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. Author manuscript; available in PMC: 2019 Jun 1.
Published in final edited form as: Biochem Pharmacol. 2018 Apr 5;152:236–243. doi: 10.1016/j.bcp.2018.04.001

Regulation of Dichloroacetate Biotransformation in Rat Liver and Extrahepatic Tissues by GSTZ1 Expression and Chloride Concentration

Stephan C Jahn 1, Marci G Smeltz 1, Zhiwei Hu 1, Laura Rowland-Faux 1, Guo Zhong 1, Ryan J Lorenzo 1, Katherine V Cisneros 1, Peter W Stacpoole 1, Margaret O James 1
PMCID: PMC5960630  NIHMSID: NIHMS957732  PMID: 29626439

Abstract

Biotransformation of dichloroacetate (DCA) to glyoxylate by hepatic glutathione transferase zeta 1 (GSTZ1) is considered the principal determinant of the rate of plasma clearance of the drug. However, several other organismal and subcellular factors are also known to influence DCA metabolism. We utilized a female rat model to study these poorly understood processes. Rats aged 4 weeks (young) and 42 to 52 weeks (adult) were used to model children and adults, respectively. Hepatic chloride concentrations, which influence the rate of GSTZ1 inactivation by DCA, were lower in rat than in human tissues and rats did not show the age dependence previously seen in humans. We found GSTZ1 expression and activity in rat brain, heart, and kidney cell-free homogenates that were age-dependent. GSTZ1 expression in brain was higher in young rats than adult rats, whereas cardiac and renal GSTZ1 expression levels were higher in adult than young rats. GSTZ1 activity with DCA could not be measured accurately in kidney cell-free homogenates due to rapid depletion of glutathione by γ-glutamyl transpeptidase. Following oral administration of DCA, 100 mg/kg, to rats, GSTZ1 expression and activity were reduced in all rat tissues, but chloride concentrations were not affected. Together, these data extend our understanding of factors that determine the in vivo kinetics of DCA.

Keywords: chloride, dichloroacetate, glutathionase, glutathione transferase, kidney, rat

Graphical Abstract

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1. Introduction

The investigational drug dichloroacetate (DCA) is dechlorinated to glyoxylate by glutathione transferase zeta 1 (GSTZ1), whose physiological function is to isomerize maleylacetone and maleylacetoacetate to fumarylacetone and fumarylacetoacetate, respectively [1]. This enzyme, predominantly localized in liver cytoplasm and, to a much lower extent, in mitochondria [2], can be irreversibly inactivated and its protein abundance drops dramatically during DCA metabolism, an effect mitigated by some anions, including chloride (Cl−) [3]. We found a mean Cl− concentration ([Cl−]) of 42 mM in human donor liver specimens [4], similar to the previously reported value of 38.3 mM measured in adult human liver [5]. However, the human liver cytoplasmic [Cl−] ranged between 75–120 mM and varied inversely with donor age, whereas the mitochondrial [Cl−] averaged less than 5 mM and was directly associated with subject age [4]. It is presently unknown whether these marked subcellular differences in hepatic [Cl−] are species-specific. Previous work reported [Cl−] in cultured rat hepatocytes as 38 mM and 30 mM [6, 7], but there are no reports of the subcellular concentrations of Cl− in rat liver.

The distribution of GSTZ1 is not confined to the liver. In 2002, Lantum et al. measured enzyme expression in several other organs and tissues harvested from 175–200 g male Fischer 344 rats [8], consistent with similar findings in humans [9, 10]. The rat study [8] employed a polyclonal antibody to human GSTZ1 to examine protein expression through immunohistochemistry and immunoblot following immunoprecipitation, and reported enzyme activity toward maleylacetone and chlorofluoroacetic acid. However, these earlier studies utilized only young rats and we have subsequently determined that subject age influences DCA plasma kinetics [11], hepatic GSTZ1 expression [12], and liver [Cl−] [4], which exerts a stabilizing effect on GSTZ1 during DCA exposure [3]. Therefore, we sought to compare GSTZ1 expression and activity as well as [Cl−] in tissues from both young and adult rats. Female rats were used since they have previously been shown as a good model for studying adverse neuropathic effects of chronic DCA treatment, the major side effect of DCA treatment in people [13]. Previous studies have shown similar hepatic cytosolic GSTZ1 activity with DCA in untreated rats of both sexes, similar loss of GSTZ1 protein following DCA treatment [2, 14], and it was seen that there are no sex-based differences in DCA clearance in humans [15]. We used a purified rabbit polyclonal antibody raised against expressed recombinant rat GSTZ1 that was much more sensitive for detecting rat GSTZ1 than an antibody raised against human GSTZ1, thus allowing for the accurate detection of lower expression levels.

2. Materials and Methods

2.1. Tissues

Organs were obtained from female Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA). The use of rats was approved by the University of Florida Institutional Animal Care and Use Committee. Rats that were 4 weeks (average weight: 95 ± 13 g) or 10–12 months of age (average weight: 406 ± 50 g) at the time of sacrifice represented young and adult rats, respectively [16]. Animals were given a 100 mg/kg dose of sodium acetate (ThermoFisher Scientific, Waltham, MA) (control group) or sodium DCA (Tocris Bioscience, Minneapolis, MN) (treated group) by oral gavage at 8 am, and groups of six control and six DCA-treated rats were sacrificed at different times after the dose. Organs were removed and portions set aside for [Cl−] analysis. Other portions were homogenized with 4 volumes of 0.25 M sucrose containing 0.02 M Hepes-NaOH (Sigma Aldrich, St. Louis, MO) pH 7.4 and 0.1 mM phenylmethylsulfonyl fluoride (Sigma Aldrich), as described earlier [2], and the 600-g supernatant (cell-free homogenate) was used for measurement of GSTZ1 expression and activity. In separate studies with control rats, kidney cytosol, mitochondria and mitochondrial subfractions were isolated as in previous studies with liver [2]. Briefly, washed mitochondria were swollen in 0.01 M Tris-HCl (BioRad, Hercules, CA) pH 7.4, followed by their shrinking by addition of 2 M sucrose (Sigma Aldrich), 100 mM Tris-HCl, pH 7.4. Preparations were centrifuged at 20,000-g for 20 minutes to yield the intermembrane space fraction as the supernatant. The pellet was frozen and thawed three times, homogenized, and sonicated, then centrifuged at 125,000-g for 60 minutes. The supernatant yielded mitochondrial matrix proteins, while the pellet contained the mitochondrial membrane proteins.

2.2 GSTZ1 expression

To quantitate GSTZ1 expression in the rat samples, a custom polyclonal antibody was raised against full-length expressed recombinant rat GSTZ1 (Cocalico Biologicals, Reamstown, PA). The antigen used to produce this antibody consisted of the rat GSTZ1 gene (NCBI accession#: NM_001109445.1), produced de novo with a N-terminal his-tag and subcloned into the pet21a vector (Bio Basic Inc., Markham, ON, CA). Protein was then produced in E. coli. and purified with a nickel column (ThermoFisher Scientific). Tissue samples, 300 μg of total cell-free homogenate protein per well, were separated on SDS-PAGE gels, transferred to nitrocellulose membranes and immunoblotted for GSTZ1 expression as described previously [2, 17]. Due to variations in actin expression among tissues, equal loading was confirmed by Coomassie staining of the acrylamide gel following transfer to the nitrocellulose membrane. Inter-sample variability in total protein loading was less than 15% from the mean.

2.3 GSTZ1 activity

GSTZ1 activity towards DCA was determined as before [17]. Samples were exchanged from sucrose buffer into 0.05 M K-phosphate (Sigma Aldrich), pH 7.4 with 1.15% KCl (Fisher Scientific) using three washes through a 10 kD molecular weight cutoff spin filter (EMD Millipore, Billerica, MA), as sucrose inhibits GSTZ1 activity [2]. Then, 350 μg of protein was incubated with saturating concentrations of glutathione (GSH, 1 mM) (Sigma Aldrich) and 14C-DCA (0.2 mM) (Perkin Elmer, Waltham, MA). HPLC analysis allowed the determination of the ratio of the DCA metabolite, glyoxylate, to unmetabolized DCA.

2.4 Calculation of percent extrahepatic activity

To calculate the percent of overall GSTZ1 activity in a rat that is derived from organs other than the liver, we calculated the total activity in the brain, heart, and kidney and divided by that value plus the total activity in the liver cytosol and mitochondria. Values for all compartments other than kidney were calculated using the measured activity and expression data in cell-free homogenates along with known organ weights and homogenization volumes. Activity measurements for the kidney were artificially low because the presence of mitochondria and brush border membrane caused very rapid consumption of GSH and inhibited GSTZ1 activity (see below). There was insufficient material to fractionate these samples. Therefore, the activity of kidney cytosolic GSTZ1, measured as nmol/min/mg GSTZ1 protein, was determined from other female Sprague-Dawley rats and was used subsequently with expression data from our experimental rats to estimate total kidney enzyme activity.

2.5 Effect of kidney fractions on GSTZ1 activity with DCA

Because activity in kidney cell-free homogenate from untreated rats was considerably lower than expected from the GSTZ1 protein expression level, we determined the effect of kidney cell-free homogenate, cytosol, mitochondria and mitochondrial sub-fractions on activity in liver cytosol. To investigate possible non-specific effects, some assays were conducted with liver cytosol and bovine serum albumin protein (BSA) (Sigma Aldrich). GSTZ1 activity measured in the presence of liver cytosol protein alone was set as 100% activity and the activity seen in incubations containing additional kidney or BSA protein was compared to that value to determine the percent activity remaining.

2.6 Glutathione depletion measurements

GSH concentrations were measured using a previously described fluorometric assay [18, 19]. Incubations with liver and kidney fractions were set up to resemble the conditions of the GSTZ1 activity assays, but with no DCA. Tubes contained 1 mM GSH, 0.1 M potassium phosphate buffer pH 8.0 containing 0.005 M EDTA (Sigma Aldrich), liver or kidney fractions, and water to 200 μL. After 15 minutes at 37 °C, the mixtures were spun through 10 kD molecular weight cutoff filters to remove proteins. Up to 120 μL of flow-through was mixed with 1.0 mL of 1 M HEPES pH 8.0, 20 μL of 0.1% o-phthalaldehyde (Sigma Aldrich) in methanol, and sufficient water to yield a 2.5 mL total volume. The amount of flow-through used was altered to ensure that all samples remained in the linear range of the detector. After standing at room temperature for 30 minutes, fluorescence of the sample was measured with an excitation wavelength of 350 nm and an emission wavelength of 420 nm. The amount of GSH was calculated from a standard curve prepared with known concentrations of GSH under the same conditions.

2.7 Determination of kinetics of GSH Consumption by Kidney Mitochondria

GSH concentration measurements used to calculate the kinetic data were determined as described in [19]. A mixture consisting of 50 μg rat kidney mitochondria washed with 1.15% KCl and 0.05 M potassium phosphate pH 7.4, GSH ranging from 1 – 10 mM, and 0.1 M sodium phosphate with 0.005 M EDTA pH 8.0 to a total volume of 200 μL was held at 37 °C for 15 minutes. Samples were diluted with water based on their expected GSH concentration to yield concentrations in the range of the prepared standard curve and incubated for 30 minutes at room temperature with 100 μg o-phthalaldehyde dissolved in methanol. Fluorescence was then determined with an excitation wavelength of 350 nm and an emission wavelength of 420 nm.

2.8 Immunoblot for brush border membrane contamination

Villin was selected as a marker for brush-border membrane [20] to assess the purity of kidney mitochondrial preparations. Protein samples were prepared for SDS-PAGE, blotted, and detected using previously described methods [2, 17]. The anti-villin antibody (sc-58897) was obtained from Santa Cruz Biotechnology (Dallas, TX). After visualization, blots were stained with Ponceau red (ThermoFisher Scientific) to confirm that protein loading was within 15% of the mean for all samples.

2.9 γ-glutamyltranspeptidase activity assay

A previously published colorimetric assay was used to determine γ-glutamyltranspeptidase (GGT) activity [21]. Mixtures consisting of 50 μg of kidney mitochondria or mitochondrial subfractions, 1 mM L-γ-glutamyl-p-nitroanilide (Sigma Aldrich), 20 mM glycylglycine (Sigma Aldrich), and 0.1 M Tris-HCl pH 8 in a total volume of 2 mL were incubated at 37 °C while the absorbance at 410 nm was recorded. A linear increase in absorbance was seen between the 1 and 4 minute time points, so this interval was used for the presented calculations. The concentration of p-nitroaniline was calculated using its molar extinction coefficient, ε = 8800 cm−1. The GGT inhibitor azaserine (Sigma Aldrich) was added to some incubations prior to addition of the kidney fraction.

2.10 Measurement of chloride ion concentration

Cl− concentrations in all tissues and subcellular fractions were measured using a HPLC-based method that relies on the conversion of pentafluorobenzyl bromide (Sigma Aldrich) to pentafluorobenzyl chloride [4, 22]. Whole tissue samples were homogenized in 4 volumes of deionized water, then sonicated to facilitate tissue disruption. Cytosol and mitochondrial samples were taken from the fractions isolated in sucrose-containing buffer as described above. To 50 μL of sample was added 400 μL of acetone (Sigma Aldrich) to precipitate proteins. After being stored at −20 °C overnight, samples were centrifuged at 17,000-g and the pellet discarded. Ten μL of pentafluorobenzyl bromide was added to each tube and the mixture was incubated at 50 °C for 30 minutes. Acetone was removed under nitrogen and 400 μL of 75:25 acetonitrile:water containing 25 μM p-hydroxybiphenyl, which served as the internal standard, was added. The final solution was passed through a 0.22 μm nylon spin filter (Costar, Tewksbury, MA) for HPLC analysis. The injection volume for reversed-phase chromatography using a 4.6 mm × 25 cm, 5 μm C18 Discovery column (Sigma Aldrich) was 20 μL for homogenate and cytosol, and 50 μL for mitochondria. The column was eluted with a binary gradient mobile phase with water as mobile phase A and acetonitrile (Sigma Aldrich) as mobile phase B. The program was 50% B for 20 min, gradient to 100% B over 4min, held at 100% B for 4 min then returned to 50% B over 4 min followed by equilibration for 6min.. Separate standard curves were created for water and sucrose buffer and both showed excellent linearity with R2 > 0.99 using the least squares method. The experimental procedure has a limit of detection, with signal to noise greater than or equal to 3, of 0.05 mM and a limit of quantitation of 0.1 mM.

The concentrations presented for liver cytosol and mitochondria were calculated to represent the concentrations present in an intact hepatocyte. This calculation assumes the average hepatocyte’s volume is 36.4% cytosol and 14.8% mitochondria [4, 23].

2.11 Data analysis

Mean values were calculated as the arithmetic mean and are presented with the standard deviation. Statistical significance was determined using Student’s unpaired t-test. Kinetics data were analyzed using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA).

3. Results

3.1 Chloride concentration in rat liver

We measured the [Cl−] in cell-free homogenates of livers and in individual cytoplasmic and mitochondrial fractions from Sprague-Dawley rats at multiple time-points following gavage administration of 100 mg/kg sodium DCA or, as a control, sodium acetate. We did not observe a consistent, time-dependent pattern in [Cl−] in the young (Figure 1A) or adult (Figure 1B) control rats. The [Cl−] in young rat mitochondria changed less than 1 mM over 24 hours (Figure 1C). Therefore, because time of sacrifice did not have a dramatic influence on [Cl−] in either the control or treated rats, all control and all DCA-treated rats were grouped for further statistical analysis. For each rat, the measured [Cl−] was highest in the cytoplasmic compartment, lowest in the mitochondria, and the cell-free homogenate fell between them as an average of the cytoplasm, mitochondria, and other organelles that likely have low [Cl−].

Fig. 1.

Fig. 1

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Chloride concentrations in rat liver (A) Cytosolic and mitochondrial fractions from young control rat livers, together with whole-liver homogenate, were assayed for [Cl−]. Data points represent the mean of 6 animals ± the standard deviation. (B) Cytosolic and mitochondrial fractions from adult control rat livers, together with whole-liver homogenate, were assayed for [Cl−]. Data points represent the mean of 6 animals ± the standard deviation. (C) [Cl−] in the young and aged mitochondrial fractions from panels A and B are shown with a smaller scale on the y-axis. Data points represent the mean of 6 animals ± the standard deviation. (D) Concentrations from all control time points were pooled to calculate a mean concentration in each compartment with respect to age. Values depict the mean and error bars represent the standard deviation. * indicates p < 0.05 and ** indicates p < 0.0001 as determined by Student’s t-test. (E) Concentrations were compared between time-matched control and treated animals. Values depict the mean and error bars represent the standard deviation. * indicates p < 0.05 as determined by Student’s t-test.

The cytosolic [Cl−] was slightly higher in young (44.9 ± 4.0 mM, mean ± S.D., n=42) than adult (43.0 ± 4.1 mM, n=40) control rats, p < 0.01, whereas mitochondrial [Cl−] in the young control rats (0.8 ± 0.3 mM) was half that in the adult control rats (1.9 ± 0.6 mM), p < 0.0001. No age dependence in [Cl−] was observed in whole liver homogenates of young (29.7 ± 2.3 mM) and adult (30.1 ± 2.9 mM) rats (Figure 1D). DCA treatment had a small, but statistically significant effect (p < 0.05) of decreasing [Cl−] in the adult rat liver cytosol and whole livers, while no change was seen in the adult rat liver mitochondria or in any of the young rat liver compartments (Figure 1E). While we cannot rule out these small changes being due to slight diurnal changes in [Cl−], we did not observe any pattern in changes over time, and any physiological changes would be expected to be the same in both the control and treated groups. It is therefore likely that any change over time between the groups is due to the treatment itself.

3.2 GSTZ1 expression in extrahepatic sites

The brain, heart, and kidneys from young and adult rats taken 30 minutes and 24 hours after dosing were processed to make cell-free homogenates. Additionally, a sample of skeletal muscle was taken from the 24 hour animals. Immunoblots of GSTZ1 in these tissues showed detectable levels of enzyme in all control tissues, with the exception of skeletal muscle. Representative blots are shown in Figure 2A. While the kidney showed the highest expression in both age groups, the brain had higher expression than the heart in young rats, and the opposite was observed in the adult rats. The relatively high enzyme expression in the kidney was quantified for comparison with hepatic GSTZ1 expression, which averages around 1–2 ng GSTZ1 per μg total protein in the adult liver cytosol and 0.02–0.1 ng GSTZ1 per μg total protein in the adult liver mitochondria (manuscript submitted). Young rat kidneys had a mean expression of 0.082 ± 0.016 ng GSTZ1 per μg of total protein (n=8), while kidneys from adult rats expressed 0.118 ± 0.030 ng GSTZ1 per μg total protein (n=8) (Figure 2B). A 30 minute DCA exposure had no observable effect on GSTZ1 levels in the extrahepatic tissues, other than in the brain of young rats, which exhibited a reduction in expression. GSTZ1 levels 24 hours after treatment were strikingly decreased in all tissues. Kidney GSTZ1 expression at 24 hours fell to 0.0028 ± 0.0003 and 0.0034 ± 0.0008 ng GSTZ1 per μg total protein in the young and adult rats, respectively (Figure 2B).

Fig. 2.

Fig. 2

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GSTZ1 expression in extrahepatic tissues (A) The indicated tissues (kidney, brain, heart) were removed from rats sacrificed 30 minutes or 24 hours after DCA dosing. Homogenates were immunoblotted for GSTZ1. C indicates a control animal while T indicates a DCA treated animal. (B) Quantitation of GSTZ1 expression in the young and adult rat kidney. Values depict the mean and error bars represent the standard deviation. * indicates p < 0.05 as determined by Student’s t-test. n=8 per age group.

3.3 GSTZ1 activity in extrahepatic sites

GSTZ1 activity in the brain and heart toward DCA closely mirrored changes in enzyme expression. Brain tissue from young rats showed higher activity levels than did brain tissue from adult animals, with activities of 44 ± 4 and 17 ± 8 pmol DCA/min/mg total protein, respectively (Figure 3A). Data are presented as mean ± S.D. with n=4. Heart tissue showed the opposite relationship, as activity increased from 44 ± 5 to 63 ± 10 pmol DCA/min/mg total protein with age. Measured GSTZ1 activity in the kidney cell-free homogenate was 50 ± 30 in young and 10 ± 6 pmol DCA/min/mg total protein in adult rats. This activity was much lower than expected, based on the expression levels of GSTZ1 found in kidney, therefore further studies were conducted to determine the cause of this discrepancy.

Fig. 3.

Fig. 3

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GSTZ1 activity in extrahepatic tissues (A) Age dependence of GSTZ1 activity in the indicated tissues. Activity is expressed as pmol glyoxylate formed/minute/mg of total protein. n=4 (B) GSTZ1 activity in young rats 30 minutes or 24 hours after dosing with DCA, compared to the time-matched control animals. (C) GSTZ1 activity in adult rats 30 minutes or 24 hours after dosing with DCA, compared to the time-matched control animals. In all panels, values depict the mean and error bars represent the standard deviation. * indicates p < 0.05 and ** indicates p < 0.01 (Student’s t-test).

In both young and adult rats, DCA treatment reduced GSTZ1 activity in the brain and heart (Figure 3B and C, respectively), 30 minutes after the DCA dose. Brain and heart from young animals had approximately 40% enzyme activity remaining 30 minutes after dosing and 10–15% remaining after 24 hours (Figure 3B).

Remaining GSTZ1 activity in the brain and heart of adult rats was similar to that measured in young rats 30 minutes after DCA administration. Both young and adult brain retained approximately the same enzyme activity 24 hours following treatment. GSTZ1 activity in the young and adult heart decreased to 12% and 5% of their control values, respectively, after 24 hour treatment. GSTZ1 activity in muscle tissue was not measured because of very low expression levels.

3.4 A glutathione consuming factor is present in kidney mitochondria

It was intriguing to see the apparent lack of correlation between high kidney GSTZ1 expression (Figure 2) and low observed activity. In an effort to determine the cause of the low GSTZ1 enzyme activities observed in the kidney preparations, we combined 100 μg liver cytosol protein and 350 μg kidney cell-free homogenate (as in section 3.3) from an adult rat and measured the activity of the resultant mixture. When the activity of the liver component by itself is defined as 100% activity, the mixture had little to no activity, while addition of an equivalent amount of bovine serum albumin protein had no effect (Figure 4A). Subcellular fractionation of the kidney identified the mitochondrial intermembrane space as the primary location of this factor, with lesser amounts in the mitochondrial membrane fraction as well as the microsomal fraction (Figure 4A). Addition of the mitochondrial matrix caused an increase in GSTZ1 activity, likely due to the lack of the inhibitory factor and the presence of GSTZ1.

Fig. 4.

Fig. 4

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Inhibition of GSTZ1 activity (A) GSTZ1 activity, expressed as % of liver only, of 100 μg liver cytosol, and liver cytosol + 350 μg kidney homogenate protein, liver cytosol + 350 μg bovine serum albumin, and liver cytosol + 350 μg of protein from various subcellular fractions: cytosol (Cyto), mitochondria (Mito), mitochondrial membrane (Mito Memb), mitochondrial intermembrane space (Mito IMS), mitochondrial matrix (Mito Matrix), and microsomes (Mics) (B) Glutathione concentration, expressed as % control (no protein), remaining after a mock (no DCA) GSTZ1 activity assay containing 100 μg of liver cytosol protein ± 350 μg of protein from the various indicated sources. In all panels, values depict the mean and error bars represent the standard deviation of duplicate measurements.

To determine whether the inhibitory effect of the kidney homogenate was a direct inhibition of GSTZ1 or an indirect inhibition due to the consumption of glutathione, a mock GSTZ1 activity assay, without DCA, was carried out and the glutathione concentration remaining after incubation was measured. Strikingly, mixtures that contained kidney homogenate had extremely low levels of glutathione while those containing no protein, liver only, or liver and bovine serum albumin showed no loss of GSH concentrations (Figure 4B). Boiling kidney homogenate prior to incubation partially rescued the glutathione concentration.

3.5 γ-glutamyl transpeptidase is present in the mitochondrial fraction of kidney

To better characterize the consumption of glutathione, we determined enzyme kinetics by incubating the kidney mitochondrial fraction with increasing concentrations of glutathione and measuring the decrease in GSH concentration after 15 minutes. Plotting these data yielded a standard Michaelis-Menten curve with Vmax of 47.00 ± 2.76 nmol GSH consumed/min/mg of mitochondrial protein and a Km of 0.57 ± 0.12 mM (Figure 5).

Fig. 5.

Fig. 5

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γ-glutamyltranspeptidase activity in the kidney mitochondrial fraction Michaelis-Menten kinetics of glutathione consumption. The indicated glutathione concentrations were incubated with 50 μg of kidney mitochondrial protein for 15 minutes. The remaining glutathione was compared to that of a sample lacking mitochondrial protein to determine the amount of glutathione consumed. Km = 0.57 ± 0.12 mM and Vmax = 47.00 ± 2.76 nmol/min/mg. Values represent the mean and error bars represent the standard deviation of measurements from 3 separate rat kidney mitochondrial preparations.

Hypothesizing that the protein responsible for this consumption was γ-glutamyltranspeptidase (GGT) due to its known high expression in the kidney [24], we directly measured GGT activity through the formation of p-nitroaniline from L-γ-glutamyl-p-nitroanilide [21]. We found an average GGT activity of 973 ± 203 nmol p-nitroaniline/min/mg mitochondrial protein. Addition of 10 mM azaserine, a known GGT inhibitor [25], resulted in roughly a 55% decrease in GGT activity, reducing it to a mean value of 434 ± 86 nmol p-nitroaniline/min/mg protein.

Since GGT is present in the brush border membrane (BBM) of the kidneys [26], and some BBM isolation protocols [27, 28] utilize centrifugation spins of comparable force compared to our mitochondrial isolation procedure, it was likely that our mitochondrial fraction contained BBM, and therefore GGT. However, the mitochondrial intermembrane space, a supernatant fraction, showed substantial GSTZ1 inhibition and GSH consumption. As BBM would not be expected to be found in the IMS, we blotted for villin, a BBM marker [20] in the three submitochondrial fractions. In subfractions from three representative rat kidneys, villin was indeed absent in the IMS and present in the matrix and membrane (Figure 6A). While the matrix is also a supernatant fraction, we believe that villin is present here but not in the IMS due to the vigorous sonication involved in separating the matrix from the membrane. A GGT assay of these fractions showed that roughly 40% of the GGT activity in rat kidney mitochondria comes from the IMS in the absence of BBM with the majority of the remaining activity coming from the membrane fraction containing BBM (Figure 6B).

Fig. 6.

Fig. 6

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The kidney mitochondrial intermembrane space fraction contains γ-glutamyltranspeptidase but not brush border membrane (A) Immunoblot analysis of villin, a marker for brush border membrane, in the kidney mitochondrial intermembrane space (IMS), matrix, and membrane, from three representative rats. (B) Results from a γ-glutamyltranspeptidase (GGT) assay on the kidney mitochondrial IMS, matrix, and membrane from the same representative rats as in panel A. Data are normalized to represent the percent of total GGT activity present in each rat’s mitochondria. Data shown are the average of those percentages. Error bars represent the standard deviation.

3.6 GSTZ1 expression and activity distribution in the rat

Because of the observed inhibition of GSTZ1 activity with DCA in the kidney cell-free homogenates, additional studies were conducted with untreated female rats to measure GSTZ1 expression and activity with DCA in the kidney cytosol fraction. Activity was 0.31 ± 0.03 nmol/min/mg cytosolic protein, mean ± S.D., n=6. This activity was used together with measured activity in liver cytosol and mitochondria, and activity in brain and heart cell-free homogenate to determine the potential contribution of extrahepatic tissues to DCA metabolism.

Calculation of total GSTZ1 activity in entire organs yielded data showing that extra-hepatic tissues combined constituted a lower percentage of GSTZ1 activity in the body of young rats (11.4 ± 2.4%, n=4) than in adult rats (19.4 ± 3.9%, n=4) (Figure 7). Twenty-four hour DCA treatment slightly increased this percentage in both young (13.5 ± 8.5%, n=4) and adult (19.7 ± 8.6%, n=4) rats, but the change did not achieve statistical significance.

Fig. 7.

Fig. 7

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Percent of total-body GSTZ1 activity contributed by extrahepatic tissues in control and treated animals 24 hours after dosing. Values depict the mean and error bars represent the standard deviation (n = 4). * indicates p < 0.05 as determined by Student’s t-test.

3.7 Chloride concentrations in extra-hepatic tissues

Because Cl− provides a protective effect to GSTZ1 through an as-yet unidentified mechanism, stabilizing it during metabolism of DCA [3], we sought to determine the [Cl−] in the organs expressing GSTZ1. Since we were concerned that different tissues may have been homogenized to different degrees, we have reported concentrations in micromoles Cl− per milligram of protein rather than in mM. For reference, we reported above that the average concentration in adult rat liver cytosol from all control rats was 43.0 ± 4.1 mM (mean ± S.D., n=40). This corresponds to 0.33 ± 0.04 μmol Cl− per milligram total cytosolic protein based on the protein concentration of each sample.

The average Cl− levels in the young rats were 0.44 ± 0.06, 0.32 ± 0.04, 0.36 ± 0.04, and 0.17 ± 0.02 μmol Cl− per milligram total protein in the brain, heart, kidney and muscle, respectively (Figure 8). The same tissues from adult rats showed levels of 0.99 ± 0.09, 0.36 ± 0.02, 0.70 ± 0.04, and 0.17 ± 0.02 μmol Cl− per milligram total protein, respectively. There were highly significant age-related differences in the brain and kidney, with the adult rats showing a roughly two-fold greater [Cl−].

Fig. 8.

Fig. 8

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Chloride concentrations in extrahepatic tissues were measured in young and adult rats (n=4). Values depict the mean and error bars represent the standard deviation. ** indicates p < 0.01 as determined by Student’s t-test.

4. Discussion

The majority of GSTZ1 is expressed in the liver; however hepatic enzyme expression is not the only factor determining DCA clearance. DCA treatment leads to the inactivation of GSTZ1 and a subsequent decrease in expression [29]. The Cl− ion provides protection from this inactivation, although its quantitative significance on whole-body DCA kinetics is difficult to determine. However, we found that Cl− levels in the rat liver cytoplasm are substantially lower (~45 mM) than in humans (~125 mM for young donors and 75 mM for adults) [4], which may contribute to the different rates of GSTZ1 inactivation reported for these species [29–31].

While our understanding of factors affecting DCA metabolism in the liver has advanced in recent years, the functional role of GSTZ1 in extra-hepatic tissues has largely been neglected. Here, we extend early work by others [8, 32] using a more sensitive, species-specific antibody probe for rat GSTZ1 to demonstrate notable GSTZ1 expression in brain, heart, and kidneys of Sprague-Dawley rats. Brain and heart expression were related to age, with young rats having higher brain expression and adult rats showing greater expression in the heart. This expression decreases with DCA treatment but is still detectable after 24 hours, showing a lesser decrease than the liver in these same animals (manuscript submitted). Kidney tissues showed considerably higher GSTZ1 expression, being roughly equivalent to what we have observed in the liver mitochondria of adult rats, though still far less than the liver cytosol. The physiological reason for the age-related expression changes in the brain and heart is likely related to its natural role as a maleylacetoacetate isomerase in the catabolism of tyrosine. Tyrosine is a major precursor for catecholamine synthesis, including norepinephrine, which plays a large role in both the brain and heart [33, 34], and is synthesized in both locations [35, 36]. Identifying the exact connection between these pathways will require future studies.

GSTZ1 activity in the cell-free homogenates of these tissues largely mirrors the expression data. The notable exception is very low activity in the kidney cell-free homogenate, which showed the highest levels of expression of the extrahepatic tissues. A previous study that used kidney homogenate found that, of several extrahepatic tissues studied (heart, brain, testis, muscle, and kidney), the kidney had the lowest activity with chlorofluoroacetic acid, and that activity was not reduced in kidney by DCA treatment, although it was reduced in other tissues [8]. Further investigation demonstrated that there is a factor present in rat kidney homogenates that is capable of inhibiting GSTZ1 activity when it is added to a liver sample with known activity. Subcellular fractionation located this factor primarily to the mitochondrial intermembrane space. We then found that this inhibition was indirect through the consumption of glutathione by the glutathionase, GGT, which is known to be highly expressed in the kidney [37], but has not previously been found in the mitochondrial intermembrane space. While the physiological relevance of this finding is not yet known, it is a factor to consider when utilizing any glutathione-dependent assay in kidney tissues. We have observed much smaller, though still measurable, GGT activities in liver tissues and are currently examining its impact (manuscript in process).

Together, these data provide insight into factors beyond hepatic GSTZ1 expression that may impact in vivo DCA metabolism and clearance. Species differences in hepatic Cl−concentrations must be considered when extrapolating results generated in rodents to humans. Moreover, although liver is the predominant site of DCA metabolism, other tissues and organs can provide meaningful activity, up to nearly 20% of all GSTZ1 enzyme activity in adult rats.

Acknowledgments

This work was funded by grants from the National Institutes of Health National Institute of General Medical Sciences [Grants 1 RO1 GM 099871, 2 R01 GM 099871].

Abbreviations

BBM

brush border membrane

[Cl−]

chloride concentration

DCA

dichloroacetate

GGT

gamma-glutamyltranspeptidase

GSH

glutathione

GSTZ1

glutathione transferase zeta 1

IMS

mitochondrial intermembrane space

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

Footnotes

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