Phenobarbital pretreatment alters the localization of CCl4-induced changes in rat liver microsomal fatty acids.

Phenobarbital treatment induces an isozyme(s) of liver microsomal cytochrome P450 susceptible to CCl4 and enhances the latter's lethality. We have now studied phenobarbital's effect on the specificity of phosphatidyl fatty acid changes in rat liver microsomes. Male Sprague-Dawley rats were pretreated with three daily ip doses of phenobarbital (50 mg/kg) or saline and then orally dosed with CCl4 (2.5 ml/kg). Liver microsomes were prepared 7.5 to 180 min after CCl4 treatment, the lipid fraction was extracted, diene conjugate content was determined, and phospholipids were separated by HPLC for fatty acid content determination. Protein, phospholipid, and phosphatidyl fatty acid residue loss occurred early (7.5 to 30 min) and in some cases later (60 to 180 min) in both pretreated groups, suggesting that two phases of CCl4-mediated injury occurred. Phenobarbital pretreatment accelerated the CCl4-induced formation of diene conjugates in the microsomal lipids. In studies on the separated phospholipids, phenobarbital alone altered microsomal fatty acid content, primarily decreasing arachidonic acid in favor of linoleate, particularly in phosphatidylserine. During the early phase of CCl4 injury, phenobarbital pretreatment shifted the major loss of arachidonic acid from phosphatidylserine to phosphatidylethanolamine. During the later phase, arachidonic acid loss was still prominent, but the most extensive CCl4-induced changes in fatty acids occurred in the neutral lipid fraction, regardless of pretreatment. These changes included loss of neutral lipid linoleic and docosahexanoic acids associated with an increase in palmitic acid. These data demonstrate that phenobarbital pretreatment is associated with a shift in the predominant phospholipid locus from phosphatidylserine to phosphatidylethanolamine for the early CCl4-induced fatty acid changes in rat liver microsomes.

Sodium cholate extraction of rat liver nuclear xenobiotic-metabolizing enzymes.

DNA is the purported target of several carcinogenic and mutagenic agents. Nuclear enzymes which could generate or detoxify reactive metabolites are of major concern. Several such enzymes have been identified within nuclei, but obtaining samples with enriched content or activity is difficult, time-consuming, and uses harsh isolation techniques. Extraction of rat liver nuclear suspensions with cholate-containing buffer results in solubilization of 25-30% of the protein. Linear extraction was obtained for total protein and cytochromes P-450 and b5, NADPH-cytochrome P-450 reductase, NADH-cytochrome b5 reductase, DT-diaphorase, and microsomal-like epoxide hydrolase with specific activities comparable to values reported for isolated nuclear membrane, while the yield was five to ten times greater. Detergent extracts of rat liver nuclei were employed to study the comparative response of microsomal and nuclear enzymes to chemical treatment. While the responses to acute inductive (phenobarbital and 3-methylcholanthrene) and toxic (carbon tetrachloride and dibromochloropropane) treatments were qualitatively similar, an initiation-promotion protocol (diethylnitrosamine with phenobarbital promotion) resulted in divergent responses between the enzymes in the two subcellular fractions. Detergent extracts of nuclei offer an efficient means of recovering xenobiotic-metabolizing enzymes from rat liver nuclei, and have been utilized to demonstrate a differential response of nuclear enzymes during preneoplastic development.

Differential inhibition of indirect immunofluorescence in rat liver sections incubated with antibodies against microsomal cytochromes P-450a, b, and c and epoxide hydrolase.

Rat liver sections were incubated with antibodies (100-1000 micrograms IgG/ml) against microsomal cytochromes P-450a, P-450b, and P-450c, and epoxide hydrolase. Inhibition of indirect immunofluorescence, which progressed with higher concentrations of primary antibody, corresponded with antigen-enriched tissue in frozen liver sections from male and female rats. It was found in liver sections from phenobarbital-treated rats incubated with anti-P-450b and anti-epoxide hydrolase and from 3-methylcholanthrene-treated rats incubated with anti-P-450c. No inhibition was found in sections from untreated rats or rats receiving treatments that did not induce the specific antigen. No inhibition was found in sections incubated with anti-P-450a. Inhibition of immunofluorescence was abolished in frozen sections subjected to dehydration-rehydration protocols known to extract antigens, and was prevented by certain solvents and detergent-wash. Inhibition of immunofluorescence provides a unique method for confirming the antigen-rich regions of the liver lobules specific for microsomal expoxide hydrolase and the cytochrome P-450s.

An ultrastructural and histochemical study of the prominent inflammatory response in D(+)-galactosamine hepatotoxicity.

The biochemical basis of the hepatitis-like liver injury produced by D(+)-galactosamine in rats is well-established and is linked to depletion of uridine nucleotides within parenchymal cells. However, the prominent inflammatory response that accompanies this lesion in vivo has been largely overlooked as a component of the hepatic damage. This study examines the cellular components of the inflammatory infiltrate of galactosamine-induced liver injury over time using histochemical and ultrastructural techniques. By 12 h after toxin administration, the infiltrate consisted largely of neutrophils and recently-mobilized monocytes. By 24 to 48 h after the toxin, when hepatocellular necrosis was maximal, few neutrophils were found in the infiltrate. At these times, the infiltrate consisted almost exclusively of large phagocytic cells, histochemically and morphologically consistent with active tissue macrophages apparently derived from circulating monocytes. The extent of the inflammatory response to this experimental hepatotoxin suggests that effects on the generation and development of the inflammatory response should be considered for treatments reported to alter the intrinsic hepatotoxicity of galactosamine.

Isolation and maintenance of monolayer hepatocytes from the livers of toxin-treated rats.

Hepatocytes were isolated from male Sprague-Dawley rats 30 min after oral challenge with carbon tetrachloride (CCl4), or 1 hr after ip injection of D(+)-galactosamine (GAL). Cell preparations of comparable yield and initial viability were obtained from toxin-treated and respective control animals with equivalent 1-hr attachment efficiencies to tissue culture plastic. Cells from toxin-treated rats exhibited significant degeneration over 48 hr in culture. This degeneration included loss of viable cell density on the monolayer and increased lactate dehydrogenase activity in the culture media compared to controls. Toxicity expressed in culture was dose dependent for both CCl4 (0.1-2.5 ml/kg) and GAL (100-400 mg/kg). Loss of cell viability in vitro and ultrastructural degeneration followed a time course consistent with hepatocellular necrosis produced by these agents in vivo. As a model for studying late occurring events in the progression from initiation of toxic cell injury to cell death, this methodology offers several potential advantages over strict in vivo or in vitro models. The analytical advantages of an in vitro cell model are incorporated with initiation of injury in vivo by physiologically relevant doses of hepatotoxins. Limited bioactivating potential of monolayer hepatocytes, and time course limitations of suspension hepatocytes for toxicity studies, are also circumvented in this model.

Whipple's disease confined to the central nervous system.

Progressive hypersomnia, memory disturbance, and vertical ophthalmoplegia developed in a 63-year-old woman. The diagnosis of Whipple's disease of the central nervous system was suggested by her presentation and results of studies using magnetic resonance imaging. Despite a one-month course of antibiotics, active Whipple's disease, localized to the central nervous system, was found at autopsy.

The major nucleoside triphosphatase of nuclear scaffold is distinct from actin.

The major nucleoside triphosphatase of rat liver nuclear scaffold, a 46 kD protein thought to participate in nucleocytoplasmic RNA translocation, is distinct from immunologically-identified scaffold actin on Western blots, has a substantially different amino acid composition, and its enzymatic activity is not affected by anti-actin antibodies. Thus, although the contractile protein actin is found in nuclear scaffold and appears to interact with RNA, it is not associated with the nucleoside triphosphatase activity in such preparations.

Immunohistochemical evidence for alterations in specific forms of rat hepatic microsomal cytochrome P-450 during acute carbon tetrachloride intoxication.

The response of specific forms of hepatic microsomal cytochrome P-450 to carbon tetrachloride was studied by immunohistochemical techniques in order to assess the localization and specificity of action for this hepatotoxin. Liver sections were taken from control, phenobarbital-pretreated, and 3-methylcholanthrene-pretreated male rats that had received acute (3 hr) treatment with carbon tetrachloride. The liver sections were examined after indirect immunofluorescent labeling with anti-P-450un, anti-P-450a, anti-P-450b, and anti-P-450c. Diminished fluorescence and loss of intracellular homogeneity of fluorescence were found in the centrilobular cells of liver sections from control rats incubated with anti-P-450un, anti-P-450a, and phenobarbital-pretreated, carbon tetrachloride-challenged rats incubated with anti-P-450b. In liver sections from 3-methylcholanthrene-pretreated, carbon tetrachloride-challenged rats incubated with anti-P-450c, fluorescence was diminished in periportal cells but did not show loss of intracellular homogeneity. These results provide immunohistochemical evidence for the differential effect of carbon tetrachloride on specific forms of cytochrome P-450.

Disturbances in hepatic heme metabolism in rats administered alkyl halides.

Liver tissue from rats administered 13 different alkyl halides and 4 other hepatotoxins were assayed for indices of hepatic heme synthesis. These included aminolevulinic acid (ALA)-dehydratase activity, porphyrin content, microsomal cytochrome P-450 and reduced glutathione content. Consistent decreases in ALA-dehydratase activity (14 of 17 compounds) and cytochrome P-450 content (14 of 17 compounds) were found. Significant changes in glutathione and porphyrin content also occurred after exposure to some compounds, but were not consistent. Alkyl halides are a class of toxicologically important compounds which have been found to exert an influence on hepatic heme synthesis.

Human prion protein cDNA: molecular cloning, chromosomal mapping, and biological implications.

A human complementary DNA whose protein product is considered to be the major component of scrapie-associated fibrils in Creutzfeldt-Jakob disease, kuru, and Gerstmann-Straussler syndrome has been identified and characterized. The extensive homology of this gene sequence to the hamster PrP 27- to 30-kilodalton prion protein complementary DNA clone, and its existence as a single copy in the human genome, leads to the conclusion that this is the human prion gene. This human prion gene has been mapped to human chromosome 20, negating a direct link between the prion protein and Down's syndrome or the amyloid of Alzheimer's disease.

NADPH-dependent and -independent loss of cytochrome P-450 in control and phenobarbital-induced rat hepatic microsomes incubated with carbon tetrachloride.

Carbon tetrachloride-mediated loss of cytochrome P-450 has been compared in hepatic microsomes from untreated and phenobarbital-treated rats. At concentrations of carbon tetrachloride greater than 2.5 mM, a direct effect (i.e., NADPH- independent) on cytochrome P-450 was observed. This apparently arose from the "solvent" properties of carbon tetrachloride as this effect could be duplicated with the physically similar alkyl halide 1,2-dibromo-3-chloropropane. NADPH-dependent loss of cytochrome P-450 occurred at lower concentrations with maximal response occurring at 2.5-5.0 mM. Residual cytochrome P-450 at these concentrations was similar in untreated and phenobarbital-treated microsomes. Mixed-function oxidase activities in phenobarbital-treated microsomes were reduced to levels below those of uninduced controls. The 52-kDa polypeptide(s) in untreated microsomes and that specifically induced in phenobarbital-treated microsomes were susceptible to NADPH-dependent carbon tetrachloride incubation. These data suggest that the susceptibility of specific forms of cytochrome P-450 to carbon tetrachloride can be duplicated in in vitro incubation. Furthermore, data on the direct action of carbon tetrachloride suggest that this route of damage must be taken into consideration when concentrations of carbon tetrachloride of 2.5 mM or greater are used in in vitro incubation systems.

Cytoplasmic RNA complementary to pre-albumin intron V.

We have detected an RNA species, containing sequences complementary to pre-albumin intron V in poly(A)+ RNA from rat liver cytoplasm (but not in nuclear RNA). Its relative abundance roughly parallels that of mature albumin mRNA, when comparing control with acute phase preparations.

Alterations in hepatocyte plasma membrane in carbon tetrachloride poisoning. Freeze-fracture analysis of gap junction and electron spin resonance analysis of lipid fluidity.

The plasmalemma of the livers of rats treated with carbon tetrachloride (CCl4) were examined by freeze-fracture and electron spin resonance probe techniques. The rodents received by mouth either mineral oil alone (0 to 4.5 hours before sacrifice) or CCl4 in mineral oil (1:1) (2.5 ml of CCl4/kg, 0 to 3 hours before sacrifice). Rats were anesthetized with ether and livers were perfused in situ with saline either at ambient temperature or at 4 degrees C. After perfusion, livers were fixed in situ and processed for freeze-fracture and electron microscopy. Hepatocytes were isolated and incubated with 12-doxylstearic acid and subjected to electron spin resonance analysis. Electron microscopy revealed greater than a 2.5-fold increase in the individual mean gap junction size when rats were treated with mineral oil alone for 4.5 hours and the livers were processed at room temperature. The mean gap junction size in rats dosed with CCl4 for 0.5 hours before sacrifice equalled those of the group treated with mineral oil for 4.5 hours. Increases in gap junction size with CCl4 were progressive with time; by 3 hours, a 3.5-fold increase over controls was observed (p less than 0.05). When livers were perfused with iced saline, rats treated with mineral oil for 1.5 hours had a slight decrease (not significant) in mean gap junction size as compared to controls, while the size in rats treated for the same amount of time with CCl4 increased almost 5-fold over controls (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Is enflurane hepatotoxic?

We evaluated 88 cases of hepatic injury that followed, and were attributed to, enflurane anesthesia. In 30 of the cases, data were insufficient to assess the role of enflurane vs other variables as causal factors. In 43 ("unlikely") patients, factors known to produce hepatic injury were clearly present; in the remaining 15 ("possible"), such factors were not evident. No consistent pathologic change was found in liver specimens from either the unlikely or the possible group of cases. A syndrome suggested to be associated with enflurane-induced hepatic injury was present in both groups and did not differ between groups. We conclude that the data do not demonstrate a causal relationship between enflurane anesthesia and subsequent liver injury and that if severe injury is caused by enflurane anesthesia, it is an extremely rare event.

In vitro release of alpha 1-acid glycoprotein RNA sequences shows fidelity with the acute phase response in vivo.

The acute phase reaction of rat liver to subcutaneous turpentine challenge results in a 20- to 100-fold increase in alpha 1-acid glycoprotein (alpha AGP) mRNA. We utilized this response to establish conditions appropriate for study of RNA transport in vitro using hybridization with 32P-labeled exon and intron alpha AGP sequences. Contamination of nuclear preparations by membrane-absorbed cytoplasmic RNA was eliminated by detergent-rinsing. The in vitro incubation conditions that most reflected the in vivo state required RNase inhibitor (purified from placenta), polyvinylpyrrolidone to prevent nuclear swelling, and addition of ATP. Under these circumstances, alpha AGP sequences were transported only from turpentine-stimulated preparations, were found only in poly(A)+ RNA, and were the same size as authentic cytoplasmic mRNA. Omission of polyvinylpyrrolidone resulted in release of some alpha AGP sequences in smaller, more heterogeneous poly(A)- RNA, and leakage of some alpha AGP sequences was observed from control preparations. Omission of ATP resulted in restriction of mature alpha AGP mRNA to the nucleus. In contrast to alpha AGP mRNA, transport of albumin mRNA was decreased 3-4X in turpentine-treated preparations. The largest alpha AGP intron was not found in RNA transported from treated nuclei in complete medium. The intron-containing fragments remained in the nucleus, largely in poly(A)- RNA of a size consistent with free intron. Some hybridization of intron sequences was observed with cytoplasmic and nuclear membrane-associated poly(A)+ RNA preparations which may represent 3'-processing catabolites; leakage of these sequences was considerably greater in the absence of PVP. On the basis of densitometric estimates, a 5-fold increase in the amount of alpha AGP exon sequences was observed in nuclear RNA, comparing treated with control animals, but transport of alpha AGP exon sequences was detectable only from treated nuclei, indicating at least a 50-fold increase in abundance of alpha AGP sequences. This suggests that a selective gating mechanism may be operative at the level of post-transcriptional nucleocytoplasmic transport during induction of alpha AGP in the acute phase response.

Structure of the rat alpha 1-acid glycoprotein gene.

The complete nucleotide sequence of the rat alpha 1-acid glycoprotein gene has been determined from an isolated lambda recombinant bacteriophage. Southern blot analysis and DNA sequencing indicate that there is only one gene per genome; it contains six exons and is located within a 3,200-base-pair fragment starting from a TATA box and extending to the polyadenylation signal AATAAA. Transcription starts 37 base pairs upstream from the beginning of the translation codon ATG. The TATA box (TATAAA) lies 26 base pairs upstream from this site. The gene contains several potential glucocorticoid receptor-binding sites, both inside and outside the structural gene.

Liver pathology accompanying chronic liposome administration in mouse.

Particulate drug carriers have a pronounced tendency to localize in the mononuclear phagocyte system and chronic administration of such carriers can result in reticuloendothelial (RE) blockade. In a previous study (Allen et al., J. Pharmacol. Exp. Therap., 229, 267, 1984) we have examined the ability of chronic i.v. administration of liposomes of a variety of compositions to cause RE blockade in mice. In this communication we report on the time course of histological changes in liver accompanying chronic liposome administration in samples collected at the time of our previous studies. The predominant histological feature was the appearance of a granulomatous reaction in liver. Granulomas were frequent in liver tissue of mice receiving 2 or more injections of sphingomyelin:phosphatidylcholine, 4:1 or distearoylphosphatidylcholine:cholesterol, 1:1 liposomes, but disappeared shortly after termination of liposome injections. In mice receiving phosphatidylcholine:cholesterol, 2:1 liposomes no granulomas in liver were apparent during the injection course (10 injections of 2 mg phospholipid each over 25 days) but granulomatous inflammation of the liver became apparent 2 weeks after the last injection and had not resolved by 9 weeks post-injection. The appearance of granulomas was correlated with depression of phagocytic index and their disappearance was correlated with normalization or stimulation of reticuloendothelial function. These observations may be related to the rate of phospholipid metabolism for the various phospholipid types or to the nature of phospholipid metabolites.

Nucleocytoplasmic RNA transport.

A number of closely related post-transcriptional facets of RNA metabolism show nuclear compartmentation, including capping, methylation, splicing reactions, and packaging in ribonucleoprotein particles (RNP). These nuclear 'processing' events are followed by the translocation of the finished product across the nuclear envelope. Due to the inherent complexity of these interrelated events, in vitro systems have been designed to examine the processes separately, particularly so with regard to translocation. A few studies have utilized nuclear transplantation/microinjection techniques and specialized systems to show that RNA transport occurs as a regulated phenomenon. While isolated nuclei swell in aqueous media and dramatic loss of nuclear protein is associated with this swelling, loss of RNA is not substantial, and most studies on RNA translocation have employed isolated nuclei. The quantity of RNA transported from isolated nuclei is related to hydrolysis of high-energy phosphate bonds in nucleotide additives. The RNA is released predominantly in RNP: messenger-like RNA is released in RNP which have buoyant density and polypeptide composition similar to cytoplasmic messenger RNP, but which have distinctly different composition from those in heterogeneous nuclear RNP. Mature 18 and 28S ribosomal RNA is released in 40 and 60S RNP which represent mature ribosomal subunits. RNA transport proceeds with characteristics of an energy-requiring process, and proceeds independently of the presence or state of fluidity of nuclear membranes. The energy for transport appears to be utilized by a nucleoside triphosphatase (NTPase) which is distributed mainly within heterochromatin at the peripheral lamina. Photoaffinity labeling has identified the pertinent NTPase as a 46 kD polypeptide which is associated with nuclear envelope and matrix preparations. The NTPase does not appear to be modulated via direct phosphorylation or to reflect kinase-phosphatase activities. A large number of additives (including RNA and insulin) produce parallel effects upon RNA transport and nuclear envelope NTPase, strengthening the correlative relationship between these activities. Of particular interest has been the finding that carcinogens induce specific, long-lasting increases in nuclear envelope (and matrix) NTPase; this derangement may underlie the alterations in RNA transport associated with cancer and carcinogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)