Hepatic Microsomal Membranes Research Articles

Responses of young energy-restricted sheep to chronically administered insulin-like growth factor I (IGF-I): evidence that IGF-I suppresses the hepatic growth hormone receptor.

We have shown previously that chronic administration (8 weeks) of insulin-like growth factor-I (IGF-I) has little growth-promoting effect in well fed sheep. The aim of this study was to investigate the anabolic effects of IGF-I in energy-restricted conditions in which circulating concentrations of IGF-I in control animals were expected to be low. Young castrate male sheep were offered chaffed lucerne at a rate equivalent to 110% maintenance and were treated by sc injection three times per day for either 8 or 12 weeks with recombinant human IGF-I (150 micrograms/kg live wt x day) or saline in a 2 x 2 factorial design (eight animals per cell). IGF-I treatment significantly increased plasma IGF-I concentrations, but reduced plasma concentrations of IGF-II, GH, urea, and creatinine. Treatment with IGF-I also decreased (P < 0.1) GH secretion in response to a GRF load, but significantly (P < 0.05) increased the nonesterified fatty acid response to an epinephrine load. The reduction in circulating GH levels was accompanied by a suppression of [125I]oGH binding to hepatic microsomal membranes. This effect, if apparent in other tissues, may act as a feedback mechanism to limit the local synthesis of IGF-I and could explain why IGF-I treatment had little effect on the growth rate of the sheep, although it did increase nitrogen digestibility of the feed consumed and decreased the fat content of the hind leg. It also differentially promoted the growth of the spleen, thymus, and mandibular salivary gland and increased blood counts of eosinophils. It is concluded that IGF-I does not have marked effects on growth rate or body composition in sheep fed a near-maintenance diet. Possible reasons include the associated suppression of GH secretion and action, which limits the ability of treated animals to repartition absorbed nutrients.

Ascorbate synthesis-dependent glutathione consumption in mouse liver

Ascorbate synthesis causes glutathione consumption in the liver. Addition of gulonolactone resulted in an increase of ascorbate production in isolated murine hepatocytes. At the same time, a decrease in reduced glutathione (GSH) level was observed. In hepatic microsomal membranes, ascorbate synthesis stimulated by gulonolactone caused an almost equimolar consumption of GSH. This effect could be counteracted by the addition of catalase or mercaptosuccinate, indicating the role of hydrogen peroxide formed during ascorbate synthesis in the depletion of GSH. The observed phenomenon may be one of the reasons why the evolutionary loss of ascorbate synthesis could be advantageous.

Growth hormone (GH) status regulates GH receptor and GH binding protein mRNA in a tissue- and transcript-specific manner but has no effect on insulin-like growth factor-I receptor mRNA in the rat

The effect of growth hormone-deficiency (GHD) and treatment with recombinant bovine GH (bGH) or human IGF-I (hIGF-I) for 10 days on the expression of GH receptor (GHR). GH binding protein (GHBP) and of insulin-like growth factor-I receptor (IGF-IR) mRNA was examined using dw dw and normal Lewis rats. Hepatic GHR and GHBP mRNA expression was significantly lower in dw dw rats in comparison to Lewis rats ( P < 0.01) while specific 125I-bGH binding to hepatic microsomal membranes was significantly higher ( P < 0.01), suggesting a reduction in hepatic GHR turnover with GHD. Treatment with bGH reduced hepatic specific 125I-bGH binding in dw dw rats, but had no effect in Lewis rats. Treatment with hIGF-I increased hepatic specific 125I-bGH binding in Lewis rats. Hepatic GHR and GHBP mRNA expression was not changed by bGH or hIGF-I treatment, suggesting that differences in hepatic specific 125I-bGH binding may be due to posttranscriptional mechanisms. GHBP mRNA expression was higher in kidney, heart, and muscle of dw dw rats in comparison to Lewis rats ( P < 0.01), while GHR mRNA abundance was not changed. Treatment of dw dw rats with hIGF-I or bGH resulted in a coordinate reduction of GHR and GHBP mRNAs in kidney ( P < 0.01). IGF-IR mRNA was not detected in liver and despite reduced plasma IGF-I levels and IGF-I mRNA expression IGF-IR mRNA abundance was not changed in nonhepatic tissues by GHD. Our data suggest that changes in plasma IGF-I levels and local IGF-I mRNA do not influence IGF-IR mRNA expression, while GHR and GHBP mRNA expression in different rat tissues are regulated independently. The increased nonhepatic GHBP mRNA expression with GHD suggests that nonhepatic GHBP may have an important physiological function distinct from that of GHBP in liver or in plasma.

The fetal somatotropic axis during long term maternal undernutrition in sheep: evidence for nutritional regulation in utero.

Nutrition is a major determinant of the somatotropic axis during postnatal life. However, little is known about the response of the fetal somatotropic axis to nutritional limitation. From day 100 of gestation (term = 147 days), singleton-bearing ewes were fed either ad libitum (control; n = 6) or 25% of the recommended energy and protein requirements (restricted; n = 7). Ewes and fetuses were chronically catheterized on day 110. On day 120, paired maternal and fetal blood samples were taken over a 6-h period at 15-min intervals. Forty-eight hours later, fetuses were given a 20-micrograms GRF bolus (i.v.), and samples were collected for 48 h. Undernourished mothers and fetuses had higher GH concentrations (P < 0.05). Although plasma GH profiles were independent in mothers and their fetuses, both maternal and fetal GH peak and nadir levels were increased (P < 0.05) by nutritional restriction, but the peak/nadir ratio and the number of pulses remained unaltered. Deconvolution analysis showed that the GH mass secreted per burst was higher in nutritionally restricted animals, whereas basal GH secretion and GH serum half-life were not influenced by undernutrition. Both maternal and fetal insulin-like growth factor-I levels were reduced (P < 0.01 and P < 0.05), whereas insulin-like growth factor-II concentrations were not influenced by the feed restriction. Fetuses from restricted mothers had higher peak GH concentrations after a GRF challenge (P < 0.001), but after correction The specific binding of [125I]ovine placental lactogen ([125I]oPL) or [125I]oGH to maternal or fetal hepatic microsomal membrane preparations was not changed by the maternal undernutrition. Maternal oPL concentrations showed considerable short term fluctuations, whereas fetal oPL levels revealed no major fluctuations. Mean maternal oPL levels tended (P < 0.06) to be elevated, whereas fetal oPL concentrations tended (P < 0.06) to be decreased in restricted animals. These results provide evidence that the somatotropic axis is functional in utero and suggest that the fetal somatotropic axis plays an active role during adaptation of the fetus to nutritional limitation.

The fetal somatotropic axis during long term maternal undernutrition in sheep: evidence for nutritional regulation in utero

Nutrition is a major determinant of the somatotropic axis during postnatal life. However, little is known about the response of the fetal somatotropic axis to nutritional limitation. From day 100 of gestation (term = 147 days), singleton-bearing ewes were fed either ad libitum (control; n = 6) or 25% of the recommended energy and protein requirements (restricted; n = 7). Ewes and fetuses were chronically catheterized on day 110. On day 120, paired maternal and fetal blood samples were taken over a 6-h period at 15-min intervals. Forty-eight hours later, fetuses were given a 20-micrograms GRF bolus (i.v.), and samples were collected for 48 h. Undernourished mothers and fetuses had higher GH concentrations (P < 0.05). Although plasma GH profiles were independent in mothers and their fetuses, both maternal and fetal GH peak and nadir levels were increased (P < 0.05) by nutritional restriction, but the peak/nadir ratio and the number of pulses remained unaltered. Deconvolution analysis showed that the GH mass secreted per burst was higher in nutritionally restricted animals, whereas basal GH secretion and GH serum half-life were not influenced by undernutrition. Both maternal and fetal insulin-like growth factor-I levels were reduced (P < 0.01 and P < 0.05), whereas insulin-like growth factor-II concentrations were not influenced by the feed restriction. Fetuses from restricted mothers had higher peak GH concentrations after a GRF challenge (P < 0.001), but after correction The specific binding of [125I]ovine placental lactogen ([125I]oPL) or [125I]oGH to maternal or fetal hepatic microsomal membrane preparations was not changed by the maternal undernutrition. Maternal oPL concentrations showed considerable short term fluctuations, whereas fetal oPL levels revealed no major fluctuations. Mean maternal oPL levels tended (P < 0.06) to be elevated, whereas fetal oPL concentrations tended (P < 0.06) to be decreased in restricted animals. These results provide evidence that the somatotropic axis is functional in utero and suggest that the fetal somatotropic axis plays an active role during adaptation of the fetus to nutritional limitation.

Characterization of ovine growth hormone (oGH) and ovine placental lactogen (oPL) binding to fetal and adult hepatic tissue in sheep: evidence that oGH and oPL interact with a common receptor

The GH receptor (GHR) plays a key role in postnatal growth regulation. Although plasma concentrations of GH are high during fetal life, its role during fetal development is not well understood. Recent data suggest that GHR are present in fetal hepatic tissue as early as 51 days gestation. However, the levels of GHR expression are markedly lower in fetal hepatic tissue compared to postnatal values, and there are conflicting data suggesting that ovine placental lactogen (oPL) and oGH share a common receptor. Given the uncertainty about whether oPL acts via the oGHR or a distinct receptor, we performed ligand binding and affinity cross-linking studies on hepatic microsomal membranes from adult castrated male, pregnant female, and fetal sheep. Ligand binding assays at a constant concentration of membranes showed that [125I]oPL yielded consistently higher (P < 0.001) specific binding (59.5 +/- 6.4%, 30.5 +/- 5.7%, and 7.6 +/- 2.4% for castrated male, pregnant female, and fetal sheep, respectively) compared to [125I]oGH (17.8 +/- 4.7%, 5.0 +/- 1.6%, and 1.2 +/- 0.4% for castrated male, pregnant female, and fetal sheep, respectively). Cross-reactivity studies showed that unlabeled oPL was consistently more potent than unlabeled oGH in displacing either of the labeled ligands. The dissociation constant (Kd) for oPL binding ranged from 0.16-0.40 nM and was not changed by solubilization with Triton X-100. Equilibrium binding analysis for oGH showed lower affinity for hepatic microsomal membranes (Kd, 1.7-3.2 nM) in each of the three groups of animals. Affinity cross-linking of microsomal membranes from castrated male and pregnant female sheep liver showed four major cross-linked complexes with both [125I]oPL and [125I]oGH, with mol wt of 150, 97, 75, and 60 kilodaltons. All four bands were identified with both ligands. Unlabeled oPL showed markedly higher potency than unlabeled oGH in reducing the signal of the [125I]oPL cross-linked complexes, whereas unlabeled oGH and oPL showed comparable potencies in reducing the signal of the [125I]oGH complexes. Immunoprecipitation of detergent-solubilized hepatic microsomal membranes from pregnant and fetal sheep using a panel of monoclonal antibodies raised against the extracellular region of the rabbit GHR showed potent immunological recognition of the [125I]oPL-receptor complexes. We suggest that oGH and oPL bind to a common or a related receptor protein(s). It is possible that differences in receptor dimerization or association with other membrane proteins are the basis of the differences in affinity and biological actions of the two hormones.

Characterization of ovine growth hormone (oGH) and ovine placental lactogen (oPL) binding to fetal and adult hepatic tissue in sheep: evidence that oGH and oPL interact with a common receptor.

The GH receptor (GHR) plays a key role in postnatal growth regulation. Although plasma concentrations of GH are high during fetal life, its role during fetal development is not well understood. Recent data suggest that GHR are present in fetal hepatic tissue as early as 51 days gestation. However, the levels of GHR expression are markedly lower in fetal hepatic tissue compared to postnatal values, and there are conflicting data suggesting that ovine placental lactogen (oPL) and oGH share a common receptor. Given the uncertainty about whether oPL acts via the oGHR or a distinct receptor, we performed ligand binding and affinity cross-linking studies on hepatic microsomal membranes from adult castrated male, pregnant female, and fetal sheep. Ligand binding assays at a constant concentration of membranes showed that [125I]oPL yielded consistently higher (P < 0.001) specific binding (59.5 +/- 6.4%, 30.5 +/- 5.7%, and 7.6 +/- 2.4% for castrated male, pregnant female, and fetal sheep, respectively) compared to [125I]oGH (17.8 +/- 4.7%, 5.0 +/- 1.6%, and 1.2 +/- 0.4% for castrated male, pregnant female, and fetal sheep, respectively). Cross-reactivity studies showed that unlabeled oPL was consistently more potent than unlabeled oGH in displacing either of the labeled ligands. The dissociation constant (Kd) for oPL binding ranged from 0.16-0.40 nM and was not changed by solubilization with Triton X-100. Equilibrium binding analysis for oGH showed lower affinity for hepatic microsomal membranes (Kd, 1.7-3.2 nM) in each of the three groups of animals. Affinity cross-linking of microsomal membranes from castrated male and pregnant female sheep liver showed four major cross-linked complexes with both [125I]oPL and [125I]oGH, with mol wt of 150, 97, 75, and 60 kilodaltons. All four bands were identified with both ligands. Unlabeled oPL showed markedly higher potency than unlabeled oGH in reducing the signal of the [125I]oPL cross-linked complexes, whereas unlabeled oGH and oPL showed comparable potencies in reducing the signal of the [125I]oGH complexes. Immunoprecipitation of detergent-solubilized hepatic microsomal membranes from pregnant and fetal sheep using a panel of monoclonal antibodies raised against the extracellular region of the rabbit GHR showed potent immunological recognition of the [125I]oPL-receptor complexes. We suggest that oGH and oPL bind to a common or a related receptor protein(s). It is possible that differences in receptor dimerization or association with other membrane proteins are the basis of the differences in affinity and biological actions of the two hormones.

The induction of hepatic somatotrophic receptors after birth in sheep is dependent on parturition-associated mechanisms

While the GH receptor (GHR) plays a key role during adult life, its role during fetal development is not well understood. Recent data suggest that GHRs are present in ovine fetal hepatic tissue at mid-gestation. However, the levels of GHR expression are markedly lower in fetal hepatic tissue in comparison with postnatal values. The present study investigates whether the neonatal induction of the hepatic GHR and plasma levels of IGF-I follow a pattern of strictly age-related development or whether birth-associated processes stimulate this increase. Stereotaxic electrolytic lesioning of the fetal paraventricular nuclei was employed to prolong gestation markedly. We compared the hepatic binding of ovine placental lactogen (oPL) and oGH and plasma levels of IGF-I in these post-mature fetuses with those in pre-term fetuses, pregnant mothers and lambs which were of the same conceptional age as the post-mature fetuses. While specific binding of both 125I-labelled oGH and 125I-labelled oPL to hepatic microsomal membranes was fully reversible in all groups, the specific binding of 125I-labelled oGH was significantly (P < 0.001) lower than specific binding of 125I-labelled oPL in all groups of animals. There was no difference in specific 125I-labelled oGH or 125I-labelled oPL binding in livers or plasma levels of IGF-I in post-mature fetuses in comparison with pre-term fetuses. In contrast, a major increase (P < 0.001) in 125I-labelled oGH and 125I-labelled oPL binding and plasma IGF-I levels was observed in lambs.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of reactive oxygen species by peritoneal macrophages and hepatic mitochondria and microsomes from endrin-treated rats

Recent studies have shown that the adminstration of endrn to rodents induced lipid peroxidation in various tissues and decreases glutathione content. These results suggest that endrin produces reactive oxygen species and/or free radicals. We have therefore examined the effects of endrin(4.5 mg/kg) on the production of reactive oxygen species by peritoneal macrophages and hepatic mitochondria and microsomes in rats. The effects of endrin on hepatic mitochondrial and microsomal lipid peroxidation and membrane fluidity as well as the incidence of hepatic nuclear DNA damage were also examined. Twenty-four hours after endrin administration, significant increases in the production of the chemiluminescence by the three tissue fractions were observed. Furthermore, peritoneal macrophages form endrin-animals resulted in 3.0- and 2.8-fold increases in cytochrome c and iodonitrotetrazolium (INT) reduction, indicating enhanced production of superoxide anion. Endrin administration also resulted in significant increases in lipid peroxidation of mitochondrial and microsomal membranes as well as decreases in the fluidity of these two membranous fractions. A significant increase in hepatic nuclear DNA single-strand breaks also occurred in response to endrin administration. The reslts indicate that macrophage, mitochondria, and microsomes produce reactive oxygen species following endrin administration, and these reactive oxygen species may contribute to the toxic manifestations of endrin.

Effects of bile acids and taurine on the lipid fluidity of hepatic microsomes in normal and bile duct-ligated rats — A spin label study

In cholestasis and obstructive jaundice, hepatic bile acids and taurine content increase significantly and bile acids change in compositions. For example, increases in cholic acid, beta-muricholic acid and taurine-conjugated bile acids occur in rats. In this study, to clarify the significance of changes in bile acid composition in obstructive jaundice, the effects of various bile acids and taurine on the fluidity of the hepatic microsomal membrane were studied by the electron spin resonance spin-label method in normal and bile duct-ligated (for 10 days) rats. The flow properties (order parameter) of the shallow and deep layers of the membrane were determined with 5- and 12-doxyl stearic acid, respectively. Bile acids mainly affected the fluidity of the shallow membrane layer. The fluidity of the hepatic microsomal membrane was decreased in bile duct-ligated rats. Conjugated cholic acid (taurine-conjugated cholic acid, glycine-conjugated cholic acid and non-conjugated beta-muricholic acid exerted effects that increased fluidity in comparison with non-conjugated cholic acid and chenodeoxycholic acid, a precursor of beta-muricholic acid. Taurine had no clear effect on fluidity. In conclusion, cholic acid conjugation and beta-muricholic acid formation may be mechanisms which protect against the decrease in lipid fluidity of hepatic biomembranes in cholestasis or obstructive jaundice. Taurine may also affect fluidity through the formation of taurine-conjugated bile acids.

Fish oil and oat bran in combination effectively lower plasma cholesterol in the rat.

Male rats were fed a semi-purified diet containing oat bran or wheat bran with or without a marine fish oil to investigate the effects of such combinations on lipid metabolism. Oat bran alone and wheat bran plus fish oil gave lower plasma cholesterol concentrations than wheat bran alone while oat bran plus fish oil gave the lowest. Oat bran increased plasma triacylglycerols compared with wheat bran but oat bran plus fish oil gave concentrations similar to those seen with wheat bran plus fish oil. Oat bran gave higher hepatic cholesterol synthesis rates and a higher activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase compared to wheat bran. The addition of fish oil to either bran diet decreased cholesterol synthesis but HMG CoA reductase activity was not reduced. Oat bran increased hepatic acyl coenzyme A:cholesterol acyl transferase (ACAT) activity and increased the ratio of esterified to unesterified cholesterol in hepatic microsomal membranes compared with wheat bran. Fish oil decreased hepatic LDL receptor activity and increased HDL binding activity when added to the wheat bran diet but these effects were not seen with oat bran. Oat bran also had no effect on hepatic lipoprotein receptor activity compared with wheat bran. These results show that fish oil and oat bran have complementary cholesterol lowering effects in the rat.

Photodynamic effects of chloroaluminum phthalocyanine tetrasulfonate are mediated by singlet oxygen: In vivo and in vitro studies utilizing hepatic microsomes as a model membrane source

Chloroaluminum phthalocyanine tetrasulfonate (Al-PcTS) is a promising photosensitizer for the photodynamic therapy (PDT) of cancer. In this study, we investigated the in vivo and in vitro photodestruction of hepatic microsomal membranes by AlPcTS and studied the role of reactive oxygen species in this process. Irradiation of hepatic microsomes prepared from AlPcTS-pretreated SENCAR mice to ≈675 nm light resulted in rapid destruction of cytochrome P450 and associated monooxygenase activities, and enhancement of lipid peroxidation in a light-dose-dependent manner. The specificity of AlPcTS and light dependency on photodestruction of microsomal membranes was confirmed by Western blot analysis. Similar results were obtained when AlPcTS was added in vitro to a suspension of hepatic microsomes prepared from control animals followed by irradiation to ≈675 nm light. Among the quenchers of singlet oxygen, superoxide anion, hydrogen peroxide, and hydroxyl radical, only the quenchers of singlet oxygen such as sodium azide, histidine, and 2,5-dimethyl furan afforded substantial protection in a dose-dependent manner against AlPcTS-mediated photodestruction of cytochrome P450 and associated monooxygenase activities, and photoenhancement of lipid peroxidation under both in vivo and in vitro conditions. These results suggest that lipid-rich microsomal membranes may be the potential targets of cell injury by AlPcTS-based PDT and that this process is mediated by singlet oxygen.

Lipid peroxidation of ultrastructural components of rat liver induced by metanil yellow and orange II: comparison with blend.

Metanil yellow and Orange II are extensively used in various industries and have not been included in the list of permitted food colours because of inadequate safety evaluation data. In this investigation the effect of Metanil yellow and Orange II on the lipid peroxidation in different subcellular fractions of liver was studied in order to understand the site of hepato-toxic potential and whether its blend has an additive, synergistic or antagonistic effect. Parenteral administration of Metanil yellow (80 mg/kg body weight) to rats for 3 days produced a highly significant (p less than 0.001) increase in NADPH-dependent enzymatic lipid peroxidation in nuclear (62%), mitochondrial (104%) and microsomal (54%) fractions. The same dose of Orange II to rats showed a relatively less but significant effect in nuclear (62%), mitochondrial (59%) and microsomal (38%) membranes. The blend of Metanil yellow and Orange II (40 mg + 40 mg/kg body weight) given intraperitoneally for 3 days resulted in either synergistic or additive effect in nuclear (122%), mitochondrial (130%), and microsomal (62%) membranes. The pro-oxidant free or FeSO4/ADP (1 mM/5 mM) or ascorbate (1 mM) dependent non-enzymatic lipid peroxidation in nuclear, mitochondrial and microsomal fractions was found to be enhanced by Metanil yellow to a greater extent as compared to Orange II, while the blend showed a synergistic or additive response. These results suggest that Metanil yellow and Orange II causes hepatic nuclear, mitochondrial and microsomal membrane damage and that the effect may be more severe with the use of blend.

Effect of benzyl viologen on the phospholipid fatty acid composition and some properties in hepatic microsomal membrane of rats

The effect of benzyl viologen (a stimulator of free radical production in cells) on lipid composition, fluidity and enzymes involved in both polyunsaturated fatty acid biosynthesis and cholesterol metabolism was studied in liver microsomal membrane of adult rats. In viologen-treated animals, a significant decrease in the levels of free cholesterol and cholesteryl esters, accompanied to a decrease at the free cholesterol/phospholipid ratio, were observed. The levels of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acyl-coenzyme A: cholesterol acyltransferase (ACAT) were also lower in viologen-treated rats than in controls. Linoleic and arachidonic acids were both severely lower while docosatetraenoic, docosapentaenoic and docosahexaenoic acids were significantly higher as compared with controls. Furthermore, a decrease in monounsaturated/saturated ratio was found. In addition, the treatment evoked a depression in the fatty acid desaturation complex, with a diminish of delta 9, delta 6 and delta 5 desaturase activities in microsomal membrane. It was concluded that changes in phospholipid microsomal fatty acid and cholesterol content could be directly responsible for changes in membrane fluidity and function, and that extensive yield of docosahexaenoic acid may serve to maintain the physical characteristics of particular domains against oxidative stress caused by benzyl viologen treatment.

Metabolism of 7,12-dimethylbenz[ a]anthracene in hepatic microsomal membranes from rats treated with isoenzyme-selective inducers of cytochromes P450

Previous work has shown that member(s) of the cytochrome P450IIC sub-family play significant roles in the formation of diols of 7,12-dimethylbenz[ a]anthracene (DMBA) and are particularly important in formation of the proximate carcinogen (DMBA-3,4-diol). To further characterize the role of members of this subfamily in DMBA-diol formation and to assess the part played by other P450s, DMBA metabolism has been investigated in microsomes prepared from animals pre-treated with isoenzyme selective inducers. The rates of formation of DMBA-diols in membranes from phenobarbital-treated rats were very low when NADH was used as reductant and rates were not altered when NADPH and NADH were used in combination rather than using NADPH alone. This suggests that cytochrome b 5 is not involved in DMBA-diol formation in these membranes. Treatment of animals with clofibrate, pyrazole and dexamethasone produced regio-selective alterations in the rates of formation of DMBA-diols at the -3,4-, -5,6- and -8,9- positions. However, none of the inducers caused increases in the rates of DMBA-diol formation of any great magnitude suggesting that the isoforms which are the major induced proteins (P450IVA1, P450IIE1 and P450IIIA1) do not play a significant role in diol formation. The content of other P450s in these membrane are also altered and these were investigated by Western blot using antibodies to P450IIC6, P450IIB1 and P450IIIA1. The results of the Western blots show that the effects of the inducing agents on DMBA-diol formation can be explained by alterations of members of the P450IIC and P450IIB subfamilies.

Dietary Magnesium Depletion: p-Nitroanisole Metabolism and Glucuronidation in Rat Hepatocytes and Hepatic Microsomal Membranes

Male Sprague-Dawley rats were pair fed either a control magnesium diet (480 mg/kg) or a magnesium-deficient diet (30 mg/kg) for 10 days to produce magnesium depletion. Serum magnesium concentration declined 60%, but tissue magnesium deficiency was not apparent. Hepatic parenchymal cells and microsomal membranes were isolated to evaluate in vitro metabolism of p-nitroanisole and conjugation of its metabolite, p-nitrophenol (pNP). O-Demethylation of p-nitroanisole was decreased slightly in both magnesium depletion hepatocytes and microsomal membranes. However, pNP glucuronide formation was specifically decreased 50% in the magnesium depletion cells at all p-nitroanisole concentrations. Similar results were found in the microsomal reaction system measuring pNP conjugation. Thus, early stages of magnesium depletion may result in a specific decrease in the pNP UDP-glucuronyl transferase activity.

Lead-induced tissue fatty acid alterations and lipid peroxidation

Previous work showed that dietary lead (Pb) increases the relative concentration of arachidonic acid (20:4) as a percentage of total fatty acids, and decreases the relative proportion of linoleic acid (18:2) to arachidonic acid (18:2/20:4) in chick liver, serum, and erythrocyte membranes. The present investigation was undertaken to examine the time-course and magnitude of the fatty acid alterations with increasing dietary Pb levels. We also examined the effects of Pb on the fatty acid composition and lipid peroxide content of hepatic subcellular organelles. In Exp. 1, chicks were fed diets containing 0, 62.5, 125, 250, 500, or 1000 ppm added Pb (as Pb acetate trihydrate) from 1 to 21 d of age. After 21 d, no growth effects were observed; however, Pb lowered the 18:2/20:4 ratio and increased 20:4 concentration in total liver and serum lipids, and in total hepatic phospholipids in a dose-dependent manner. Hepatic mitochondrial membrane fatty acids were not altered, nor was there any increase in hepatic lipid peroxidation. In Exp.2, chicks were fed diets containing 0, 500, 1000, or 2000 ppm added Pb from 1 to 21 or 22 d of age. Pb depressed growth in a dose-dependent manner. In addition, Pb lowered the 18:2/20:4 ratio and increased 20:4 concentration in total liver lipids and in hepatic mitochondrial and microsomal membranes in a dose-dependent manner. Total hepatic lipid peroxidation was increased over control values by 1000 ppm Pb, and hepatic microsomal lipid peroxidation was increased by dietary Pb levels of 1000 and 2000 ppm. In Exp. 3, body weight, hepatic microsomal lipid peroxidation, and fatty acid composition were determined in 4-, 9-, 14-, 18-, and 23-d-old chicks fed 0 or 1500 ppm added Pb. Body weights of Pb-treated chicks were significantly lower than those of control chicks by day 18. Microsomal 20:4 concentration and peroxidation increased, and the 18:2/20:4 ratio decreased with age in both groups, but the changes were of greater magnitude in the Pb-treated chicks. The results suggest that some of the manifestations of Pb toxicity may be a reflection of increased concentration of 20:4 in specific membranes. Further, since the Pb-induced alterations in fatty acid composition were noted in the absence of any growth depression, we propose that fatty acid composition is more sensitive than growth rate to the presence of lead in the diet.

Evidence for the induction of fatty acid desaturation in proliferating hepatic endoplasmic reticulum in response to treatment with polychlorinated biphenyls. Are fatty acid desaturases cytochrome P-450-dependent monooxygenases?

1. 1. Polychlorinated biphenyls (PCBs) are abundant and persistent pollutants in the ecosystem which accumulate in biological systems. 2. 2. We have shown previously (Borlakoglu et al., 1990; Ew. J. Biochem. 118, 327–332) that 120 hr after treating pigeons and rats with 1.5mmol Aroclor 1254/kg body weight, hepatic microsomal membranes showed significant increases in the proportion of arachidonate (20:4 5,8,11,14), in the concentration of cytochrome P-450 and in the activities of a wide range of cytochrome P-450-dependent enzymes involved in the metabolism of drugs and other xenobiotics. 3. 3. After treating pigeons and rats in vivo with Aroclor 1254, linoleate desaturases activity increased significantly 3.35-, 4.35-, 5.83- and 8.61-fold 24,48,68 and120 hr for pigeons and 2- and 7-fold for rats respectively 48 and 120 hr post treatment. The total activity of linoleate desaturases in the whole liver of pigeons and rats increased 40- and 10-fold respectively. 4. 4. There were excellent correlations between the concentrations of cytochrome b 5 and cytochrome P-450 and the activity of pigeon linoleate desaturases. Extrapolation of the concentration of cytochrome P-450 to zero is coincident with zero linoleate desaturase activity. 5. 5. Evidence is presented to suggest the novel concept that linoleate desaturation is dependent upon the catalytic cycle of these monooxygenases.

Influence of Hyperalimentation on Rat Hepatic Microsomal Fluidity and Function

Total parenteral nutrition with an amino acid-glucose solution has previously been shown to decrease rat hepatic drug metabolism compared with drug metabolic activity observed in rats receiving the same solution enterally and chow-fed animals. Because changes in membrane fluidity and lipid composition are reported to influence activity of a number of liver enzymes, effects of parenteral and enteral nutrition on hepatic microsomal membrane fluidity and lipid composition were assessed and compared with hepatic mixed-function oxidase activity. Both parenteral and enteral hyperalimentation produced a significant decrease in microsomal membrane fluidity (fluorescence anisotropy = 0.155 +/- 0.003 in both experimental groups versus 0.129 +/- 0.003 for microsomes from chow-fed animals). However, meperidine demethylase activity was significantly decreased compared with chow-fed experiments only in hepatic microsomes from parenterally hyperalimented animals, whereas ethoxyresorufin deethylase activity was significantly reduced only in the enteral-nutrition group. Inclusion of lipid in the parenterally administered hyperalimentation solution normalized microsomal membrane fluidity and lipid profile to those of chow-fed animals but did not increase hepatic meperidine demethylation. Both parenteral and enteral nutrition produce significant changes in physical state and lipid composition of rat hepatic microsomal membranes, but these changes are not responsible for the altered hepatic drug metabolism observed during hyperalimentation.

Polychlorinated biphenyls increase fatty acid desaturation in the proliferating endoplasmic reticulum of pigeon and rat livers

1. Polychlorinated biphenyls (PCB) are abundant and persistent pollutants in the ecosystem. Commercial mixtures (e.g. Aroclor 1254) can contain up to 80 different isomers and congeners, many of which accumulate in biological systems by the ingestion of PCB-contaminated lipid components of food chains. 2. Commercial mixtures of PCB induce, in hepatic microsomal membranes in vivo, a variety of different forms of the cytochrome P-450 components of enzyme systems involved in the metabolism of drugs and other xenobiotics, and can also induce the proliferation of this membrane. Since these microsomal enzyme systems share a number of the requirements of microsomal fatty acid desaturases, we have investigated whether the induction by PCB in vivo of cytochrome-P-450-linked enzymes in the proliferating hepatic microsomal membrane of the pigeon and the rat is accompanied by increased proportions of polyunsaturated fatty acids in this membrane. 3. The most striking changes observed 120 h after treating pigeons and rats with 1.5 mmol Aroclor 1254/kg body mass were 2.2-fold and 1.6-fold increases, respectively, in the proportion of arachidonic acid in the hepatic microsomal membrane. When the effects of this treatment on the proliferation of this membrane and increase in liver mass are taken into account, the amount of arachidonic acid in the total microsomal membrane of pigeon and rat livers increased 6.7-fold and 1.9-fold, respectively. 4. These changes were accompanied by very significant increases in pigeons and rats of the concentration of hepatic microsomal cytochrome P-450, and in the activity in microsomal protein of a wide range of cytochrome P-450-dependent enzyme involved in the metabolism of drugs and other xenobiotics. 5. This effect of PCB, of increasing in vivo the degree of unsaturation of fatty acids of hepatic microsomal membrane, appears to be a novel finding, and does not seem to have been investigated for other drugs and xenobiotics. Preliminary results have shown that the effect is accompanied by substantial increases in the total activity of delta 6 and delta 5 microsomal fatty acid desaturases converting 18:2 (9, 12) (linoleic acid) to 20:4 (5, 8, 11, 14) (arachidonic acid) [Borlakoglu, J.T., Dils, R.R., Edwards-Webb, J.D. & Walker, C.H. (1988) Biochem. Soc. Trans. 16, 1072]. 6. It is postulated that there is a significant link between increased fatty acid desaturation and the induction of cytochrome-P-450-linked enzymes, and this is discussed in terms of the mechanisms involved in the metabolism of foreign compounds.