Effect of Nutritional Status and Ozone Exposure on Rat Brain Serotonin

Archives of Medical Research 33 (2002) 15–19

ORIGINAL ARTICLE

Effect of Nutritional Status and Ozone Exposure on Rat Brain Serotonin M. Gerardo Barragán-Mejía, Luis Castilla-Serna, David Calderón-Guzmán, J. Luis Hernández-Islas, Norma A. Labra-Ruiz, R. Antonio Rodríguez-Pérez and Daniel Santamaría-Del Angel Laboratorio de Neuroquímica, Torre de Investigación Dr. Joaquín Cravioto, Instituto Nacional de Pediatría (INP)–Secretaría de Salud (SSA), Mexico City, Mexico Received for publication May 4, 2000; accepted July 20, 2001 (00/067).

Background. Ozone is an environmental pollutant that has widely documented deleterious effects on exposed organisms. In Mexico City, this pollutant frequently reaches concentrations that surpass safe health limits. In addition, it has been reported that the prevalence of malnutrition remains high in our childhood population. This experiment was carried out to determine whether malnutrition is a factor contributing to an increase in the risk of damage associated with ozone exposure. Methods. Using an experimental animal model, 21-day-old rats fed normally or with induced malnutrition were subchronically exposed to 0.5 ppm of ozone or fresh air, respectively, for 30 days. At the end of this period and using HPLC, serotonin concentrations were measured in four areas of the brain: cortex, hemispheres, cerebellum, and medulla oblongata. Results. Malnourished animals had a significant weight deficit beginning at 28 days with respect to well-fed animals. Among the well-fed animals, this phenomenon is seen at 35 days in exposed and non-exposed animals. In the four regions of the brain, malnourished animals show low serotonin concentrations with respect to well-nourished animals. In the cerebellum, there was an interaction between the nutritional factor and ozone exposure, while in the medulla oblongata both factors acted independently. Conclusions. Our results suggest a multiplicative effect from the nutritional factor and ozone exposure in the changes observed concerning serotonergic metabolism. © 2002 IMSS. Published by Elsevier Science Inc. Key Words: Malnutrition, Ozone, Brain serotonin.

Introduction Ozone is an allotropic oxygen isotope capable of generating free radicals due to its physical-chemical characteristics upon coming into contact with biological systems. It has been associated with a wide range of conditions in organisms exposed to its action (1–4). In the atmosphere of large urban centers, this pollutant frequently reaches concentrations over the set norm. Hicks and colleagues (5) reported that as a consequence of photochemical processes, ozone levels in Mexico City’s atmosphere rose during 1987–1992. Address reprint requests to: M. Gerardo Barragán Mejía, Torre de Investigación Dr. Joaquín Cravioto, INP-SSA, Av. Imán #1, Col. Insurgentes Cuicuilco, Coyoacán, 04530 México, D.F., México. Tel.: (52) (55) 5606-5026, exts. 429 and 441; FAX: (52) (55) 5606-9455; E-mail: [email protected]

In humans, damage to the respiratory system such as nasal mucosal and inflammatory response changes, as well as reduction of ventilatory capacity, has been reported as a consequence of ozone exposure (6–8). In animals, there is evidence that exposure to ozone may cause changes in the central nervous system (CNS) (9,10), specifically on serotonin (5-HT) brain levels (11) that actively participate in the previously proven regulation of physiologic, behavioral, and learning processes. It has also been demonstrated that litters from mothers exposed to ozone during gestation have lower body weights than litters from non-exposed mothers. Changes are observed in sleep patterns (12,13), the regulation of which is thought to be controlled by serotonin (14), whose brain levels are susceptible to the animal’s nutritional state (15). In this sense, it has been demonstrated that a low protein-high carbohydrate diet induces a decrement in

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Barragán-Mejía et al./ Archives of Medical Research 33 (2002) 15–19

the serotonin levels in cortex, hippocampus, and hypothalamus (16). Ozone pollution is not seen in all urban centers in Mexico. However, malnutrition is a highly prevalent condition in our childhood population. As has already been proven, malnutrition produces adverse effects on mental development, behavior, and learning, mainly when occurring in early stages of life (17). Additionally, it has been demonstrated that deficient intake of the amino acid tryptophan induces alterations in serotoninergic metabolism, which are related to regulation of sleep patterns in humans. At the national level, there is a prevalence of malnutrition of between 30 and 42.7% in children 5 years of age (18,19). A survey conducted in 1995 on Mexico City’s urban population revealed that a significant number of preschoolers have a certain degree of malnutrition (20). Therefore, it is expected that urban centers with high ozone pollution indexes, such as in Mexico City, contain a sector of individuals with nutritional deficiencies who are exposed to the effects of this pollutant. The purpose of this study was to document the contribution of the nutritional condition to the production of adverse effects by ozone exposure on serotonin brain levels using an experimental animal model. Materials and Methods Twenty male Wistar strain 21-day-old rats from mothers normally fed during pregnancy were randomly selected, comprising four groups, i.e., animals with induced malnutrition exposed to 0.5 ppm of ozone (MNO3) or fresh air (MNA), and animals without malnutrition induction exposed to ozone (WNO3) or air (WNA). Malnutrition was induced beginning at day 21 of age, feeding the animals a 7% protein content diet (Table 1). Animals without induced malnutrition were fed regular lab rodent bioterium feed containing a minimum of 23% protein (Lab Rodent Diet 5001, Purina Milk Co., PMI Feeds, Inc., St. Louis, MO, USA). All groups were fed and given water freely and were kept under normal light-dark cycles. Animal weight was recorded weekly. Ozone exposure. Ozone exposure was carried out using the method previously validated at our laboratory (21). We used highly resistant acrylic chambers measuring 60  50  30 cm (length, width, height) divided into two compartments with a capacity for 12 animals each. Malnourished and wellnourished animals were placed in one of the compartments. These animals were exposed 6 h per day to 0.5 ppm of ozone in air at a flow rate equivalent to 2 L/min. The other compartment contained animals under the same conditions and exposed to filtered fresh air. Ozone was obtained using an ozone generator (model W-10, Tecnozono de México, Mexico City). Its concentration was constantly monitored using a Dasibi 1008 PC (Glendale, CA, USA) monitor pre-

viously calibrated by the Mexican Ministry of the Environment, Natural Resources, and Fishing. The animals were subjected to these conditions for 30 consecutive days. Serotonin measurement. After 30 days of exposure, the animals were sacrificed by decapitation; their brains were removed and dissected into four areas: cortex; hemispheres (hypothalamus, striatum, hippocampus, and mid-brain); cerebellum, and medulla oblongata according to Glowinski’s technique (22). Each area was homogenized in 10 volumes of 0.1 N cold perchloric acid and centrifuged at 15,000 rpm (13,000 g) for 10 min. Twenty microliters of the supernatant was injected into a high pressure liquid chromatograph (Perkin-Elmer series 3B, Perkin-Elmer Co., Norwalk, CT, USA) equipped with a Bondapack C18 column, using 0.01 M sodium acetate as the mobile phase buffer at a pH of 4 and methanol in a 85:15 ratio at a 1.5 mL/min flow. Detection was carried out using an ultraviolet (UV1000) detector with Winner Windows 2.0 software (Thermo Separation Products, San Jose, CA, USA) at 280 nm. The concentration of the compound at hand in each region was estimated using the interpolation of the area in a curved graph using known 5-HT concentrations (y  0.011  1.00x, r  0.999) and reported in g/g of wet tissue. Some quality control data of serotonin determination are as follows: recovery 86  0.7%; linearity 12–360 ng, and detection limit 5 ng, CV 4%. Statistical analysis. The data are shown as mean values  standard deviations (SDs). The strategy used for statistical analysis consisted of the following: comparing weights between groups using one-way analysis of variance (ANOVA); serotonin concentration for each area between groups using one-way ANOVA with multiple comparisons (Bonferroni method), and evaluating the influence of nutritional and ozone exposure by using two-way analysis of variance. A p value of 0.05 was considered significant (23). Results Figure 1 shows changes in animal body weights during 30 days of exposure. Beginning on day 28, malnourished animals gained less weight until they reached a final deficit of approximately 60% with respect to well-fed animals. This phenomenon was seen beginning on day 35 in well-fed animals. Animals exposed to ozone showed a final weight deficit of approximately 13% with respect to those exposed to air, a significant difference (p 0.01). Table 2 shows means and SD values for the 5-HT concentration in the four studied areas. Analysis by one-way ANOVA with multiple comparisons allowed us to observe that in cortex the WNO3 group shows the higher concentration of 5-HT, which is significantly different regarding the WNA and MNA groups. In hemispheres, we observed differences between WNA and MNA among the WNO3, MNA, and MNO3 groups in favor of the WNO3 group, and

Nutritional Status, Ozone Exposure, and Rat Brain Serotonin

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Figure 1. Weight evolution of the four groups of animals during the 30 days of the study. ANOVA ap 0.01 from this point between well-nourished and malnourished animals; bp 0.05 from this point between exposed and non-exposed animals.

within the WNA group with respect to the MNO3, in which the lowest concentration of 5-HT is observed. With regard to the cerebellum, we observed a significant difference between WNA and MNA groups; in this particular area, we found that well-nourished animals exhibit significantly higher 5-HT levels than malnourished animals, and that the MNO3 group is again the group presenting the lowest value. In medulla oblongata, the difference is observed between the well-nourished groups and the MNO3 group; in this area, the fact that stands out is the statistically significant difference observed between the MNA and MNO3 groups. In general, the well-nourished groups show the highest 5-HT values while the MNO3 exhibited the lowest. The difference observed between the WNA and WNO3 groups in cortex indicates that under the conditions of this study ozone exposure induces an increment in the serotonin level, while a decrement was observed in hemispheres and cerebellum due to malnutrition. Analysis by two-way ANOVA (Table 3) revealed that in hemispheres and cerebellum, the observed differences are

explained by the nutritional factor; however, in cerebellum interaction is appreciated between the two factors. With regard to cortex, both factors apparently act in an independent fashion although the nutritional factor alone reaches statistical significance. Finally, in medulla oblongata it is clear that the two factors act independently. Discussion The weight difference observed in well-nourished animals exposed to ozone is a common finding and has been previ-

Table 2. Mean  SD of 5-HT levels in the four studied areas Region

Group

n

Mean

SD

Prosencephalus

WNA WNO3 MNA MNO3 WNA WNO3 MNA MNO3 WNA WNO3 MNA MNO3 WNA WNO3 MNA MNO3

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

1.48876 1.70001a 1.32609 1.44955 1.71314b 1.78150b 1.46290 1.33555 2.12365c 2.42643c 1.55165 1.23596 1.27313d 1.13276d 1.17060 0.64748e

0.08867 0.05175 0.24286 0.24511 0.11872 0.07851 0.23058 0.13663 0.13849 0.30586 0.028499 0.046861 0.021245 0.021881 0.21528 0.26643

Diencephalus Table 1. Composition of diet fed to animals with induced malnutrition Diet ingredients Purina Starch Bran Vitaminsa Mineralsb

Percentage (%)

Mesencephalus

3.926 58.445 37.629 0.05 0.10

Metencephalus

a Vitamins: A, 125,000 IU; D3, 41,500 IU; B12, 500 g; E, 40 IU; B2, 90 mg; B1, 100 mg; B6, 50 mg; C, 100 mg; K, 100 mg; Pantothenic, 400 mg; Nicotinamide, 3 mg; Folic acid, 100 g; bMinerals: P, 17.5%; Na, 5%; Ca, 6%; Mg, 4%; Zn, 1%; S, 0.4%; Fe, 0.3%; Cu, 0.25%; Mn, 0.20%; I, 0.05%; Co, 0.01%; Se, 0.01%.

a

Statistically different with regard to WNA and MNA groups; b statistically different from MNA and MNO3 groups; c significantly different from MNA and MNO3 groups; d statistically different with regard to MNO3; esignificantly different from the MNA group.

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Barragán-Mejía et al./ Archives of Medical Research 33 (2002) 15–19

Table 3. Factor interaction analysis by two-way analysis of variance Region Prosencephalus

Diencephalus

Mesencephalus

Metencephalus

Factor

F

p

Power

Nutrition Ozone Nutrition  ozone Nutrition Ozone Nutrition  ozone Nutrition Ozone Nutrition  ozone Nutrition Ozone Nutrition  ozone

6.011 4.191 0.0778 26.314 0.189 2.079 37.557 0.002 4.625 8.213 10.464 3.482

0.026 0.057 NS 0.784 NS 0.000 0.670 NS 0.169 NS 0.0001 0.965 NS 0.047 0.011 0.005 0.080 NS

0.674 0.498 0.081 0.998 0.690 0.273 1.000 0.050 0.524 0.767 0.859 0.419

NS  not significant.

ously reported. The explanation suggested is that O3 causes a decrease in consumption of food and water by these animals (24,25). In this experiment, well-nourished animals exposed to ozone show a significant 5-HT increase compared with well-nourished non-exposed animals in cortex. Other authors reported a 5-HT increment in pons as a consequence of 1.5 ppm ozone exposure for 24 h; this indicates that acute and chronic ozone exposure induces changes in 5-HT metabolism in specific brain areas of well-fed animals (26,27). In animals subjected to malnutrition, the panorama is entirely different. According to our data analysis, differences found in hemispheres and cerebellum between well-nourished and malnourished groups in 5-HT levels are explained to a great degree by the nutritional factor. In this experiment, deficient protein intake appears to be implicated in the reduction of 5-HT concentration in some brain areas. This is a result contrary to that reported for animals submitted to in utero malnutrition (15,28) in which high 5-HT levels are recorded even after nutritional recovery. The explanation proposed by these authors is that elevation in serotonin concentration is due to an increase in the passage of free tryptophan, an immediate precursor of 5-HT to the brain, and to the high activity of tryptophan-5-hydroxylase (29). However, it has been reported that 5-HT concentration decreases in animals that consume lowprotein diets ad libitum while increasing in animals consuming limited amounts of this diet (30). The authors propose that limited intake of low protein diets intensifies the passage of circulating tryptophan toward the brain, thereby increasing serotonin levels. In this sense, it has been shown that animals submitted to different malnutrition regimens show a decrease in 5-HT concentrations in the hypothalamus (31) and diencephalus (32), as well as in the circadian rhythm of 5-HT and circulating tryptophan (33). Our results agree with the previously mentioned authors, suggesting that the feeding scheme and the period during which malnutrition was induced were determinants in the production of the differences observed in serotonin levels. However, interaction of the factors found for the cerebellum and the independent action of both in the medulla oblongata suggests the participation of

ozone in the decrease of serotonin levels observed in these regions. In well-nourished animals, it has been proposed that the mechanism by which ozone induces changes in serotonin metabolism involves the participation of free radicals and lipid peroxidation, because both factors increase as a consequence of ozone exposure (10,34,35). These are products to which the nervous system is highly susceptible. It has been shown that the activity of oxidative stress protective enzymes changes as a consequence of dietetic manipulation in rats (36,37) and due to malnutrition in humans (38). In humans exposed to ozone due to environmental pollution, there have been reports concerning the decrease of superoxide dismutase activity (39). It would then appear that changes in 5-HT levels found in this study, especially in the cerebellum and medulla oblongata, are due to the fact that the mechanism involves the participation of both malnutrition and ozone exposure. Although we are unable to state with total assuredness what the nature of this mechanism is, there is evidence that the consumption of an unbalanced protein diet induces a decrease in 5-HT and other neurotransmitters together with an increase in the activity of monoamine-oxidase MAO (40), an effect reported as one of the first findings of CNS ozone exposure (41). Nonetheless, further studies should be conducted to explore the specific activity of MAO under these conditions with the purpose of further clarifying this matter. The results of this study allow us to conclude that lack of intake of nutrients together with exposure to environmental pollutants that cause oxidative stress may result in important changes in serotonergic metabolism with implied consequences on the behavior and functioning of the CNS.

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