Synergistic neurotoxic effects of styrene oxide and acrylamide: glutathione-independent necrosis of cerebellar granule cells.

Conjugation with glutathione (GSH) is a mechanism of detoxification of acrylamide (ACR); hence, prior depletion of GSH might be expected to exacerbate ACR's neurotoxicity. GSH levels in female rats were reduced by ip administration of styrene oxide (SO; 250 mg/kg), diethylmaleate (DEM; 0.5 ml/kg), or 2-vinylpyridine (VP; 100 mg/kg) 1.5 or 2 hr prior to a single dose of ACR (100 mg/kg). The time course of GSH depletion following treatment with SO/ACR, DEM/ACR, or VP/ACR showed that all three regimens were equally effective in reducing GSH in liver, cerebellum, cerebral cortex, and hippocampus. GSH levels in the liver were reduced to 4-22% of control levels between 2 and 4 hr after treatment and to 38-57% of control levels in all brain regions between 4 and 8 hr. ACR alone (100 mg/kg) reduced both brain and liver GSH to about 60% of normal. The administration of a second dose of ACR (also 100 mg/kg) 12 hr later further depleted brain and liver GSH to 33% of control. Brains were examined 2, 4, 7, 14, and 30 days after treatment by light and electron microscopy. The administration of SO plus ACR (in either order) produced lesions consisting of pyknotic granule cells confined to the anterior portions of the cerebellum and some of the small neurons of lamina II and III of the cerebral cortex. Electron microscopy revealed condensation of the granule cell chromatin and dissolution of the cytoplasm with the formation of large pericellular spaces. The granule cell lesion was not produced when the time between SO and ACR injections was either 4 or 24 hr. No pathology was observed following treatment with DEM/ACR, VP/ACR, ACR/ACR, vehicle (peanut oil), SO, or ACR alone. It appears that the neurotoxicity in animals treated with SO plus ACR is not directly the result of reduced cellular GSH levels per se, but may involve other detoxification pathways of ACR and SO.

The polyamines putrescine, spermidine and spermine in human gingival crevicular fluid.

In human periodontal disease, there may be periods of exacerbation and remission. Definition of the homeostatic mechanisms in the periodontium may therefore be important in understanding the natural history of this disorder. The polyamines are biologically active amines involved in the regulation of cell growth, regeneration of tissue and modulation of inflammation. Their occurrence was examined in gingival crevicular fluid (GCF). Fifteen sites were evaluated in four patients with moderately advanced periodontitis before and after root planing and scaling, and 15 sites were evaluated in four patients with mild inflammatory gingivitis and no attachment loss. Polyamine analysis was by high-performance liquid chromatography. GCF from untreated sites in periodontitis patients contained the highest concentration of putrescine (10(4) greater than serum). This polyamine was detected in all periodontitis samples and 12 of 15 gingivitis samples. Significant differences were seen when the amount of putrescine/30 s sample was compared: periodontitis sites before treatment 1005.7 +/- 106.1 pmol; periodontitis sites after treatment 504.7 +/- 89.2 pmol; gingivitis sites 186.7 +/- 40.1 pmol. In contrast, spermidine and spermine were detected only occasionally. Thus putrescine may play an important homeostatic role in the periodontium.

Lactate dehydrogenase activity in gingival crevicular fluid collected with filter paper strips: analysis in subjects with non-inflamed and mildly inflamed gingiva.

A previous study of lactate dehydrogenase (LDH) in gingival crevicular fluid (GCF) suggested that the concentration is 10 to 25 times that of serum (means = 2300 international units/1 versus 100 IU/1 for serum). That study used capillary tubes to collect microliter amounts of GCF. Since invasive collection techniques can influence GCF flow, we evaluated LDH activity in GCF collected by filter strips. GCF was collected in a standardized fashion from 10 subjects with mild inflammation (GI = 0.5-1.0) and 10 subjects without evidence of gingival inflammation (GI = 0). Our results indicate that LDH volume activity was greater for subjects with GI = 0 (means = 105,529 IU/1) than for subjects with GI = 0.5-1.0 (means = 77,661 IU/1), but the difference was not significant. LDH total unit activity was significantly greater in subjects with GI = 0.5-1.0 versus GI = 0 (means = 0.048 IU versus means = 0.0242 IU, P less than 0.0001). The relationship of LDH volume activity to GCF volume, the regression lines fit to the data, and calculation of LDH total unit activity were important for analysis of enzyme activity in GCF.

Polyamine biosynthesis in the developing rabbit palate.

Levels of putrescine, spermidine, and spermine and their biosynthetic enzymes, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) were measured in the developing rabbit palate between day 14 and day 18 of gestation. DNA, RNA, and protein synthesis were also measured during this time period to determine if a correlation exists between polyamine biogenesis and macromolecular synthesis. ODC activity was found to be twice as high on day 14 as on the succeeding days of gestation, while SAMDC activity did not change significantly. Levels of putrescine and spermine were higher on day 14 by 22% and 30%, respectively, than levels on day 18. Spermidine concentration did not change. DNA synthesis remained relatively constant between days 14 and 18 of gestation, suggesting that there is no peak in cell proliferation during this period. RNA synthesis was elevated significantly on day 14 and protein synthesis was significantly higher on both days 14 and 16. This data indicates that there is no correlation between polyamine synthesis and cell proliferation during this period of palatal development, but polyamines could play a regulatory role in RNA and/or protein synthesis.

Folate deficiency and in vitro palatogenesis II: Effects of methotrexate on rabbit palate fusion, folate pools, and dihydrofolate reductase activity.

Folate deficiency during pregnancy induced by dietary deprivation or folate antagonists is teratogenic. Methotrexate is a commonly used antifolate drug that produces congenital defects, with the craniofacial complex being especially vulnerable. The molecular action of this compound, therefore, was examined in vitro using fetal rabbit palates in an organ culture system. Dihydrofolate reductase activity was measured in these palates on the 4 days preceding fusion. There was no significant fluctuation in activity with gestational age. Methotrexate treatment of the palates in vitro resulted in significant reduction of enzyme activity and also decreased total folate concentration. However, the in vitro fusion ability of these palates was not altered by exposure to methotrexate. These results suggest that since methotrexate is a potent inhibitor of DNA synthesis and has its maximal effect on rapidly dividing systems, the peak in cellular proliferative activity in the fetal rabbit palate occurs very early in its development.

Folate deficiency and in vitro palatogenesis I: effects of a folate-deficient culture on rabbit palate fusion and folate pools.

Folate compounds are essential for nucleic acid metabolism and data from in vitro experiments have shown that folatee deficiency during pregnancy induced by dietary deprivation or folate antagonists is teratogenic. The craniofacial complex is a main target of this teratogen, with the lip and palate being especially vulnerable. The direct effect of folate deficiency on a target teratogenic tissue was, therefore, examined in vitro using fetal rabbit palates in an organ culture system. Folate pool sizes in the palates on the 4 days preceding fusion were determined by competitive binding assay. There was a sharp decrease in folate levels from 2.63 +/- 0.86 ng/mg wet wt on Day 15 of gestation to 1.23 +/- 0.35 ng/mg on Day 16 and 0.59 +/- 0.12 ng/mg on Day 17. The folat pool size on Day 14 was found to be quite variable with a mean of 1.93 ng/mg wet wt. Comparable results were obtained when the folate pool sizes were expressed as nanograms of folate per milligram of protein. The effect of a folate-deficiency culture medium on in vitro fusion and folate pool sizes was examined. The results of incubating palatal shelves excised on Days 14, 15, 16, and 17 of gestation for 5, 4, 3, and 2 days, respectively, show that a folate-depleted medium neither prevented nor delayed fusion. An examination of the palatal pool size after incubation in folate-deficient media revealed that while there was some decrease in folate levels, the palates apparently retained sufficient folate to support cellular activity.

Secondary palatal development in the New Zealand white rabbit: a scanning electron microscopic study.

Examination of surface topography in prefusion stages of secondary palatal development in rabbit embryos reveals a sequence of alterations in the surface cells of the epithelium along the medial margins of the palatal processes. A progressive increase in cellular protrusions resembling lamellipodia and filopodia, as well as cellular necrosis, is observed in those areas that undergo fusion. The changes precede fusion and are restricted to fusion sites. Prior to and at the time of epithelial contact between palatal processes, many long slender cellular protrusions are seen bridging the gap between the approximating tissues. The localization of the epithelial alterations and the appearance of similar cellular morphology in other embryonic epithelial fusion events strongly suggest: either an active role of the epithelial cells in the fusion of the secondary palate, or some common fundamental biochemical events that may facilitate or are responsible for the initial adhesion of such tissues.

Relationship between biotin-binding proteins from chicken plasma and egg yolk.

The plasma of laying hens contains a specific biotin-binding protein that appears to be identical with an egg-yolk biotin-binding protein. Both proteins are saturated with biotin and require elevated temperatures to effect the exchange of [14C]biotin for the protein-bound vitamin. The heat-exchange curve in each case is the same and differs sharply from that of avidin, the egg-white biotin-binding protein. On Sephadex G-100 gel filtration, plasma and yolk biotin-binding proteins were each eluted slightly ahead of avidin (mol.wt. 68,000), suggesting that they are of similar molecular weight. Plasma and yolk biotin-binding proteins required the same ionic strength to be eluted from a phosphocellulose ion-exchange column. Both the plasma and yolk biotin-binding proteins had a pI of 5; avidin has a pI of 10. Plasma biotin-binding protein cross-reacted with antiserum to yolk biotin-binding protein and showed a precipitin line of identity with purified yolk biotin-binding protein. It is suggested that biotin-binding plays an important role in mediating the transport of the vitamin from the bloodstream to the developing oocyte.

The mitochondrial malic enzymes. I. Submitochondrial localization and purification and properties of the NAD(P)+-dependent enzyme from adrenal cortex.

Rat and calf adrenal cortex homogenates were found to contain three different malic enzymes. Two were strictly NADP+-dependent and were localized, one each, in the cytosol and the mitochondrial fractions, respectively. These two enzymes appear to be identical to those described by Simpson and Estabrook (Simpson, E. R., and Estabrook, R. W. (1969) Arch. Biochem. Biophys. 129, 384-395). The third was NAD(P)+-linked and was present in the mitochondrial fraction only. All three malic enzymes separated as distinct bands during electrophoresis on 5 percent polyacrylamide slab gels at pH 9.0. Marker enzymes and the mitochondrial malic enzymes migrated together in intact mitochondria during sucrose density gradient centrifugations despite changes in the equilibrium position of the mitochondria promoted by energy-dependent calcium phosphate accumulation. In adrenal cortex mitochondria subfractionated by the method of Sottocasa et al. (SOTTOCASA, G.L., KUYLENSTIERNA, B., ERNSTER, L., and BERGSTAND, A. (1967) J. Cell Biol. 32, 415-438), both malic enzymes were associated with the inner membrane-matrix space. Sonication solubilized the two malic enzymes along with the matrix space marker enzymes. The NAD(P)+-dependent malic enzyme was purified 100-fold from calf adrenal cortex mitochondria. The final preparation was free of malic dehydrogenase, fumarase, the strictly NADP+-linked malic enzyme and adenylate kinase. Either Mn24 orMg2+ was required for activity and 1 mol of pyruvate was formed for each mole of NAD+ and NADP+ reduced. The pH optima with NAD+ and NADP+ were 6.5 tp 7.0 and 6.0 to 6.5, respectively. Michaelis-Menten kinetics were observed on the alkaline side. Fumarate, succinate, and isocitrate were positive and ATP and ADP were negative modulators of the regulatory enzyme. The modulators did not influence the stoichiometry and they were not metabolized during the reaction. Under Vmax conditions the ratios for the rate of NAD+:NADP+ reduction were 1.76 and 1.15 at pH 7.4 and 6.0, respectively. The apparent Michaelis constants also differed depending on the pH and the coenzyme. At pH 7.4 (in the presence of 5 mM fumarate) and at pH 6.0 (no fumarate) the Km values for (-)-malate, NAD+, and Mn2+ were 1.7, 0.16, and 0.15 mM, and 0.31, 0.06, and 0.09 mM, respectively. At pH 7.4 (5MM fumarate) and pH 6.0 (no fumarate), the Km values for (-)-malate, NADP+, and Mn2+ were 6.5, 0.62, and 0.59 mM, and 0.68. 0.12, and 0.31 mM, respectively. The apparent Ki values for ATP with NAD+ and NADP+ as coenzyme were 0.42 and 0.27 mM, respectively.