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Introduction Human placenta mitochondria produces huge amounts of progesterone necessary for maintaining the pregnancy. Lipid peroxidation in human placental mitochondria inhibits progesterone synthesis and that inhibition can be reversed by superoxide dismutase and other antioxidants. Paraquat (PQ) a highly toxic herbicide generates superoxide radical inside cells and induces lipid peroxidation. Hence, it is supposed to stimulate lipid peroxidation in human placental mitochondria and in consequence to inhibit a placental mitochondrial steroidogenesis. Methods Placentas were obtained from normal pregnancies. All experiments were done using isolated human placental mitochondria. Mitochondrial lipid peroxidation was determined as tiobarbituric acid reactive substances (TBARS). A conversion of cholesterol to pregnenolone or pregnenolone to progesterone was measured using radiolabeled steroids and thin layer chromatography. Results PQ enhanced the iron-dependent lipid peroxidation as also PQ heightened the inhibitory action of this process on progesterone synthesis in isolated human placental mitochondria. Paradoxically, a superoxide dismutase (SOD) reversed the inhibition of progesterone synthesis only minimally although it strongly inhibited PQ stimulated iron-dependent lipid peroxidation. When iron was absent, PQ stimulated only negligible lipid peroxidation but strongly inhibited progesterone synthesis. SOD had no effect on inhibition of progesterone synthesis by PQ. PQ strongly inhibited of the conversion of cholesterol to pregnenolone but had not got any influence on the enzymatic activity of mitochondrial 3β-hydroxysteroid dehydrogenase. PQ strongly decreased the efficiency of NADPH-dependent cytochrome P450 reduction as well as it promoted the rapid oxidation of the pre-reduced mitochondrial cytochrome P450. However PQ has not inhibited combined activity of adrenodoxin reductase and adrenodoxin. Discussion We conclude that the most important reason of the inhibition of progesterone synthesis by PQ is the escape of electrons from cytochrome P450scc to that compound what leads to cytochrome oxidation and, in consequence the inhibition of the reaction catalyzed by it. The action of PQ described here should be considered as potentially harmful for pregnancy and fetal development.

During pregnancy the isoform composition of human placental AMP-deaminase changes. This may reflect the adaptation of enzyme to changing metabolic requirements of the growing fetus. In this paper kinetic and regulatory properties of AMP-deaminase purified from human preterm (∼25 week of gestation) placenta were described and compared with these of the enzyme purified from term placenta. AMP-deaminase from preterm placenta was less sensitive to pH changes and in contrast to the enzyme from the term organ, at low range of substrate concentrations was not inhibited but activated by physiological concentrations of orthophosphate. This may significantly improve the catalytic efficiency of enzyme at early phase of the pregnancy.

In pregnant females, placenta is the most important source of lipid hydroperoxides and other reactive oxygen species (ROS). The increased production of lipid peroxides is often linked to preeclampsia. In our study, we revealed that NADPH- and iron-dependent lipid peroxidation in human placental microsomes (HPM) occurred. In the presence of Fe2+ ion, HPM produced small amounts of thiobarbituric acid-reactive substances (TBARS) - a final product of lipid peroxidation. NADPH caused a strong increase of iron stimulated TBARS formation. TBARS formation was inhibited by superoxide dismutase, butylated hydroxytoluene and alpha-tocopherol but not by mannitol or catalase. TBARS and superoxide radical production was inhibited in similar manner by cytochrome P450 inhibitors. The results obtained led us to the following conclusions: (1) microsomal lipid peroxidation next to mitochondrial lipid peroxidation may by an important source of lipid hydroperoxides in blood during pregnancy and (2) superoxide radical released by microsomal cytochrome P450 is an important factor in NADPH- and iron-dependent lipid peroxidation in HPM.

Time-dependent changes in the cell death mode from apoptosis to necrosis were studied in cultured 143B cells treated with menadione, an anti-cancerous drug, excluding a possible involvement of "secondary necrosis." The population of apoptotic cells judged by FITC-Annexin V and propidium iodide (PI) double staining reached its maximum at 6 hours after 100 microM menadione treatment followed by an abrupt decrease thereafter, while that of necrotic cells continuously increased reaching 90% at 24 hours. Electron microscopically, cells attached to the culture dish at 6 hours after the treatment consisted of two different types of cells: cells with typical apoptotic features occupying the major population and those with condensed nuclei and swollen cytoplasm. Cells attached to the culture dish at 8 hours after the treatment consisted exclusively of those with condensed nuclei and swollen cytoplasm. Mitochondria in these cells showed various structural changes: those swollen to various degrees with deposition of flocculent densities, or those with highly condensed matrix. Distinct decreases both in intracellular levels of ATP and caspase-3-like activities and remarkable elevations of intracellular levels of superoxide, which were partly suppressed by NAD(P)H oxidase inhibitors, occurred at 6 hours after the treatment. These results may suggest that distinct increases of the intracellular level of superoxide derived from plasma membrane NAD(P)H oxidase besides that from mitochondria have triggered the transition of cell death mode from apoptosis to necrosis. Transition of highly condensed mitochondria to extremely swollen ones may reflect necrotic processes in menadione-treated cells. The present study strongly suggests that time-dependent study is essential using the electron microscopic technique to analyze detailed processes in the changes of the cell death mode.

The effects of ascorbate and a-tocopherol as antioxidants and as co-operative factors against NADPH-dependent lipid peroxidation in human placental mitochondria have been studied. The addition of ascorbate at low concentration (up to 50 μM) to the NADPH-generating system resulted in increasing lipid peroxidation and Fe3+ to Fe2+ reduction. High concentration of ascorbate (150 μM), which produced maximal rate of ascorbate-dependent lipid peroxidation, was found to inhibit almost completely NADPH-dependent lipid peroxidation by maintaining too much iron in its reduced form. Either stimulatory or inhibitory effect of ascorbate on NADPH-dependent lipid peroxidation depends on the appropriate Fe3+/Fe2+ ratio. α-Tocopherol caused a decrease of NADPH-dependent lipid peroxidation, inhibiting completely this process at 150 μM concentration. The inhibitory effect of α-tocopherol increased rapidly with the increasing ascorbate concentration, almost complete inhibition of NADPH-dependent lipid peroxidation being obtained at 25 μM α-tocopherol and 50 μM ascorbate. This strong inhibitory combined effect of α-tocopherol and ascorbate was independent of the Fe3+/Fe2+ ratio, as a-tocopherol is not able to reduce Fe3+ to Fe2+ under the conditions employed. These findings suggest that antioxidant effects of ascorbate in placental mitochondria are mediated by recycling of a-tocopherol rather than by strong reduction of Fe3+ to Fe2+. On the basis of the results obtained, we assume that adequate concentrations of a-tocopherol and ascorbate in placental tissue may prevent the release of lipid peroxide from placental mitochondria and therefore could be protective against the development of preeclampsia.

Thiophene-2,3-dione derivatives 2 were prepared in the reaction of β-aminovinylthioamides 1 with ethyl oxalyl chloride. Treatment of compounds 2b,c with malonodinitrile led to the ring transformation of the thiophene-2,3-dione system to pyrrolo[3,2-c]pyridine derivatives 4b,c.

Different natural and synthetic organic hydroperoxides have been found to stimulate TBARS formation in human term placental mitochondria. The levels of TBARS were lower than arising from NADPH-dependent lipid peroxidation. BHT, Mn2+ and DMPO counteracted TBARS formation in the presence of cumene hydroperoxide implicating involvement of free radicals in this process. On the other hand superoxide dismutase, catalase and EDTA while being inhibitory in NADPH-dependent lipid peroxidation did not inhibit cumene hydroperoxide-dependent TBARS formation. Amphenone B and SKF-525A, inhibitors of cytochrome P-450, strongly inhibit both NADPH- and cumene hydroperoxide-dependent lipid peroxidation. These data provide evidence that cytochrome P-450SCC is involved in both these processes. However NADPH-dependent lipid peroxidation and the cumene hydroperoxide have been found to inactivate placental mitochondrial cytochrome P-450SCC. The presence of cumene hydroperoxide resulted in a more rapid inactivation of cytochrome P-450SCC and consequently inhibited NADPH-dependent lipid peroxidation. It has been observed for the first time that progesterone biosynthesis can be inhibited by cumene hydroperoxide. Protective effect of Mn2+ and DMPO on progesterone biosynthesis indicates the importance of free radicals as transient products of cytochrome P-450SCC-dependent cumene hydroperoxide metabolism. In contrast to progesterone formation from cholesterol, the conversion of pregnenolone to progesterone was not affected by cumene hydroperoxide. This suggests that inhibition of progesterone synthesis from cholesterol by hydroperoxide may be ascribed to its effect on the desmolase activity of cytochrome P-450SCC in placental mitochondria. On the basis of the results obtained, we propose that the inhibition of progesterone biosynthesis by naturally occurring hydroperoxides may contribute to the development of preeclampsia.

11-beta-hydroxysteroid dehydrogenase type I (11-beta-HSD1) in the white adipose tissue (WAT) of rats catalyses the conversion of 11-dehydrocorticosterone to corticosterone, a more active glucocorticosteroid. Glucocorticosteroids in WAT stimulate adipocytes differentiation and increase adipocytes size. The aim of this study was to examine the association between expression of 11-beta-HSD1 in the WAT of male and female rats and adipose tissue mass as well as body mass.Perirenal WAT from male and female Wistar rats aged three months, and ovariectomized females of the same age, was used in the study. 11-beta-HSD1 gene expression was assayed in the perirenal WAT of rats by real-time PCR.11-beta-HSD1 gene expression in the perirenal WAT of male rats was higher than in female rats. The WAT and body mass of male rats was also higher than in females. 11-beta-HSD1 gene expression in the perirenal WAT as well as WAT mass and body mass increased simultaneously after ovariectomy.The results presented in this paper suggest that higher 11betaHSD1 gene expression in the WAT is associated with higher body and adipose tissue mass. Moreover, our results suggest that oestradiol can modulate 11-beta-HSD1 gene expression in the WAT of rats.

Pathomechanism of vitamin D intoxication is still unclear. Experiments on animals showed that intoxication is accompanied by distinctly elevated serum level of vitamin D precursor [25(OH)D3] but not by that of hormonally, metabolic active form of it [1alpha,25(OH)2D3]. The most probable explanation of this phenomenon is that vitamin D precursor releases hormonal form of vitamin from its binding with serum transporting protein (DBP) stimulating in this way transcription of genes in cells of target tissues.

Summary The effect of glutamine and glutamate on progesterone biosynthesis in humanplacental mitochondria has been investigated. Both amino acids stimulates this process, but glutamate to a greater extent. At pH 8.2 activity of placental mitochondrial glutaminase and progesterone biosynthesis in the presence of glutamine depend on inorganic phosphate concentration in the same manner. This suggests that glutaminase at first hydrolyses glutamine to glutamate and further metabolism of glutamate supports progesterone biosynthesis. Results of experiments in which progesterone biosynthesis has been measured at pH 7.2 and 8.2 indicate the possible involvement of NADP-dependent glutamate dehydrogenase, NAD(P)-dependent malic enzyme, and NADP-dependent isocitrate dehydrogenase in providing the reducing equivalents (NADPH) for progesterone biosynthesis from glutamine and glutamate. Data reported in this paper suggest that in human placental mitochondria metabolism of glutamine and glutamate may be closely correlated to progesterone biosynthesis.

A C34T mutation in the AMPD1 gene is proposed to cause local or systemic augmentations in blood adenosine level and improvement of prognoses in heart diseases like congestive heart failure or heart ischemic disease. This study examines some physico-chemical properties of AMP-deaminase isolated from cardiac muscle of a 10-year-old boy heterozygote for this mutation.

Human placental mitochondria might be a significant source of NADPH- and iron-dependent production of reactive oxygen species (ROS). Preeclampsia is believed to be a consequence of overproduction of ROS in human placenta. The experimental results presented here show that melatonin inhibits NADPH- and iron-dependent lipid peroxidation of human placental mitochondria in a concentration-dependent manner. At 1.5 mm concentration, melatonin suppressed this process nearly completely. Melatonin does not influence significantly the iron oxidation at this conditions, indicating that free radical scavenging rather than metal-chelating phenomenon is the basis of its antioxidant action. The fact of inhibition of lipid peroxidation by melatonin at conditions excluding iron participation also supports this hypothesis. Elucidation of the nature of common interaction among melatonin, ascorbate, and alpha-tocopherol in human placental mitochondria was the main aim of this study. In presence of 90 mum ascorbate, the inhibition of lipid peroxidation by melatonin was strong and had a feature of synergistic interaction. At presence of 30 mum ascorbate, which stimulated lipid peroxidation, melatonin caused a loss of pro-oxidant effect of ascorbate. While the interaction of melatonin with ascorbate indicated synergism, the joint action of melatonin and alpha-tocopherol was additive. When all three antioxidants were applied together, the strongest inhibition of lipid peroxidation was observed. The experimental results presented here indicated that melatonin could be considered as an effective component of antioxidant treatment of preeclampsia, allowing the use of reduced doses of vitamin C and E owing to elevated efficiency of their antioxidant activity in placenta when used in combination.

Human placental mitochondria might be a significant source of NADPH- and iron-dependent production of reactive oxygen species (ROS). Preeclampsia is believed to be a consequence of overproduction of ROS in human placenta. The experimental results presented here show that melatonin inhibits NADPH- and iron-dependent lipid peroxidation of human placental mitochondria in a concentration-dependent manner. At 1.5 mm concentration, melatonin suppressed this process nearly completely. Melatonin does not influence significantly the iron oxidation at this conditions, indicating that free radical scavenging rather than metal-chelating phenomenon is the basis of its antioxidant action. The fact of inhibition of lipid peroxidation by melatonin at conditions excluding iron participation also supports this hypothesis. Elucidation of the nature of common interaction among melatonin, ascorbate, and alpha-tocopherol in human placental mitochondria was the main aim of this study. In presence of 90 mum ascorbate, the inhibition of lipid peroxidation by melatonin was strong and had a feature of synergistic interaction. At presence of 30 mum ascorbate, which stimulated lipid peroxidation, melatonin caused a loss of pro-oxidant effect of ascorbate. While the interaction of melatonin with ascorbate indicated synergism, the joint action of melatonin and alpha-tocopherol was additive. When all three antioxidants were applied together, the strongest inhibition of lipid peroxidation was observed. The experimental results presented here indicated that melatonin could be considered as an effective component of antioxidant treatment of preeclampsia, allowing the use of reduced doses of vitamin C and E owing to elevated efficiency of their antioxidant activity in placenta when used in combination.

During pregnancy the activity of AMP-deaminase in developing human placenta gradually decreases, being in homogenates of mature, term placenta (approximately 40 week of gestation) one fourth to one third of that in homogenates of immature (approximately 25 week of gestation) organ. The gradual decrease of activity correlates inversely with the increasing presence of the form of enzyme predominating in homogenates of the mature placenta. The discrepancy observed indicate that isozymic pattern of AMP-deaminase in developing human placenta changes.

In an in vitro system consisting of human term placental mitochondria and an NADPH-generating system plus Fe2+, significant lipid peroxidation was observed along with a concomitant inhibition of progesterone biosynthesis. This inhibition could be markedly blocked by Mn2+, superoxide dismutase and dimethylfuran, inhibitors of NADPH-dependent lipid peroxidation. In addition, it has been found that malondialdehyde formation is accompanied by a corresponding decrease in placental mitochondrial cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in cell-free systems. These measurements provide the first evidence that the inhibition of progesterone biosynthesis by a NADPH-dependent lipid peroxidation in placental mitochondria is a consequence of cytochrome P-450 degradation due to lipid peroxidation.

The main target of acetaminophen application is bifunctional enzyme--prostaglandin endoperoxide H2 synthase (PGHS)--which has cyclo-oxygenase and peroxidase activities and synthesizes initial intermediates in prostanoid synthesis. The reaction catalyzed by PGHS is radical-based and it is initiated and then maintained by the constant presence of peroxides especially peroxynitrate, which generate so-called "peroxide tone" in the enzyme surrounding. Currently it is known that inhibitory effect of acetaminophen on PGHS activity is directly connected with the elimination of "peroxide tone". High concentrations of reactive compounds (e.g. peroxynitrate and lipid peroxides)--produced by cellular defending mechanism at inflammatory sites--significantly decrease inhibitory impact of acetaminophen on PGHS activity. Such observation allows explanation of weak antiinflammatory effect of acetaminophen together with its strong analgesic and antipyretic properties.

The NADPH-dependent lipid peroxidation in human placental mitochondria has been found to be inhibited strongly by amphenone B, aminoglutethimide and carbon monoxide, inhibitors of cytochrome P-450-mediated reactions, but was hardly affected by respiratory chain inhibitors. Cytochrome c, an exogenous electron acceptor which is known to compete with cytochrome P-450 for the reducing equivalents, showed an inhibitory effect on NADPH-dependent lipid peroxidation. The observed NADPH-dependent superoxide generation was also strongly inhibited by amphenone B and aminoglutethimide. Moreover, the lipid peroxidation in placental mitochondria was demonstrated to be stimulated by xanthine/xanthine oxidase added as superoxide generating system. This peroxidation was not affected by amphenone B and aminoglutethimide. On the other hand, the superoxide dismutase was found to inhibit both the xanthine oxidase- and NADPH-dependent lipid peroxidation. These data provide evidence that cytochrome P-450 is involved in NADPH-dependent mitochondrial lipid peroxidation. It is suggested that superoxide liberated from cytochrome P-450, in combination with iron, may be responsible for initiation of NADPH-dependent lipid peroxidation in human placental mitochondria.

Background AMP-deaminase (EC 3.5.4.6) and 5'-nucleotidase (EC 3.1.3.5) are enzymes responsible for the maintenance of cellular adenine nucleotides pool. Both exist in several isoforms that differ in kinetic properties and tissue distribution. Profile of isoforms of these enzymes in human placenta has not been analyzed so far while this could be important for understanding of pathology of placental ischemia such as in preeclampsia. Our aim was therefore to analyze expression of AMPD and CN-I genes in human term placenta. Methods RT-PCR analysis was used for determine expression of AMPD1, AMPD2, AMPD3 and CN-I. Results and conclusion The experimental results presented here indicate that genes coding "AMP-preferring", cytosolic isozyme of 5'-nucleotidase (cN-I) as well as "muscle-type" isozyme of AMP-deaminase (AMPD1) are not expressed in human term placenta. Among other AMPD family genes, only these coding "liver-type" isozyme (AMPD2) and, in lesser degree, "erythrocyte-type" isozyme (AMPD3) of AMP-deaminase are expressed in this organ. The expression level of AMPD3 was a half of that presented by AMPD2. We conclude that high abundance of AMP-deaminase 2 transcript suggest that this particular isoform is a predominant pathway of adenine nucleotides degradation in human term placenta that follows liver-type regulation of this process.

In the result of liver detoxification, xenobiotics change into more water soluble and thus easier for excretion from the body. It is convenient to consider this process as occurring in two phases. In phase I, the major reactions involved are hydroxylation, catalyzed by monoxygenases. In phase II, the preliminary modified xenobiotics after conjugation with some specific metabolites are transformed into less toxic and more soluble end-products. Recently, antiporter activity of MDR1 (MultiDrug Resistence) gene products in enterocytes was recognized as important stage in detoxification of xenobiotics, and definied as phase III of this process.

Estrogens control a large range pivotal life functions as reproductive development and fertility, bone growth and sexual behavior. Aromatase is a key enzyme of estrogen biosynthesis. The property, structure and reaction mechanism of aromatase as well as detailed structure of human aromatase cytochrome P450 gene (CYP19) was discussed in this article. It was pointed that unique human CYP19 gene expression results from presence of many tissue specific promoters and alternative splicing. The molecular mechanism of control aromatase cytochrome P450 gene expression in various species ovaries, testes and human adipose tissue and placenta was discussed in details. Because of a very important role of estrogen in breast cancer a molecular base of aberrant expression CYP19 gene in breast tumor and adipose tissue proximal to breast tumor and potential possibility of pharmacological silencing of this gene expression was discussed in the article.

Conflicting results have been reported regarding the effect of triiodothyronine (T(3)) on serum leptin and adipose tissue leptin gene expression in human and animals. The aim of the present study was to evaluate the effect of administration of increasing doses of T(3) on serum leptin concentration and on leptin mRNA abundance in white adipose tissue of rats. The results presented in this paper indicate that administration of single different doses of T(3) to euthyroid rats resulted dose dependent increases of serum total T(3) concentrations which are associated with a decrease in white adipose tissue leptin mRNA level. The leptin mRNA level in white adipose tissue was negatively correlated with serum total T(3) concentration (r=-0.8, p0.001). Like white adipose tissue leptin mRNA level, serum leptin concentration decreased after T(3) administration, and was also negatively correlated with the serum T(3) concentration (r=-0.8, p0.001). In contrast, administration of T(3) to the same rats led to a significant increase in white adipose tissue expression of the malic enzyme gene (malic enzyme activity and malic enzyme mRNA level), a known target gene for T(3). The results indicate that T(3) exerts a selective inhibitory effect on white adipose tissue leptin gene expression in vivo. A conclusion is that T(3) decreases rat serum leptin concentration by down-regulation of leptin gene expression in white adipose tissue.

Carbon monoxide is a side product of enzymatic degradation of heme--a reaction catalyzed by heme oxygenase (HO). There are three independent molecular forms (isozymes) of heme oxygenase in man. Isoforms HO-2 and HO-3 are constitutive. More detailed studied isoform HO-2 is especially abundant in the human brain. In contrast to isoforms: HO-2 and HO-3, isoform HO-1 is an inducible species of heme oxygenase. Seen in other tissues, expression of HO-1 is also manifested in the endothelium and smooth muscle of blood vessels. Accumulated experimental data indicate more and more clearly on the possible role of endogenous carbon monoxide in cell cytoprotection, and shows it as a probable factor participating in modulation of the vascular tonus in hypoxia.

Previously, we have shown that the age-related changes in 6-phosphogluconate dehydrogenase (6PGDH) activity depend on sex, and that oestradiol is playing a crucial role in the regulation of 6PGDH gene expression in rat liver, but not in other tissues [Pankiewicz, A., Sledzinski, T., Nogalska, A., Swierczynski, J., 2003. Tissue specific, sex and age-related differences in the 6-phosphogluconate dehydrogenase gene expression. Int. J. Biochem. Cell Biol. 35, 235-245.]. To complete the knowledge on the influence of sex hormones on 6PGDH activity, experiments have been performed on the effect of testosterone on 6PGDH gene expression in rat white adipose tissue and liver. The results presented here disclosed that in young male rats high serum testosterone concentration was associated with high white adipose tissue 6PGDH activity. After orchidectomy, a decrease in serum testosterone concentration (both in young and old rats) was observed. In contrast, no changes in white adipose tissue and liver 6PGDH activity were found. In female rats, both young and old, serum testosterone concentration was below the limit of detection, whereas 6PGDH activity was much higher in young than in old animals. Moreover, the testosterone administration to 9-month old male rats (which displayed much lower serum testosterone concentration that young animals) resulted in no effect on 6PGDH activity either in WAT or in the liver. In conclusion, the results presented in this paper indicate that testosterone does not play any role in the age- and gender-related differences in 6PGDH gene expression in white adipose tissue.

Mitochondrial dysfunction plays a crucial role in cell types that exhibit necrosis-like death after activation of their death program. Tumour necrosis factor (TNF) induces abnormal, perinuclear clustering of mitochondria from an evenly spread distribution throughout the cytoplasm. The mitochondria withdraw from the cell periphery and aggregate in a unipolar perinuclear cluster. TNF-induced mitochondrial clustering is caused by impaired kinesin-mediated transportation of mitochondria. In this report, we describe a novel activity of menadione (MEN), namely the induction of an altered spatial distribution of mitochondria in the choriocarcinoma JAR cells. Strikingly, 2 hours of cell exposition to menadione did not disrupt the integrity of the plasma membrane, while the intracellular ATP level significantly decreased. Control (untreated) cells displayed a typically scattered distribution of filamentary mitochondria inside the cell. After 2 hours of MEN treatment the spatial distribution of the mitochondria was markedly altered to an asymmetric perinuclear clustered distribution. Menadione-stressed cells displayed a highly asymmetrical perinuclear clustered distribution of the mitochondria. The exposure of cells to MEN also results in a change in shape of the mitochondria into a population of enlarged granular structures. The results of our study demonstrate that in JAR cells menadione causes mitochondria to translocate from the cell periphery into the perinuclear region several hours before disruption of cell membrane integrity and cell death.

Thymocytes exposed to the pro-oxidant tert-butyl-hydroperoxide (ButOOH) display a number of dramatic changes in morphology similar to those observed in the case of dexamethasone-treated cells. Both reagents induce nuclear chromatin peripheral aggregation below the nuclear membrane. Some nuclei themselves break up producing two or more fragments. ButOOH-treated cells are morphologically characterised by cell shrinkage, extensive surface blebbing and, finally, fragmentation into membrane-bound apoptotic bodies composed of cytoplasm and tightly packed with or without nuclear fragments. An increased level of lipid hydroxyperoxides was detected after exposure of thymocytes to ButOOH. Both oxidative stress markers and morphological damage to cells were prevented by the antioxidant 4-OH-TEMPO.

Characterization of free radical-induced cell injury processes of placenta cells is of vital importance for clinical medicine for the maintenance of intrauterine fetal life. The present study has analyzed cell injury processes in cells of the choriocarcinoma cell line JAR treated with menadione, an anticancer drug, and H(2)O(2) in comparison to osteosarcoma 143B cells using electron microscopic and flow cytometric techniques. Flow cytometry on JAR cells exposed to 100 muM menadione and double-stained with Annexin V and propidium iodide (PI) detected apoptotic cells reaching the maximum after 4 h of incubation with a rapid decrease thereafter. Viable cells became decreased to 46% of the control after 2 h of incubation, reaching 5% after 4 h. Cells stainable with both Annexin V and PI began to increase distinctly after 2 h of incubation, reaching 55% after 4 h. Electron microscopy showed that cells stainable with both dyes specified above had condensed nuclei and swollen cytoplasm, suggesting that they were undergoing a switch of the cell death mode from apoptosis to necrosis. On the other hand, 90% of 143B cells remained intact after 4 h of menadione treatment although the intracellular levels of superoxide were always higher than those of JAR cells treated with the drug. In contrast, JAR cells were more resistant than 143B cells to H(2)O(2)-induced cytotoxicity. These results may suggest that cytotoxicity of menadione cannot be explained simply by oxygen free radicals generated from the drug. The resistance of JAR cells to oxygen free radical-induced cytotoxicity may be advantageous for intrauterine fetal life.

Mitochondrial fraction prepared from human term placentae were incubated with [14C]-cholesterol in the presence of citrate, cis-aconitate or isocitrate. A stimulatory effect of these metabolites on cholesterol to progesterone conversion was found. In contrast to the other metabolites tested, citrate supported progesterone biosynthesis was found to be inhibited by manganese ions. This effect occurred in intact mitochondria only and was accompanied by a decreased citrate consumption.

Mitochondria from human term placenta convert pregnenolone to progesterone. It was found that the soluble 3β-hydroxysteroid dehydrogenase, Δ4,5-isomerase complex from placental mitochondria uses both NAD+ and NADP+ as coenzymes. A much higher affinity for NAD+ than for NADP+ was noted. The reduced form of phosphopyridine nucleotides inhibited the biosynthesis of progesterone from pregnenolone. NADH exhibits a stronger inhibitory effect than NADPH. The physiological importance of these facts was discussed.

Cytosolic malic enzyme ( l -malate: NADP oxidoreductase decarboxylating, EC 1.1.1.40) has been isolated and purified from postmitochondrial supernatant 0f human term placenta by ammonium sulphate fractionation, chromatography on diethylaminoethyl- (DEAE-)cellulose, Sepharose 6B, ADP-Sepharose 4B and Ultrogel AcA-34 to apparent homogeneity as judged from polyacrylamide gel electrophoresis (PAGE). The specific activity of the purified enzyme was 24.0 αmol. min -1 . mg -1 protein, which corresponds t0 about 7500-fold purification. The molecular weight of the native enzyme was determined by gel filtration to be about 250 000. Sodium dodecyl sulphate- (SDS-)PAGE showed one polypeptide band of molecular weight 63 000. It appears that the native protein is a tetramer composed 0f sub its of identical molecular weight. The isoelectric point of the purified malic enzyme was pH 5.55. The enzyme was shown t0 carboxylate pyruvate in the presence 0f high concentrations 0f pyruvate and bicarbonate at about 8o per cent of the rate of the forward reaction. The optimum pH for the carboxylation reaction was pH 7.3, and that for the decarboxylation reaction varied with malate concentration. The Km values, determined at pH 7.2, for malate, NADP + , Mn 2+ , and Mg 2+ were 81 μ M , 10 μ M , 2.5 μ M and 0.6 m M , respectively. The K m values for pyruvate, NADPH and bicarbonate were 4 m M , 25 μ M and 20 m M , respectively. The enzyme converted malate to pyruvate (at pH 6.3) in the presence of 5 m M NAD + at approximately 8o per cent of the maximum rate with NADP + . It exhibited oxaloacetate decarboxylase activity at about 10 per cent of the rate of oxidative decarboxylation of malate (with NADP + as coenzyme) and pyruvate reductase activity at about 4 per cent of the rate of oxidative decarboxylation of malate with NADP + . The oxidative decarboxylation of malate was inhibited by malate at lower values of pH of incubation medium. This inhibition gradually decreased as the pH of the incubation medium increased. No inhibition was observed at pH 8.2. The addition of purified cytoplasmic malic enzyme, pyruvate, bicarbonate and NADPH generating system (consisting of NADP + , glucose 6-phosphate, glucose 6-phosphate dehydrogenase) stimulated about twof old progesterone biosynthesis by the isolated human placental mitochondria. This stimulation was abolished by arsenite and fluorocitrate. A possible role for the cytosolic malic enzyme in the regulation of progesterone biosynthesis in human placenta is discussed.

We have found that NADPH-dependent lipid peroxidation in bovine adrenal cortex mitochondria is strongly inhibited by paraquat. The site of the inhibition of the lipid peroxidation by paraquat has been examined. Paraquat neither inhibits NADPH-2,6-dichlorophenolindophenol nor NADPH-cytochrome c reductase activities. However, paraquat is able to retard the rate of reduction of cytochrome P-450 by NADPH. The spectrophotometric measurements provide the first evidence that lipid peroxidation in adrenal cortex mitochondria involves cytochrome P-450 and that the inhibitory effect of paraquat on lipid peroxidation is due to reoxidation of reduced cytochrome P-450 by the reagent.

Mitochondria isolated from human term placenta were able to form citrate from malate as the only added substrate. While mitochondria were incubated in the presence of Mn2+ the citrate formation was stimulated significantly both by NAD+ and NADP+ and was inhibited by hydroxymalonate, arsenite, butylmalonate and rotenone. It is concluded that NAD(P)-linked malic enzyme is involved in the conversion of malate to citrate in these mitochondria. It has also been shown that the conversion of cholesterol to progesterone by human term placental mitochondria incubated in the presence of malate was stimulated by NAD+ and NADP+ and inhibited by arsenite and fluorocitrate. This suggests that the stimulation by malate of progesterone biosynthesis depends not only on the generation of NADPH by NAD(P)-linked malic enzyme, but also on NADPH formed during further metabolism of pyruvate to isocitrate which is in turn efficiently oxidized by NADP+-linked isocitrate dehydrogenase.

We have investigated the effect of paraquat (methyl viologen) on lipid peroxidation in bovine adrenal cortex mitochondria. Incubation of a buffered aerobic mixture of mitochondria in the presence of Fe2+ or NADPH resulted in the formation of lipid peroxides whose accumulation could be followed at 532 nm as malondialdehyde. Fe2+ stimulates lipid peroxidation in normal mitochondria and those in which enzymes have been inactivated with heat. In contrast, NADPH has a stimulatory effect only in normal mitochondria, but not in heat-treated mitochondria. These results indicate that NADPH-dependent lipid peroxidation is an enzymatic process. Paraquat strongly inhibits this enzymatic lipid peroxidation, but has no effect on the non-enzymatic Fe2+-dependent process. The chemiluminescence that accompanies the NADPH-dependent lipid peroxidation is also markedly decreased in the presence of paraquat. Superoxide dismutase, which removes Superoxide anion efficiently, does not inhibit malondialdehyde production. The mechanism of the inhibition of the lipid peroxidation by paraquat has been examined. Paraquat has no effect on NADPH-2,6-dichlorophenolindophenol reductase and on NADPH-cytochrome c reductase activities in bovine adrenal cortex mitochondria. However, paraquat strongly inhibits the NADPH-dependent reduction of cytochrome P-450. These results suggest that the inhibitory effect of paraquat on NADPH-dependent lipid peroxidation in adrenal cortex mitochondria is due to a decrease in the level of reduced cytochrome P-450 probably by diverting electrons from cytochrome P-450. Cytochrome c, which can compete with P-450 for available electrons from adrenodoxin, like paraquat had an inhibitory effect on NADPH-dependent lipid peroxidation. Lipid peroxidation was also strongly inhibited by steroid hydroxylase inhibitors e.g., amphenone B, aminoglutethimide and metyrapone.

It has been shown that the conversion of cholesterol to progesterone by human term placental mitochondria incubated in the presence of malate or fumarate was inhibited by hydroxymalonate--an inhibitor of malic enzyme. No inhibition was observed when mitochondria were incubated in the presence of citrate or isocitrate. The degree of inhibition by hydroxymalonate of partly purified NAD(P)-linked malic enzyme activity was identical to that of both malate dependent pyruvate and progesterone formation by intact mitochondria. These data strongly support a previous suggestion that malic enzyme plays an important role in the malate dependent progesterone biosynthesis by human placental mitochondria.

1. 1. The interrelationship between progesterone (from cholesterol) biosynthesis and oxidative phosphorylation in human placental mitochondria was examined. 2. 2. ADP and ATP stimulated the malate, succinate and α-ketoglutarate-supported progesterone biosynthesis probably via the energy-dependent pyridine nucleotide transhydrogenase activation. The effect of ADP was abolished by rotenone and antimycin in the presence of malate or α-ketoglutarate. 3. 3. In the non-energized state of mitochondria malate may supported progesterone biosynthesis by the malic enzyme-dependent pathway. 4. 4. The inhibitory effects of antimycin or cyanide, and the stimulatory effect of rotenone on the succinate-supported progesterone biosynthesis indicate that the succinate to malate conversion is a necessary condition for the stimulation of progesterone biosynthesis from cholesterol. 5. 5. α-Ketoglutarate plus malonate did support progesterone biosynthesis also in the presence of ADP or ATP and to a lesser degree in the presence of DNP and rotenone. Arsenate in the presence of α-ketoglutarate, malonate, dinitrophenol and rotenone did not affect significantly progesterone biosynthesis. These results indicate that NADPH may be generated also by a non-energy-dependent transhydrogenation in placental mitochondria.

Incubation of human term placental mitochondria with Fe2+ and a NADPH-generating system initiated high levels of lipid peroxidation, as measured by the production of malondialdehyde. Malondialdehyde formation was accompanied by a corresponding decrease of the unsaturated fatty acid content. This NADPH-dependent lipid peroxidation was strongly inhibited by superoxide dismutase and singlet oxygen scavengers, markedly stimulated by paraquat, but was not affected by hydroxyl radical scavengers. Catalase enhanced the production of malondialdehyde by placental mitochondria. The effects of catalase and hydroxyl radical scavengers suggest that the initiation of NADPH-dependent lipid peroxidation is not dependent upon the hydroxyl radical produced via an iron-catalyzed Fenton reaction. These studies provide evidence that hydrogen peroxide strongly inhibits NADPH-dependent mitochondrial lipid peroxidation. The inhibitory effect of superoxide dismutase and stimulatory effect of paraquat, which was abolished by the addition of superoxide dismutase, suggests that superoxide may promote NADPH-dependent lipid peroxidation in human placental mitochondria.

The relationship between malate dependent conversion of cholesterol to progesterone and citrate biosynthesis in human term placental mitochondria has been investigated. It has been shown that ADP and ATP (but not AMP) stimulate, significantly, both progesterone and citrate formation. The stimulatory effect of these adenine nucleotides was dependent on the presence of Mn2+ in the incubation medium. When Mn2+ was omitted or replaced by Mg2+ only negligible stimulatory effect of ADP and ATP was observed. Atractyloside and oligomycin were without effect on ADP and ATP stimulated progesterone and citrate production. Other dinucleotides tested as: GDP, UDP and CDP stimulated both progesterone and citrate formation only slightly. In all the experiments presented the rate of progesterone biosynthesis was found to be significantly correlated with the rate of citrate production. The experimental results presented in this paper suggest that the stimulatory effect of ADP and ATP on malate dependent progesterone biosynthesis is a consequence of an increased conversion of malate to tricarboxylic Krebs cycle intermediates. The possible mechanism by which ATP and ADP stimulate the citrate formation in human placental mitochondria is discussed.

The relationship between NADPH-dependent lipid peroxidation and the degradation of cytochrome P-450 has been studied in bovine adrenal cortex mitochondria. Malondialdehyde formation is accompanied by a corresponding decrease in total cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of P-450. To differentiate between cytochrome P-450(11)beta and P-450scc, steroid-induced difference spectra were used to evaluate P-450 degradation. These measurements provide the first evidence that both P-450's are degraded during NADPH-dependent lipid peroxidation with P-450(11)beta being much more susceptible to this process.