Ergothioneine distribution in bovine and porcine ocular tissues.

Ergothioneine (ERT), is a low molecular weight, sulfur-containing antioxidant occurring in up to millimolar amounts in mammalian tissues. Using an improved HPLC assay, ERT levels have been measured and compared in bovine and porcine eyes and erythrocytes. The rank order of ERT levels in bovine ocular tissue was lens > retina = cornea > pigmented retinal epithelium (RPE) > aqueous humor (AQ) > vitreous humor (VIT) > sclera. In porcine ocular tissue, the rank order was retina > AQ > VIT > RPE > cornea > lens > sclera. ERT levels in bovine lens were about 250 x > that in porcine lens. Porcine erythrocyte levels were 5.5 x > bovine levels. Species differences were also observed in the retina, VIT and AQ where porcine levels were 2 to 10-fold greater than bovine levels. ERT in bovine lens and cornea was 35 and 14 times greater than the corresponding level of reduced glutathione (GSH). Porcine lens had 45 times more GSH than ERT. Values for ERT and GSH in other tissues from both species were of the same order of magnitude. These results are consistent with a role for ERT in prevention of oxidative damage to the eye.

Protein levels in the vitreous of rats with streptozotocin-induced diabetes mellitus.

The vitreous is a neural extracellular space separated from the blood-vascular compartment by the blood-retinal barrier. Study of the appearance of serum proteins in this space have been carried out in rats with streptozotocin-induced diabetes mellitus, a condition associated with barrier dysfunction. A vitreous sampling technique that avoids contamination with surrounding tissue was employed. In rats 1 month after administration of streptozotocin (fasting serum glucose > or = 375 mg/dl), significant increases in vitreous protein were observed in the absence of discernible eye pathology. Two-dimensional isoelectric focusing and SDS-polyacrylamide gel analysis of the soluble fraction demonstrated 85 polypeptides, 28 of whose electrophoretic positions coincided with positions of serum polypeptides. The remainder were unrelated to serum polypeptide loci. Overall patterns of soluble protein from the vitreous of streptozotocin-injected and normoglycemic-uninjected control animals were virtually identical. Results support a system for selective transfer for certain proteins into the extraneural vitreous space as suggested by Chen and Chen (6).

Insulin in the vitreous of the normal and streptozotocin-induced diabetic rat.

Insulin has been detected by ELISA in the vitreous of the normal and streptozotocin-diabetic rat at levels for both about 1% of those in serum. 131I-labeled insulin, administered to conscious rats via an indwelling cannula in the right atrium, was found to cross the blood-ocular barrier into the vitreous. Autoradiographic gel analysis showed the peptide was transferred as an intact molecule. Vitreous insulin levels reflected serum levels as seen in relatively constant vitreous-to-serum insulin ratios over a wide range of serum insulin concentrations. The rate of blood-to-vitreous passage of insulin was about the same in normal as in diabetic rats (fasting serum glucose greater than or equal to 21 mM). At least a portion of vitreous insulin is therefore of pancreatic origin, and retinal tissue in the normal and diabetic animal is thus accessible to circulating hormone. The blood-ocular barrier is unaltered in streptozotocin diabetes with regard to insulin passage.

DNA modification in vivo by derivatives of glucose: enhancement by glutathione depletion.

When BHK or HTC cells are cultured for 20 min with [U-14C]glucose in the presence of agents that deplete reduced glutathione, DNA banded from the cells in cesium salt gradients containing guanidium HCl is radioactively labeled. This depletion-dependent labeling required live cells. It was not caused by reactive contaminants in the radioactive glucose preparations, by carbohydrate or protein comigration into the DNA band, or by metabolism of glucose into deoxyribose. Labeling levels are similar whether depletion is achieved by oxidation (with the drug diamide) or by inhibition of synthesis (with methionine sulfoximine). A temporal association between GSH repletion and the appearance of D-lactate, the putative unique product of GSH-dependent glyoxylase action on pyruvaldehyde, suggests possible involvement of 3-carbon dicarbonyls.

Nonenzymatic glycosylation of vitreous proteins in vitro and in the streptozotocin-treated diabetic rat.

Nonenzymatic glycosylation has been shown to affect collagen elsewhere in the body in diabetic patients. A fluorescent product of nonenzymatic glycosylation of protein was found to accumulate in the vitreous of rats 2 months after diabetogenic doses of streptozotocin. The same product could be demonstrated in the same time frame after in vitro incubation of glucose with insoluble protein constituents isolated from vitreous. These in vitro and in vivo studies show that nonenzymatic glycosylation of vitreous collagens does occur. Further studies to examine whether nonenzymatic glycosylation affects vitreous collagen function should be performed.

Immunoquantitation of cytochrome b5 and methylcholanthrene-induced cytochromes P-450.

The enzyme-linked immunosorbent assay (ELISA) has been investigated for its ability to quantitate hydrophobic proteins like cytochromes b5 and P-450 at the subnanogram level. Issues encountered that have broad significance not only for ELISA, but for other qualitative and quantitative immunoassays as well, include the effects of detergent, the discriminatory capacity of ELISA, and the method for determining an assay's selectivity.

Purification and characterization of a previously unreported form of cytochrome P-448 from the liver of 3-methylcholanthrene-pretreated rats.

At least four hepatic isoenzymes of cytochrome P-450 were purified and characterized from rats treated with 3-methylcholanthrene. A monoclonal antibody developed against one of the forms (designated cytochrome P-450 MC-B) and polyclonal antibodies against others were used to demonstrate that form MC-B is immunologically distinct from other methylcholanthrene-inducible forms. Limited N-terminal amino acid sequencing showed that cytochrome P-450 MC-B has a primary structure that differs from the N-terminal sequences of other established rat isoenzymes. Cytochrome P-450 MC-B has a minimum Mr of 53,000, a CO-reduced spectral maximum at 448 nm, a Soret maximum of 417 nm in the absolute oxidized spectrum and a pattern of substrate preferences that differs from those of the other methylcholanthrene-induced forms. The other forms (MC-A, MC-C and MC-D) share characteristics with isoenzymes previously reported by other investigators.

Assessment of internal primary structure of polypeptides newly translated in vitro by reticulocyte lysate: a study with cytochrome b5.

A modified peptide mapping technique is described that allows the survey of the primary structure of proteolytic fragments of particular newly translated proteins in reticulocyte lysate. The technique is demonstrated with rat liver cytochrome b5.

Hepatotoxicity of acetaminophen in neonatal and young rats. I. Age-related changes in susceptibility.

The susceptibility of neonatal (11 days) and young rats (19 and 33 days) to acetaminophen-induced hepatic necrosis was examined. Acetaminophen-induced lethality (LD50) was slightly lower in 19-day-old animals (840 mg/kg) compared to 11- and 33-day-old animals (1220 and 1580 mg/kg, respectively). A toxic dose of the drug ( LD20 ) produced elevated serum glutamate-pyruvate transaminase and lactate dehydrogenase activities 20-24 hr after drug administration only in 19- and 33-day-old animals. Serum enzyme elevation was not observed after a toxic dose of acetaminophen ( LD20 or LD50) in 11-day-old rats. Histological evaluation showed that both 19- and 33-day-old rats developed extensive hepatic centrilobular damage, whereas morphological parameters in 11-day-old animals given acetaminophen were not different from controls. It appears that high doses of acetaminophen are lethal to young rats, but that 11-day-old animals are different from 19-day-old and older rats in that the neonatal animals lack susceptibility to the hepatotoxic effects of the drug. Lower susceptibility of the neonatal rat liver to the hepatic effects of two other hepatotoxicants (bromobenzene and tannic acid) was also observed.

Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay.

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.

An enzyme-linked immunoadsorbent assay for measuring cytochrome b5 and NADPH-cytochrome P-450 reductase in rat liver microsomal fractions. Evidence for functionally inactive protein.

Immunoreactive cytochrome b5 and NADPH-cytochrome P-450 reductase (EC 1.6.2.4) from rat liver microsomal fractions were measured by using an enzyme-linked immunoadsorbent assay (e.l.i.s.a.) as a function of age, sex and type of inducer (phenobarbital or 3-methylcholanthrene), and the values were compared with those obtained by spectral measurement (for cytochrome b5) or enzymic assay (for reductase). In untreated animals, there was more cytochrome b5 and NADPH-cytochrome P-450 reductase when measured by an e.l.i.s.a. than was seen spectrally or enzymically. However, for microsomal preparations from phenobarbital-pretreated animals, spectrally obtained values for cytochrome b5 and immunoreactive-cytochrome b5 values were similar. Values from control animals suggest that there is about 20-30% more immunoreactive cytochrome b5 than that which is spectrally detectable.

Cleavage of newly translated NADPH-cytochrome P-450 reductase in a rabbit reticulocyte lysate cell-free system in the absence of exogenous membranes.

In a rabbit reticulocyte lysate cell-free system and using double antibody immunoprecipitation method, newly translated rat liver NADPH-cytochrome P-450 reductase (E.C. 1.6.2.4) was shown to be cleaved in the absence of exogenous membranes. Reductase having a Mr = 78,000 was shown to be converted to a Mr = 67,000 upon incubation with either lysate or antisera. Peptide maps of 78,000 dalton reductase and 67,000 dalton protein were identical except for three additional peptides present in the 78,000 dalton reductase map. The cleavage activity associated with the antisera could be prevented by using the IgG fraction, while that associated with the lysate was inhibited by using the protease inhibitors leupeptin, pepstatin or bestatin.

Unscheduled DNA synthesis in rat liver after gastric administration of halothane.

Unscheduled DNA replication occurred in response to halothane administered to rats by stomach tube. This response was observed under a narrow range of experimental conditions that included pretreatment with phenobarbital and a short interval between barbiturate and anesthetic administration. Under the conditions of the experiment, hepatic necrosis was not demonstrable.

Iron-induced DNA damage and synthesis in isolated rat liver nuclei.

Incubation of iron with isolated rat liver nuclei stimulated fragmentation of single-stranded DNA, incorporation of [3H]thymidine into DNA and the binding of 59Fe to DNA. FeCl2 was about twice as active as FeCl3. Lipid peroxidation took place in nuclei incubated with FeCl2, but not with FeCl3. Generation of reactive forms of oxygen was required for iron-mediated DNA damage, but evidence for direct interaction of reactive oxygen with DNA was not found. Apparent adducts of iron bound to DNA seemed to be formed by an enzymic mechanism.