Microsomal Protein Synthesis Research Articles

Sublethal Toxic effects of spent Oil Based Drilling Mud and Cuttings to Earthworm Aporrectodea Longa.

Sublethal toxic effects of spent oil based drilling mud collected from an abandoned oil drilling site in Mpanak, Akwa Ibom State, Nigeria were assessed in the earthworm Aporrectodea longa . The test annelid was exposed to sub-lethal Concentration of 0ppm SPP; 62,500ppm SPP; 125, 000ppm SPP; 250,000ppm SPP and 500,000ppm SPP of drilling mud for 28 days. The procedure for the exposure was according to the Organisation for Economic Co-operation and Development (OECD) recommended method for testing chemicals No 207. There were three replicate jars per treatment and 10 earthworms per jar of soil spiked with drilling mud. Control groups were also set up. At the end of the exposure, bioaccumulation of the chemical constituents of the drilling mud (heavy metals and TPH) increased (p<0.01) with increase drilling mud concentration. Mean weights of earthworms increased in the 62,500 and 125,000SPP and decreased in the 250,000 and 500,000SPP. Glucose levels increased with increase in the drilling mud concentration with the exception of 62,500ppm SPP. The elevated glucose levels could be due to hyperglycemia induced by the toxicity of the drilling mud. Protein levels increased with increase in drilling mud concentration except in the 125,000ppm SPP treatment. The increase in protein content could be due to enhancement of microsomal protein synthesis prompted by the toxicity of the drilling mud to test organisms. The results of this study have shown that drilling mud and cuttings could cause serious health risk to Aporrectodea longa. The discharge of drilling mud into the terrestrial ecosystems should be discouraged. © JASEM

Effect of chronic intoxication with (+)-amphetamine on its concentration in liver and brain and on (14C) leucine incorporation into microsomal and cytoplasmic proteins of rat liver.

Abstract Rats were treated with increasing concentrations of (+)-amphetamine sulphate in drinking water for 90 days. The ingested dose of amphetamine was found to increase from 16 mg kg−1 on the first day up to 90 mg kg−1 on the 32nd day of treatment. The rats were maintained on the highest dose regime for a further 58 days without any deaths, which showed that tolerance to the overall toxicity of the drug developed. The concentrations of [3H]amphetamine in liver and brain of chronically treated rats were significantly higher than those of controls. Chronic treatment with amphetamine significantly reduced body and liver weight of rats, but did not influence the relative liver to body weight. A marked inhibition of [14C]leucine incorporation into liver microsomal and cytoplasmic proteins was observed after 90 days of treatment with amphetamine. The relation between inhibition of microsomal protein synthesis and the increase of amphetamine concentrations in liver and brain is discussed.

Subclinical kidney injury incites endotoxin hyperresponsiveness

gentamicin, an aminoglycoside discovered in 1963 from micromonospora, is one of the most commonly used antibiotics against Gram-negative infections. Aminoglycosides achieve bacterial killing by binding to bacterial ribosomes, causing blockade of protein synthesis through failure of initiation and

Book Review

Book Review

Induction of Microsomal Membrane Proteins in Roots of an Aluminum-Resistant Cultivar of Triticum aestivum L. under Conditions of Aluminum Stress.

Three-day-old seedlings of an Al-sensitive (Neepawa) and an Al-resistant (PT741) cultivar of Triticum aestivum were subjected to Al concentrations ranging from 0 to 100 [mu]M for 72 h. At 25 [mu]M Al, growth of roots was inhibited by 57% in the Al-sensitive cultivar, whereas root growth in the Al-resistant cultivar was unaffected. A concentration of 100 [mu]M Al was required to inhibit root growth of the Al-resistant cultivar by 50% and resulted in almost total inhibition of root growth in the sensitive cultivar. Cytoplasmic and microsomal membrane fractions were isolated from root tips (first 5 mm) and the adjacent 2-cm region of roots of both cultivars. When root cytoplasmic proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no changes in polypeptide patterns were observed in response to Al stress. Analysis of microsomal membrane proteins revealed a band with an apparent molecular mass of 51 kD, which showed significant accumulation in the resistant cultivar following Al exposure. Two-dimensional gel analysis revealed that this band comprises two polypeptides, each of which is induced by exposure to Al. The response of the 51-kD band to a variety of experimental conditions was characterized to determine whether its pattern of accumulation was consistent with a possible role in Al resistance. Accumulation was significantly greater in root tips when compared to the rest of the root. When seedlings were subjected to Al concentrations ranging from 0 to 150 [mu]M, the proteins were evident at 25 [mu]M and were fully accumulated at 100 [mu]M. Time-course studies from 0 to 96 h indicated that full accumulation of the 51-kD band occurred within 24 h of initiation of Al stress. With subsequent removal of stress, the polypeptides gradually disappeared and were no longer visible after 72 h. When protein synthesis was inhibited by cycloheximide, the 51-kD band disappeared even when seedlings were maintained in Al-containing media. Other metals, including Cu, Zn, and Mn, failed to induce this band, and Cd and Ni resulted in its partial accumulation. These results indicate that synthesis of the 51-kD microsomal membrane proteins is specifically induced and maintained during Al stress in the Al-resistant cultivar, PT741.

Dissociation of decreases in renal cellular energetics and recovery of renal microsomal translation during chronic cyclosporine a administration

Male Sprague-Dawley rats were given oral cyclosporine A or control vehicle, and renal protein synthesis and renal ATP levels were examined. Acute oral cyclosporine A at 5, 10, 25 and 50 mg/kg/day for 6 days reduced [ 3H] L-leucine incorporation by isolated renal microsomes to 76.2, 56.8, 44.3 and 29.5% of control incorporations, respectively. No significant changes in renal ATP levels were detected by NMR spectroscopy after acute oral cyclosporine A administration at the doses indicated. However, during chronic exposure to cyclosporine A at doses of 5 and 25 mg/kg day for 30, 60 and 90 days, there was a recovery of renal microsomal protein synthesis by day 30 at 5 mg/kg/day, and by day 45 at 25 mg/kg/day. NMR spectroscopy of the kidneys of these rats demonstrated decreases in renal ATP level by day 60 in animals given cyclosporine A at 25 mg/kg/day. Cyclosporine A administration produced a renal acidosis and up to a 40% decrease in renal ATP level by day 90 in rats fed cyclosporine A at 25 mg/kg. No apparent histologic abnormalities were observed in the ATP deficient renal tissue by NMR imaging. Reductions in renal ATP level suggest that the recovery of renal microsomal protein synthesis is aberrant in the continued presence of cyclosporine A, or that mitochondria are direct sites of cyclosporine A toxicity.

Cyclosporine-induced renal dysfunction

The main adverse reaction to the immunosuppressive drug cyclosporine is dose-dependent renal dysfunction. Although renal vasoconstriction without major tubular dysfunction is usually noted, recent studies have demonstrated an inhibition of renal cortical microsomal protein synthesis. Sprague-Dawley rats and appropriate pair-fed controls were given cyclosporine orally in doses of 5, 10, 25, and 50 mg/kg/day for periods up to 10 days. A dose-dependent decline in glomerular filtration rate and effective renal plasma flow was maximal by day 3 and did not worsen despite continued dosing. Microsomal protein synthesis as measured by [3H]leucine incorporation was also depressed in a dose-dependent fashion; however, inhibition did not reach the nadir until day 4, 1 day after renal dysfunction was established. When cyclosporine was discontinued, microsomal protein synthesis was normalized by 4 days after drug withdrawal; in contrast, the return of glomerular filtration rate and effective renal plasma flow to normal required 8 days after drug discontinuation. Tubular function as measured by fractional excretion of lithium, enzymuria, and urinary osmolality was well maintained despite the depression of renal hemodynamics. There was no evidence of tubular necrosis by light or electron microscopy. Although cyclosporine produces reductions in renal microsomal protein synthesis, measured by "run-off" translation assays, these effects appear unlikely to be the direct cause of acute renal dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of IFN-α-Con 1 on hepatic cytochrome P-450 and protein synthesis and degradation in hepatic microsomes

Interferon and its inducers are well known to depress drug biotransformation in the liver by decreasing the levels of cytochrome P-450 in that organ. We now report that IFN-α-Con 1, which was constructed from the most frequently observed amino acid sequences in human α-interferon subtypes, causes a loss in cytochrome P-450 which could be prevented by pretreating animals with either puromycin or actinomycin D. This suggests that the loss in drug biotransformation is mediated via the production of an intermediate protein. When the turnover of microsomal protein was examined this interferon appeared to depress the synthesis of proteins with molecular weights 46–60 kd and had little effect on the synthesis of other proteins. The in vitro translation of proteins of molecular weights 45–60 kd was also depressed in an in vitro translation system using mRNA isolated from the livers of interferon treated hamsters. Interferon had no effect on the degradation of microsomal proteins of all molecular weights. It is concluded that interferon probably depresses the levels of cytochrome P-450 in the liver by decreasing the synthesis of the apoprotein and that interferon has little effect on the degradation of the hemoprotein.

A comparison of hepatic p450 induction in rat and trout (Oncorhynchus mykiss): Delineation of the site of resistance of fish to phenobarbital-type inducers

1. A comprehensive approach was taken to delineate the site of refractivity of trout to phenobarbital-type (PB-type) hepatic monoxygenase (MO) inducers. 2. Model inducers β-naphthoflavone (BNF; 3-MC-type), and PB as well as the polychlorinated biphenyl isomers, 3,4,5,3',4',5'-hexachlorobiphenyl (3,4,5-HCB; 3-MC-type) and 2,4,5,2',4',5'-hexachlorobiphenyl (2,4,5-HCB; PB-type) were used to assess MO activities, total cytochromes P450, and [ 35S]-methionine incorporation into de novo synthesized microsomal protein in both trout and rats. 3. In rainbow trout immunodetection of P450 § § Throughout this paper, the suggested nomenclature for the P450 gene superfamily (Nebert et al., 1987, 1989) has been used except when reference is made to an earlier study where the original nomenclature is used. P450 inducers: β-naphthoflavone(BNF); 3,4,5, 3',4',5' hexachlorobiphenyl(PCB); 2,4,5,2',4',5' Hexachlorobiphenyl(PCB); phenobarbital(PB). isozymes and nucleic acid hybridization of rainbow trout P 1450 mRNA using pfP 1450-3' (trout 3-MC-inducible, P450IA1 gene) and genomic DNA using pfP 1450-3' or pSP450-oligo (rat PB-inducible, P450IIB1 gene) cDNAs were carried out. 4. In rainbow trout, PB and 2,4,5-HCB do not increase hepatic MO activities, total cytochromes P450, de novo synthesis of microsomal protein, levels of P450 isozymes, or levels of P 1450 mRNA. 5. Rainbow trout have, within their genome, DNA with sequence(s) similar to rat P450IIB1, but inducibility of this P450 in trout by PB-type inducers is lacking.

A new proposal for the mechanism of cyclosporine a nephrotoxicity. Inhibition of renal microsomal protein chain elongation following in vivo cyclosporine a

In this paper, we report experiments examining the effect of cyclosporine A on “run-off” translation in microsomes isolated from tissues of Sprague-Dawley rats. In microsomes isolated from rat brain, kidney and thymus, cyclosporine A added in vitro in concentrations of up to 100 μg/ml did not reduce [ 3H] l-leucine incorporation relative to controls. A small dose-dependent reduction in [ 3H]leucine incorporation was observed in microsomes isolated from rat liver when cyclosporine A was added in high concentrations (5 and 6% at 25 and 100 μg/ml). However, when cyclosporine A was injected at 50 mg/kg/day for 10 days. [ 3H] L-leucine incorporation was inhibited 99.9% in microsomes isolated from kidney. The oral administration of cyclosporine A at 50 mg/kg/day for 6–10 days produced a 75% inhibition of incorporation by isolated renal microsomes. These changes were observed in the absence of measurable reductions in “run-off” transcription measured as [ 3H]UTP incorporation by renal nuclei exposed to cyclosporine A in concentrations of up to 100 μg/ml in vitro or isolated from animals given oral cyclosporine A at 50 mg/kg/day for 6 days. Cross-over experiments were performed using microsomes and microsomal supernatant fractions (cell saps) from tissues of animals treated with cyclosporine A and control vehicle. Renal cell sap from cyclosporine A treated animals inhibited [ 3H] l-leucine incorporation by microsomes isolated from the kidneys or other tissues of animals treated with control vehicle. These experiments demonstrated that a translation inhibitor was present in the cell sap of cyclosporine A treated animals which could directly block translation elongation in microsomes from control animals. When renal cell sap from both control and cyclosporine A treated animals was added to control microsomes, inhibition was still prominent, suggesting the presence of an inhibitor rather than the absence of an elongation factor. Oral administration of cyclosporine A at 50 mg/kg/day for 6 days depressed renal microsomal [ 3H] l-leucine incorporation equally in male and female rats to 25% of control. The dose-response relationship for microsomal protein synthesis inhibition after 6 days of oral cyclosporine A administration was: 5 mg/kg, 73.7% of control; 10 mg/kg, 64.1% of control; 25 mg/kg 54.9% of control and 50 mg/kg, 24.1% of control. Renal microsomal protein synthesis following oral cyclosporine A at 50 mg/kg/day was reduced to 54% of control by day 2 and was maximally inhibited at 25–30% of control by day 4. When cyclosporine A was stopped after 10 days, renal microsomal protein synthesis returned to 59% of control by day 2 and to 99.5% of control by day 4. Oral administration of 50 mg/kg/day cyclosporine A for 10 days was associated with whole blood through levels of 4.1 μg/ml cyclosporine A and metabolites and with reductions in renal function. Results of our studies are consistent with the hypothesis that a cyclosporine A metabolite, or a product produced in the cell in response to cyclosporine A, directly interferes with translation or the regulation of translation. Translation inhibition may explain the nephrotoxic actions of cyclosporine A.

Modulation of DNA synthesis in cultured muscle cells by 1,25-dihydroxyvitamin D-3

Biphasic effects of 1,25-dihydroxyvitamin D-3 on DNA synthesis were shown in primary cultured (24 h) chick embryo myoblasts exposed to physiological concentrations of the hormone. The sterol stimulated [3H]thymidine incorporation into DNA in proliferating myoblasts, e.g., at early stages of culture prior to cell fusion or in high serum-treated cells. The opposite effects were observed during the subsequent stage of myoblast differentiation in low-serum media. The mitogenic effect of 1,25-dihydroxyvitamin D-3 was correlated with an increase in c-myc mRNA and a decrease in c-fos mRNA levels, whereas its inhibitory action on DNA synthesis was accompanied by increased myofibrillar and microsomal protein synthesis and an elevation of creatine kinase activity, the latter suggesting a stimulation of muscle cell differentiation by the sterol. These data are in agreement with the results of previous morphological studies. Treatment of myoblasts with the calcium ionophore X-537 A or the phorbol ester TPA caused only a transient stimulation of [3H]thymidine incorporation into DNA, which occurred earlier than the response elicited by 1,25-dihydroxyvitamin D-3, suggesting that changes in intracellular Ca2+ and kinase C activity are not major mediators of the hormone effects. A similar temporal profile of changes in calmodulin mRNA levels as that of [3H]thymidine incorporation into DNA was observed after treatment of myoblasts with the sterol, in accordance with the role of calmodulin in the regulation of cell proliferation. 1,25-dihydroxyvitamin D-3 may play a function in embryonic muscle growth and differentiation.

Microsomal protein synthesis inhibition: an early manifestation of gentamicin nephrotoxicity

Aminoglycoside antibiotics achieve bacterial killing by binding to bacterial ribosomes and inhibiting protein synthesis. To examine whether similar mechanisms could be present in renal tubular cells prior to the onset of overt proximal tubular necrosis due to these drugs, we isolated microsomes from Fischer rats given 20 mg/kg gentamicin every 12 h subcutaneously for 2 days and from vehicle-injected controls. Concomitant studies of renal structure, function, and mitochondrial respiration were carried out. [3H]leucine incorporation into renal microsomes of treated animals was reduced by 21.9% (P less than 0.01), whereas brain and liver microsomes from the same animals were unaffected. Gentamicin concentration in the renal microsomal preparation was 56 micrograms/ml, a value 7- to 10-fold above concentrations necessary to inhibit bacterial growth. Conventional renal function studies were normal (blood urea, serum creatinine, creatinine clearance). Treated animals showed only a mild reduction of inulin clearance, 0.71 compared with 0.93 ml.min-1.100 g-1 in controls (P less than 0.05), and an increase in urinary excretion of N-acetylglucosaminidase of 20 compared with 14.8 units/l (P less than 0.05). Renal slice transport of p-aminohippuric acid, tetraethylammonium, and the fractional excretion of sodium were well preserved. There was no evidence, as seen by light microscopy, of proximal tubular necrosis. Mitochondrial cytochrome concentrations were normal and respiratory activities only slightly reduced. Processes similar to those responsible for bacterial killing could be involved in experimental gentamicin nephrotoxicity before overt cellular necrosis.

Comparison of xenobiotic biotransformation enzymes in kidney and liver of rainbow trout ( Salmo gairdneri)

The microsomal cytochrome P-450 content in kidney of rainbow trout ( Salmo gairdneri) was approximately 5-fold lower than the content in liver. The renal ethoxycoumarin- and ethoxyresorufin- O-deethylase activities calculated on a per-cytochrome P-450 basis were, however, found to be about 10-fold higher than the hepatic activities. The patterns of time-dependent increase and subsequent decrease of microsomal cytochrome P-450-dependent monooxygenase activities after a single injection of β-naphthoflavone (BNF) were similar in the kidney and liver. The microsomal ethoxyresorufin- and ethoxycoumarin- O-deethylase activities were maximally induced in liver (120- and 10-fold, respectively) by a single BNF injection (50 mg/kg body wt), whereas in kidney the maximal levels of induction (135- and 21-fold, respectively) were reached after three injections with BNF. The induction of cytochrome P-450 systems was associated with synthesis of a new microsomal protein of 58,000 Da in both kidney and liver. UDP-glucuronosyl transferase activity toward p-nitrophenol was about 8-fold lower in kidney than in liver. A significant 2.5-fold elevation in microsomal UDP-glucuronosyltransferse activity was found in the kidney 14 days after a single injection with BNF (50 mg/kg). In the liver, a 2-fold increase of this activity was seen 3 days after the treatment. The results indicate that the rainbow trout kidney in addition to the liver is of great importance in biotransformation of lipophilic xenobiotics.

Effects of tetrachlorvinphos on hepatic microsomal enzymes in the rat.

The inducement of the hepatic microsomal enzymes in male rats following oral administration of tetrachlorvinphas (TCVP) for 7 days with the dosage of 150 mg/kg and the mechanism of its inducing effect were studied. It was confirmed that TCVP could enhance significantly the activity of hepatic microsomal enzymes, such as aniline hydroxylase, P-nitroanisole O-demethylase and uridine diphosphoglucuronyltransferase (UDP-GAT). Besides, it could also increase contents of microsomal cytochrome P-450 and microsomal protein. In rats it shortened pentobarbital sleeping time. These results demonstrated that repeated administration of TCVP might induce the activity of hepatic microsomal enzymes in rats. The inducing effect of TCVP could be blocked by DL-ethionine, an inhibitor of aminoacids. Furthermore, TCVP failed to increase enzymic activity in vitro. These findings suggested that the inducing effect of TCVP on the activity of hepatic microsomal enzymes could be explained by accelerating synthesis of microsomal protein.

Inhibitory effects of gentamicin and ethacrynic acid on mammalian microsomal protein synthesis

The ototoxic antibiotic gentamicin, and the ototoxic diuretic ethacrynic acid, both produce inhibitory effects on protein synthesis in microsomes isolated from rat brain. Inhibitory effects appear to be essentially independent of each other. The inhibitory dose-response curve for gentamicin is logarithmic, while that for ethacrynic acid is linear. The dose-response curves make gentamicin the predominant inhibitor when the drugs are combined at low concentrations and ethacrynic acid the predominant inhibitor when the drugs are combined at high concentrations. Accumulation of aminoglycoside by tissues of the inner ear may result in inhibition of protein synthesis in spite of low and transient plasma levels of the drug. Further inhibition of translation by ethacrynic acid could account, in part, for the ototoxic interaction of aminoglycosides and high ceiling diuretics.

Gentamicin administered in vivo reduces protein synthesis in microsomes subsequently isolated from rat kidneys but not from rat brains.

Gentamicin injected in vivo, in doses equivalent to those used therapeutically in humans, produced a decrease in the protein synthesizing capacity of microsomes subsequently isolated from rat kidneys, but not of microsomes isolated from rat whole brains. The decreases in eukaryotic cell protein synthesis observed following in-vivo administration of gentamicin were similar to those observed following the addition of gentamicin to microsomes in vitro.

Inhibition of mammalian microsomal protein synthesis by aminoglycoside antibiotics.

The aminoglycoside antibiotics gentamicin, kanamycin and netilmicin produce a dose-dependent inhibition of amino acid incorporation in microsomes isolated from human liver and rat brain, kidney and liver. Inhibitory effects on microsomal protein synthesis occur at concentrations that have been shown to accumulate in rodent and human renal cortex and perilymph following therapeutic administration. Inhibition of translation in those tissues which specifically accumulate aminoglycoside antibiotics may, in part, explain toxicities observed following exposure to aminoglycosides.

Prevention of lithocholate - induced cholestasis by cycloheximide, an inhibitor of protein synthesis

Our previous investigations have shown that lithocholic acid (LCA)-induced cholestasis is associated with an increased synthesis of microsomal cholesterol which is transported with LCA and incorporated in the bile canalicular membrane. As the significance of these changes remains unknown the effect of interference with microsomal protein synthesis and/or with the cellular transport of cholesterol was studied. Male Wistar rats were injected i.p. with cycloheximide at the dose of 15 μg/100 g BW 3 times over a 24-hour period. After cannulating the common bile duct and collecting bile for one hour, the animals were either injected i.v. with 12 μmoles C 14-LCA/100 g BW or with a 7.5% albumin solution and bile was collected for another hour. LCA injection in untreated animals reduced bile flow by more than 90% of control values. In contrast, bile flow in the group treated with cycloheximide and LCA was normal and did not differ from that of animals given cycloheximide alone. Bile salt secretion rate was increased in the cycloheximide-LCA group over the control groups. This was mainly due to the secretion of more than 80% of the injected LCA and was confirmed by the distribution of the radioactivity. By electron microscopy, the liver in the cycloheximide-LCA group did not show any of the well defined changes associated with LCA-induced cholestasis. These data suggest that microsomes play an important role in the pathogenesis of LCA cholestasis and that inhibition of microsomal protein synthesis can prevent its development.

Effects of phenobarbital, beta-naphthoflavone, dexamethasone, and formamidoxime on the turnover of inducible microsomal proteins in cultured hepatocytes.

Microsomal proteins from cultured chick embryo hepatocytes were separated by polyacrylamide gel electrophoresis and their rate constants of degradation (Kd) were estimated using double isotope techniques. The proteins were found to be heterogeneous in their turnover rates, proteins, or subunits, with higher molecular weights being more rapidly degraded than those with lower molecular weights. The Kd values of three selected microsomal proteins with Mr 52,000, 54,000, and 56,000 presumed to represent cytochrome P-450 apoproteins were found unaltered following treatment of cultured hepatocytes with inducers of cytochrome P-450 such as phenobarbital or beta-naphthoflavone. Based on their Kd values, the half-lives of these inducible microsomal proteins were estimated to be 10.4, 9.6, and 11.3 h. The response to phenobarbital was markedly modified when cells were either pretreated with dexamethasone or formamidoxime, agents inhibiting replicative DNA synthesis. Under these conditions, the phenobarbital-mediated de novo synthesis of several microsomal proteins was enhanced. Our turnover data may offer an explanation for the rapid changes in cytochrome P-450 concentration and function following drug exposure of cultured chick embryo hepatocytes.

Protective effect of methionine against vinyl chloride-mediated depression of non-protein sulphydryls and cytochrome P-450

Potentiality of a protective effect of methionine against hepatotoxic action of vinyl chloride (VC) was assessed on rats, induced with phenobarbital and thereafter exposed to 50 000 ppm VC for 5 h. This extremely high dosage of VC warranted the appearance of drastic symptoms of liver injury in the course of 20 h postexposure. dl-Methionine in a dose of 1 g/kg given by gavage prior to the VC-exposure counteracted both the BC-provoked depletion of non-protein sulphydryls and destruction of cytochrome P-450 in the liver. In the methionine-pretreated rats the phenobarbital-induced synthesis of microsomal proteins in the livers was maintained during the post-exposure period, contrary to the non-pretreated animals. It may be justified to apply methionine as a prophylactic agent in cases of occupational exposure to VC.