Data supporting the involvement of the adenine nucleotide translocase conformation in opening the Tl+-induced permeability transition pore in Ca2+-loaded rat liver mitochondria

There we made available information about the effects of the adenine nucleotide translocase (ANT) ‘c’ conformation fixers (phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside) as well as thiol reagent (4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS)) on isolated rat liver mitochondria. We observed a decrease in A540 (mitochondrial swelling) and respiratory control rates (RCRADP [state 3/state 4] and RCRDNP [2,4-dinitrophenol-uncoupled state/basal state or state 4]), as well as an increase in Ca2+-induced safranin fluorescence (F485/590, arbitrary units), showed a dissipation in the inner membrane potential (ΔΨmito), in experiments with energized rat liver mitochondria, injected into the buffer containing 25–75 mM TlNO3, 125 mM KNO3, and 100 µM Ca2+. The fixers and DIDS, in comparison to Ca2+ alone, greatly increased A540 decline and the rate of Ca2+-induced ΔΨmito dissipation. These reagents also markedly decreased RCRADP and RCRDNP. The MPTP inhibitors (ADP, cyclosporin A, bongkrekic acid, and N-ethylmaleimide) fixing the ANT in ‘m’ conformation significantly hindered the above-mentioned effects of the fixers and DIDS. A more complete scientific analysis of these findings may be obtained from the manuscript “To involvement the conformation of the adenine nucleotide translocase in opening the Tl+-induced permeability transition pore in Ca2+-loaded rat liver mitochondria” (Korotkov et al., 2016 [1]).

permeability transition pore in Ca 2 þ -loaded rat liver mitochondria" (Korotkov et al., 2016 [1] Liver was extracted from Wistar male (250-300 g). Rat liver mitochondria were isolated by a dual sequential isolation, and the resulting protein was used for the observational data assay Data source location

St. Petersburg, Russian Federation
Data accessibility Data is within the article.

Value of the data
The scientific data can be referenced by other scientists investigating the effects of Tl þ on cells and mitochondria.
The findings can provide comprehensive toxicological analysis of the effects of thallous salts on animal organisms.
Effects of tBHP, PAO, and DIDS in the new in vitro model of the K þ surrogate Tl þ -induced MPTP can be the basis in searching new inducers and inhibitors of mitochondrial permeability transition pores in the inner membrane.
These data may be helpful in evaluating the combined action of thallium and other sulfhydryl toxicants such as heavy metals and industrial oxidants.

Data
This manuscript contains additional information to the research of [1]. The use of swelling technique as the change in A 540 tests changes in mitochondrial volume. The respiratory control ratios (RCR ADP ¼state 3/state 4 and RCR DNP ¼DNP-uncoupled respiration/basal state or state 4) give information about enzymes, involved in oxygen consumption and oxidative phosphorylation processes, correspondingly. The safranin uptake of energized rat liver mitochondria allows to do assertion about the change in the inner membrane potential (ΔΨ mito ).

Experimental design, materials and methods
The research was used male Wistar rats (250-300 g) of 9-12 months old which were kept at 20-23°C under 12-h light/dark cycle with free access to water ad libitum and the standard rat diet. All treatment procedures of rats were carried out according to the Animal Welfare act and the Institute Guide for Care and Use of Laboratory Animals.

Isolation of rat liver mitochondria
Rat liver mitochondria were isolated accordance the standard protocol [2]. Male rat was decapitated and the liver was quickly extracted and placed into ice-cold isolation buffer containing 250 mM sucrose, 3 mM Tris-HCl (pH 7.3), and 0.5 mM ethylene glycol tetraacetic acid (EGTA). The decapitation procedure of fasted animals is mandatory in isolating rat liver mitochondria. Then the liver was minced with scissors, washed out by the medium, transferred into a Potter-Elvehjem glass homogenizer and homogenized using a teflon pestle. The liver homogenate was centrifuged at 800 Â g for 7.5 min, then the pellet has been thrown out and the supernatant was centrifuged at 10,000 Â g for 10 min. The mitochondrial pellet was twice washed out with a buffer containing 250 mM sucrose and 3 mM Tris-HCl (pH 7.3) and centrifuged at 10,000 Â g for 10 min. The final pellet was resuspended in 950 μl of the wash buffer and kept on ice during the experiment. The whole process of mitochondrial isolation was carried out on ice. The mitochondrial protein content was determined by Bradford [3] and was within the range of 50-60 mg/ml.

Swelling of mitochondria
The early mention about suitability to use millimolar Tl þ concentrations was made in research of Melnick et al. and Saris et al. which applied swelling and polarographic techniques in experiments with isolated mitochondria (see more detail [1]). The applicability of such experimental model in toxicological studies using isolated mitochondria and buffers containing thallous salts has been earlier substantiated by us in more detail [4]. Mitochondrial swelling was measured as a decrease in A 540 at 20°C using a SF-46 spectrophotometer (LOMO, St. Petersburg, Russia). Mitochondria (1.5 mg protein/ml) were injected into a 1-cm cuvette with 1.5 ml of 400 mOsm buffer containing 200 mM KNO 3 (Table 1) or 75 mM TlNO 3 and 125 mM KNO 3 (Tables 1-4) as well as 5 mM succinate, 5 mM Tris-NO 3 (pH 7.3), 2 μM rotenone, and 1 μg/ml of oligomycin. The following chemicals were added into the medium before mitochondria: phenylarsine oxide (PAO), tert-butyl hydroperoxide (tBHP), N-ethylmaleimide (NEM), 4,4 0 -diisothiocyanostilbene-2, 2 0 -disulfonate (DIDS), ADP, cyclosporin A (CsA), bongkrekic acid (BKA), and carboxyatractyloside (CATR). Ca 2 þ (where indicated) was injected to the buffer at one min after mitochondria. The swelling, oxygen consumption rates, and ΔΨ mito were carried out in 400 mOsm media in order to verify the comparability and consistency between findings in different experiments.

Oxygen consumption assay
Respiration (oxygen consumption rate) was tested using Expert-001 analyzer (Econix-Expert Ltd., Moscow, Russia) in a 1.3-ml closed thermostatic chamber with magnetic stirring at 26°C. Mitochondria (1.5 mg protein/ml) were administrated into 400 mOsm buffer containing 25 mM TlNO 3 , 100 mM sucrose, 3 mM Mg(NO 3 ) 2 , and 3 mM Tris-PO 4 ( Table 5) or 75 mM TlNO 3 and 1 μg/ml of oligomycin (Table 6) as well as 125 mM KNO 3 , 5 mM Tris-NO 3 (pH 7.3), 5 mM succinate, and 2 μM rotenone. In some cases, we used buffers containing glutamate with malate and free of rotenone ( Fig. 1). The following reagents (Table 5) were added in the buffer at one minute after mitochondria: PAO, tBHP, DIDS, and NEM. ADP at 130 μM and DNP at 30 μM were correspondingly injected into the buffer after 2 min to induce state 3 and after 4 min to record DNP-uncoupled respiration. The following reagents (Table 6) were added in the buffer one min after mitochondria: PAO, tBHP, and DIDS. If the MPTP inhibitors (ADP plus CsA or NEM alone) were injected into the buffer one min after mitochondria, the first reagents (PAO, tBHP, and DIDS) and Ca 2 þ at 100 μM were correspondingly added into the buffer one and two min latter the inhibitors. Further, DNP at 30 μM was administrated one minute later the reagents or Ca 2 þ (Table 6). The respiratory control ratio (RCR ADP ¼state 3/state 4) that shows the quality of rat liver mitochondria (RLM) was measured in standard buffer containing 100 mM KCl, 20 mM Tris-HCl (pH 7.3), 3 mM MgCl 2 , and 3 mM Tris-PO 4 , 5 mM Tris-succinate, and Table 1 Effects of PAO and tBHP on A 540 in suspension of succinate-energized rat liver mitochondria in nitrate buffers.  Table 2 Effects of PAO, DIDS, and tBHP on change of A 540 in suspension of Ca 2 þ -loaded succinate-energized rat liver mitochondria. The absorbance changes (ΔA 540 ) were accordingly detected within three minute interval after administration of mitochondria ("À Ca 2 þ " columns) or 100 mM Ca 2 þ to mitochondria ("þ100 mM Ca 2 þ " columns) and this is presented as Means 7SEM. The number of experiments showed in parentheses. P-values in experiments free of Ca 2 þ (" À Ca 2 þ " columns) are calculated to experiments free additions (a dash in the P value columns). P-values with Ca 2 þ -loaded mitochondria ("þ 100 mM Ca 2 þ " columns) are calculated to experiments with Ca 2 þ alone (a dash in the P value columns). Asterisks indicate that statistical difference between appropriate ΔA 540 values is not statistically significant.
2 mM rotenone. The RCR ADP for succinate-energized RLM was equal 6.657 0.21 (n ¼14) (Fig. 1). Wherein, the DNP-dependent respiratory control ratio (RCR DNP ) was calculated as a ratio of DNPuncoupled respiration to state 4 (Fig. 1). The RCR DNP for succinate-energized RLM was equal 9.187 0.49 (n¼ 14) (Fig. 1). Table 5 shows the RCR ADP under above experimental conditions in TlNO 3  The absorbance changes (ΔA 540 ) were detected within three minute interval after administration of 100 mM Ca 2 þ to mitochondria and presented as Means 7 SEM. The number of experiments showed in parentheses and corresponding P-values calculated to experiments free of above additions (a dash in the P value columns). Asterisks indicate that statistical difference between appropriate ΔA 540 values is not statistically significant.

Table 4
Effect of NEM on A 540 in suspension of Ca 2 þ -loaded succinate-energized rat liver mitochondria. The absorbance changes (ΔA 540 ) in experiments free of Ca 2 þ (" À Ca 2 þ " columns) were detected within six minute interval and P-values are calculated to experiments free of NEM (a dash in the P value columns). The absorbance changes with CaRLM ("þ100 mM Ca 2 þ " columns) were detected within three minute after administration of Ca 2 þ to mitochondria and they are presented as Means 7SEM. P-values with Ca 2 þ -loaded mitochondria are calculated to experiments with Ca 2 þ alone (a dash in the P value columns). Asterisks indicate that statistical difference between appropriate ΔA 540 values is not statistically significant. The absorbance changes in experiments free of NEM (2*) were detected within six minute after Ca 2 þ administration to mitochondria. P-values (2*) are calculated to experiments free of additions (a dash with asterisk in the P value columns). buffers. The DNP-dependent respiratory control ratio (RCR DNP ) in above TlNO 3 buffers was accordingly determined as a ratio of DNP-uncoupled respiration to state 4 (Table 5) or a basal state respiration (Table 6).

Mitochondrial membrane potential
The ΔΨ mito induced in succinate-energized on the IMM of RLM (Table 7) was evaluated according to Waldmeier [5] by the intensity of safranin fluorescence (arbitrary units) in the mitochondrial suspension with magnetic stirring at 20°C using a Shimadzu RF-1501 spectrofluorimeter (Shimadzu, Japan) at 485/590 nm wavelength (excitation/emission). Mitochondria (0.5 mg protein/ml) were placed into a quartz cuvette of four clear walls with 3 ml of a buffer containing 20 mM TlNO 3 , 125 mM KNO 3 , 110 mM sucrose, 5 mM Tris-NO 3 (pH 7.3), 1 mM Tris-P i , 3 μM safranin, 2 μM rotenone, and 1 μg/  ml of oligomycin. In addition, the next chemicals were added in the medium before mitochondria: PAO, tBHP, DIDS, ADP, and CsA (where indicated). Succinate, Ca 2 þ , and DNP were administrated into the medium after mitochondria. Temperature conditions used in the research were standard for experiments with isolated mitochondria in vitro.

Statistics
The statistical differences and P-values of experimental results in Tables 1-7 are correspondingly evaluated using the two population t-test (Microcal Origin, Version 6.0, Microcal Software).