Barbara J. Keller

Several nongenotoxic carcinogens uncouple mitochondrial oxidative phosphorylation

A number of plasticizers and lipid-lowering drugs induce peroxisomes and cause hepatocellular carcinoma in rodents by mechanisms which remain unknown. In this study, seven structurally dissimilar peroxisome proliferating agents were shown to uncouple oxidative phosphorylation in isolated rat liver mitochondria. For example, perfluorooctanoate (0.5 mM) increased succinate-induced (state 4) mitochondrial respiration by over 50% while stimulation of state 3 respiration with ADP was minimal (i.e., uncoupling occurred). Interestingly, compounds which are potent carcinogens in vivo (e.g., Wy-14,643 and perfluorooctanoate) were more powerful uncouplers of oxidative phosphorylation in vitro than weak tumor-causing agents (e.g., valproate). Uncoupling also occurred in vivo. Basal rates of oxygen uptake in perfused livers from chronically treated rats were increased from 137 +/- 7 mumol g-1/h in pair-fed controls to 153 +/- 5 mumol g-1/h after 2.5 months of feeding Wy-14,643 (0.1% w/v in diet). Concomitantly, rates of urea synthesis from ammonia, a process highly dependent on ATP supply, were reduced almost completely from 104 +/- 10 mumol g-1/h to 13 +/- 6 mumol g-1/h. Bile flow, another energy-dependent process, was also reduced significantly by treatment with Wy-14,643 in vivo for 24 h. Taken together, these data indicate that energy supply for cellular processes such as urea synthesis and bile flow was disrupted in vivo due to uncoupling of oxidative phosphorylation by Wy-14,643. It is proposed that peroxisomal proliferators accumulate in the liver where they uncouple mitochondrial oxidative phosphorylation and interfere with cellular energetics.

Inhibition of mitochondrial respiration and oxygen-dependent hepatotoxicity by six structurally dissimilar peroxisomal proliferating agents.

The purpose of this study was to test the hypothesis that a variety of structurally dissimilar peroxisomal proliferators inhibited O2 uptake and caused O2-dependent hepatotoxicity in the perfused rat liver. Aspirin, valproate, ethylhexanol, clofibric acid, ciprofibrate and perfluorooctanoate were selected as a representative group of weak, moderate, and potent peroxisomal proliferators, respectively. All compounds studied inhibited state 3 but not state 4 rates of oxygen uptake in isolated mitochondria (perfluorooctanoate greater than ciprofibrate greater than ethylhexanol greater than clofibric acid greater than aspirin greater than valproate; half maximal inhibition occurred at concentrations ranging from 0.6 to 3.2 mM depending on the compound). Clofibric acid, ethylhexanol and aspirin inhibited oxygen uptake only in upstream, oxygen-rich periportal regions of the perfused liver lobule by 30-40%. Perfusion with the six agents studied caused release of lactate dehydrogenase into the effluent perfusate in a dose-dependent manner and caused damage predominantly in periportal regions of the lobule as reflected by trypan blue uptake. A strong correlation between the concentration of compound needed to inhibit respiration in isolated mitochondria and cause hepatotoxicity in the perfused liver was observed. We propose that peroxisomal proliferators accumulate in the liver due to their lipophilicity where they inhibit actively respiring mitochondria in periportal regions of the liver lobule and cause local toxicity.

Wy-14,643 stimulates hepatic protein kinase C activity

The mechanism by which hypolipidemic drugs and industrial plasticizers cause hepatic tumors in rodents remains unknown. Protein kinase C is elevated during hepatic cell turnover, and sustained cellular replication has been shown to correlate with an increase in hepatic tumors. Therefore, the effect of [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) on protein kinase C activity was examined. Female Sprague-Dawley rats were given 100 mg/kg Wy-14,643 in olive oil (i.g.), while control rats received equal volumes of oil vehicle. After 24 h, the activity of protein kinase C was estimated in isolated hepatic fractions by measuring the binding of 3H-phorbol-12,13-dibutyrate, a specific ligand for protein kinase C. Administration of Wy-14,643 significantly increased protein kinase C activity nearly 2-fold in microsomal fractions. Thus, it is possible that Wy-14,643 increases cell proliferation and causes tumors by mechanisms involving protein kinase C.

2-ethylhexanol uncouples oxidative phosphorylation in rat liver mitochondria

2-Ethylhexanol (70 microM), a non-genotoxic carcinogen and peroxisome proliferator, stimulated oxygen uptake in the perfused rat liver by about 10% during the first 10 min of infusion. Perfusion with a higher, hepatotoxic dose of ethylhexanol (3 mM) led to a transient increase in oxygen uptake followed by a rapid inhibition of respiration of over 50% in 10 min. Lactate dehydrogenase (LDH) release, indicative of irreversible cell death, was detected in the effluent perfusate after 20 min. After 10 min of perfusion with ethylhexanol, livers were freeze-clamped, acid extracts were prepared and adenine nucleotides were measured by high-pressure liquid chromatography. Ethylhexanol decreased the ATP/ADP ratio from 2.5 to 0.9. Thus, marked decreases in hepatic energy state due to inhibition of respiration preceded cell death. To attempt to understand this phenomenon, the effect of ethylhexanol on isolated mitochondria was studied. Similar to classical uncoupling agents, ethylhexanol stimulated state-4 rates of respiration, diminished coupled rates of respiration, and decreased the P/O ratio in a dose-dependent manner in isolated mitochondria. Ethylhexanol also decreased uptake of radiolabeled 45CaCl2 by isolated mitochondria 4- to 5-fold. Therefore, we hypothesize that ethylhexanol initially uncouples oxidative phosphorylation leading to diminished ATP synthesis and collapse of ion gradients across the mitochondrial membrane.

Effect of hepatotoxic chemicals and hypoxia on hepatic nonparenchymal cells: impairment of phagocytosis by Kupffer cells and disruption of the endothelium in rat livers perfused with colloidal carbon.

Kupffer cells play an important role in liver function and phagocytosis of foreign particles in the hepatic portal tract. Therefore, the purpose of this study was to investigate the influence of several hepatotoxic chemicals (allyl alcohol, ethylhexanol, and menadione) and hypoxia on phagocytic activity of Kupffer cells in perfused rat liver. A recently developed optical method was used to determine rates of phagocytosis of carbon particles by Kupffer cells in periportal and pericentral regions of the liver lobule based on changes in reflected light from the liver surface (te Koppele, J.M., and Thurman, R.G. 1990. Am. J. Physiol. 259, G814-G821). With all chemicals studied, a rapid (10-30 min) decline in the rate of phagocytosis preceded parenchymal cell death as assessed from release of lactate dehydrogenase. These chemicals impaired parenchymal cell energy status as indicated by inhibition of O2 uptake and bile flow prior to cell death. Livers swell when they are damaged, a process which increases perfusion pressure and could theoretically damage the endothelium and lead to nonspecific uptake of carbon. In perfusions with a hepatotoxic concentration of allyl alcohol (350 microM), carbon particles accumulated in swollen livers after 70 min of perfusion. Histological studies revealed that carbon particles were localized predominantly in periportal regions of the liver lobule in perfusions with all hepatotoxicants studied. When perfusion pressure was elevated to 20 cm H2O in the absence of hepatotoxicants, carbon particles detected optically accumulated in upstream regions of the liver lobule (periportal or pericentral regions in perfusions in the anterograde or retrograde directions, respectively). In scanning electron microscopy of nonswollen livers, the endothelium remained intact. In swollen livers, however, the endothelium was disrupted and carbon was detected bound nonspecifically to parenchymal cells. Fifteen minutes after addition of allyl alcohol, bile canaliculi were dilated and endothelial fenestrations were enlarged. After 2 hr of perfusion with allyl alcohol, hepatic ultrastructure was severely disrupted. Thus, it is concluded that perfusion with hepatotoxic chemicals or hypoxia results in a rapid decrease of particle phagocytosis by Kupffer cells followed by changes in endothelial cell ultrastructure.

Wy-14,643 but not 2-ethylhexanol increases intracellular free calcium in cultured Kupffer cells

Hypolipidemic drugs and phthalic ester plasticizers induce peroxisomes and cause hepatocellular carcinoma in rodents by mechanisms which remain unknown. Recent evidence from this laboratory suggests that many agents in this class of chemicals are uncouplers of mitochondrial oxidative phosphorylation both in vitro and in vivo. Uncoupling of oxidative phosphorylation decreases ATP required for ion pumps and could thereby indirectly increase intracellular free calcium. The goal of these experiments, therefore, was to compare the effect of the potent uncoupler and non-genotoxic carcinogen Wy-14,643 with the weaker agent 2-ethylhexanol on intracellular free calcium in cultured Kupffer cells. Kupffer cells, the resident hepatic macrophages, are activated by calcium and release a variety of mitogenic growth factors that may modulate cell proliferation. In this study, the cytosolic free calcium concentration in Fura-2-loaded cultured Kupffer cells was increased significantly from 78 +/- 11 to 838 +/- 112 nM following incubation with Wy-14,643 (1.25 mM). The increase in intracellular calcium due to Wy-14,643 was both time- and dose-dependent. At equimolar concentrations, ethylhexanol had no effect on intracellular calcium (65 +/- 20 nM). However, at higher concentrations (3 mM), ethylhexanol also increased intracellular calcium. These data suggest that elevation of intracellular calcium in Kupffer cells may be involved in the mechanism of action of this interesting class of non-genotoxic carcinogens.

Hepatotoxicity due to clofibrate is oxygen-dependent in the perfused rat liver.

Toxicity of clofibrate, a hypolipidemic drug, was assessed in livers from fasted rats perfused in both the anterograde and the retrograde directions. Oxygen uptake decreased steadily following infusion of clofibrate (15 mM) and was diminished by about 40% in 15 min. Cell damage, assessed by the appearance of lactate dehydrogenase (LDH) in the effluent perfusate, began within 20 min. Maximal values for LDH release into perfusate were around 250 U/g/hr after perfusion with clofibrate for 40 min. Inhibition of oxygen uptake and release of LDH into the perfusate was dose-dependent (half-maximal effect = ca. 12 mM clofibrate). Nearly 90% of hepatocytes in oxygen-rich, periportal regions but only about 30% in oxygen-poor, pericentral areas took up trypan blue, an indicator of irreversible cell death, following perfusion with clofibrate in the anterograde direction. In contrast, when livers were perfused in the retrograde direction, 85% of cells in upstream, oxygen-rich pericentral regions were damaged whereas only about 30% in downstream areas were stained. When local oxygen tension was lowered by reducing the flow rate to one-quarter of normal, trypan blue uptake in periportal areas was diminished nearly completely (ca. 5% of cells were stained). Incubation in vitro of isolated cylinders of periportal and pericentral tissue with clofibrate at 800 or 200 microM oxygen led to about three times greater LDH release in incubations carried out at high than at low oxygen tension. This experiment led us to rule out the involvement of clofibrate delivery in the mechanism of zone-specific toxicity. Subsequently, local rates of oxygen uptake were measured using miniature oxygen electrodes placed on the liver surface. Clofibrate decreased oxygen uptake about 30% in oxygen-rich, periportal regions of the liver lobule, yet had no effect on respiration in downstream, pericentral areas. These phenomena can best be explained by a direct effect of clofibrate on active mitochondria in periportal regions of the liver lobule where oxygen uptake predominates, since state 3 but not state 4 rates of respiration were inhibited by clofibrate in isolated mitochondria (half-maximal effect = ca. 1.8 mM clofibrate). Thus, toxicity of clofibrate in upstream, periportal areas of the liver lobule is dependent on local oxygen tension and affects actively respiring mitochondria. This may lead to local cell death and be responsible for initiating a sequence of events leading to the well-known carcinogenic effects of this compound.

OXYGEN TENSION IS A MAJOR DETERMINANT OF HEPATOTOXICITY DUE TO 2-ETHYLHEXANOL IN ISOLATED TISSUE CYLINDERS FROM PERIPORTAL AND PERICENTRAL REGIONS OF THE LIVER LOBULE FROM PHENOBARBITAL-TREATED RATS

2-Ethylhexanol, a metabolite of the commonly used plasticizer di(ethylhexyl)phthalate, was shown to cause toxicity exclusively to periportal regions of the perfused liver (Keller et al., 1990, J. Pharmacol. Exp. Ther. 252, 1355-1360.) To determine whether this toxicity was due to local oxygen tension or to drug delivery, isolated cylinders (plugs) of periportal and pericentral regions of the liver lobule from rats pretreated with phenobarbital were collected with a micropunch following brief perfusion of the organ. Plugs were 0.2 mm wide and 0.5 mm long and weighed between 0.5 and 1 mg each. Following incubation for at least 2 hr in Eagles medium, they were judged viable based on production of urea at high rates and minimal leakage of lactate dehydrogenase (LDH). Plugs could be cultured for up to 24 hr with minimal loss of activity. Urea synthesis from ammonium chloride (3 mM) by plugs incubated in Krebs-Henseleit buffer equilibrated with 95% O2:5% CO2 was proportional to protein concentration and was linear with time for up to one hour at rates around 75 mumol/g/hr. Incubation of plugs with 2-ethylhexanol (0.1 to 3 mM) diminished urea synthesis in a dose-related manner (half-maximal effect = 0.5 mM). Ethylhexanol also caused extensive cell damage assessed from LDH leakage in incubations at 800 microM O2 but significantly less injury at 200 microM O2. Concomitantly, urea synthesis was inhibited by ethylhexanol by over 80% at 800 microM O2 but less than 50% at 200 microM O2. Plugs isolated from both regions of the liver lobule were affected similarly by ethylhexanol and O2. Taken together, these data indicate that ethylhexanol toxicity is dependent on oxygen tension in isolated sublobular regions of the liver lobule, and therefore it is unlikely that drug delivery can explain the selective injury to periportal regions in studies with the perfused liver.