Reduced mitochondrial membrane potential and metabolism correspond to acute chloroform toxicity ofin vitro hepatocytes

Abstract

Chloroform is a non-genotoxic compound that is present in drinking water and ambient air as a result of water chlorination but whose carcinogenic mechanism in humans is unknown. Chloroform targets the liver in humans, where cytochrome P-450-dependent biotransformation to phosgene results in mitochondrial damage and cell death. The purpose of this study is to investigate the relationship between cell death, loss of mitochondrial membrane potential (MMP) and reduction of metabolic rates for in vitro cultured mouse hepatocytes after acute exposure to two doses of chloroform. Immediately following a 2-h exposure, culture viabilities were 70% and 54% for concentrations of 7.0 and 8.8 mM, respectively, in contrast with 90.0% for controls. Interestingly, the viabilities of these cultures decreased further, to 6% and 12%, respectively, over the next 24-h period despite being placed in fresh, chloroform-free medium. Measurement of MMP for viable cells at the end of the exposure revealed a decrease in Rhodamine 123 uptake, which indicates a loss of MMP. Additionally, glucose consumption and lactate production rates were reduced during the 6-h period following the exposure. These results support the hypothesis that a subpopulation of cells at the end of an acute exposure may be activated for apoptosis, suggesting a role for apoptosis markers during risk assessment for chloroform.

Targeting Mitochondria: A New Promising Approach for the Treatment of Liver Diseases

“…The ADP/ATP gradient across the inner membrane is balanced by the ANT activity, which extrudes ATP from the mitochondria in exchange for ADP. Other drugs have been demonstrated to induce acute liver injury by causing hepatic mitochondrial dysfunction and ATP depletion, such as diclofenac [43] or chloroform [44]. After being specifically transported from cytosol to mitochondrial matrix, pyruvate (1) and free fatty acids (2) undergo oxidation providing reducing equivalents which in turn are oxidised to water by the respiratory chain (3).…”

“…Oxidative stress has been implicated in several druginduced acute liver diseases, such acetaminophen, diclofenac or chloroform [43,44,71]. Epirubicin therapy results in acute hepatotoxicity by impairing catalase and superoxide dismutase [72].…”

The mitochondrial fluorescent dye rhodamine 123 is a high‐affinity substrate for organic cation transporters (OCTs) 1 and 2

“…This preferential sensibility to inhibitors of hOCT2-mediated transport when compared with that related to hOCT1 has also been reported for other substrates. It is therefore noteworthy that caution has likely to be considered when using rhodamine 123 for investigating membrane potential in hOCT1-or hOCT2-positive cells, knowing moreover that the concentrations of rhodamine 123 used for investigating mitochondrial potential in living cells can be in the 2-10 lM range [36,37], for which carrier-mediated transport of the dye is the major way of uptake in HEK-hOCT1 and HEK-hOCT2 cells (Figure 4). In the same way, various OCT1/2 inhibitors such as disopyramide, dipyridamole, imipramine, tacrine, and cimetidine inhibited membrane transport of the cationic dye 4-(4-(dimethylamino)styryl)-N-methylpyridinium in a more potent manner in hOCT2-transfected cells than in hOCT1-transfected counterparts [30].…”

Application of Key Events Analysis to Chemical Carcinogens and Noncarcinogens

“…The mechanism of cell death has yet to be clearly determined, but there are data suggesting that it involves mitochondrial dysregulation leading to impairment of the mitochondrial membrane potential via effects on mitochondrial permeability transition (Hartig et al, 2005; Burke et al, 2007). …”

“…No evidence for renal toxicity is found in females. Low levels of damage are not sufficient to cause cytotoxicity (Hartig et al, 2005), as repair and/or resynthesis of damaged proteins enables cellular recovery (Fig. 8).…”