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Hexachlorobenzene Treatment on Hepatic Mitochondrial Function Parameters and Intracellular Coproporphyrinogen Oxidase LocationDepartamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina Correspondence: Address correspondence to Horacio A. Sancovich Navarro, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 4557 (C1419HUM) CABA, Ciudad Autónoma de Buenos Aires, Argentina. E-mail:sancovic{at}qb.fcen.uba.ar
These studies try to elucidate why isocoproporphyrin appears in hexachlorobenzene-poisoned rats’ feces. Chronic exposure of hexachlorobenzene to rats produces an experimental model for human porphyria cutanea tarda. After 8 weeks of treatment, rats showed high porphyrin excreta and 50% inhibition of liver uroporphyrinogen decarboxylase activity. Uroporphyrin plus heptacarboxylic porphyrin exceeded coproporphyrin in urine, whereas in feces, isocoproporphyrin, from abnormal pentacarboxylic porphyrinogen III oxidative decarboxylation by liver coproporphyrinogen oxidase, became the main porphyrin. Trypsin-treated mitochondria showed that the outer and inner membrane permeability barrier was highly conserved after hexachlorobenzene intoxication. In digitonin-treated hexachlorobenzene mitochondria, coproporphyrinogen oxidase was free in the mitochondrial intermembrane space, whereas in normal mitochondria, 30% to 50% remained anchored to the inner membrane. Hexachlorobenzene led to a decrease in respiratory control and ADP/O ratios (uncoupled mitochondria). Albumin restored oxidative phosphorylation, indicating no irreversible inner membrane damage. Normal and hexachlorobenzene mitochondria oscillatory studies exhibited similar damping factor values, showing that hexachlorobenzene had no significant effect on membrane fluidity and elasticity. Mitochondrial uncoupling could explain the free state of the enzyme within the intermembrane space. The free state of the enzyme makes it more flexible and would allow pentacarboxylic porphyrinogen III, whose levels are increased, to compete with coproporphyrinogen III and being transformed into dehydroisocoproporphyrinogen, the liver forerunner of fecal isocoproporphyrin.
Key Words: Coproporphyrinogen Oxidase • Experimental Porphyria • Hexachlorobenzene • Isocoproporphyrin • Mitochondria The formation of tetrapyrroles and their subsequent modification into heme is a multistep process that has been highly conserved throughout evolution (Beale and Weinstein 1990; Wyckoff and Kushner 1994). In eukaryotes, the biosynthetic pathway leading to heme involves a sequence of eight enzymatic steps. The fifth of these steps is catalyzed by the cytosolic enzyme uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37). URO-D catalyzes the sequential decarboxylation of uroporphyrinogen (Uro’gen) III to coproporphyrinogen (Copro’gen) III. Theoretically, this transformation may involve 24 possible routes with 14 porphyrin structures. We have demonstrated that URO-D takes only one route, and this involves three porphyrinogen intermediates: heptacarboxylic (Hepta’gen) d, hexacarboxylic (Hexa’gen) ad, and pentacarboxylic (Penta’gen) abd (the letters indicate the position of methyl groups) (Jackson et al. 1976a, 1976b; Jackson, Sancovich, and Ferramola de Sancovich 1980; Smith et al. 1976). At this stage, Copro’gen III is transferred into the mitochondria and undergoes two stepwise oxidative decarboxylations by the enzyme coproporphyrinogen oxidase (CPO; EC 1.3.3.3). This enzyme catalyzes the removal of the carboxyl group and the two hydrogens from the propionic acid side chains of Copro’gen III to form vinyl groups at positions 2 and 4, leading to protoporphyrinogen (Proto’gen) IX. In intact mitochondria, CPO is loosely attached to the inner membrane surface surrounding the intermembrane space (Elder and Evans 1978b). Porphyrias are disorders caused by marked enzymatic deficiencies in the heme pathway. Porphyria cutanea tarda (PCT) is the most common, and it is caused by a heterozygous deficiency of URO-D (Elder 1998). Estimates of its prevalence range from 1 in 25 000 in the U.S. population (Elder 1998) and 1 in 37 000 in Argentina (Méndez et al. 2005). PCT is biochemically characterized by a marked increase in porphyrin excretion: uroporphyrin (Uro) and heptacarboxylic porphyrin (Hepta) in urine, and isocoproporphyrin (Isocopro) and smaller amounts of coproporphyrin (Copro) in feces (Elder 1972). Chronic exposure to hexachlorobenzene (HCB) may generate a toxic cutaneous porphyria in rats. In several aspects, this form of porphyria resembles human PCT (Stornard 1974). It seems to be a dose-related response to the absorption of the porphyrogenic chemical, but it is not influenced by individual susceptibility (Elder 1972). HCB intake was followed initially by a moderate increase of Copro and later by an increase of highly carboxylated porphyrins, such as Uro and Hepta, in urine (Elder, Evans, and Matlin 1976) and the presence of Isocopro and smaller amounts of Copro in feces (Elder 1972). Isocopro excreta is the result of increased quantities of Penta’gen III (Penta’gen abd) (Jackson et al. 1976a, 1976b; Jackson, Sancovich, and Ferramola de Sancovich 1980; Smith et al. 1976), which is formed in the cytosol and competes with Copro’gen III for CPO decarboxylation (Elder 1972; Kennedy et al. 1970), producing dehydroisocopro’gen, which is not further metabolized in the liver. The former porphyrinogen is then hydrogenated by intestinal bacteria and before being excreted is oxidized to Isocopro. It is still not clear whether an increase in Penta’gen abd–Copro’gen III ratio, caused by URO-D impairment, is the only reason for this abnormal transformation. It may be speculated that alterations in mitochondria could play an essential role or, at least, contribute to dehydroisocopro’gen biosynthesis. To test this hypothesis, comparative studies on mitochondria properties and function parameters were carried out in normal (N) and HCB-intoxicated (HCB) rats. Results emphasize the notion that abnormal dehydroisocopro’gen biosynthesis catalyzed by CPO depends not only on increased cytosol hepatocyte Penta’gen abd levels, but also on other mitochondrial alterations. It was observed that the main difference between N and HCB mitochondria was the location of CPO. In HCB mitochondria, CPO was mostly free in the mitochondrial inner membrane space (MIMS), whereas in N mitochondria, most enzyme activity was anchored to the outer face of the mitochondrial inner membrane (MIM) in the MIMS. Thus, HCB treatment would let CPO be more flexible, allowing Penta’gen abd to compete with Copro’gen III for the CPO catalytic site, and to be transformed into dehydroisocopro’gen, the liver precursor of fecal Isocopro.
Chemicals HCB (commercial grade) (95% HCB, 5% tetra- and pentachlorobenzene) was generously provided by Compañía Química S.A. (Buenos Aires, Argentina). Copro III and Uro III were obtained from Porphyrin Products (Logan, UT, USA). Porphyrinogens were prepared with sodium amalgam, according to Mauzerall and Granick (1958). L-malic acid, L-glutamic acid, L-malonic acid, ADP (grade I), valinomycin, rotenone, EDTA, digitonin, trypsin, soybean trypsin inhibitor, Percoll, DMSO, and crystalline bovine serum albumin (BSA) were obtained from Sigma Chemical Co. (St Louis, MO, USA). Sephadex G-25 was obtained from Pharmacia (Uppsala, Sweden). All other reagents were of commercially available analytical grade.
Animals Animals received humane care and were treated in accordance with guidelines established by the Animal Care and Use Committee of the Argentine Association of Specialists in Laboratory Animals (AADEALC). The experimental protocols were also approved by the Local Committee of Animal House in the School of Sciences, University of Buenos Aires, Argentina.
Analysis of Porphyrins in Urine and Feces
Tissue Fractions
Enzyme Assays
URO-D activity was determined according to Spinelli et al. (1997) with some modifications. The reaction was stopped adding HCl (final 5%  / ) and DMSO (final 21% /). Porphyrinogens were exposed to light and centrifuged at 10,000 rpm, for 10 min. Porphyrins were analyzed by HPLC; linear gradient from 10% acetonitrile (/) to 30% acetonitrile in 1 M ammonium acetate buffer, pH 5.16, for 15 min, followed by isocratic elution in 30% acetonitrile for another 10 min. Flow rate was 1 ml/min. A fluorescence detector was used. URO-D activity was expressed in nmol Uro’gen III transformed/h/mg protein.
CPO activity was assayed according to Sorianello and Mazzetti (2000) with some modifications. The reaction was stopped by adding 1 ml 10% HCl. Porphyrinogens were exposed to light and centrifuged at 10,000 rpm for 10 min. Porphyrins were analyzed by HPLC: 23% methanol (
Marker Enzyme Activities Citrate synthase (CS) activity was determined spectrophoto-metrically according to the method of Siu et al. (2003). Malate dehydrogenase (MD) activity was determined in a continuous assay, recording decreases or increases in absorbance at 340 nm according to Mullinax et al. (1982). Sulfite oxidase (SO) activity was routinely assayed using a direct spectrophotometric method following the decrease in absorbance at 212.5 nm according to Cohen and Fridovich (1971).
Mitochondrial Isolation and Purification
Mitochondrial Disruption
Trypsin Treatment
Digitonin Method for Mitochondrial Fractionation For each fraction, enzyme activity recovery was calculated as the sum of total activity in each fraction in relation to total activity in nonfractioned material (disrupted mitochondria). Enzyme activity of each fraction was expressed as a percentage of total recovered activity.
Mitochondrial Respiration Assay
Mitochondrial Oscillation
Damping Factor Calculation The damping factor is defined as the ratio of successive oscillation amplitudes of peaks and troughs of tracing damped harmonic oscillatory variations. Peak damping factors were obtained from the a/b, b/c, c/d, and d/e ratios, and trough damping factors from the 1/2, 2/3, and 3/4 ratios (Figure 1).
Protein Concentration
Statistical Analysis
Effect of HCB on Porphyrin Rat Excreta To detect the onset of HCB intoxication, urine and feces were collected weekly from animals in N and HCB groups. The evolution of porphyrin excreta (Figures 2 A and 3A) showed that HCB intoxication increased porphyrin content of both urine and feces in function of time of the treatment. During the first 5 weeks of treatment, both HCB and N rats showed very low porphyrin content in urine and feces, and Copro III as the main tetrapyrrole component in excreta. From the 6th week onwards, an increase in porphyrin excreta was observed. In urine, a decrease of Copro III percentage was paralleled to a concomitant increase of highly carboxylated porphyrins, mainly Uro III and Hepta III (Figure 2B). From the 7th week onwards, Uro III plus Hepta III proportions exceeded Copro III content. Between weeks 7 and 11, the pattern of porphyrin excreta remained nearly constant. In feces, concomitant with a high increase in porphyrin excreta, Copro III and Isocopro, another tetracarboxylated porphyrin, were accompanied by smaller amounts of penta-, hexa-, and heptacarboxylated porphyrins (Figure 3B). After the 8th week, the highly carboxylated porphyrin fraction was barely noticeable and Isocopro became the main product.
Time Course of Uro-D Inhibition Rat liver URO-D activity was monitored over 11 weeks of HCB treatment (Figure 4). Liver URO-D activity seemed to fall from the beginning of the experiment. It was significantly inhibited from the 2nd week of HCB intoxication, but maximum inhibition (over 50%) was not observed until 5 to 6 weeks later. Then, the level of URO-D activity remained nearly constant until the end of the treatment. No significant differences (NSD) in values were observed from week 8 until the end of the experiment.
Trypsin Treatment and Digitonin Action on Isolated Mitochondria Figure 5 shows that, in HCB-treated rats, CPO and marker enzyme activities, with different location in mitochondria, were not affected by trypsin treatment. After mitochondrial digitonin treatment, nearly 100% CPO, AK, and SO activities were recovered in the supernatant. In supernatant and sediment trypsin treatments, no CPO, AK, and SO activities were detected in any preparation. However, CS and MD (matrix enzyme) activities were not modified (Figure 5B to F).
Marker enzyme activities in N rats (data no shown) showed NSD with HCB data in all the treatments assayed. The only difference was observed in the digitonin treatment, in which 28% CPO activity appeared joined to the sediment (mitoplasts), whereas the remnant CPO activity was found in the supernatant. After trypsin treatment, CPO activity disappeared from both fractions (supernatant and sediment) (Figure 5A). When N intermembrane mitochondrial space proteins were released, modifying the pH during digitonin treatment, different results for CPO location were obtained. Figure 6 shows the percentages of mitochondrial enzyme activities in the sediment of digitonin treatments at pHs 7.4 to 6.4. No differences were found in matrix marker enzyme activities of N (Figure 6A) and HCB (Figure 6B) rats at all the pHs studied. However, N mitochondria showed that when pH decreased in digitonin treatment, CPO activity attached to the MIM increased, reaching almost 50% at the lower pH assayed (pH 6.4). In HCB mitochondria, on the other hand, no differences in CPO activity were observed at the different digitonin treatment pHs assayed (7.4 to 6.4).
Effect of HCB on Rat Liver Mitochondrial Respiration Table 1 shows NSD between N and HCB mitochondrial Vstate3 with malate-glutamate or succinate as substrate. Likewise, no changes were detected in the presence of BSA (1 mg/ml). HCB treatment with malate-glutamate or succinate as substrate triggered a significant increase in Vstate4 and a significant decrease of the means of RCR and the ADP/O ratio. When BSA was used in these determinations, respiration at rest (state 4) significantly fell in relation to values obtained without this compound. Likewise, RCR and ADP/O mean ratios were higher than in studies without BSA, and similar to data from N rats.
Effect of HCB on Mitochondrial Oscillation
The time of onset of HCB experimental porphyria showed that increase in porphyrin excreta was a function of time of the intoxication (Figures 2A and 3A). In urine, Uro III plus Hepta III proportions exceeded Copro III content from the 7th week onwards (Figure 2B), and this pattern remained nearly constant up to the end of the treatment. In feces, Isocopro became the main excreta porphyrin after the 8th week (Figure 3B). HCB seemed to have no effect on rat erythrocytes, because only traces of Copro were detected in N and HCB-intoxicated rat erythrocytes (data not shown). Kinetic studies with HCB (Figure 4) demonstrated that liver URO-D activity fell from the beginning of the experiment. However, maximum inhibition (over 50%) was not observed until the 7th week. Such decrease seemed not to be caused by lower liver URO-D synthesis (Elder and Sheppard 1982). Smith and Francis (1983) reported that rodents exposed to long HCB treatment developed massive accumulation of porphyrins in the liver. This accumulation was detected at the early intoxication stages (Masini et al. 1984). Uro and Hepta predominated, but considerable amounts of Copro, Hexa, and Penta were also observed (Doss, Schermuly, and Koss 1976). The aim of this work was to find why Isocopro appear in HCB poisoned rat feces. It is still not clear whether the increase in the ratio between Penta’gen abd and Copro’gen III, caused by URO-D impairment, would be the only reason for CPO to develop an abnormal Penta’gen abd transformation. It may be speculated that mitochondrial alterations would play an essential role in dehydroisocopro’gen biosynthesis or at least contribute to it. To test this hypothesis, comparative studies on mitochondrial properties and function parameters were carried out in N and HCB-intoxicated rats. According to the results obtained, all further studies were performed on HCB-treated rats for at least 2 months (Figures 2 to 4). HCB increases lipid metabolism of the lipogenic enzymes glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malic enzyme (Kleiman de Pisarev, Ferramola de Sancovich, and Sancovich 1995; Loaiza-Pérez et al. 1999), and lipid peroxidation levels (Fernández Tomé et al. 2000). Mitochondrial functions depend on membrane phospholipid and fatty acid composition, and membrane-bound enzyme functions and interactions within the membrane depend on the physical properties of phospholipids or, at least, on the nature of their fatty-acyl components (McMurchie et al. 1983). Both in intact N and HCB mitochondria, the insensitivity to the proteolytic attack of MIMS enzymes (CPO, AK, and SO) exerted by trypsin was higher than 98% and lower than 2% in disrupted samples (Figure 5A to D). This demonstrated that in both treated groups, mitochondrial outer membranes were preserved. The permeability barrier of the external membrane against the release of MIMS enzymes, particularly CPO, and the accessibility of trypsin remained highly conserved even after HCB treatment. Digitonin, which reacted, and formed complexes, with cholesterol molecules, was useful to selectively enhance the permeability of the external membrane of mitochondria, leaving the MIM, virtually devoid of cholesterol, almost completely intact (Janski and Cornell 1980). Studies showed that the main difference between HCB and N mitochondria after digitonin treatment was CPO location. In HCB mitochondria, the enzyme was entirely located in the soluble fraction of MIMS (Figure 5B), and was not altered at the different pHs assayed (Figure 6B). In N mitochondria and depending on the pH assayed, 30% to 50% CPO was anchored to the outer surface of the MIM (Figures 5A and 6A). Matrix enzyme activities (CS and MD) from submitochondrial particles (mitoplasts) were not affected by trypsin treatment (Figure 7E and F). Thus, even after digitonin exposure, MIM from N and HCB mitochondria also kept their permeability barrier. In order to assess other damages caused by HCB treatment to the MIM, certain mitochondrial function properties from HCB rats were compared with those from N animals. RCR values have been important in the study of mitochondrial integrity with respect to oxidation and phosphorylation coupling. Effects on electron transport and oxidative phosphorylation in HCB-treated rats were compared with those of N animals (Table 1). During active metabolism (state 3), NSD in respiratory velocities were detected between N and HCB mitochondria. Data obtained may be considered normal standard values for this parameter. In N rats, BSA did not impair mitochondrial respiration parameters (Table 1). HCB treatment led to a substantial modification in the efficiency of the oxidative phosphorylation process. A significant decrease in RCR and ADP/O mean ratio was observed after HCB treatment (Table 1). Thus, respiration in HCB mitochondria was not completely coupled to phosphorylation, and membrane potential would be dissipated. Masini et al. (1984) reported that induced experimental porphyria resulted in partial and constant uncoupling of oxidative phosphorylation in liver mitochondria. During HCB administration, pentachlorophenol was the main metabolite recovered in rat tissues (Debets, Strik, and Olie 1980). HCB uncoupling can be attributed to the action of this metabolite, but albumin can substantially reverse this uncoupling (Masini et al. 1984; Trenti et al. 1986). Table 1 also demonstrated that BSA fully reversed the uncoupling of the oxidative phosphorylation process in liver HCB mitochondria. These results indicated that no irreversible damage occurred, and that MIM potential could be reestablished. Alterations in membrane lipid fatty-acid composition resulted in a shift in transition temperatures, suggesting that membrane fluidity can be modulated, altering fatty-acyl chain components of membrane phospholipids (Innis and Clandinin 1981). To assess HCB effects on mitochondrial fluidity and elasticity, changes in mitochondrial oscillations were studied. Gooch and Packer (1974) found that in vitro mitochondria showed oscillatory phenomena with concomitant volume changes, and they suggested the damping factor was a good parameter to measure mitochondria oscillatory responses. The higher ratios corresponded to smaller damped mitochondrial oscillatory responses, usually due to lower membrane elasticity caused by volume changes. Studies with N and HCB-intoxicated rat liver mitochondria showed a clearly visible similarity in their oscillatory variations (Figure 7). Mean damping factor values for both groups of mitochondria were also very similar (Figure 8). This would indicate that HCB treatment have no marked influence on mitochondrial membrane fluidity and elasticity. It has been reported that oxidative phosphorylation uncouplers such as 2,4-dinitrophenol enhance the damping factor, increasing passive membrane permeability to protons (Gooch and Packer 1974). Our data do not agree with these results, maybe because the HCB metabolite (pentachlorophenol) had a weaker uncoupling effect at the concentration levels in the experiments. The final steps of the heme biosynthetic pathway in higher eukaryotes are catalyzed by three enzymes: CPO, protoporphyrinogen oxidase (PPO), and ferrochelatase, which are either bound to the MIM or associated with it. These enzymes may participate of the multienzyme complex in a stable or dynamic manner, following a spatially close orientation in the MIM that does not allow significant accumulation of intermediates to occur (Grandchamp, Pung, and Nordmann 1978; Deybach et al. 1985; Ferreira and Dailey 1988; Ferreira et al. 1988; Proulx, Woodard, and Dailey 1993; Olsson et al. 2002). Peripheral (extrinsic) proteins like CPO may bind reversibly to the outer face of the MIM through electrostatic interactions and hydrogen bonding to membrane lipids and intrinsic proteins. They can be released by relatively mild treatments that interfere with electrostatic interactions or by breaking hydrogen bonds. The techniques used to locate CPO could influence our results, affecting the actual CPO location. Data obtained, as previously stated by Grandchamp, Pung, and Nordmann (1978), do not rule out the possibility that CPO from in vivo N mitochondria could be entirely bind to the external face of the MIM. Thus, there would be a direct transfer of Proto’gen and Proto IX between catalytic sites. Should this not happen and due to their poor solubility in water, they could be intercalated into the surrounding mitochondrial membrane, rather than diffused into the bulk aqueous phase (Proulx, Woodard, and Dailey 1993). In HCB-treated rat livers, data clearly demonstrated that CPO would be mainly free in the MIMS, as AK and SO are (Figure 5B to D). Thus, Copro’gen III would have a better accessibility to CPO catalytic site than in N mitochondria, in which it would be attached to the MIM, partially impeding porphyrinogen traffic into the CPO catalytic center. As Grandchamp, Pung, and Nordmann (1978) proposed, when Copro’gen III crosses the outer membrane, a free enzyme-substrate complex would be formed, which would induce and/or help it to bind to a specific MIM destination, near or on PPO. The binding of an enzyme and a substrate involves conformational changes, which are essential to enzymatic functions. HCB treatment would grant CPO more flexibility. Even subtle changes in CPO three-dimensional structure would allow Penta’gen abd, whose levels are increased in liver, to compete with Copro’gen III for the CPO catalytic site and for being transformed into dehydroisocopro’gen, the liver forerunner of fecal Isocopro. This hypothesis is based on the notion that experimental Penta’gen abd oxidative decarboxylation by CPO was only achieved in disrupted mitochondria (Elder and Evans 1978a) or highly purified human CPO (Cooper et al. 2005). CPO is synthesized outside the mitochondria and must be imported to its final location. A proton-motive force across the MIM (with electric and pH gradients provided by the electron transport chain) would be required for its translocation and could be indeed essential for CPO-MIM interactions. Because mitochondria from HCB-intoxicated rats are uncoupled, the electrochemical potential of the membrane is dissipated. Therefore, it would be important to keep CPO free in the MIMS.
The studies of this work were supported through a grant from the University of Buenos Aires (UBACYT). A.M.F. de S. and H.A.S. are Scientific Research Carrier Members of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Manuscript received March 25, 2008. Accepted for publication December 1, 2008.
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International Journal of Toxicology, Vol. 27, No. 6,
455-465 (2008)
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Abstract
MATERIALS AND METHODS
A520/mg protein (
