In vitro effects of eight-carbon fatty acids on oxidations in rat liver mitochondria

Sodium valproate, a commonly used anticonvulsant agent, is a simple branched-chain fatty acid which interferes with beta-oxidation and ammonia metabolism in most patients, with hepatotoxic consequences in some cases. Rat liver mitochondria incubated with valproate displayed time-dependent inhibitions of state 3 oxidation rates with all the substrates tested, but most markedly with glutamate, pyruvate, alpha-ketoglutarate and acylcarnitines (Ki = 125 microM with glutamate and palmitoylcarnitine, and 24 microM with pyruvate). The inhibition of glutamate appeared to be specifically directed against the glutamate dehydrogenase pathway of this oxidation. Valproate was less effective when added to uncoupled mitochondria, suggesting the formation of an inhibitory species by an ATP-dependent mechanism. Mitochondria from clofibrate-treated rats were less sensitive to valproate inhibition. Neither fasting nor the presence of 1 mM L-carnitine affected the inhibition of beta-oxidation. The branched-chain isomer, 2-ethylhexanoic acid, had similar effects to valproate, but the straight-chain octanoic acid was totally different in its spectrum of actions on mitochondria. The data support the theory that valproate may inhibit by sequestration of CoA as valproyl-CoA, but also suggest that there are other mechanisms responsible for some of the inhibitions. Furthermore, it argued that while mitochondrial respiration is decreased, valproate is not an inhibitor of oxidative phosphorylation per se.     

The effects of miconazole on rat liver mitochondria

The antifungal agent Miconazole nitrate has been shown to cause uncoupling and at higher concentrations an inhibition of oxidative phosphorylation in isolated rat liver mitochondria, with concurrent damage to the mitochondrial membranes.     

Effect of phthalate esters on energy coupling and succinate oxidation in rat liver mitochondria

Isolated rat liver mitochondria were exposed to mono- and di-n-butyl phthalate (MBP and DBP) and mono- and di(2-ethylhexyl)phthalate (MEHP and DEHP) and examined for effects on mitochondrial energy-dependent processes, including oxidative phosphorylation and active K+ uptake. Additional studies on the effects of these phthalate esters on succinate oxidation and on mitochondrial membrane integrity are also included. DBP and MEHP stimulated succinate state 4 respiration, impaired K+-valinomycin induced swelling with succinate, ascorbate, or ATP as the energy sources, and inhibited succinate state 3 respiration and succinate cytochrome c reductase activity. MEHP was found to act as a non-competitive inhibitor of succinate dehydrogenase activity, with an apparent Ki = 2.4 X 10(-4) M. At concentrations which uncouple energy linked reactions, MEHP and DBP produced only slight energy-independent swelling and release of soluble proteins from isolated mitochondria. MBP caused only slight stimulation of state 4 respiration and impairment of K+-valinomycin induced swelling with each of the 3 energy sources, however, of the 4 phthalate esters, it produced the greatest energy-independent swelling and led to the greatest release of soluble mitochondrial proteins. DEHP had no apparent effect on any of these processes except for slight impairment of ATP-dependent K+-valinomycin induced swelling. It is concluded that phthalate ester toxicity in liver mitochondria is due to uncoupling of energy linked reactions and/or inhibition of succinate dehydrogenase activity. Uncoupling by MBP may involve disruption of mitochondrial membrane integrity, while uncoupling by DBP and MEHP is probably due to an increase in membrane permeability to H+ and other small ions.     

The hepatic sinusoid in aging and cirrhosis: effects on hepatic substrate disposition and drug clearance

The fenestrated sinusoidal endothelium ('liver sieve') and space of Disse in the healthy liver do not impede the transfer of most substrates, including drugs and oxygen, from the sinusoidal lumen to the hepatocyte. Plasma components transfer freely in both directions through the endothelial fenestrations and into the space of Disse. The endothelium is attenuated, there is no basement membrane and there is minimum collagen in the space of Disse, thus minimising any barriers to substrate diffusion. Both cirrhosis and aging are associated with marked structural changes in the sinusoidal endothelium and space of Disse that are likely to influence bulk plasma transfer into the space of Disse, and diffusion through the endothelium and space of Disse. These changes, termed capillarisation and pseudocapillarisation in cirrhosis and aging, respectively, impede the transfer of various substrates. Capillarisation is associated with exclusion of albumin, protein-bound drugs and macromolecules from the space of Disse, and the progressive transformation of flow-limited to barrier-limited distribution of some substrates. There is evidence that the sinusoidal changes in cirrhosis and aging contribute to hepatocyte hypoxia, thus providing a mechanism for the apparent differential reduction of oxygen-dependent phase I metabolic pathways in these conditions. Structural change and subsequent dysfunction of the liver sieve warrant consideration as a significant factor in the impairment of overall substrate handling and hepatic drug metabolism in cirrhosis and aging.     

The influence of maternal cadmium exposure or fetal cadmium injection on hepatic metallothionein concentrations in the fetal rat

The ability of Cd to induce the synthesis of fetal hepatic metallothionein (MT) was investigated in rat fetuses exposed to Cd throughout gestation via the mother's drinking water or injected directly with Cd through the uterine wall on Day 18 of gestation. On Day 21 all dams were killed and fetal and maternal tissues were removed. Tissue MT, Zn, Cu, and Cd concentrations were measured. Fetal hepatic Cd concentration was increased only at the high maternal Cd exposure, whereas Zn concentration was significantly reduced by Cd exposure. Both fetal liver and kidney MT were reduced following maternal Cd exposure. Unlike maternal hepatic MT, fetal hepatic MT was not increased after maternal Cd exposure nor did the direct injection of Cd into the 18 days of gestation fetus induce fetal MT synthesis. These data suggest that fetal rat liver is incapable of synthesizing MT in response to Cd, possibly because Cd is not transported to the site of MT synthesis in the fetal system. Furthermore, neither the route of exposure, the duration of prenatal Cd exposure, nor the stage of gestation appear to account for the differences observed between fetal and adult hepatic MT induction by Cd.     

Intestinal and hepatic binding of cadmium in the neonatal rat

At 18 h after intragastric administration of 0.1 mg cadmium (Cd)/kg body weight to the 8-day-old rat, systemic absorption is small and at least 95% of the body burden (70% of the dose) is retained in the intestine. Most (75% of the total) of this Cd is bound in the soluble fraction of the intestine by the endogenous copper (Cu)-complex, which normally appears to function in the regulation of Cu absorption in the neonate. After an intragastric dose of 1.0 mg Cd/kg body weight, intestinal retention at 18 h is reduced to about 50% of the body burden and 40% of the dose is present in the carcass. The distribution pattern of Cd at this time suggests that, at this higher dose level, the binding capacity of the Cu-complex for Cd is saturated without, however, any appreciable loss of Cu. After intraperitoneal injection, Cd is transported rapidly to the intestine and at 2 h after a dose of either 0.1 or 1.0 mg Cd/kg body weight, 15–20% of the body burden is located therein. At 18 h after the administration of the higher dose about 50% of the intestinal Cd is bound to the Cu complex.Most of the Cd retained by the liver of the 8-day-old rat at 18 h after intraperitoneal injection of 0.1 or 1.0 mg Cd/kg body weight is bound by the endogenous metallothionein. The distribution of Cd in the liver, however, alters with both dose and time since, between 2 and 18 h after administration of the higher, but not of the lower dose, the ratio of the Cd concentrations in the metallothionein and high molecular weight protein components in the hepatic soluble fraction increases approximately 15-fold. Although the metallothionein binds 80% of the Cd incorporated into the liver of the 8-day-old rat after an intragastric dose of 1.0 mg Cd/kg body weight, it does not cause excessive Cd accumulation in this organ. Most of the Cd that crosses the intestine is located elsewhere in the residual carcass (i.e., after removal of the liver, kidneys, gastrointestinal tract and blood) and hepatic uptake at 18 h is small (less than 1% of the body burden). These unidentified sites in the carcass also retain about 40% of the body burden at 2 or 18 h after intraperitoneal injection of Cd. It seems, therefore, that the intestinal Cu complex is of much greater significance than the hepatic metallothionein in the protection of the newborn rat against the toxicity of orally and parenterally administered Cd.     

Evidence for singlet oxygen involvement in rat and human cytochrome P450-dependent substrate oxidations

Recently, we proposed that singlet oxygen ((1)O(2)) plays an essential role in microsomal cytochrome P450 (P450)-dependent p-hydroxylation of aniline and O-deethylation of 7-ethoxycoumarin. We then examined whether the role of (1)O(2) is general in the P450-dependent substrate oxidations. In the present study, we examined omega- and (omega-1)-hydroxylations of lauric acid, O-demethylation of p-nitroanisole, and N-demethylation of aminopyrine in rat liver microsomes. The addition of beta-carotene and NaN(3) significantly suppressed these reactions in a concentration-dependent manner, and (1)O(2) during the reactions was detected by ESR spin-trapping using 2,2,6,6-tetramethyl-4-piperidone (TMPD) as a (1)O(2)-spin trapping reagent, where the addition of (1)O(2) quenchers, SKF-525A as a P450 inhibitor, or p-nitroanisole decreased ESR signal intensities due to TMPD-(1)O(2) adduct. Next, we examined the effect of (1)O(2) quenchers on P450-dependent reactions in the human liver microsomes, and (1)O(2) was also indicated to be an active species in substrate hydroxylations and dealkylations such as nifedipine oxidation by CYP3A4. On the basis of the results, we concluded that (1)O(2) is an essentially important active oxygen species in both rat and human P450-dependent substrate oxidations.     

Acute effect of cadmium on hepatic drug-metabolizing enzymes in the rat

Single ip injections of cadmium chloride at doses of 2.5 or 3.75 mg/kg (equivalent to 1.5 or 2.3 mg of Cd, respectively) into male Wistar rats of mean body weight of 226 ± 17 g produced significant inhibition (25–60%) of aniline hydroxylase and nitroreductase activity and also lowered the microsomal cytochrome P-450 content to 50% of the control value. These effects are in contrasto those on O-demethylase activity, which was not inhibited by either dose of cadmium chloride when enzyme activities were subsequently measured in a 0.05 m phosphate-buffered incubation system. However, the opposite conclusion was reached for the effect of these same doses of cadmium chloride when O-demethylase activity was assessed in comparable Tris-buffered systems. There appears to be a definite “buffer effect” in operation here. While the higher dose of this toxic metal salt significantly reduced the activities of the phenobarbital-induced hepatic microsomal drug-metabolizing enzymes and of cytochrome P-450, the lower dose was without significant effect on this induction, with the one exception of aniline hydroxylase when assessed in a Tris-buffer system.     

The effects of dietary selenium on cadmium binding in rat kidney and liver

In acute studies, approximately 70–90% of cytosolic cadmium in liver and kidney has been shown to be bound to metallothionein, a low-molecular weight protein. In this study, we report on the influence of dietary selenium on the distribution of cadmium in rat kidney and liver. Contrary to the findings of most acute studies, our results indicate that only a relatively small proportion of cadmium (approximately 14% in the kidney and 44% in the liver) is bound to metallothionein when cadmium is administered for 7 weeks in the diet and via osmotic minipumps to selenium-deficient rats. Feeding rats the same diet supplemented with 1.0 ppm selenium results in no detectable cadmium-metallothionein peak in the kidney, and only about 10% of the cytosolic cadmium elutes as cadmium bound to metallothionein in the liver. In animals fed the selenium-supplemented diet, the bulk of the cadmium is recovered in the low-molecular weight fraction. Dietary selenium did not significantly affect the distribution of zinc and copper in the kidney or liver.Supported by NIEHS Center Grant ES-00159 and a grant from the Selenium — Tellurium Development Association     

鲨鱼肝提取物促肝细胞增殖及对大鼠肝细胞线粒体的保护作用

目的 研究鲨鱼肝提取物的促肝细胞增殖及对线粒体的保护作用。方法 利用[^3H]-TdR掺入的方法，测定HepG2细胞的增殖指数；利用D-半乳糖建立大鼠急性肝损伤模型．提取肝脏细胞线粒体，测定以苹果酸-谷氨酸，琥珀酸为底物，ADP诱导的氧消耗、呼吸控制率（RCR）、ADP／O、氧化磷酸化率（OPR）等指标。结果与结论 鲨鱼肝提取物能够提高肝源细胞的增殖指数并对线粒体具有明显的保护作用。     

氯化镉对大鼠肝线粒体和微粒体膜功能的影响

体外观察ＣｄＣｌ２对大鼠肝线粒体和微粒体膜流动性，膜结合酶活性，脂质过氧化产物丙二醛（ＭＤＡ）生成的影响以及维生素Ｃ的拮抗作用。结果表明，Ｃｄ２＋可在较短时间内引起膜流动性的降低和ＭＤＡ的增高，并伴有膜结合酶活性的抑制；维生素Ｃ处理不能改善膜流动性的降低。但能完全拮抗ＭＤＡ的增高，并部分缓解Ｃｄ２＋对葡萄糖－６－磷酸酶的抑制，提示Ｃｄ２＋膜毒性并非单纯由脂质过氧化所致，其中包括了对膜脂双层的直接作用。     

Effects of dietary pectin and cellulose on hepatic and intestinal mixed-function oxidations and hepatic 3-hydroxy-3-methylglutaryl-coenzyme a reductase in the rat

The aim of this study was to determine if feeding dietary fiber (cellulose or pectin) to male rats could influence hepatic and intestinal mixed-function oxidation. We simultaneously compared hepatic drug-oxidizing activity with the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme for cholesterol biosynthesis. Three groups of six animals were fed a purified diet containing by weight either 10.4% cellulose or 10.4% pectin, or a standard cereal-based diet containing 4.5% crude fiber; the caloric contributions by carbohydrate, protein and fat in the three diets were similar. In the cellulose-fed rats, the hepatic microsomal cytochrome P-450 content and the activities of ethylmorphine N-demethylase and aniline hydroxylase were significantly lower when compared with those of rats fed pectin or the cereal-based diet. The hepatic microsomal cytochrome P-450 content and the activities of ethylmorphine N-demethylase and aniline hydroxylase were similar in the pectin-fed and cereal diet-fed rats. Hepatic HMG-CoA reductase activity, hepatic microsomal cytochrome b₅ content, and intestinal benzo[a]pyrene hydroxylase activity were comparably lower in rats fed the purified diet with either dietary fiber when compared to those fed the cereal diet. It is concluded that dietary pectin and cellulose exert distinctly different influences on the hepatic microsomal mixed-function oxidase system for drug metabolism, but not on liver cholesterol synthesis or intestinal benzo[a]pyrene hydroxylation, suggesting that different physiological mechanisms control these enzyme systems.     

The effects of propiconazole on hepatic xenobiotic biotransformation in the rat

Propiconazole, a foliar fungicide used for agricultural purposes was studied for its effects on the hepatic xenobiotic biotransformation in the rat. Rats were given an intraperitoneal injection of 0.1, 1, 10 or 100 mg/kg in corn oil for seven consecutive days. Induction was seen for cytochrome P-450, ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, aldrin epoxidase, aminopyrine N-demethylase and microsomal expoxide hydrolase activities. Aniline p-hydroxylase and cytosolic glutathione S-transferase activities were unchanged. All responses occurred at only 100 mg/kg, except for that of aminopyrine N-demethylase which also occurred at the 10 mg/kg dose. SDS polyacrylamide gel electrophoresis showed increased staining of a protein band of molecular weight 54,000 corresponding to cytochrome P-450b and/or P-450d. Collectively these results suggest that cytochromes P-450b and P-450d have been induced after exposure of rats to propiconazole.     

The effects of oral cadmium exposure on passive avoidance performance in the adult rat

Step-down passive avoidance acquisition and 24 h retention performance were examined in adult rats given daily doses of either 0, 1, or 5 mg/kg cadmium (Cd) (as CdCl2) via the diet. Results indicated that subjects exposed to the 5 mg/kg Cd diet were less likely to step off a safe platform onto an electrified grid floor than controls. The 1 mg/kg subjects did not differ from controls on this acquisition task. None of the groups showed differential performances on a retention test administered 24 h after acquisition training. These results are discussed in terms of recent claims that Cd exposure enhances emotional reactivity in animals.     

The acute and subchronic effects of ketoconazole on hepatic microsomal monooxygenases in the rat

Female adult Wistar rats were treated with single or repeated doses of ketoconazole ranging from 10 mg/kg to 100 mg/kg. Single dose treatment produced inhibition of hepatic microsomal ethoxyresorufin O-deethylation (EROD) and aldrin epoxidation (AE) 2 hr after oral dosing. Twenty-four hours after a single dose, inhibition was still demonstrable after the low dose of 10 mg/kg, but at higher doses increased microsomal activity was apparent. After 7 days repeated dosing liver weight and microsomal protein content were increased in a dose-dependent fashion. EROD and AE were induced at all doses after repeated treatment when the increase in liver size was considered. These effects were seen at doses within the antimycotic therapeutic range and add support to the suggestion that reported drug interactions with ketoconazole in man are due to the effects of this drug on hepatic microsomal activity.     

The effects of cephem antibiotics and related compounds on the aldehyde dehydrogenase in rat liver mitochondria

The effects of cephem antibiotics and their related compounds on aldehyde dehydrogenase obtained from rat liver mitochondria were studied. A pH of 8.8 and reaction temperature 24 degrees were the conditions for measurement of enzyme activity. The apparent Michaelis constant Km values for NAD, acetaldehyde and propionaldehyde were 3.8 X 10(-5) M, 4.0 X 10(-5) M and 2.5 X 10(-5) M, respectively. Cefamandole, cefoperazone and cefmetazole, having a 1-methyl-5-thiotetrazol group at position 3 of the cephem ring, caused a relatively potent inhibition of aldehyde dehydrogenase. Cefmetazole and cefoperazone also showed a significant inhibition on highly purified yeast aldehyde dehydrogenase; the extent of inhibition on yeast enzyme was almost the same as that on rat mitochondrial aldehyde dehydrogenase. The decrease in enzyme activity effected by 1-methyl-1H-tetrazol-5-thiol (MTT) was greater than those of 1H-tetrazol (TZ), 1H-tetrazol-5-thiol and 1-(2-dimethylaminoethyl)-1H-tetrazol-5-thiol, but was, of course, less than that of disulfiram. Cefamandole, cefmetazole and MTT showed competitive inhibition with NAD, while TZ was uncompetitive inhibitor with respect to both NAD and acetaldehyde. Enzyme inhibition caused by disulfiram, cefmetazole and MTT increased time-dependently and the addition of 2-mercaptoethanol into the medium effectively and completely restored enzyme inhibition. These results suggest that thiol group at position 5 of 1H-tetrazol ring is responsible for the type of inhibition with NAD, and methyl group at position 1 of 1H-tetrazol ring is important to exhibit a potent inhibition on aldehyde dehydrogenase.     

Reversibility effects on renal and hepatic gluconeogenic enzymes in rats from chronic exposure of cadmium

The reversibility of cadmium chloride (CdCl2) effects on renal and hepatic gluconeogenic enzymes in rats was studied by removal of CdCl2 from the diet. Adult, male Sprague-Dawley rats were continuously treated with 0, 25, 50, and 75 ppm CdCl2 mixed in powdered diet for 180 d. After this period of chronic exposure, all treated rats were kept on CdCl2-free diet for another period of 180 d. The CdCl2 induced serum glucose and activities of glucose 6-phosphatase, fructose 1, 6-bisphosphatase, and phosphoenolpyruvate carboxykinase reversed to the levels of control values 120 d after the rats were fed with CdCl2-free diet. There were no significant differences in measured enzyme activities either 120 or 180 d after the cessation of CdCl2 treatment. The data indicate that CdCl2 effects on gluconeogenesis will gradually recover with time when the metal is removed from the diet.     

Induction of hepatic metallothionein in the immature rat following administration of cadmium

Hepatic metallothionein (MT) levels, as measured indirectly by total metal-binding capacity, are approximately 8.5-fold higher in the 7-day-old rat than in the 28-day-old rat where levels are barely detectable. To stydy how the presence of MT might influence the toxicity of cadmium, a single subcutaneous injection of cadmium chloride was administered at dose levels of 0, 1, 2, 3, and 6 mg Cd/kg body weight, 48 hr prior to sacrifice on Day 7 or Day 28. In both the 7- and 28-day-old groups, there was a dose-related increase in the amount of cadmium bound to MT. There were no significant age-related differences in the amount of cadmium bound to MT at the various doses, with the exception of the 6 mg/kg dose, where 7 day levels were higher. The 28-day-old rats responded to cadmium exposure with induction of MT and subsequent binding of both cadmium and zinc at all doses. The 7-day-old appeared to have sufficient levels of MT to handle cadmium doses at or below 3 mg/kg without induction; however, at the 6 mg/kg dose induction was observed. Despite the presence and inducibility of hepatic MT at Day 7, 30% of the rats treated with 6 mg Cd/kg died within 48 hr of exposure compared with only 4% at Day 28.     

Effect of cadmium and/or selenium on liver mitochondria and rough endoplasmic reticulum in the rat.

This study was conducted to investigate the effect of cadmium (Cd) and/or selenium (Se) on hepatic mitochondria and rough endoplasmic reticulum (RER). Male Sprague Dawley derived rats (150-200 g) received sodium acetate (NaAc; 1.23 mg/kg), Cd (0.84 mg/kg), and/or Se (1.6 mg/kg) intraperitoneally. Livers were perfused with 2% glutaraldehyde at 48 h, 72 h, or 96 h after treatment and prepared for electronmicroscopy. Results indicate that Cd and/or Se are capable of inflicting mitochondrial and RER structural changes, and the damage by Cd alone is more severe than Se alone or with Cd.