Subfractionation of smooth microsomes from rat liver.

Total smooth microsomes from rat liver isolated on a Cs(+)-containing sucrose gradient were concentrated and subsequently fractionated by zone centrifugation on a stabilizing sucrose gradient. The prerequisite for fractionation is to prepare total smooth microsomes in a nonaggregated condition, as well as to utilize a procedure which counteracts enzyme inactivation. The median equilibrium density of the various smooth microsomal vesicles ranges from 1.10 to 1.18. The phospholipid/protein ratio is identical in all subfractions, but cholesterol, on a PLP basis, is enriched in the subfractions with the highest sedimentation velocity. The enzyme distribution pattern reveals a pronounced heterogeneity. A number of NADH- and NADPH-oxidizing enzymes are concentrated in the upper part of the gradient and exhibit a certain degree of separation from G6Pase. Mg(++)-ATPase and AMPase are enriched in the lower part of the gradient. No specific enrichment of newly synthesized NADPH-cytochrome c reductase activity occurs in any of the subfractions after phenobarbital treatment. These data demonstrate that smooth microsomes, by adequate fractionation procedure, can be separated into subfractious of heterogeneous composition.

Evidence for the participation of the Golgi apparatus in the intracellular transport of nascent albumin in the liver cell.

A comparative biochemical and radioautographic in vivo study was performed to identify the site of synthesis and route of migration of albumin in the parenchymal liver cell after labeling with leucine-(14)C or leucine-(3)H via the portal vein. Free cytoplasmic ribosomes, membrane-bound ribosomes, rough- and smooth-surfaced microsomes, and Golgi membranes were isolated. The purity of the Golgi fraction was examined morphologically and biochemically. After administration of leucine-(14)C, labeled albumin was extracted, and the sequence of transport was followed from one fraction to the other. Approximately 2 min after the intravenous injection, bound ribosomes displayed a maximal rate of leucine-(14)C incorporation into albumin. 4 min later, a peak was reached for rough microsomes. Corresponding maximal activities for smooth microsomes were recorded at 15 min, and for the Golgi apparatus at approximately 20 min. The relative amount of albumin, calculated on a membrane protein basis, was higher in the Golgi fraction than in the microsomes. By radioautography the silver grains were preferentially localized over the rough-surfaced endoplasmic reticulum at the 5 min interval. Apparent activity in the Golgi zone was noted 9 min after the injection; at 15 and 20 min, the majority of the grains were found in this location. Many of the grains associated with the Golgi apparatus were located over Golgi vacuoles containing 300-800 A electron-opaque bodies. It is concluded that albumin is synthesized on bound ribosomes, subsequently is transferred to the cavities of rough-surfaced endoplasmic reticulum, and then undergoes migration to the smooth-surfaced endoplasmic reticulum and the Golgi apparatus. In the latter organelle, albumin can be expected to be segregated together with very low density lipoprotein in vacuoles known to move toward the sinusoidal portion of the cell and release their content to the blood.

Studies on the synthesis and intracellular transport of lipoprotein particles in rat liver.

Lipoprotein particles (d less than 1.03 g/ml) were isolated from rough and smooth microsomes and from the Golgi apparatus of rat liver, and were characterized chemically and morphologically. The rough endoplasmic reticulum (ER) particles were rich in protein (50%) and contained phospholipids (PLP) and triglycerides (TG) in smaller amounts, whereas the lipoprotein particles emanating from the smooth ER, and especially the Golgi apparatus, were rich in TG and PLP, resembling very low density lipoproteins (VLDL) of serum. The difference in chemical composition among the particles was associated with change in size both in situ and in isolated lipoprotein fractions. The rough ER particles were 200-800 A in diameter (mean similar to 420 A); the smooth er particles 200-900 A (mean similar to 520 A); the Golgi particles 350-950 A (mean similar to 580A); and serum VLDL 300-800 A (mean similar to 450 A). Generally, lipoprotein particles were rare in the rough ER, frequent but diffusely dispersed in smooth ER, and occurring mainly in clusters in "secretory vesicles" of the Golgi complex. They were seldom observed in the cisternal compartments of the Golgi complex. At short intervals (less than 15 min), intravenously injected radioactive glycerol was preferentially channelled into TG, whereas at later time points the majority of the isotope was recovered in the PLP. Three TG pools were distinguished: (a) a cytoplasmic pool with a slow turnover rate; (b) a membrane-associated TG pool; and (c) a pool corresponding to the TG moiety of lipoprotein particles, which showed the highest initial rate of labeling and fastest turnover. When, after pulse labeling, the appearance of incorporation of radioactive glycerol into TG or PLP of isolated lipoproteins was followed from one subcellular fraction to the other, a sequence of labeling was noted. During the first interval, TG from both rough and smooth microsomal lipoproteins displayed a high rate of labeling with peak value at 6 min, followed by a quick fall-off, while the Golgi lipoproteins reached maximal level at 10-20 min after administration. There was an interval of 10-15 min before the appearance of labeled VLDL in serum. It is concluded that the assembly of the apoproteins and lipid moieties into lipoprotein particles-presumed to be precursors of liver VLDL-begins in the rough ER and continues in the smooth ER. Also, there is a parallel change in chemical composition and size of the lipoprotein particles as they make their way through the ER and the Golgi apparatus. Some remodeling of the particles may take place in the Golgi apparatus before discharge into the circulation.

Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization.

The induction of autophagy caused by vinblastine (VBL) has been found to be concomitant with a stimulation of proteolysis in a mitochondrial-lysosomal (ML) fraction from the rat liver (Marzella and Glaumann, 1980, Lab. Invest., 42: 8-17. Marzella and Glaumann, 1980, Lab. Invest., 42:18-27). In this fraction the enhanced proteolysis is associated with a threefold increase in the relative fractional volume of autophagic vacuoles (AVs). In an attempt to isolate the AVs, we subfractionated the ML suspension at different intervals after the induction of autophagy by VBL by centrifugation on a discontinuous Metrizamide gradient ranging from 50% to 15%. The material banding at the 24 to 20% and the 20 to 15% interphases was collected. Morphological analysis reveals that 3 h after induction of autophagy these fractions consist predominantly (approximately 90%) of intact autophagic vacuoles. These autophagic vacuoles contain cytosol, mitochondria, portions of endoplasmic reticulum, and occasional very low density lipoprotein, particles either free or in Golgi apparatus derivatives, in particular secretory granules. The sequestered materials show ultrastructural signs of ongoing degradation. In addition to containing typical autophagic vacuoles, the isolated fractions consist of lysosomes lacking morphologically recognizable cellular components. Contamination from nonlysosomal material is only a few percent as judged from morphometric analysis. Typical lysosomal "marker" enzymes are enriched 15-fold, whereas the proteolytic activity is enriched 10- to 20-fold in the isolated AV fraction as compared to the homogenate. Initially, the yield of nonlysosomal mitochondrial and microsomal enzyme activities increases in parallel with the induction of autophagy but, later on, decreases with advanced degradation of the sequestered cell organelles. Therefore, in the case of AVs the presence of nonlysosomal marker enzymes cannot be used for calculation of fraction purity, since newly sequestered organelles are enzymatically active. Isolated autophagic vacuoles show proteolytic activity when incubated in vitro. The comparatively high phospholipid/protein ratio (0.5) of the AV fraction suggests that phospholipids are degraded more slow than proteins. Is it concluded that AVs can be isolated into a pure fraction and are the subcellular site of enhanced protein degradation in the rat liver after induction of autophagy.

Isolation of liver lysosomes by iron loading. Ultrastructural characterization.

In summary, the data demonstrate that, by the use of repeated injections of an iron sorbitol complex, it is possible to isolate a fraction highly enriched in hydrolytic enzymes (60 times over the homogenate) and in well preserved lysosomes emanating almost entirely from liver parenchymal cells. The advantage of adding fixative to the bottom of the gradient and of using en bloc staining with uranyl acetate is also demonstrated.

Acetaminophen: potentially toxic metabolite formed by human fetal and adult liver microsomes and isolated fetal liver cells.

A reactive metabolite of acetaminophen is hepatotoxic in humans when the drug is ingested in large overdoses. The ability of the human fetal and adult liver to oxidize acetaminophen by trapping the potentially toxic metabolite as a glutathione conjugate has been measured. Oxidation by fetal liver was approximately ten times slower than by adult liver. However, there was a definite increase in acetaminophen oxidation with fetal age. Isolated human fetal liver cells conjugated acetaminophen with sulfate but not with glucuronic acid. The results indicate that the human fetal liver is able to detoxify acetaminophen by conjugation. However, it also catalyzes the formation of an active metabolite of acetaminophen through oxidation. Hence the fetus remains at risk should a large dose of the drug cross into the fetal circulation.

Lower-than-standard dose peg-IFN alfa-2a for chronic hepatitis C caused by genotype 2 and 3 is sufficient when given in combination with weight-based ribavirin.

Mono-therapy with pegylated interferon (peg-IFN) has shown that a lower-than-standard dose yields the same sustained viral response (SVR) rates as standard doses for chronic hepatitis C virus (HCV) infection caused by genotypes 2 or 3. Our aim was to see if a fixed, lower-than-standard dose of peg-IFN alfa-2a (135 microg weekly) in combination with ribavirin 11 mg/kg daily for 24 weeks yields sufficient SVR rates for genotypes 2 or 3. Hundred consecutive patients with a mean age of 44 years (range 20-69 years), 59 with genotype 3 and 41 with genotype 2, were studied. Rapid viral response (RVR) with HCV-RNA <15 IU/mL at treatment week 4 and SVR were calculated. RVR was achieved by 28/40 (70%) patients with genotype 2 and 41/58 (71%) with genotype 3. Significantly more genotype 2 patients with RVR achieved SVR 27/28 (96%) than genotype 2 patients who failed to achieve RVR, 8/12 (66%), P = 0.009. The corresponding figures for genotype 3 patients were 39/41 (95%) vs 11/17 (65%), respectively, P = 0.002. In total, SVR was achieved by 35/41 (85%) patients with genotype 2 and 51/59 (86%) patients with genotype 3, respectively. We found that 135 microg peg-IFN alfa-2a weekly was sufficient for treatment of genotype 2 and 3 chronic hepatitis C when combined with RBV dosed daily according to body weight. This combination yielded high SVR rates (85-86%) and may be cost-saving.

The hepatitis C virus and immune evasion: non-structural 3/4A transgenic mice are resistant to lethal tumour necrosis factor alpha mediated liver disease.

BACKGROUND: The hepatitis C virus (HCV) establishes chronic infection by incompletely understood mechanisms. The non-structural (NS) 3/4A protease/helicase has been proposed as a key complex in modulating the infected hepatocyte, although nothing is known about the effects this complex exerts in vivo. AIM: To generate mice with stable and transient hepatocyte expression of the HCV NS3/4A proteins to study its effects in vivo. METHODS: NS3/4A expression was determined by western blot and immunohistochemistry. Two independent pathologists determined the liver histology. Hepatic immunity was characterised by quantifying intrahepatic immune cell subsets. Liver damage was induced using carbon tetrachloride (CCl(4)), lipopolysaccaride (LPS), tumour necrosis factor alpha (TNFalpha), and anti-Fas antibody. RESULTS: Expression of NS3/4A was restricted to the cytoplasm of hepatocytes, and did not cause liver cancer or any spontaneous liver pathology. However, the presence of NS3/4A modulated the intrahepatic immunity, as follows: first, the CD4+ T cell and type I/II dendritic cell subsets were reduced in transgenic livers; second, NS3/4A protected hepatocytes from liver damage mediated in vivo by CCl(4), LPS, TNFalpha, but not FAS; and third, both stable and transiently NS3/4A transgenic mice were resistant to lethal doses of liver targeted TNFalpha, and the resistance could be reverted by treatment with a p38 mitogen activated protein kinase inhibitor (MAPK). CONCLUSIONS: Hepatic expression of NS3/4A does not induce spontaneous liver disease. NS3/4A does, however, alter the intrahepatic immune cell subsets and protects hepatocytes against TNFalpha induced liver damage in vivo. The TNFalpha resistance can be reverted by treatment with a p38 MAPK inhibitor. This represents a new immune evasion strategy conferred by NS3/4A.

Immunochemical detection and quantitation of microsomal cytochrome P-450 and reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase in the rat ventral prostate.

Treatment with beta-naphthoflavone (BNF) was found to induce 7-ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities about 500-fold in the microsomal fraction of the rat ventral prostate but had no effect on aminopyrine N-demethylase or reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activities. Phenobarbital (PB) treatment did not alter any of these enzyme activities. Antibodies raised in rabbits against rat liver cytochrome P-450 reductase (P-450 reductase) and against P-450 BNF-B2 and P-450 PB-B2, the major forms of P-450 isolated from liver microsomes of BNF- and PB-treated rats, respectively, were used to characterize the P-450-dependent monooxygenase system in the rat ventral prostate. Anti-P-450 reductase immunoglobulin G inhibited reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activity in prostatic microsomes, and anti-P-450 BNF-B2 but not anti-P-450 PB-B2 immunoglobulin G inhibited the BNF-induced prostatic microsomal 7-ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities. A highly sensitive immunoblotting method was used to quantitate P-450 BNF-B2, P-450 PB-B2, and P-450 reductase in prostatic microsomes. Using this technique, prostatic P-450 reductase with a molecular weight corresponding to that of purified liver P-450 reductase was detected at a level of 0.02 nmol/mg of microsomal protein. In the liver, the same enzyme amounts to 0.2 nmol/mg of microsomal protein. P-450 BNF-B2 was not detected in prostatic microsomes from control or PB-treated rats, whereas a protein band with a molecular weight corresponding to that of purified liver P-450 BNF-B2 was found in prostatic microsomes from BNF-treated rats at a level of 0.05 nmol P-450 per mg microsomal protein. P-450 PB-B2 was not detected in prostatic microsomes from either control, PB-treated, or BNF-treated animals.

Immunohistochemical localization of cytochrome P-450 and reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase in the rat ventral prostate.

Rabbit antibodies raised against the major isozymes of cytochrome P-450 isolated from hepatic microsomes of beta-naphthoflavone- (BNF) and phenobarbital-treated rats (cytochrome P-450 BNF-B2 and cytochrome P-450 PB-B2, respectively) and against rat liver NADPH-cytochrome P-450 reductase were used to localize these enzymes immunohistochemically in the rat ventral prostate. Using the unlabeled antibody peroxidase-antiperoxidase technique, NADPH-cytochrome P-450 reductase was detected exclusively in the epithelial cells of the gland to the same magnitude in untreated, phenobarbital-, and BNF-treated rats. Cytochrome P-450 BNF-B2-like immunoreactivity was exclusively present in the glandular epithelium in BNF-treated rats, whereas staining could not be visualized in untreated or in phenobarbital-treated rats. The staining for NADPH-cytochrome P-450 reductase was more uniformly distributed within the epithelium than was the cytochrome P-450 BNF-B2-like immunoreactivity. Cytochrome P-450 PB-B2-like immunoreactivity was not found, regardless of animal pretreatment. These findings support our previous results (Haaparanta, T., Halpert, J., Glaumann, H., and Gustafsson, J-A., Cancer Res. 43: 5131-5137, 1983) demonstrating the presence of constitutive NADPH-cytochrome P-450 reductase in the prostate and that an isozyme of cytochrome P-450 is highly inducible by BNF in this gland. The significance of these findings are discussed in view of the essentially unknown etiology of human prostatic cancer.

Incorporation of purified components of the rabbit liver microsomal hydroxylase system into phospholipid vesicles.

Methods are described for incorporation of purified forms of rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450LM2, P-450LM3 and P-450LM4 (LM, liver microsomes) into phospholipid vesicles. It was found that each cytochrome could individually be incorporated into preformed phospholipid vesicles in the absence of cholate. However, NADPH-cytochrome P-450 reductase prevented incorporation of P-450 by this method, a phenomenon possibly inherent in the formation of complexes between P-450 and the reductase in solution. Using the cholate-gel filtration technique it was possible to prepare monolamellar phosphatidylcholine vesicles containing any of the cytochromes and P-450 reductase in good yields. It was found that P-450LM3-containing vesicles had a mean diameter of 47 nm, whereas vesicles formed under the same conditions but containing P-450LM4 were much smaller (mean diameter 33 nm). Vesicles formed with P-450LM2 were homogeneous in density (1.04 g/cm3) according to isopycnic centrifugation in Ficoll but not in size (44-72 nm). These findings, taken together with results obtained from treatment of the cytochromes in soluble form and in reconstituted vesicles with the non-penetrating reagent, p-diazobenzene sulphonate, indicate a unidirectional, relatively peripheral orientation of P-450LM4 with the major part localized on the outside of the vesicles. Experiments with trypsin and cytochrome c-reduction demonstrated a unidirectional orientation of P-450 reductase towards the outside of the vesicles.

Fractionation of the rat ventral prostate with respect to isolation and exocytosis of the prostatic secretion protein.

The ventral prostate was fractionated into one mitochondrial and three microsomal fractions. The different fractions were characterized morphologically and chemically. An interesting finding was that upon homogenization the endoplasmic reticulum membranes often turned 'inside-out' giving rise to microsomes with ribosomes attached to the inside of the vesicles. The secretion of the prostatic secretion protein was studied by means of isotopic pulse labeling using radioactive leucine. Peak radioactivity in the microsomal fraction was obtained at 2 h after injection with a relatively rapid fall. The radioactivity in the secretory fluid displayed a continuous increase up to 8 h followed by a plateau. When prostatic secretion protein was purified from secretory fluid and microsomes using a Con A-Sepharose column it showed a typical precursor-product relationship with an early peak at 60 min in microsomal prostatic secretion protein followed by a peak in secretory fluid at 4 h. Vinblastine blocked the release of labeled secretion protein into the secretory fluid, a phenomenon characteristic for secretory proteins which are exocytosed by means of fusion between secretory granules and the plasma membrane. Following intravenous injection of [3H]estramustine, accumulation was seen in the secretory fluid. Some estramustine probably binds to newly synthesized prostatic secretion protein and follows the same route of intracellular transport and extracellular discharge as does prostatic secretion protein.

Unspecific and sequence-specific deoxyribonucleic acid binding of the partially purified human progesterone receptor.

The progesterone receptor (PR) was partially purified from T47D human breast cancer cells by sequential chromatography on phosphocellulose, heparin-Sepharose, and DNA-cellulose. Heparin-Sepharose chromatography resulted in an efficient conversion of the receptor to a DNA-binding form (activation) since more than 85% of the 3H-R5020 labeled eluate from heparin-Sepharose was retained on DNA-cellulose and since the cytosolic 8S receptor was converted to a 4S moiety after chromatography on heparin-Sepharose. The 3H-R5020 labeled human PR eluted from DNA-cellulose as a single symmetrical peak at 0.2 M NaCl; after photoaffinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this species was shown to consist of about equal amounts of two proteins of Mr approximately equal to 96,000 and 120,000 (the so called A- and B-subunits, respectively). This partially purified receptor preparation (SA 490 pmol/mg protein) did not contain any glucocorticoid receptor (GR) as shown by immunoblotting with a monoclonal antirat GR antibody that cross-reacts with the human GR. Therefore, this preparation was used to compare the specific DNA-binding properties of the human PR with those of the purified rat GR. The human PR bound specifically to the promoter region of mouse mammary tumor virus (MMTV) at a molar ratio between receptor and DNA similar to the molar ratio between GR and DNA needed for binding of rat GR to MMTV, indicating that the PR was purified in a biologically active form.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal regulation of estrogen receptor messenger ribonucleic acid in T47Dco and MCF-7 breast cancer cells.

Using a combination of hormone-binding assays, immunologic techniques, and mRNA hybridizations we have measured the estrogen receptor (ER) content and studied the hormonal regulation of ER mRNA in one estrogen responsive and one estrogen unresponsive breast cancer cell line, MCF-7 and T47Dco, respectively. Estradiol binding could be detected in cytosol from MCF-7 cells but not in T47Dco cells. However, when measured by an enzyme-linked immunosorbent assay, T47Dco cells were found to contain approximately 15 fmol ER/mg cytosolic protein or 10% of the ER content in MCF-7 cells. Immunologically reactive ER in T47Dco cells was indistinguishable in size (approximately equal to 68 KD) from the ER in MCF-7 cells, as shown by Western blotting using a monoclonal antihuman ER antibody. Quantification of ER mRNA in MCF-7 and T47Dco cells indicated that T47Dco cells contained approximately 50% of the ER mRNA levels found in MCF-7 cells. This basal level of ER mRNA in T47Dco cells was not decreased by estradiol treatment, as opposed to in MCF-7 cells where estradiol caused 40-60% decrease in the ER mRNA expression. Also, estradiol did not increase the progesterone receptor (PR) mRNA levels in T47Dco cells whereas in MCF-7 cells an approximately 5-fold increase of the PR mRNA levels occurred after estradiol treatment. However, incubation of the cells with the synthetic progestin R5020 decreased the ER mRNA levels to approximately the same degree in both cell lines. In conclusion, we have shown that estrogen down-regulates ER mRNA and up-regulates PR mRNA in MCF-7 cells. Neither of these estrogenic effects were seen in T47Dco cells. It appears that the steroid-resistance in T47Dco cells does not occur as a consequence of a complete absence of ER mRNA or protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term follow-up of successful hepatitis C virus therapy: waning immune responses and disappearance of liver disease are consistent with cure.

BACKGROUND: A sustained viral response (SVR) after interferon-based therapy of chronic hepatitis C virus (HCV) infection is regarded to represent a cure. Previous studies have used different markers to clarify whether an SVR truly represents a cure, but no study has combined a clinical work-up with highly sensitive HCV RNA detection, and the determination of immune responses. AIM: To determine clinical, histological, virological and immunological markers 5-20 years after SVR. METHODS: In 54 patients, liver biochemistry, histology and elastography were evaluated. Liver biopsies, plasma and peripheral blood mononuclear cells (PBMCs) were tested for minute amounts of HCV RNA. HCV-specific T-cell responses were monitored by ELISpot and pentamer staining, and humoral responses by measuring HCV nonstructural (NS)3-specific antibodies and virus neutralisation. RESULTS: Liver disease regressed significantly in all patients, and 51 were HCV RNA-negative in all tissues tested. There was an inverse association between liver disease, HCV-specific T-cell responses and HCV antibody levels with time from SVR, supporting that the virus had been cleared. The three patients, who all lacked signs of liver disease, had HCV RNA in PBMCs 5-9 years after SVR. All three had HCV-specific T cells and NS3 antibodies, but no cross-neutralising antibodies. CONCLUSIONS: Our combined data confirm that a SVR corresponds to a long-term clinical cure. The waning immune responses support the disappearance of the antigenic stimulus. Transient HCV RNA traces may be detected in some patients up to 9 years after SVR, but no marker associates this with an increased risk for liver disease.

Characterization and endocrine regulation of the cytochrome P-450 dependent microsomal hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol in the rat ventral prostate.

Of 11 structurally related steroids, 5 alpha-androstane-3 beta,17 beta-diol was the one most efficiently hydroxylated to polar metabolites by microsomes isolated from the rat ventral prostate. The metabolites were identified by gas chromatography-mass spectrometry as 6 alpha-,7 alpha-, and presumably 6 beta-hydroxylated 5 alpha-androstane-3 beta,17 beta-diol. The hydroxylated metabolites were separated and quantitated by high performance liquid chromatography using an aminopropylsilane hypersil column. Maximum velocity (V max) values of 0.36, 0.12, and 0.044 nmol/min X mg of microsomal protein were determined for the 6 alpha-,7 alpha-, and 6 beta-hydroxylase activities, respectively, with an apparent Michaelis-Menten constant (Km) of 30 microM for all hydroxylases. Hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol in prostatic microsomes was NADPH dependent and was inhibited by several cytochrome P-450 inhibitors in vitro and by antibodies against rat liver NADPH-cytochrome P-450 reductase. The enzyme activities were stimulated by treatment of the rats with phenobarbital, beta-naphthoflavone, 5 alpha-androstane-3 beta,17 beta-diol,5 alpha-dihydrotestosterone, and methyltrienolone, whereas the activities were suppressed by ovine PRL, human GH, estradiol benzoate, cholesterol, andrenalectomy , hypophysectomy, and castration. It is concluded that the prostatic 5 alpha-androstane-3 beta,17 beta-diol hydroxylase activities represent a constitutive cytochrome P-450 form in the rat ventral prostate with a high steroid substrate specificity. Endocrine factors are involved in the regulation of the activity of the enzyme. The ratio between the three hydroxylated metabolites was constant in almost all experiments suggesting that only one cytochrome P-450 species was involved in the hydroxylation.

Ontogeny of a human polychlorinated biphenyl-binding protein. Level of expression in tracheal aspirates in bronchopulmonary dysplasia.

A human lung polychlorinated biphenyl binding protein (PCB-BP,M(r) 13 kd) has recently been purified from lavage fluid. Polyclonal monospecific antibodies against PCB-BP were produced and used for immunohistochemical staining in sections of human lung tissue. PCB-BP was found to localize preferentially to the nonciliated (Clara) cells of the lung, whereas the alveolar cells and ciliated cells of the larger airways were devoid of staining. Tracheal aspirates from infants receiving mechanical ventilation were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and Western immunoblotting. The antibodies to human PCB-BP detected a single band of the expected molecular weight, and a quantitative analysis of the ontogeny of PCB-BP in tracheal aspirates was performed by construction of Western immunoblot standard curves. A significant increase in the levels of PCB-BP in late gestation (gestational weeks 39 to 41) was demonstrated. In similar experiments, levels of PCB-BP in tracheal aspirates obtained from infants with bronchopulmonary dysplasia (BPD) at the postconceptional age of less than 38 weeks were found to be significantly elevated as compared with a normal study group of similar gestational age (21.8 +/- 4.8 vs 3.1 +/- 0.8 ng of PCB-BP per microgram of total protein, p < 0.005). It is suggested that the high levels of PCB-BP at the postconceptional age of less than 38 weeks observed in infants with BPD may reflect inflammatory injury and regeneration of airway epithelium, associated with proliferation of Clara cells.

Induction of cytochrome P-450 dependent reactions in the rat ventral prostate by beta-naphthoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The purpose of this study was to characterize drug metabolism in the rat ventral prostate in untreated, beta-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated animals. Intraperitoneally administered [3H]benzo[a]pyrene (BP) was recovered in about equal quantities in rat ventral prostate, lungs and testes. The relatively high uptake of [3H]BP in the ventral prostate might possibly be related to the occurrence of a macromolecule in the gland binding TCDD and 3-methylcholanthrene (MC) with a relatively high affinity (Kd 1.5 and 5.0 microM, respectively). BNF, BP and MC competed with the TCDD binding site of the macromolecule. The prostatic cytochrome P-450 dependent microsomal enzyme activities aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase are very low in untreated animals (about 1 pmol/min/mg of microsomal protein). BNF- or TCDD treatment of the animals induced these activities about 500-fold, whereas aminopyrine N-demethylase was enhanced to a smaller degree. Maximal enzyme activities of AHH and 7-ethoxyresorufin O-deethylase were obtained 24 h after a single intraperitoneal injection of BNF and the activities decayed relatively rapidly after this peak. The magnitude of enzyme induction was much higher in the prostate than in the liver. Mutagenic BP metabolites were formed in vitro by induced prostatic microsomes as revealed by HPLC analysis. The activity of NADPH-cytochrome c reductase was 35 pmol/min/mg and was not changed by BNF- or TCDD treatment.

Induction of cytochrome P-450 in the rat liver after exposure to xylenes, dose-response relationship and dependence on endocrine factors.

Male rats were exposed to different concentrations of xylenes for 3 days. Small but statistically significant increases in cytochrome P-450 content, NADPH-cytochrome c reductase activity and O-deethylation of 7-ethoxyresorufin in liver microsomes were detected already at an exposure level of 75 ppm. Morphological studies of livers from rats exposed to relatively high concentrations of xylenes for 5 days showed marked proliferation of smooth ER with little changes of the rough ER. No pathological alterations were observed. Castration of male rats influenced the response to xylene exposure only to a minor extent. Hypophysectomy alone was shown to cause significant increases in cytochrome P-450 and cytochrome b5 content and epoxide hydrolase activity. Induction of cytochrome P-450 dependent enzymatic activities after exposure to xylenes was reduced but qualitatively similar to that obtained with normal male rats whereas the induction of epoxide hydrolase activity was prevented. The difference in response to exposure to xylenes between male and female rats was mainly quantitative in nature. Polyacrylamide gel electrophoresis of liver microsomes from animals exposed to xylenes in the presence of SDS showed increases in protein bands comigrating with cytochrome P-450 PB-B2 and epoxide hydrolase purified from phenobarbital treated rats. It is concluded that in the rat exposure to xylenes mimics the effects of phenobarbital treatment as judged by both biochemical and morphological criteria and that the pituitary seems to have a regulatory function with regard to the induction of several drug-metabolizing enzymes.

Polychlorinated terphenyls: alterations in liver morphology and induction of cytochrome P-450.

Exposure of male rats to the polychlorinated terphenyl (PCT) mixtures Aroclor 5460 and Aroclor 5432 containing 60% and 32% (w/w) of chlorine, respectively, showed that the PCT mixture with a low degree of chlorination, Aroclor 5432, was a potent inducer of liver microsomal cytochrome P-450, aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase whereas Aroclor 5460 and the unchlorinated isomers o-, m- and p-terphenyl were weak inducers. Ultrastructurally, proliferation of SER but not RER, frequent occurrence of lysosomes containing partially degraded lipid material as well as an increased number and size of cytoplasmic lipid droplets were observed. These changes were most pronounced in Aroclor 5432 treated rats. Competition experiments with [3H]2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD) for binding to the cytosolic receptor protein indicated the presence in Aroclor 5432, but not in Aroclor 5460, of components with receptor affinity. These components represent only a minor fraction of the mixture as judged by the 1900-fold excess necessary to displace 50% of the specific [3H]TCDD binding. Based on the biochemical results and the ultrastructural findings it is concluded that the PCT mixture Aroclor 5432 is a mixed type inducer of hepatic cytochrome P-450 in the rat. The presence in Aroclor 5432 of compounds capable of inducing AHH in vivo and of binding to the TCDD-receptor might be highly relevant with regard to the potential toxicity in view of the apparent correlation between affinity for the TCDD-receptor, induction of AHH activity and toxic properties for chlorinated aromatic hydrocarbons.