steidemann, Michelle

Data in Brief

PubMed

Elsevier - Publisher Connector 

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Data in Brief
Data in Brief 8 (2016) 93–97http://d
2352-34
(http://c
DOI
n Corr
48824,
E-mjournal homepage: www.elsevier.com/locate/dibData ArticleData of enzymatic activities of the electron
transport chain and ATP synthase complexes
in mouse hepatoma cells following exposure
to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
Hye Jin Hwang a,b, Michelle Steidemann c,d, Taylor K. Dunivin e,
Mike Rizzo c,f, John J. LaPres a,b,n
a Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824,
United States
b Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI 48824,
United States
c Institute for Integrative Toxicology, Michigan State University, East Lansing, MI 48824-1319, United States
d Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824,
United States
e Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824,
United States
f Cell and Molecular Biology Graduate Program, Michigan State University, East Lansing, MI 48824,
United Statesa r t i c l e i n f o
Article history:
Received 31 March 2016
Received in revised form
3 May 2016
Accepted 13 May 2016
Available online 20 May 2016
Keywords:
AHR
Aryl hydrocarbon receptor
TCDD
Electron transport chain
Mitochondriax.doi.org/10.1016/j.dib.2016.05.018
09/& 2016 The Authors. Published by Else
reativecommons.org/licenses/by/4.0/).
of original article: http://dx.doi.org/10.1016
esponding author at: Department of Bioch
United States.
ail address: lapres@msu.edu (J.J. LaPres).a b s t r a c t
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most widely studied
ligand of the aryl hydrocarbon receptor (AHR). The AHR-dependent
TCDD-inducedmitochondrial hyperpolarization (Tappenden et al., 2011)
[1] and reduced oxygen consumption rate of intact mouse hepatoma
cells (Huang et al., in press) [2] in the previous studies suggest that these
alterations can be related to enzymatic activities of the electron trans-
port chain (ETC) and ATP synthase in oxidative phosphorylation
(OXPHOS) system. Here, we evaluated the activity of each complex in
the OXPHOS system using in vitro enzymatic assays. The calculated
enzymatic activity of each complex was normalized against the activity
of citrate synthase. To combine each value from an independent
experiment, normalized enzyme activities from cells exposed to TCDD
were converted to fold changes via comparison to the activity relative tovier Inc. This is an open access article under the CC BY license
/j.taap.2016.04.005
emistry and Molecular Biology, Michigan State University, East Lansing, MI
S
M
T
H
D
E
E
D
H.J. Hwang et al. / Data in Brief 8 (2016) 93–9794time-matched vehicle control. The averaged fold change for each
treatment suggests more replicates are needed in order to clearly
evaluate a difference between treatments.
& 2016 The Authors. Published by Elsevier Inc. This is an open access
article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).Speciﬁcations Tableubject area Biochemistry, Toxicology
ore speciﬁc sub-
ject areaEnzyme kineticsype of data Graph
ow data was
acquiredSpectrometry (SpectraMax M2 spectrophotometer, Molecular Devices,);ata format Analyzed
xperimental
factorsHepa1c1c7 and Hepa-C12 cells were exposed to 30 nM 2,3,7,8-tetra-
chlorodibenzo-p-dioxin (TCDD) or 0.01% DMSO for 6 and 24 h.xperimental
featuresMitochondria were isolated from each cell line following DMSO/TCDD treatment
and used for each enzymatic assay of the electron transport chain (ETC) and ATP
synthase.ata source
locationEast Lansing, MI, USAata accessibility Data are within this articleD
Value of the data These in vitro data can be used to help explain other TCDD-induced changes in mitochondrial
function.
 These data can help explain the direct impact of TCDD and the AHR on the respiratory chain.
 This data can help understand the role of mitochondria in AHR-dependent TCDD-induced toxicity.1. Data
We evaluated AHR-dependent changes in enzyme activity of the OXPHOS system in mouse hepatoma
cells following exposure to TCDD (30 nM) for 6 and 24 h. Each enzymatic activity was normalized against
citrate synthase activity, which represents an assessment of mitochondrial amount and integrity for each
sample. Data is presented as a fold change by re-normalizing citrate synthase normalized enzymatic
activities from cells exposed to TCDD against the activity relative to a time-matched vehicle (DMSO)
control (Fig. 1). The variability of this data suggests more replicates than are obtained here (n¼4) are
needed to clearly understand AHR-dependent TCDD-induced mitochondrial dysfunction.2. Experimental design, materials and methods
2.1. Cell culture
The mouse hepatoma cell line, hepa1c1c7 (AHR-expressing) and hepac12 (AHR-deﬁcient), were
grown as described previously [1,2].
H.J. Hwang et al. / Data in Brief 8 (2016) 93–97 952.2. TCDD exposure
Hepa1c1c7 and c12 cells were exposed to 30 nM TCDD or 0.01% DMSO as a vehicle control for 6 or
24 h when cells had reached 70% conﬂuency.
2.3. Preparation of mitochondrial proteins
TCDD or DMSO-treated cells were harvested and mitochondrial fractions were isolated using
protocols as described previously [1,2].ESnaeM
1C1C7 C12 1C1C7 C12
6 hrs TCDD 0.832 1.63 0.202 0.440
24 hrs TCDD 0.877 1.50 0.402 0.267
0.0
0.5
1.0
1.5
2.0
2.5
6 hrs TCDD 24 hrs TCDD
Fo
ld
 C
ha
ng
e 
Complex I1C1C7
C12
ESnaeM
1C1C7 C12 1C1C7 C12
6 hrs TCDD 0.930 1.05 0.282 0.235
24 hrs TCDD 1.75 1.14 0.802 0.187
0.0
0.5
1.0
1.5
2.0
2.5
3.0
6 hrs TCDD 24 hrs TCDD
Fo
ld
 C
ha
ng
e 
Complex II
1C1C7
C12
ESnaeM
1C1C7 C12 1C1C7 C12
6 hrs TCDD 1.07 1.10 0.173 0.200
24 hrs TCDD 0.804 1.14 0.091 0.288
0.0
0.5
1.0
1.5
2.0
6 hrs TCDD 24 hrs TCDD
Fo
ld
 C
ha
ng
e
Complex II + III1C1C7
C12
ESnaeM
1C1C7 C12 1C1C7 C12
6 hrs TCDD 0.981 1.09 0.177 0.234
24 hrs TCDD 0.834 1.04 0.132 0.235
0.0
0.5
1.0
1.5
6 hrs TCDD 24 hrs TCDD
Fo
ld
 C
ha
ng
e
Complex III1C1C7
C12
ESnaeM
1C1C7 C12 1C1C7 C12
6 hrs TCDD 0.994 1.15 0.273 0.190
24 hrs TCDD 0.926 1.07 0.097 0.105
0.0
0.5
1.0
1.5
6 hrs TCDD 24 hrs TCDD
Fo
ld
 C
ha
ng
e
Complex IV 1C1C7
C12
ESnaeM
1C1C7 C12 1C1C7 C12
6 hrs TCDD 0.775 0.586 0.204 0.116
24 hrs TCDD 1.20 0.977 0.335 0.349
0.0
0.5
1.0
1.5
2.0
6 hrs TCDD 24 hrs TCDD
Fo
ld
 C
ha
ng
e
Complex V
1C1C7
C12
Fig. 1. Activities of ETC complexes and ATP synthase.The activities of the ETC complexes and ATP synthase were measured with
isolated mitochondria from hepa1c1c7 and c12 cells exposed to 30 nM TCDD or vehicle control (DMSO 0.01%) for 6 and 24 h.
The activity of each complex (A. Complex I, B. Complex II, C. Complex IIþ III, D. Complex III, E. Complex IV, and F. Complex V)
was normalized against the activity of citrate synthase. The fold change was calculated by re-normalization of enzyme activity
from cells exposed to TCDD with the activity relative to time-matched vehicle control. The bars represent mean7the standard
errors (n¼4).
H.J. Hwang et al. / Data in Brief 8 (2016) 93–97962.4. Enzymatic assay of OXPHOS system
Each enzyme solution was prepared by suspension of mitochondrial pellets either in a hypotonic
buffer [25 mM potassium phosphate (KPO4) (pH 7.4) and 5 mMMgCl2] for citrate synthase, complex I,
complex II, complex IV, and complex V or in mitochondrial buffer for complex IIþ III and complex III.
After three cycles of freezing/thawing, protein concentrations were determined using Bio-Rad
Bradford assay kit (Hercules, CA). Typical protein concentrations were 1.52 μg/μL. The activities of
the individual ETC complexes, ATP synthase and citrate synthase were determined as previously
described with slight modiﬁcations [3,4]. The enzyme activities were determined at 37 °C for complex
I and V and at 30 °C for the other ETC complexes and citrate synthase. The enzyme activity was
calculated by
(ΔAbs/min) (total assay volume)/[ε (mitochondrial volume) (mitochondrial concentration)]
with units of micromoles per min per milligram (ε: extinction coefﬁcient). The enzymatic activity
was calculated as a ratio, dividing each activity in micromoles per min per milligram protein by the
citrate synthase activity. The absorbance for each enzymatic activity was measured using a Spec-
traMax M2 spectrophotometer (Molecular Devices, Sunnyvale, CA). In all cases, cytochrome c was
bovine heart cytochrome c.
2.4.1. Citrate synthase assay
5 μg of each enzyme solution was incubated with citrate synthase buffer [50 mM KPO4 (pH 7.4),
and 0.1 mM 5,50-Dithiobis(2-nitrobenzoic acid)] containing 100 μM acetyl CoA, in a cuvette for 5 min.
The change of absorbance was measured at 412 nm for 2 min for reference. After addition of 100 μM
oxaloacetate, the change in absorbance at 412 nm was recorded for 3 min. Enzyme activity was cal-
culated with ε for the thionitrobenzoate anion (13.6 mM1 cm1).
2.4.2. Complex I assay
50 μg of each enzyme solution was mixed with complex I buffer [50 mM KPO4 (pH 7.4), 140 μM β-
Nicotinamide adenine dinucleotide (NADH), 1 mM potassium cyanide (KCN), 10 μM antimycin A, 0.1%
BSA, and 50 μM 2,6-dichlorophenolindophenol (DCPIP)] with 1% ethanol and 50 μM Coenzyme Q1
(CoQ1) in a cuvette. The change in absorbance at 340 nm was recorded for 3 min. Reference was
measured in the presence of 2.5 μM rotenone (dissolved in ethanol). Enzyme activity was calculated
with ε for the NADH (6.22 mM1 cm1).
2.4.3. Complex II assay
10 μg of each enzyme solution was incubated with complex II buffer [50 mM KPO4 (pH 7.4), 10 mM
succinate, 1 mM KCN, 2.5 μM rotenone, and 10 μM antimycin A] for 10 min in a cuvette. After addition
of 50 μM DCPIP, the change in absorbance at 600 nmwas recorded for 2 min for reference. The change
in absorbance at 600 nmwas then recorded for 3 min in the presence of 50 μM CoQ1. Enzyme activity
was calculated with ε for the DCPIP (19.1 mM1 cm1).
2.4.4. Complex IIþ III assay
10 μg of each enzyme solution was incubated with complex IIþ III buffer [50 mM KPO4 (pH 7.4),
10 mM succinate, 1 mM KCN, 2.5 μM rotenone, 0.1% BSA, 0.075% EDTA, and 1 mM ATP] in a cuvette for
5 min. Upon addition of 32 μM cytochrome c, absorbance at 550 nm was recorded for 5 min. The
reference was measured in the absence of cytochrome c. Enzyme activity was calculated with ε for the
reduced cytochrome c (19.6 mM1 cm1).
2.4.5. Complex III assay
5 μg of each enzyme solution was mixed with complex III buffer [50 mM KPO4 (pH 7.4), 1 mM n-
dodecyl maltoside, 1 mM KCN, 2.5 μM rotenone, and 0.1% BSA] with 100 μM decylbenzolquinol and
30 μM cytochrome c in a cuvette. The change in absorbance at 550 nm was recorded for 3 min.
Cytochrome c was fully reduced at the end of the measurement with dithionite. The reference was
measured without enzyme solution. Enzyme activity was calculated with ε for the reduced cyto-
chrome c (19.6 mM1cm1).
H.J. Hwang et al. / Data in Brief 8 (2016) 93–97 972.4.6. Complex IV assay
Reduced cytochrome c was prepared using sodium dithionite. 10 μg of each enzyme solution was
mixed with complex IV buffer [40 mM KPO4 (pH 6.8), 0.5% Tween 80, and 0.4 mg/mL reduced
cytochrome c] in a cuvette. The change in absorbance at 550 nm was recorded for 2 min. 5 mM
potassium ferricyanide was added to fully oxidized cytochrome c at the end of the measurement. The
reference was measured without enzyme solution. Enzyme activity was calculated with ε for the
reduced cytochrome c (19.6 mM1 cm1).
2.4.7. Complex V assay
Complex V buffer [40 mM Tris-HCO3 (pH 8.0), 1 mM EGTA, 5 mM MgCl2, 0.2 mM NADH, 2.5 mM
phosphoenolpyruvate, 0.5 μM antimycin A, 15 μM Carbonyl cyanide 3-chlorophenylhydrazone,
50 μg/mL lactate dehydrogenase, and 50 μg/mL pyruvate kinase] was incubated with 2.5 mM ATP for
2 min in a cuvette. After 10 μg of each enzyme solution was added to the above mixture, the change in
absorbance at 340 nm was recorded for 5 min. The reference was measured in the presence of 2 μM
oligomycin for 5 min. Enzyme activity was calculated with ε for the NADH (6.22 mM1 cm1).Acknowledgments
We would like to thank Dr. Shelagh Ferguson-Miller in the Michigan State University for gener-
ously providing bovine heart cytochrome c.Appendix A. Supplementary material
Supplementary data associated with this article can be found in the online version at http://dx.doi.
org/10.1016/j.dib.2016.05.018.References
[1] D.M. Tappenden, S.G. Lynn, R.B. Crawford, K. Lee, A. Vengellur, N.E. Kaminski, R.S. Thomas, J.J. LaPres, The aryl hydrocarbon
receptor interacts with ATP5alpha1, a subunit of the ATP synthase complex, and modulates mitochondrial function, Toxicol.
Appl. Pharmacol. 254 (2011) 299–310.
[2] H.J. Hwang, P. Dornbos, M. Steidemann, T.K. Dunivine, M. Rizzoc, J.J. LaPres, Mitochondrial-targeted aryl hydrocarbon
receptor and the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin on cellular respiration and the mitochondrial proteome,
Toxicol. Appl. Pharmacol. (2016), In Press.
[3] A.E. Frazier, D.R. Thorburn, Biochemical analyses of the electron transport chain complexes by spectrophotometry, Methods
Mol. Biol. 837 (2012) 49–62.
[4] A. Barrientos, F. Fontanesi, F. Diaz, Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system
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Data of enzymatic activities of the electron transport chain and ATP synthase complexes in mouse hepatoma cells following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most widely studied ligand of the aryl hydrocarbon receptor (AHR). The AHR-dependent TCDD-induced mitochondrial hyperpolarization (Tappenden et al., 2011) [1] and reduced oxygen consumption rate of intact mouse hepatoma cells (Huang et al., in press) [2] in the previous studies suggest that these alterations can be related to enzymatic activities of the electron transport chain (ETC) and ATP synthase in oxidative phosphorylation (OXPHOS) system. Here, we evaluated the activity of each complex in the OXPHOS system using in vitro enzymatic assays. The calculated enzymatic activity of each complex was normalized against the activity of citrate synthase. To combine each value from an independent experiment, normalized enzyme activities from cells exposed to TCDD were converted to fold changes via comparison to the activity relative to time-matched vehicle control. The averaged fold change for each treatment suggests more replicates are needed in order to clearly evaluate a difference between treatments. Keywords: AHR, Aryl hydrocarbon receptor, TCDD, Electron transport chain, Mitochondri

Hye Jin Hwang

Michelle Steidemann

Taylor K. Dunivin

Mike Rizzo

John J. LaPres

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Data of enzymatic activities of the electron transport chain and ATP synthase complexes in mouse hepatoma cells following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

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Data of enzymatic activities of the electron transport chain and ATP synthase complexes in mouse hepatoma cells following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

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