Elsevier

Toxicology

Volume 200, Issues 2–3, 5 August 2004, Pages 123-133
Toxicology

Chlorpropham induces mitochondrial dysfunction in rat hepatocytes

https://doi.org/10.1016/j.tox.2004.03.012Get rights and content

Abstract

The metabolism and action of chlorpropham (isopropyl N-(3-chlorophenyl)carbamate; CIPC, a post-harvest agent) and its metabolites were studied in freshly isolated rat hepatocytes and isolated rat hepatic mitochondria, respectively. The exposure of hepatocytes to CIPC caused a concentration (0.25–1.0 mM)- and time (0–3 h)-dependent cell death accompanied by loss of cellular ATP and adenine nucleotides. CIPC at a weakly toxic level (0.5 mM) was metabolized to isopropyl N-(3-chloro-4-hydroxyphenyl)carbamate (4OH-CIPC) and subsequently to its glucuronide and sulfate conjugates (major metabolites) or alternatively to a minor metabolite 3-chloroaniline (3CA). The addition of SKF-525A (50 μM), an inhibitor of microsomal monooxygenase, enhanced the CIPC (0.5 mM)-induced cytotoxicity accompanied by loss of ATP and 4OH-CIPC and inhibited the decrease in the concentration of the parent compound. CIPC led to a strong decrease in cellular ATP content compared to its metabolites, 4OH-CIPC and 3CA. On the other hand, the exposure of isolated hepatic mitochondria to CIPC reduced State 3 respiration with a FAD-linked substrate (succinate plus rotenone) and/or with a NAD+-linked substrate (pyruvate plus malate), whereas State 3 respiration with ascorbate plus tetramethyl-p-phenylendiamine (cytochrome oxidase-linked respiration) was not affected markedly by CIPC. Further, the addition of CIPC caused an increase in the rate of State 4 oxygen consumption, indicating an uncoupling effect, and a decrease in the rate of State 3 oxygen consumption in a concentration-dependent manner, respectively. In contrast, the addition of neither 4OH-CIPC nor 3CA markedly affected the rate of states 3 and/or 4 oxygen consumption. These results indicate that CIPC-induced cytotoxicity is mediated by the parent compound rather than by its metabolites such as 4OH-CIPC and 3CA, and that the toxicity is associated with a rapid depletion of ATP via impairment of mitochondrial function related to oxidative phosphorylation.

Introduction

Chlorpropham (isopropyl N-(3-chlorophenyl)carbamate; CIPC) is used to control weeds in various food and non-food crops, and as a sprouting suppressant and/or for a post-harvest treatment of potatoes during storage and/or transport. Even if consumption in the diet is low, the potential toxicity of CIPC has been studied in vivo and in vitro to assess its various toxicologic properties. Although CIPC was not potently toxic in genotoxic, mutagenic, and immunotoxic studies and in some short- and/or long-term dietary studies (Van Esch and Kroes, 1972, Larson et al., 1960, Boyd and Carsky, 1969, Van Engelen, 2001), the liver, kidney, spleen and erythrocytes were identified as targets of CIPC-induced toxicity in rats and mice. Oral administration of CIPC to rats or mice caused dose-related increases in liver and spleen weight, accompanied by the acceleration of hematopoiesis and/or hemosiderosis in both organs. The serum concentrations of bilirubin and cholesterol, as parameters of hepatic damage, in rats were significantly increased at a high dose (2200 mg/kg per day) in a 28-day study (Van Engelen, 2001). The hepatic damage in mice was accompanied by focal necrosis (Fujitani et al., 2000). In the kidney, intracytoplasmic hyaline resportion bodies were found in the tubules, with the effect being more pronounced in males than in females (Fujitani et al., 1997, Fujitani et al., 2000, Van Engelen, 2001). In addition, it has been reported that CIPC is well absorbed from the gastrointestinal tract when administered via the oral route in rats and mice, undergoes extensive metabolism, and is excreted in the urine and that the metabolism of CIPC proceeds either via aromatic 4-hydroxylation followed by conjugation of the intermediate with glucuronic acid and sulfate as a major pathway, or via oxidation of the isopropyl side-chain and carbamate hydrolysis, followed by conjugation of these intermediates as minor pathways (Fang et al., 1974, Carrera et al., 1998).

The metabolic pathway and toxic effects of CIPC have been studied in vivo and/or in vitro (Fang et al., 1974, Carrera et al., 1998), whereas the relationship between metabolism and toxicity has not been clarified. The freshly isolated rat hepatocyte system which retains intact membranes and has high levels of various drug-metabolizing enzymes and their cofactors associated with Phase I and Phase II reactions is useful for the study of uptake, intracellular target sites, and temporal sequences leading to cell injury by chemicals and/or their metabolites via biotransformation (Moldéus et al., 1978). In the present study, we investigated the metabolism and cytotoxic effects of CIPC on isolated rat hepatocytes.

Section snippets

Materials

The chemical compounds used were obtained from the following companies: CIPC (purity of >99.7%) from Hodogaya Chemical Industry (Tokyo, Japan); 3-chloroaniline (3CA) and 3-chloro-4-hydroxyaniline from Tokyo Kasei Kogyo Co., Ltd. (Tokyo); β-glucuronidase, sulfatase (type VI, β-glucuronidase-free Aerobacter aerogenes), N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid) (HEPES), 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF-525A) and bovine serum albumin from Sigma Chemical Co.

Toxic effects of CIPC on rat hepatocytes

The exposure of rat hepatocytes to CIPC caused a concentration (0.25–1.0 mM) and time-dependent (0–3 h) cell death accompanied by a depletion of intracellular ATP and total adenine nucleotides (Fig. 2). CIPC at a concentration of 1.0 mM elicited a rapid depletion of total nucleotide pools with abrupt lethality, whereas the loss of ATP was reflected by concomitant increases in the levels of ADP and AMP (data not shown). The concentrations of malondialdehyde, an index of lipid peroxidation, in

Discussion

The results obtained in the present study show that in isolated rat hepatocytes, CIPC induced a concentration- and time-dependent loss of cell viability, followed by decreases in intracellular levels of ATP and total adenine nucleotide pools. The cytotoxicity of CIPC may depend on the initial concentration rather than on that of its major intermediate 4OH-CIPC, because (a) the pretreatment of hepatocytes with the inhibitor of microsomal monooxygenase enhanced CIPC-induced cytotoxicity

References (29)

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