Journal of UOEH 20(4):315-322,1998

[Original]

Effects of Pentachlorophenol, Pentylenetetrazol and Carnitine on Mitochondria

Zhengping YU, Yoshihisa IRYO, Masato MATSUOKA and Hideki IGISU

Department of Environmental Toxicology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Japan.

Yahatanishi-ku, Kitakyushu 807-8555, Japan

Abstract: Pentachlorophenol (PCP) increased oxygen consumption and lowered the respiratory control ratio (RCR) in mitochondria from rat liver. These effects of PCP were lessened by 1 mM L-carnitine but not by D-carnitine. In contrast, up to 150 mM of pentylenetetrazol (PTZ) added at state 4 of respiration did not accelerate oxygen consumption. When mitochondria were incubated with 3.3 mM of PTZ, oxygen consumption, RCR and ADP/O ratio were all decreased. Moreover, these could not be suppressed even by high concentrations (~ 20 mM) of L-carnitine. Thus, while L-carnitine could suppress effects of PCP, it could not counteract PTZ in mitochondria. It appears that anticonvulsive effects of carnitine in PTZ-induced seizures may not be due to mitochondrial protection.

Keywords: pentachlorophenol, pentylenetetrazol, carnitine, seizure, anticonvulsive.

Carnitine (beta-hydroxy-gamma-N-trimethylbutyrate) is widely distributed among tissues including the brain. In extraneural tissues, carnitine is an essential cofactor for the transport of fatty acids through the inner mitochondrial membrane [1]. In the brain, however, fatty acids are not utilized as an energy source, and physiological functions of carnitine are unknown. Previous studies indicated that carnitine can protect the brain from various insults such as hyperammonemia [2,3] and severe ischemia [4]. We have observed that carnitine can also suppress convulsions induced by pentylenetetrazol (PTZ) [5], which is one of the most commonly used epileptogenic agents. However, its mechanism is not clear. We therefore examined effects of PTZ and carnitine on mitochondria comparing them with those of pentachlorophenol (PCP) [6-8] which is a typical chemical that impairs biological membranes including mitochondria.

Chemicals. L-Carnitine (inner salt), D-carnitine (inner salt), pentachlorophenol (PCP) and pentylenetetrazol (PTZ) were purchased from Sigma (St. Louis, Mo). All other chemicals were reagent grade.

Mitochondria. Livers from male Wistar rats were minced quickly in a solution containing 250 mM sucrose, 0.5 mM EDTA and 10 mM Tris-HCl (pH 7.4), and homogenized in a Potter-Elvehjem homogenizer with a teflon pestle. The homogenate was centrifuged at 800 x g for 10 min to obtain supernatant which was centrifuged at 10000 x g for 8 min. After the sediment was resuspended in the solution and centrifuged at 8000 x g for 8 min, the supernatant was discarded. These procedures were all done at 4^oC. Protein of the mitochondiral suspension was determined by the method of Lowry et al [9].

Oxygen consumption. The solution used to measure oxygen consumption (3 ml) contained 225 mM sucrose, 10 mM KCl, 5 mM MgCl₂, 5 mM potassium phosphate buffer (pH 7.4), 0.5 mM EDTA, 20 mM Tris-HCl (pH 7.4) and mitochondrial suspension (4.5 mg of protein). A YSI Model 5300 Biological Oxygen Monitor (Yellow Spring Instrument, Yellow Spring, Ohio) equipped with a Clark-type oxygen electrode was used and the changes were recorded with a Hitachi 056 recorder. The temperature of the reaction mixture (30^oC) was controlled with a Lauda RM3 high precision circulating water bath (Lauda-Köningshofen). Unless otherwise stated, mitochondrial suspension was first incubated with or without carnitine (L or D-form) for 10 min at 30^oC, and then with or without a chemical (PCP or PTZ) for another 10 min. The reaction was monitored after adding 5 l of 80 mM ADP and 10 l of 200 mM -ketoglutarate. When necessary, chemicals were added through the port of the reaction chamber. PCP was first dissolved in ethanol and then added to the reaction mixture. The final concentration of ethanol was less than 0.3% which did not affect the reaction.

The respiratory control ratio (RCR) was calculated as (oxygen consumption at state 3 of respiration)/(oxygen consumption at state 4), and ADP/O ratio as ADP added/oxygen consumed.

PCP dose-dependently increased oxygen consumption and lowered RCR (Fig. 1). These changes were less marked when 1 mM L-carnitine was present. In addition, L-carnitine dose-dependently suppressed effects of PCP while D-carnitine did not (Fig. 2).

On adding PTZ to the reaction mixture successively over a wide range of concentrations (3, 6, 9, 12 and 15 M; 30, 60, 90, 120 and 150 M; 3, 6, 9, 12 and 15 mM; 30, 60, 90, 120 and 150 mM) at state 4 of respiration, no increase of oxygen consumption was observed. When mitochondira were incubated with 3.3 mM of PTZ for 10min, oxygen consumption, RCR and ADP/O ratios were all decreased (Fig. 3). L-Carnitine even at 20 mM did not suppress these effects of PTZ (Fig. 4).

Although PCP has been used widely, it can be toxic to humans and even fatal cases of intoxication with PCP have been reported [7]. The mechanisms of the toxicity have not been fully clarified but PCP has been known to disrupt biological membranes including mitochondria and erythrocyte membrane; PCP is a potent uncoupler of oxidative phosphorylation in mitochondria [6], and it can hemolyze erythrocytes [7]. The increase of oxygen consumption and decrease of RCR in mitochondria seen in the present experiments are consistent with the uncoupling of oxydative phosphorylation by PCP. And these could be suppressed by L-carnitine. This indicates that L-carnitine can protect mitochondria not only in hyperammonemia [10] or damage induced by octanoic acid [11] but also from impairment caused by PCP. Hence, it is of interest that mitochondrial dysfunction caused by different kinds of chemicals can be ameliorated by L-carnitine which plays an important physiological role in inner mitochondrial membrane. Furthermore, it is also of interest that, in the case of PCP as seen in the present experiments, only L-carnitine exerted protective effects while D-carnitine did not because D-carnitine is believed not to occur naturally and has been shown to interfere with some effects of L-carnitine [12]. However, much more work is necessary before any conclusion is drawn concerning whether or not carnitine is effective in prevention or treatment of intoxication with PCP.

PTZ is one of the most commonly used epiletogenic agents to evaluate possible anticonvulsive drugs. Although the mechanism of epileptogenesis by PTZ has not been well understood, drugs effective in suppressing PTZ-induced seizures often affect GABAergic systems [13]. On the other hand, it was reported that GABA caused swelling of mitochondria in vitro [14]. Furthermore, ATP content in the brain decreased prior to convulsions induced by chemicals including PTZ [15]. And we previously observed that L-carnitine could suppress seizures induced by PTZ as well as alterations of high energy phosphate compounds in the brain in mice [5]. Hence, it would be of interest to examine effects of PTZ and carnitine on mitochondria in vitro, particularly because the mechanism of the anticonvulsive effects of carnitine is unclear, and because protective effects of carnitine on mitochondria have been suggested in ammonium intoxication and in octanoic acid-induced brain damage [10,11]. Nevertheless, we observed no changes indicating that PTZ uncoupled oxidative phosphorylation in mitochondria i.e. we saw no increase of oxygen consumption by PTZ. But we did observe that PTZ could impair mitochondrial functions; oxygen consumption, RCR and ADP/O ratio were lowered by PTZ. However, these effects of PTZ are most likely to be nonspecific because only high concentrations of PTZ could induce them and because all indices were lowered. Furthermore, while toxicities of PCP could be suppressed by L-carnitine, no protection against PTZ toxicities were provided by L-carnitine, even at high concentrations. Thus, at least in vitro, while L-carnitine could suppress effects of PCP, it did not show any protective effects on mitochondria from PTZ. Hence, it is likely that the anticonvulsive effects of carnitine in PTZ-induced seizures may not be due to mitochondrial protection.

This study was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture, Japan. Zhengping Yu was a visiting scientist supported by Sasakawa Fellowship.

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2. Matsuoka M, Igisu H, Kohriyama K & Inoue N (1991): Suppression of neurotoxicity of ammonia by L-carnitine. Brain Res 567: 328-331

3. Matsuoka M & Igisu H (1993): Comparison of the effects of L-carnitine, D-carnitine and acetyl-L-carnitine on the neurotoxicity of ammonia. Biochem Pharmacol 46: 159-164

4. Matsuoka M & Igisu H (1992): Preservation of energy metabolites by carnitine in the mouse brain under ischemia. Brain Res 590: 334-336

5. Yu ZP, Iryo Y, Matsuoka M, Igisu H & Ikeda M (1997): Suppression of pentylenetetrazol-induced seizures by carnitine in mice. Naunyn-Schmiedeberg's Arch Pharmacol 355: 545-549

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Figure legends

Fig 1: Effects of pentachlorophenol (PCP) and L-carnitine on oxygen consumption and respiratory control ratio (RCR) in mitochondria from rat liver (mean SE, n=6). Mitochondria were incubated with PCP and L-carnitine (1 M or 1 mM) or without it for 10 min at 30^oC, and changes of oxygen in the solution were monitored. Oxygen consumption is expressed as nmol oxygen consumed/mg protein/min. Asterisks indicate difference from the control (no carnitine) (**p<0.01, by Student t-test).

Fig 2: Effects of L- and D-carnitine on pentachlorophenol (PCP)-induced alterations of oxygen consumption and respiratory control ratio (RCR) in mitochondria from rat liver (mean SE, n=6). Mitochondria were incubated with carnitine (L- and D-form) for 10 min at 30^oC and then with PCP (3 M) or without it for 10 min and changes of oxygen in the solution were monitored. Asterisks indicate difference from the effect of D-carnitine (**p<0.01, ***p<0.001, by Student t-test).

Fig 3: Effects of pentylenetetrazol (PTZ) on oxygen consumption, respiratory control ratio (RCR) and ADP/O ratio in mitochondria from rat liver (mean SE, n=6). After mitochondria were incubated with PTZ or without it for 10 min at 30^oC, ADP and -ketoglutarate were added and changes of oxygen in the solution were measured. Asterisks indicate difference from the control (without PTZ) (*p<0.05, **p<0.01, ***p<0.001, by Student t-test).

Fig. 4: Effects of pentylenetetrazol (PTZ) and L-carnitine on oxygen consumption, respiratory control ratio (RCR) and ADP/O ratio in mitochondria from rat liver (mean SE, n=6). After mitochondria were incubated with PTZ and L-carnitine for 10 min at 30^oC, the reaction was started by adding ADP and -ketoglutarate. Similar results were obtained when mitochondria were first incubated with L-carnitine (2 and 10 mM) at 30^oC for 10 min and then for another 10 min with PTZ (3.3 mM). Asterisks indicate difference from the control (without PTZ and carnitine) (***p<0.001, by Student t-test).

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ペンタクロロフェノール、ペンチレンテトラゾール、カルニチンのミトコンドリアへの影響

余　争平、井料　佳久、松岡　雅人、伊規須　英輝

産業医科大学産業生態科学研究所環境中毒学

要旨：ペンタクロロフェノール（ＰＣＰ）は、ラット肝臓より得たミトコンドリアで酸素消費増加と呼吸調節比（ＲＣＲ）低下をもたらした。これらのＰＣＰの効果は、1 mMのＬ−カルニチンによって抑制されたが、Ｄ−カルニチンは無効であった。これと対照的に、ペンチレンテトラゾール（ＰＴＺ）は、状態４呼吸時に１５０mMまで加えても、酸素消費を亢進させなかった。ミトコンドリアを３．３mMのＰＴＺとインキュベートすると、酸素消費、ＲＣＲ、ＡＤＰ／Ｏ比いずれも低下した。しかも、これらは高濃度（２０mM以下）のＬ―カルニチンによって抑制され得なかった。すなわち、Ｌ−カルニチンはＰＣＰの影響に対しては明確な保護効果を示したが、ＰＴＺの作用からミトコンドリアを保護することはなかった。ＰＴＺ誘発けいれんにおけるカルニチンの抗けいれん作用は、ミトコンドリア保護によるのではない可能性が考えられる。

<最終更新日： 2007.11.21.＞　＜文責：環境中毒学＞

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