Quantitative detection of DNA damage in the neuronal cells of the cerebellum and cerebrum by the analysis of Feulgen hydrolysis curves

Summary Touch smears of the cerebellum and cerebrum of ageing rats were fixed with methanol, hydrolyzed with 2N HCl at various temperatures and for various periods, and stained with pararosaniline-Schiff reagent. The hydrolysis curves were determined by fluorescence cytophotometry and were computer fitted to the Bateman function to determine the kinetic parameters, the initial yield of apurinic acid or single-stranded DNA (y ₀), and the rate constants for depurination or denaturation (k ₁) and depolymerization (k ₂). The values for k ₁ (1/k ₁ is correlated with the degree of chromatin condensation) and k ₂ (which reflects the degree of DNA instability) steadily increased with age. The values for y ₀, which may indicate the degree of DNA denaturation or damage present before acid hydrolysis, also increased with age in both the cerebellum and cerebrum; however, this value was lower in the cerebellum untill 15 weeks, with the situation being reversed after 35 weeks, the cross-over time being at about 25 weeks. The values of lnk ₁ and lnk ₂ were plotted as the function of the reciprocal of the absolute temperature (T) (Arrhenius plot) for both the cerebellum and cerebrum of 15- and 74-week-old rats, and the activation energies (E) for depurination and depolymerization were calculated from the slopes. In particular, the values of E for k ₂ decreased much more quickly with age and were smaller in cerebellum. In conclusion, the degree of DNA damage and DNA instability steadily increases in both the cerebellum and cerebrum of ageing rats, and this process is much faster in the cerebellum.In honour of Prof. P. van Duijn     

Computer analysis of sub-parameters of depurination and depolymerization rate constants in Feulgen DNA hydrolysis

Feulgen DNA hydrolysis curves derived from cytofluorometry at various temperatures and HCl concentrations were computer analyzed with least squares fit to Bateman function. By comparing the depurination (k1) and depolymerization (k2) rate constants at different hydrolysis conditions, it was found that the two parameters of temperature and HCl concentration can be expressed as k = AN2 X exp (-B/T), where A and B are constants, N is the HCl concentrations, and T is the absolute temperature. From the analysis of Feulgen hydrolysis curves with 2N HCl at various temperatures, it was calculated that A = 5.3590 X 10(14) and B = 12133.543, for k1, and A = 6.2401 X 10(14) and B = 12181.660, for k2 for mouse 4C hepatocytes fixed with absolute methanol. Computer generated theoretical hydrolysis curves using the above k1 and k2 values were compared with experimental curves at various temperatures and HCl-concentrations. The two types of hydrolysis curves coincided with each other when 1-3 N HCl was used at temperatures between 30-40 degrees C. The peak times of hydrolysis curves at different conditions determined by experimental analysis and theoretical estimations also coincided reasonably well with each other. The physico-chemical phenomena underlying the equation designating k1 and k2 values are discussed.     

Accumulation of Copper Induces DNA Strand Breaks in Brain Cells of Long-Evans Cinnamon (LEC) Rats, An Animal Model for Human Wilson Disease.

Copper accumulation and induction of DNA strand breaks were investigated in the brain of Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease that is a heritable disease of copper accumulation and copper toxicity in the liver, kidney and brain. Copper contents in the brain of LEC rats increased from 20 weeks of age and were approximately 3.5 to 6 folds higher than those in the brain of WKAH rats at 24 weeks of age. Hepatic copper contents in LEC rats increased from 4 to 12 weeks of age in an age-dependent manner, and then decreased from 16 to 20 weeks of age. Thus, we consider that copper accumulated in the liver was released from severely damaged hepatocytes and deposited in the brain, although copper contents in the brain were 1/20-fold lower than those in the liver. We also evaluated the amounts of DNA single-strand breaks (SSBs) in the brain by comet analysis. The proportions of nuclei in the cerebrum and cerebellum without DNA damage decreased, and nuclei with severe DNA damage appeared in LEC rats at 24 weeks of age. The comet scores of cerebrum and cerebellum cells significantly increased in LEC rats and were significantly higher than those in WKAH rats at 24 weeks of age. The results show that SSBs in LEC rat brain cells are induced at a lower concentration of copper than are SSBs in hepatic cells.     

Cytotoxic effects of methylnitrosourea on developing brain

The effect of methylnitrosourea (MNU) on cerebellar and cerebral DNA, RNA, protein, lysosomal enzymes (acid DNase, RNase, phosphatase, and beta-glucuronidase), and 2',3'-cyclic nucleotide 3'-phosphohydrolase (2',3'-CNPase) activities was studied in rats from birth through 12 days of age. Subcutaneous injection of MNU in a dose of 0.625 mmol/kg caused a suppression of increase in weights and content of DNA, RNA, and protein of cerebellum, but no changes in those of the cerebrum or in body weight. Ratios of protein and RNA to DNA were substantially elevated by MNU in the cerebellum but not in the cerebrum. Acid DNase and acid RNase activities of MNU-treated rats were significantly elevated beyond the increase of these activities in controls in the cerebellum, but no change in these activities by MNU was observed in the cerebrum. A slight elevation in acid phosphatase activity was observed in the cerebellum but not in the cerebrum after MNU pretreatment. Beta-glucuronidase and 2',3'-CNPase activities were not changed in the cerebellum or in the cerebrum. These results suggest that in the developing brain, especially in the cerebellum at the mitotic stage, MNU caused cell damage and inhibited cell mitosis.     

Effect of cortisol on the cellular proliferation and maturation in the cerebrum and the cerebellum of the rat: Importance of the age of the animals at the beginning of treatment]

Young rats were given either a single subcutaneous injection (1 mg at 0, 1, 4 or 8 days), or four consecutive daily injections (0.2 mg/day between 0 and 3 days; 0.4 mg/day between 4 and 7 days; 0.6 mg/day between 8 and 11 days) of cortisol acetate in order to test the influence of age on the action of corticosteroids on the biochemical maturation of the cerebrum and cerebellum in terms of their DNA, RNA, and protein contents. The results showed that: 1 The diminution of the DNA content at 35 days was greater in the cerebellum (- 16 to - 32%) than in the cerebrum (- 9 to 20%); the DNA content of the cerebrum was more affected by treatment at birth, whereas that of the cerebellum was more affected by the delayed treatments. Results were different when expressed in terms of reduction of the normal increase: the gain of DNA decreased more in the cerebrum (-70%) than in the cerebellum (-40%); but the most delayed treatment induced a greater effect in both organs. These abnormalities were not always accompanied by a significant decrease of the body weight. 2 Generally, the treatments led to an increase of the mean cell territory, expressed either in terms of decrease of the DNA concentration, or in terms of increase of the organ weight/DNA ratio. Moreover, the increase of the RNA/DNA and the protein/DNA ratios constituted an indication of an accelerated cellular maturation.     

Toxic effect of lithium in mouse brain

The effect of lithium ion on glucose oxidation in the cerebrum and cerebellum of mice was measured in vitro by the conversion of isotopic glucose into 14CO2/mg wet weight. Glucose utilization is unaffected by lowest lithium dosage but is inhibited by high lithium concentrations (197-295 mM). Chronic administration of lithium to adult mice decreased the DNA content of the cerebrum and cerebellum at concentrations of 80 and 108 mM. The DNA content of selected postnatal stages of cerebrum and cerebellum was measured starting on Day 1 or 2. This served as another parameter to evaluate glucose oxidation studies at these ages. On the basis of wet weight, both brain parts of neonates of ages 1 and 10 days were approximately one-half that of the adult counterparts. On the basis of DNA content, the cerebrum enhanced its glucose utilization twofold from Day 1 to Day 10 and tripled its utilization from Day 10 to Day 20. The glucose utilization by cerebrum at Day 20 is similar to adult values. In contrast, glucose oxidation in the cerebellum remained relatively constant throughout the postnatal growth. The relative susceptibility of the two brain parts is discussed.     

Circadian rhythm in acetylcholinesterase activity during aging of the central nervous system

The circadian rhythm of acetylcholinesterase (AChE) activity was investigated in the cerebrum, cerebellum, medulla and optic lobes of rats aged 1 day and 3, 13, 44 and 87 weeks. The rhythm was found to be age-dependent. Animals aged 1 day exhibited a bimodal rhythm in all the four regions of the brain studied. At 3 and 13 weeks the activity was unimodal. The peak occurred during the light-on phase at 3 weeks and during the light-off phase at 13 weeks. At 87 weeks the rhythms in the medulla and cerebrum were similar to those of 44 week animals. By contrast the cerebellum had a bimodal rhythm with peaks at intervals of 12 h. In the optic lobes there was a shift from a bimodal pattern at 44 weeks to a unimodal one at 87 weeks. The times of onset of the light-on and light-off peaks in different regions of the brain differed with age.     

Studies on the synthesis and degradation of DNA in developing and old chick cerebellum

The levels of DNA, RNA, protein and acid and alkaline DNase were studied in developing and old chick cerebellum. The in vitro synthesis of DNA, by both chick cerebrum and cerebellum was also studied, by following the incorporation of [3H]thymidine into DNA. It was observed that the increase in DNA content of chick cerebellum continued well beyond adult stages of life span. Maximal DNA synthesis, as judged by the [3H]thymidine incorporation, was noticed during the early embryonic development but decreased with advancement of age. There was, however, another peak of activity, although smaller, at about 9 months of age. Both cerebrum and cerebellum showed similar patterns. The highest specific activity of acid DNase was also found during the early period of cerebellar development, that is at a time when rapid cellular proliferation was occurring. The activity steadily declined with the aging and in 2-year-old cerebellum very little activity could be detected. Alkaline DNase, on the other hand, not only exhibited high activity during the early development but also remained at a significant level even in old cerebellum. It is concluded that acid DNase shows a positive correlation to the early embryonic DNA synthesis but not to the cell increase occurring in old age.     

Effect of hyperphenylalaninemia induced during suckling on brain DNA metabolism in rat pups

We studied DNA metabolism (synthesis and degradation) in brain to investigate the effect of hyperphenylalaninemia induced in rats by treatment with PCPA or MPA plus PHE during suckling (4th–20th days of postnatal age) on cell proliferation and naturally occurring cell death. The incorporation of¹⁴C in DNA as percent of total radioactivity in the tissue, 30 min after administration of [¹⁴C]thymidine served as a measure of DNA synthesis in vivo, and the amount of radioactivity recovered in DNA as percent of total¹⁴C in the tissues of 21 day old rats, injected with [¹⁴C]thymidine on 2nd day after birth, indicated the turnover (degradation) of DNA. The results showed that the DNA content of cerebellum as well as cerebrum was reduced by treatment with PCPA plus PHE, while treatment with MPA plus PHE had no effect on DNA content in cerebellum but reduced the levels in cerebrum. Treatment with PCPA or MPA plus PHE reduced the synthesis of DNA in cerebrum of 11 day old rats but not in 21 day old rats, and the treatments did not affect DNA synthesis in cerebellum of either 11 or 21 day old rats. The turnover (degradation) of DNA was increased in both cerebellum and cerebrum from rats treated with PCPA plus PHE but MPA plus PHE treatment did not alter the DNA turnover either in cerebellu or in cerebrum. The activity of acid DNase was reduced in both cerebellum and cerebrum from 11 as well as 21 day old rats treated with PCPA plus PHE, but the enzyme activity was not altered in the tissues from rats of both ages treated with MPA plus PHE. The data thus indicate that in rats treated with PCPA plus PHE the reduction in cerebral DNA levels occurs due to reduced synthesis and/or increased turnover (degradation) of DNA but that the reduction in cerebellar DNA may occur only as a result of increased turnover (degradation), and that in rats treated with MPA plus PHE the reduction in cerebral DNA must occur due to reduced synthesis. This suggests that treatment of rats with PCPA plus PHE during suckling inhibits cell proliferation and/or increases naturally occurring cell death in both cerebellum and cerebrum while treatment with MPA plus PHE inhibits only cell proliferation and in cerebrum alone.     

Influence of Novel Gallium Complexes on the Homeostasis of Some Biochemical and Hematological Parameters in Rats

Chronic overexposure to cobalt (Co) may result in neurotoxic effects, but the mechanism of Co-induced neurotoxicity is not yet well established. Our study was conducted to determine whether Co is associated to the induction of central nervous system damage in pregnant rats and their progeny. Twelve pregnant female rats were randomly divided into 2 groups: group I served as controls and group II received Co (350 mg/L, orally). Treatments started from the 14th day of pregnancy until day 14 after delivery. Co concentration in plasma was higher in the treated groups than in the controls. Exposure to Co also increased the levels of MDA, PCO, H₂O₂, and AOPP, while Na⁺K⁺-ATPase and Mg²⁺-ATPase, AChE, and BuChE activities decreased in the cerebrum and cerebellum of suckling pups. A smear without ladder formation on agarose gel was also shown in the cerebrum and cerebellum, indicating random DNA degradation. A reduction in GPx, SOD, CAT, GSH, NPSH, and vitamin C values was observed. The changes were confirmed by histological results. In conclusion, these data showed that the exposure of pregnant and lactating rats to Co resulted in the development of oxidative stress and the impairment of defense systems in the cerebrum and cerebellum of their suckling pups.     

ONTOGENETIC DEVELOPMENT OF ADENYL CYCLASE AND PHOSPHODIESTERASE IN RAT BRAIN

Abstract— The activities of adenyl cyclase and phosphodiesterase were determined in homogenates of cerebrum, cerebellum and brain stem of rats of 1 day to 9 weeks of postnatal age. The activity of cerebral and brain stem adenyl cyclase measured either in the absence or presence of sodium fluoride increased rapidly for 2 weeks, reached at 20 days a maximum about three times (brain stem) or six times (cerebrum) that seen on day 1 and then declined slightly during the next several weeks. In contrast, activity of cerebrellar adenyl cyclase increased more slowly and reached a maximum at about 32 days. Activity of phosphodiesterase in cerebrum and brain stem increased several-fold during brain maturation; however, enzymic activity in the cerebellum decreased during the entire 9 week period.
In the pineal gland, adenyl cyclase activity measured in the absence of norepinephrine or sodium fluoride did not change significantly with age. However, enzymic activity measured in the presence of these agents increased with the age of the rat. Bilateral removal of the superior cervical ganglia at 1 day of age is known to arrest the sympathetic innervation of the pineal gland but did not prevent the development of this adenyl cyclase system activated by catecholamines or fluoride.     

Effect of alkylation on the secondary structure of DNA

S1 nuclease hydrolysis and bezoylated naphthoylated DEAE-cellulose (BND-cellulose) chromatography have been used to demonstrate that alkylation of DNA by dimethyl sulfate at neutral pH leads to the production of partially denatured molecules under conditions where no significant depurination occurs. DNA was alkylated with increasing concentrations of the alkylating agent, and subjected to enzymatic degradation and binding to BND cellulose. An increasing degree of DNA hydrolysis and adherence to BND cellulose was seen. On hydroxyapatite chromatography the alkylated DNA still eluted at the position of double-stranded molecules suggesting the presence of partially denatured regions. The presence of salt had a preventive effect on such denaturation.     

Preventive Effect of CuCl2 on Behavioral Alterations and Mercury Accumulation in Central Nervous System Induced by HgCl2 in Newborn Rats

This study investigated the benefits of Cu preexposition on Hg effects on behavioral tests, acetylcholinesterase (AChE) activity and Hg, and essential metal contents in the cerebrum and cerebellum of neonate rats. Wistar rats received (subcutaneous) saline or CuCl₂·2H₂O (6.9 mg/kg/day) when they were 3 to 7 days old and saline or HgCl₂ (5.0 mg/kg/day) when they were 8 to 12 days old. Mercury exposure reduced the performance of rats in the negative geotaxis (3–13 days) and beaker test (17–20 days), inhibited cerebellum AChE activity (13 days), increased cerebrum and cerebellum Hg (13 days), cerebrum Cu (13 days), and cerebrum and cerebellum Zn levels (33 days). The performance of rats in the tail immersion and rotarod tests as well as Fe and Mg levels were not altered by treatments. Copper prevented all alterations induced by mercury. These results are important to open a new perspective of prevention and/or therapy for mercury exposure.     

Ameliorating effect of selenium on chromium (VI)-induced oxidative damage in the brain of adult rats

Chromium is known for its wide toxic manifestations. This experiment aims to evaluate the effect of selenium against oxidative stress induced by chromium in the cerebrum and cerebellum. Female Wistar rats were randomly divided into four groups of six each: group I served as controls which received the standard diet; group II received drinking water K(2)Cr(2)O(7) alone (700 ppm); group III received both K(2)Cr(2)O(7) and Se (0.5 mg Na(2)SeO(3)/kg of diet); and group IV received Se (0.5 mg/kg of diet) for 3 weeks. The exposure of rats to K(2)Cr(2)O(7) promoted oxidative stress in the cerebrum and cerebellum with an increase in malondialdehyde and a decrease of nonenzymatic antioxidant levels such as glutathione, nonprotein thiol, and vitamin C. An increase of enzyme activities like catalase, glutathione peroxidase, and superoxide dismutase activities was also observed. Acetylcholinesterase activity was inhibited after treatment with K(2)Cr(2)O(7). Co-administration of Se restored the parameters cited above. The histopathological findings confirmed the biochemical results.     

A neurochemical study of developmental impairment of the brain caused by the administration of cytosine arabinoside during the fetal or neonatal period of rats

Injection of pregnant rats with cytosine arabinoside (ara-C) (280 mg/kg) on day 15 of gestation caused a significant rise (about two times the control value) in monoamine concentrations (norepinephrine, dopamine, and serotonin) accompanied by a decrease (about 60% of the control) in the brain weight and DNA content in the cerebrum of the offspring at 60 days of age. When neonatal rats were injected with ara-C (30 mg/kg/day) for four consecutive days from the fourth to seventh days after birth, a decrease of DNA content per cerebellum and an elevation of monoamine concentrations in the cerebellum were found. However, the total content of each monoamine per cerebrum or cerebellum showed no difference from the control. These results suggest that monoaminergic neurons may remain intact, with normal monoaminergic synapses compressed into a small brain volume. The neonatal administration of ara-C caused an elevation of 2, 3-cyclic nucleotide 3-phosphodiesterase (CNPase) (EC 3.1.4.37) activity and myelin protein content in the cerebellum, suggesting a relative increase in myelin concentration as a result of hypoplasia of granule cells.     

Neuroprotective effects of zinc on antioxidant defense system in lithium treated rat brain

With a view to find out whether zinc affords protection against lithium toxicity the activities of antioxidant enzymes and lipid peroxidation profile were determined in the cerebrum and cerebellum of lithium treated female Sprague Dawley rats. Lipid peroxidation was significantly increased in both the cerebrum and the cerebellum of animals administered with lithium for a total duration of 4 months as compared to the normal control group. On the contrary, the activities of catalase and glutathione-s-transferase (GST) were significantly reduced after 4 months of lithium treatment. The activity of superoxide dismutase (SOD) was significantly increased in the cerebrum after 4 months lithium administration, whereas in the cerebellum the enzyme activity was unaffected. No significant change in the levels of reduced glutathione (GSH) was found in either cerebrum or cerebellum after 2 months of lithium treatment. However, 4 months lithium treatment did produce significant changes in GSH levels in the cerebrum and in the cerebellum. Zinc supplementation for 4 months in lithium-treated rats significantly increased the activities of catalase and GST in the cerebellum, showing that the treatment with zinc reversed the lithium induced depression in these enzyme activities. Though, zinc treatment tended to normalize the SOD activity in the cerebrum yet it was still significantly higher in comparison to normal levels. From the present study, it can be concluded that the antiperoxidative property of zinc is effective in reversing the oxidative stress induced by lithium toxicity in the rat brain.     

Effects of corticosterone on protein, RNA, DNA and tubulin contents in developing male and female rat brains

The effects of corticosterone treatment on chemical components and tubulin content were studied in the cerebrum, cerebellum and hypothalamus from male and female rats during early life. A dual effect of corticosterone treatment was observed in the cerebellum during the course of growth. In the cerebellum from 10-day-old rats, total soluble protein. DNA, and tubulin content (mg per g wet tissue) increased in the hormone-treated male organ, but RNA, DNA, and tubulin content (mg per g wet tissue) increased in the hormone-treated female. On the other hand, the cerebellum from 20-day-old rats, RNA and tubulin content (mg per g wet tissue) and relative tubulin content (mg per g total protein) decreased in the hormone-treated male organ, but the female cerebellum exhibited a decrease in total protein and tubulin content (mg per g wet tissue), and relative tubulin content after corticosterone administration. Only a few effects of the corticosterone treatment were observed in the cerebrum and hypothalamus from both sexes. It is likely that corticosterone has marked effects on the cerebellum among the three brain-regions in early life, and the dual effect of the hormone in the cerebellum appears to be due to the different responsiveness in the developmental stages of nerve cells, at which the treatment was started.     

Effects of L-triiodothyronine on tubulin content in developing male and female rat brain

The tubulin content and biochemical components were determined in the cerebrum, cerebellum and hypothalamus from intact and T3-treated male and female rats during early life. T3-treatment between 0 and 9 days of age increased soluble protein, RNA DNA and tubulin content (mg per g tissue) in the 10-day-old male cerebellum but not in the cerebrum and hypothalamus except for soluble protein and tubulin (mg per g tissue), respectively. Intracellular tubulin content (mg per mg DNA) was increased by the T3-treatment in the 10-day-old male hypothalamus but not the other regions. When T3 was administered between 10 and 19 days, there was little effect of the treatment; increased tubulin (mg per g tissue) in the cerebrum and decreased RNA (mg per g tissue) and a ratio of tubulin to protein in the cerebellum from 20-day-old males. Less response to T3-treatment was observed in female cerebrum and hypothalamus but not in the cerebellum, compared with the male. These results suggest that the effect of T3-treatment on brain is modified by several factors such as tissue specificity, age-dependency and sexual differences. Modification by these factors might depend, at least in part, on changes in the number of T3-receptors due to the hormone treatment.     

Antioxidative effects of docosahexaenoic acid in the cerebrum versus cerebellum and brainstem of aged hypercholesterolemic rats

Female Wistar rats (100 weeks old) were divided into two groups; one group was fed a high-cholesterol diet (HC) and the other a high-cholesterol diet plus docosahexaenoic acid (HC-fed DHA rats). Fatty acid concentrations in brain tissues were analyzed by gas chromatography. In the HC-fed DHA rats, brain catalase (CAT), GSH, and glutathione peroxidase (GPx) increased in the cerebrum but not in the brainstem or cerebellum. The rate of increase was 23.0% for CAT, 24.5% for GSH, and 26.3% for GPx compared with that in the HC animals (p < 0.05). In the cerebrum of the HC-fed DHA rats, CAT and GPx increased, with an increase in the ratio of DHA to arachidonic acid. The cerebrum, unlike the other areas of the brain, seems to be more sensitive to DHA in stimulating CAT and GPx. We suggest that DHA plays an important role in inducing an antioxidative defense against active oxygen by enhancing the cerebral activities of CAT, GPx, and GSH.     

Kinetics of formation of 8-oxy-2'-deoxyguanosine-5'-monophosphate under the effect of heat: determination of rate constants and activation energy]

Rate constants of 8-oxy-dGMP (8-hydroxy-dGMP) formation upon incubating dGMP in H2O solutions at different temperatures were determined with differential UV-spectroscopy. Extrapolation of rate constant values obtained at elevated temperatures to 37 degrees C gives k = 5.8 x 10(-10) s-1.M-1. The activation energy for the process was estimated to be 24 kcal/mole. In D2O solutions essential lowering of the activation energy (13 kcal/mole) and rising of rate constant (k = 3.7 x 10(-9) s-1.M-1 at 37 degrees C) were observed. The strong influence of D2O on the process points to the possible participation of singlet oxygen in a heat-induced formation of 8-oxy-dGMP. The obtained values of rate constants and activation energy induced by heat show that of all types of DNA damages currently known such as single strand scission, depurination, cytosine deamination and oxidation of guanyl residues to the 8-oxo-derivatives- the last process seems to be the strongest damage of DNA resulting in such biological consequences as mutagenesis, carcinogenesis and aging.