G Omotoso, O Akinola, E Caxton-Martins, R Kadir, A Akinyinka
alp, cigarette, dna, g6pdh, ldh, nissl bodies, superior colliculus.
G Omotoso, O Akinola, E Caxton-Martins, R Kadir, A Akinyinka. Histoenzymic Effects Of Cigarette Smoke On The Superior Colliculus Of Rattus Norvegicus.. The Internet Journal of Toxicology. 2009 Volume 8 Number 1.
The study was conducted using twenty adult Wistar rats that were randomly grouped into a treatment and a control group comprising both sexes. The control group was exposed to smoke from cotton wool, while the treatment group inhaled smoke from a completely burnt cigarette containing 0.735 g tobacco, once daily for 14 days. The rats were sacrificed by cervical dislocation and the superior colliculi (SC) excised, weighed, homogenized and assayed for the activities of lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PDH), and alkaline phosphatase (ALP). SC tissues for histological studies were fixed in formol calcium and subsequently processed using Feulgen reaction for nucleic acids and Cresyl violet stain for Nissl bodies studies. There was an increase in the enzyme activities in the treated group, compared to the control; and this increase was more in the female rats although not statistically significant. The derangements observed in the enzyme studies led to various histoarchitectural changes in the SC of the rats exposed to tobacco smoke, and a gradual depletion of DNA positive cells and Nissl bodies in the neurons.
Many of the 4 000 constituents of tobacco smoke are known to be toxic to the body, affecting the brain, cardiovascular and respiratory systems, among other systems and organs of the body1. Many of these components are volatilized during burning, and others, including the aromatic hydrocarbons, are formed during combustion of the leaf components 2. By virtue of the short-term actions of nicotine on the cholinergic system, it has positive effects on certain cognitive domains, and has been said to be neuroprotective in a way3. The chief pharmacologically active ingredients are nicotine (acute effects) and tar (chronic effects) 4. A completely burned cigarette stick usually contains between 1-6 mg of nicotine, apart from the 1-5 % carbon monoxide present in the smoke 5.
The superior colliculus (SC) functions in visual attention and centering of the visual image on the retina, thereby serving essentially as a visual relay centre6. It transforms both visual and non-visual sensory signals into motor commands that control orienting behaviors7. Studies by Yildiz
Tobacco smoke, as an exogenous source of reactive oxygen species, has a broad spectrum of oxidant-ionising radiation which generates free radicals in exposed tissues9. Due to a low oxygen tension, anaerobic glycolytic pathway is utilized to supply the brain with its ATP requirement. The conversion of lactate to pyruvate is catalyzed by lactate dehydrogenase (LDH). Following nicotine administration, LDH leakage through the membranes is elevated, due to its effects on some membrane markers, thereby increasing the level of this enzyme 8,10.
Glucose-6-phosphate dehydrogenase (G-6-PDH) is important in the production of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and the production of ribose, which are vital in RNA and DNA replication11.
Alkaline phosphatase (ALP), a membrane marker, facilitates transport across cell membranes, causing the breakdown of ATP to ADP and inorganic phosphate, thereby making free energy available for metabolic processes12. The present work was designed to study the effect of nicotine on the morphology of the superior colliculus (especially in relation to DNA and Nissl bodies) and certain enzymes of glucose metabolism in a model of Wistar rats.
Materials and Methods
Following appropriate ethical guidelines, twenty (20) adult Wistar rats of both sexes with an average weight of 219.5 g were randomly divided into a control and a treated group. They were allowed to acclimatize, and housed in different cages at normal room temperature, at the Anatomy Department Animal House, University of Ilorin. Feeds and water were available
The animals were sacrificed by cervical dislocation 18 hours after the last exposure. The cranium was gently opened to expose the brain, and the superior colliculi (SC) were identified and excised. Immediately after, the SC were weighed and placed in 0.25 M sucrose solution, and homogenized using a homogenizer. The homogenate was centrifuged at 500 rpm for 5 min using a centrifuge (Gallenkomp, England). The supernatants collected, using Pasteur pipettes, were stored at -20°C, and thereafter enzyme study was carried out for the activities of lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-PDH) and alkaline phosphatase (ALP). Through spectrophotometry (Colorimetric method), the activities of LDH and (G-6-PDH) were determined in the homogenates using the RANDOX’s Kits (Antrim, UK)14, and the
The Student’s t-test was used in analyzing the biochemical data collected, and p values of < 0.05 were considered significant.
Increased levels of enzyme activity were noticed in the SC of animals treated with cigarette smoke compared with those exposed to cotton wool smoke. The average LDH activity in the SC of the male treated group was 1262.5 U/L, higher than the control value of 1050.0 U/L. The level of LDH activity in the female rats was significantly higher (1817.5 U/L: p<0.05) in the treated group compared with the control (1135.0 U/L) (Figure 1); and there was statistically significant difference between the treatment groups of both male and female rats (p<0.05).
G-6-PDH activity was slightly higher in the male treated group (1415.0 U/ml homogenate) than the control (1394.0 U/ml homogenate) (Figure 2). However, more increase was noted in the female treated group (1437.5 U/ml homogenate; p<0.05) than the control (1295.0 U/ml homogenate); and, there was a significant difference between the male and female treatment subgroups (p<0.05).
Exposure to cigarette smoke produced higher levels of ALP in the treated groups of both male and female rats (85.5 IU/L:-male and 130 IU/L:-female) than the control groups (71 IU/L:-male and 55 IU/L:-female) (Figure 3), with the females having a more than two-fold increase (p<0.05).
The histological findings of the superior colliculus of the male Wistar rats revealed numerous neurons, majority of which were multipolar, with preserved architecture, and presence of Nissl bodies and DNA-positive cells, in the control groups (Figures 4, 7); while the treated groups showed sparsely dispersed neurons, vacuolated spaces, and progressive reduction in intensity of staining for Nissl bodies and DNA (Figures 5, 6, 8, 9) compared with the control.
Nicotine affects the process of hormone secretion, enzyme activities, and other cellular mechanisms8. To maintain brain functions during hypoxia, its energy requirement is generated via anaerobic glycolysis, where LDH catalyses the conversion of lactate to pyruvate.
During cellular oxidative stress, whether endogenous in origin or initiated by drugs, or environmental chemicals, NADPH is critical for maintaining glutathione (GSH) in its reduced form, which is essential for detoxification of reactive free radicals and lipid hydroperoxidase 11. Hence, NADPH is important for its role in the detoxification of hydrogen peroxide. The production of ribose by hexose monophosphate shunt (HMS; for which (G-6-PDH) is the rate-limiting enzyme) is relevant to the synthesis of nucleotides used in RNA and DNA replication, and hence, cell division and possibly DNA repair11.
Administration of nicotine causes an increased LDH activity, with an attendant disturbance in cellular membrane integrity8, and chronic cigarette smoking in man enhances the activity of LDH in the serum16.
Carbon monoxide from cotton wool smoke and cigarette smoke (which also contain carbon monoxide) can both induce oxidative stress in the rat’s brain, resulting in the increased activity of the enzymes of carbohydrate metabolism following the initiation of anaerobic glycolysis. Consequently, the level of activities of these enzymes were observed in this study to be higher in animals exposed to smoke from completely burnt cigarettes than those exposed only to cotton wool smoke.
The increased LDH level following exposure to cigarette smoke was much more higher among the female rats compared with their control, than it was for the male groups. Similarly, the level of ALP, which is responsible for transport across cell membranes, was especially higher in the female subgroup exposed to cigarette smoke, consequently altering the integrity of the membrane and its ability to mediate normal membrane transport. As a result of this also, leakage of LDH through the membranes was encouraged10. Although tobacco has been noted to inhibit ovarian functions17,18, the statistically significant higher levels of enzyme activities observed in the superior colliculus of the female Wistar rats might also be due to their lower body weights, compared to the male animals with higher body weights, at the time of sacrifice. At the commencement of the experiment, both male and female Wistar rats had very close body weights, but the rate of growth and increase in body weight was much faster in the male animals compared to the females.
The synthesis of proteins is critical to the growth and development of any cell, and nucleic acids as well as Nissl bodies are crucial in this19. Nissl bodies are strongly basophilic inclusions found in the cell body of the neuron, containing numerous ribosomes. The histological findings in the superior colliculus of the male rats showed a sparse distribution of neurons in the animals exposed to cigarette smoke, and various forms of cellular distortions and degeneration. The longer the number of days the treated groups were exposed, the more the reduction in the number of neurons, the more the degree of architectural distortions, cellular degeneration and vacuolation. There was a gradual decline in the staining intensity of both DNA-containing cells and Nissl bodies, which was associated with prolonged duration of exposure to cigarette smoke. The depletion of the cellular constituents in the neurons could adversely affect protein synthesis and consequently, vital cellular processes and mechanisms.