Disclosure: There is no conflict of interest.
Metabolic profile and genotoxicity in obese rats exposed to cigarette smoke
Article first published online: 13 MAY 2013
Copyright © 2012 The Obesity Society
Volume 21, Issue 8, pages 1596–1601, August 2013
How to Cite
Damasceno, D. C., Sinzato, Y. K., Bueno, A., Dallaqua, B., Lima, P. H., Calderon, I. M.P., Rudge, M. V.C. and Campos, K. E. (2013), Metabolic profile and genotoxicity in obese rats exposed to cigarette smoke. Obesity, 21: 1596–1601. doi: 10.1002/oby.20152
- Issue published online: 22 AUG 2013
- Article first published online: 13 MAY 2013
- Accepted manuscript online: 6 NOV 2012 08:13AM EST
- Manuscript Accepted: 24 OCT 2012
- Manuscript Received: 9 FEB 2012
Experimental studies have shown that exposure to cigarette smoke has negative effects on lipid metabolism and oxidative stress status. Cigarette smoke exposure in nonpregnant and pregnant rats causes significant genotoxicity (DNA damage). However, no previous studies have directly evaluated the effects of obesity or the association between obesity and cigarette smoke exposure on genotoxicity. Therefore, the aim of the present investigation was to evaluate DNA damage levels, oxidative stress status and lipid profiles in obese Wistar rats exposed to cigarette smoke.
Design and Methods
Female rats subcutaneously (sc) received a monosodium glutamate solution or vehicle (control) during the neonatal period to induce obesity. The rats were randomly distributed into three experimental groups: control, obese exposed to filtered air, and obese exposed to tobacco cigarette smoke. After a 2-month exposure period, the rats were anesthetized and killed to obtain blood samples for genotoxicity, lipid profile, and oxidative stress status analyses.
The obese rats exposed to tobacco cigarette smoke presented higher DNA damage, triglycerides, total cholesterol, free fatty acids, VLDL-c, HDL-c, and LDL-c levels compared to control and obese rats exposed to filtered air. Both obese groups showed reduced SOD activity. These results showed that cigarette smoke enhanced the effects of obesity.
In conclusion, the association between obesity and cigarette smoke exposure exacerbated the genotoxicity, negatively impacted the biochemical profile and antioxidant defenses and caused early glucose intolerance. Thus, the changes caused by cigarette smoke exposure can trigger the earlier onset of metabolic disorders associated with obesity, such as diabetes and metabolic syndrome.
Cigarette smoking and the lifestyle factors underlying obesity appear to play a major role in excess medical morbidity and mortality . Both can trigger hyperlipidemia, coronary artery disease, Diabetes mellitus and cancer. Most of these comorbid conditions affect quality of life . In 2000, 4.8 million premature deaths worldwide were attributed to smoking, and this number is expected to increase to 10 million per year by 2030. Furthermore, several studies have shown that smoking leads to a greater number of deaths from cardiovascular and degenerative diseases .
Obesity is a metabolic disorder that affects individuals of all ages and social classes. It is related to hyperlipidemia, changes in lipoprotein distribution and systemic oxidative stress in humans  and laboratory animals . The World Health Organization (WHO) reported that 300 million adults worldwide are obese . Because of its high social costs, the understanding of the mechanisms underlying the development of obesity and its metabolic consequences are of primary importance for public health .
Studies to explore the mechanisms responsible for these changes in humans are limited not only by ethical considerations but also by the multiplicity of uncontrolled variables such as socioeconomic characteristics, nutritional status and genetic factors. Thus, there is a need for appropriate animal models . Experimental studies in animals have shown that exposure to cigarette smoke has negative effects on lipid metabolism  and oxidative stress status . Studies in our laboratory have shown that cigarette smoke exposure enhances genotoxicity (DNA damage) in non-pregnant and pregnant rats . However, no previous studies have directly evaluated the effects of obesity or the association between obesity and cigarette smoke exposure on genotoxicity. Therefore, the aim of the present investigation was to evaluate DNA damage levels, oxidative stress status and lipid profiles in obese Wistar rats exposed to cigarette smoke.
Materials and Methods
Six-week-old female Wistar rats weighing ∼190 g were obtained from the Center of Biological Investigation (CEMIB, UNICAMP, Campinas, São Paulo State, Brazil). During the acclimation (2 weeks) and experimental periods, the rats (five per cage) were maintained in an experimental room under controlled temperature (22°C ± 2°C), humidity (50% ± 10%), and light (12 h light/dark cycle) with ad libitum access to a commercial diet (Purina® Rat Chow, Sao Paulo State, Brazil) and tap water. The Local Experimental Ethical Committee for Animal Research approved the protocols used in this study. These female rats were mated with healthy male rats, and experimental obesity was induced in the female offspring.
Obesity experimental design
Female offspring received a subcutaneous (sc) injection of monosodium glutamate (MSG; Sigma, G-1626, St. Louis, MO) diluted in buffer solution at a dosage of 4.0 mg g−1 body weight at days 2, 4, 6, 8, and 10 of life. The obesity parameter was estimated according to the Lee index for each animal at 90 days of life. This index was calculated by the cube root of body weight (g) × 10/nasoanal length (mm), for which a value equal to or lower than 0.300 was classified as normal. Rats with values higher than 0.300 were classified as obese and included in this experiment . The animals were distributed into two experimental groups: Obese exposed to filtered air (n = 09 rats) (Obese), and obese exposed to tobacco cigarette smoke from 90 to 150 days old (n = 10 rats) (Smoking obese). Nonobese rats (n = 10) (Control) that received saline solution (2.0% NaCl) in the same experimental procedures were used as controls for both obese groups. The Lee index was also measured in adult rats at the beginning and the end of filtered air or cigarette smoke exposure period (Timepoint 1 (90 days of life) and Timepoint 2 (150 days of life), respectively).
Cigarette smoke exposure
A commercially available nonfilter cigarette was used. Each cigarette contained 10 mg of tar, 0.80 mg of nicotine and 10 mg of carbon monoxide. A stream of smoke was generated by a mechanical smoking device and delivered into a chamber for the cigarette exposure group. At the first timepoint (cigarette smoke adaptation period), the smoke-exposed obese group rats (∼90 days old) were placed into whole-body exposure chambers to adapt to filtered air or to cigarette smoke for 30 min (minutes) once per day (5 cigarettes/day) for 7 days. After adaptation to cigarette smoke exposure, the animals were placed into a chamber and exposed to the smoke from 20 cigarettes/day for 30 min, 2 times each day for 60 days. Each cigarette was puffed 15 times for 3 min at a rate of 5 puffs/min. Fresh air inhalation was allowed for 1 min after every 3 min of cigarette smoke exposure. During the experiment, carbon monoxide was controlled at 195 ppm, the temperature was maintained at 22-25°C, and the relative humidity was ∼40%. The control and obese rats were exposed to filtered air. This exposure level corresponds to ∼3-4 packs/day in humans, based on carboxyhemoglobin levels .
Insulin tolerance test (ITT)
The insulin tolerance test was performed 4 days before the end of experimental period for all groups, after 12 h of fasting overnight. The ITT consisted of a bolus injection of insulin solution (3.33 U mL−1) in the subcutaneous dorsal region (30 mU/100 g body weight). Blood samples were obtained from a cut tail tip for serum glucose determinations (glucose oxidase) at 0, 30, 60, and 120 min after insulin administration .
Oral glucose tolerance test (OGTT)
The oral glucose tolerance test was performed 2 days before the end of the experimental period in all groups of this study. After 12 h of fasting overnight, a glucose solution (200 g L−1) was administered into the stomach of the rats through a gastric catheter at a final dose of 2.0 g kg−1 body weight. Blood samples were obtained from a cut tip tail for glycemic determinations (glucose oxidase) at 0, 30, 60, and 120 min after glucose administration. Glucose responses during the glucose tolerance test were evaluated by estimating the total area under the curve (AUC) .
Collection of blood samples
At the end of the 2-month exposure period (60 days of experiments using cigarette smoke), each rat was anesthetized and humanely sacrificed to obtain blood samples. Blood samples were used for genotoxicity, oxidative stress status and lipid level analyses.
A 10 μl volume of peripheral blood cell suspension was mixed with 100 μl of 0.5% low melting point agarose at 37°C, layered onto pre-coated slides with normal melting point agarose, and immediately covered with a cover-slip. The slides were left at 4°C for 5 min to allow the agarose to solidify. The cover-slip was gently removed, and the slides were immersed in ice-cold freshly prepared lysis solution. After 1 h in the dark at 4°C, the slides were briefly washed in PBS to remove the excess lysis solution and placed on a horizontal electrophoresis unit filled with fresh alkaline electrophoresis buffer. Electrophoresis was conducted at 4°C for 20 min at 25 V and 300 mA. All steps were carried out under minimal illumination. The slides were neutralized in a buffer, stained and analyzed with a Nikon fluorescence microscope connected to a charge-coupled device (CCD) camera and a personal computer-based analysis system (Comet Assay II, Perceptive Instruments, UK) to determine the extent of DNA damage. The results were expressed as the percentage of DNA in the tail (amount of DNA in the tail divided by the amount of DNA in the cell multiplied by 100 – tail intensity; % tail), tail moment (the product of the tail and the mean distance of migration in the tail; arbitrary units), and tail length (distance from the middle of the estimated perimeter of the comet head to the last visible signal in the tail in μm). One hundred randomly selected cells (50 from each of two replicate slides) were scored per blood sample .
Lipid profile determination
Blood samples were collected from each rat into anticoagulant-free test tubes, maintained at a low temperature for 30 minutes (min) and then centrifuged at 1,300g for 10 min at 4°C. The supernatant was collected as serum and stored at –80°C for further determination of biochemical parameters. Serum concentrations of total cholesterol, triglycerides and high-density lipoprotein (HDL-c) were determined enzymatically using Wiener® assay kits (Rosario, Argentina). Serum very-low-density lipoprotein (VLDL-c) and low-density lipoprotein (LDL-c) levels were calculated based on the previously measured lipid concentrations (cholesterol, triglycerides, and HDL-c levels), and the results were expressed in milligram (mg) per deciliter (dL) . The free fatty acid concentration (FFA) was determined in according to Reogun's modified procedure, and the results were expressed in milliequivalents (mEq) per liter (L) .
Oxidative stress analysis
For oxidative stress analysis, blood samples were transferred to anticoagulant tubes (sodium heparin, Cristalia, São Paulo, Brazil) and then centrifuged at 90g for 10 min at 25°C. All of these biomarkers were measured in the washed erythrocytes. The oxidative stress biomarkers measured were superoxide dismutase activity (SOD), thiol groups (SH group), glutathione peroxidase activity (GSH-Px) and thiobarbituric acid reactive substances (malonaldehyde, MDA). MDA was used as a lipid peroxidation index. Briefly, 1.0 mL of washed erythrocytes were added to the test tube containing 1.0 mL of 3.0% sulfosalicylic acid (Sigma, S-7432, São Paulo, Brazil), agitated for 10 seconds (s), centrifuged at 1,300g for 3 min and allowed to rest for 15 min. The sample was diluted to 500 μL with 0.67% thiobarbituric acid solution (2-TBA, T5500, São Paulo, Brazil). The mixture was heated to 80°C for 30 min, and the absorbance was measured at a wavelength of 535 nm. The results were expressed as nmoL of MDA per gram of hemoglobin (nmol/g Hb). SOD activity was determined from its ability to inhibit the auto-oxidation of pyrogallol. The reaction mixture (1.0 mL) consisted of 5.0 mmol L−1 tris (hydroxymethyl) aminomethane (pH 8.0), 1.0 mmol L−1 EDTA, double distilled water and 20 μL of the sample. The reaction was initiated by the addition of pyrogallol (final concentration of 0.2 mmol L−1) (PRG; Sigma, P0381, São Paulo, Brazil), and absorbance was measured by a spectrophotometer with a wavelength of 420 nm (25°C) for 5 min. The unit of enzymatic activity was defined as SOD units able to produce 50% pyrogallol oxidation inhibition. All data were expressed in units of SOD per mg of hemoglobin. The thiol groups were enzymatically determined using 5,50-dithiobis-2-nitrobenzoic acid (DTNB; Sigma, D8130, São Paulo SP, Brazil) and glutathione reductase in the presence of β-nicotinamide adenine dinucleotide phosphate, (NADPH, N5130, Sigma, São Paulo, Brazil) to form 2-nitro-5-thiobenzoic acid. A mixture consisting of 1,290 μl of distilled water, 200 μl of Tris/HCl buffer (1 mol/l, pH 8.0, 5 mmol/l EDTA), 200 μl of 10 IU mL−1 glutathione reductase (GSH-Rd, G9297, Sigma, S-7432, São Paulo SP, Brazil), 200 μl of 2 mmol l−1 NADPH and 100 μL of 12 mmol L−1 of DTNB was added to 10 μL of the sample. Activity was measured as absorbance at 412 nm on a spectrophotometer. One unit of activity was equal to μmol of substrate reduced per gram of hemoglobin. GSH-Px content was measured by monitoring NADPH oxidation. The reaction mixture consisted of 1,300 μL of distilled water, 200 μL of Tris/HCl buffer (EDTA 1 mol L−1; pH 8.0; 5 mmol L−1), 200 μL of 10 IU m−1 of GSH-Rd, 200 μl of NADPH (2 mmol L−1), 40 μL of GSH (0.1 mol L−1) and 40 μl of hemolysate. The mixture was agitated in a vortex mixer for 10 s. Next, 20 μL of t-butyl hydroperoxide (7 mmol L−1) (TBH70X, 458139, Aldrich, St. Louis, MO) was added, and the reaction mixture was incubated at 37°C for 10 min. Absorbance was determined by a spectrophotometer at a wavelength of 340 nm. GSH-Px activity was expressed in enzymatic activity units per gram of hemoglobin (IU/g Hb).
Data were presented as the mean ± standard deviation (SD) for biochemical data and standard error of the mean (SEM) for the comet assay. ANOVA followed by the Tukey multiple comparison test and t test were used to compare the variables between groups. Differences were considered statistically significant at P < 0.05.
The obesity rates at an early timepoint and at the end of the experiment are presented in Table 1. The rats in the control group presented Lee index values lower than 0.300 and were classified as nonobese. The obese rats, regardless of cigarette smoke exposure, showed an experimental obesity profile with Lee index values higher than 0.300 (P < 0.05).
|Control (n = 10)||Obese (n = 09)||Smoking obese (n = 10)|
|Lee index (timepoint 1)||0.288 ± 0.019||0.319 ± 0.025a||0.330 ± 0.024a|
|Lee index (timepoint 2)||0.256 ± 0.026||0.352 ± 0.022a||0.359 ± 0.023a|
|Total cholesterol (mg dL−1)||114.24 ± 39.40||173.60 ± 37.23a||242.26 ± 51.60a, b|
|Triglycerides (mg dL−1)||114.43 ± 40.79||157.31 ± 34.85a||346.99 ± 47.79a, b|
|VLDL-c (mg dL−1)||22.89 ± 8.16||31.46 ± 2.32a||69.92 ± 3.32a, b|
|HDL-c (mg dL−1)||28.96 ± 6.00||34.46 ± 7.77||49.76 ± 10.82a, b|
|LDL-c (mg dL−1)||64.20 ± 33.23||107.68 ± 42.28a||132.42 ± 43.48a|
|Free fatty acid (mEq L−1)||1.02 ± 0.93||1.46 ± 0.78a||2.21 ± 0.47a, b|
|MDA (nM/g Hb)||176.79 ± 77.66||331.74 ± 207.15||328.80 ± 229.17|
|SOD (UI/mg Hb)||6.78 ± 3.12||2.89 ± 1.59a||2.31 ± 1.03a|
|Thiol groups (μM/g Hb)||0.76 ± 0.22||0.66 ± 0.37||0.81 ± 1.34|
|GSH-Px (UI/g Hb)||0.18 ± 012||0.18 ± 0.08||0.16 ± 0.11|
The ITT, OGTT, and AUC were evaluated to estimate glycemic profile and insulin action in peripheral tissues; these data are presented in Figure 1 (ITT = 1A, OGTT = 1B, and AUC = 1C). The ITT of control rats showed a decrease in glucose levels at 30 and 60 min after insulin administration (time point 0) (P < 0.05). The comparison of ITT data among the experimental groups showed that blood glucose levels were higher in the obese groups, regardless of cigarette smoke exposure, at the 30 and 60 min timepoints compared to control rats (P < 0.05). In the OGTT analyses, all groups showed higher blood glucose levels at 30 and 60 min than at 0 min (fasting period measurement), but only the smoke-exposed obese group showed a higher blood glucose level at 120 min (P < 0.05). Higher levels of glycemia at the 60 and 120 min timepoints after glucose administration were observed in the smoke-exposed obese group compared to the other groups (P < 0.05). The cigarette smoke-exposed obese group also showed a 26% increase in blood glucose levels in the area under the curve (AUC) compared to other groups (P < 0.05).
The lipid profile determinations were presented in the Table 1. The total cholesterol, triglycerides, free fatty acids (FFA), and all lipoproteins (HDL-c, VLDL-c, LDL-c) levels were increased (P < 0.05) in smoke-exposed obese females compared to those results of control and obese groups. These biochemical parameters were also higher (P < 0.05) in the unexposed obese rats compared to the control rats, except in relation to HDL-c levels (P > 0.05). The concentrations of LDL-c were higher (P < 0.05) in the obese rats, regardless of cigarette smoke exposure, compared to those in the control group. Both groups of obese rats also exhibited lower (P < 0.05) SOD activity compared to control rats. The exposure to cigarette smoke had no statistically significant effect (P > 0.05) on glutathione peroxidase activity (Table 1).
The smoke-exposed obese rats presented higher (P < 0.05) levels of comet tail moment, intensity and length compared to those of control and obese rats. Additionally, the obese rats exhibited a greater comet tail length than the control rats (Table 2).
|Control (n = 10)||Obese (n = 09)||Smoking obese (n = 10)|
|Tail moment||0.34 ± 0.02||0.69 ± 0.04||3.33 ± 0.16a, b|
|Tail intensity||4.34 ± 0.04||4.45 ± 0.22||13.57 ± 0.50a, b|
|Tail length||17.42 ± 0.33||30.85 ± 0.56a||44.87 ± 0.63a, b|
Several methodologies have been used to induce experimental obesity in rodents in the literature [16, 17]. As shown in Table 1, all animals treated with neonatal MSG were classified as obese at 90 days of age according to the Lee index, confirming the efficacy of this methodology.
Peripheral insulin resistance is found in both humans and experimentally obese animals, and it is related to high tissue fat accumulation . Regardless of cigarette smoking exposure, our data demonstrated that both obese groups developed insulin resistance, i.e., their blood glucose levels were not decreased after insulin administration (Figure 1A), showing that the exogenous insulin apparently does not have any effect on glucose uptake in insulin-dependent tissues, suggesting Akt phosphorylation disorders in peripheral tissues in obesity state .
In the OGTT (Figure 1B), the blood glucose levels were increased at 30 and 60 min after glucose oral administration in all groups, suggesting normal glucose absorption in the intestines. Our results showed that obese rats did not exhibit glucose intolerance compared to the control rats. This result can be related the insulin resistance in obese rats occurs before the onset of the glucose intolerant state . It is known that environmental, metabolic and genetic factors influence the onset and progression of obesity and carbohydrate disorders, such as glucose intolerance, especially environmental factors and aging [12, 21]. Our previous study verified these results via an oral glucose tolerance test in obese female Wistar rats, showing glucose intolerance only at 7 months of life . However, this study shows that cigarette smoke exposure associated with obesity exacerbates the defects in glucose uptake in peripheral tissues (Figure 1C), and these changes are developed earlier than they would be otherwise.
The present study showed that the triglycerides, total cholesterol, FFA, VLDL-c, and HDL-c levels were higher in the obese rats regardless of cigarette smoke exposure. However, the smoke-exposed obese rats presented a further increase in these parameters compared to non-smoke-exposed obese rats. The metabolic events leading to high HDL-c levels may be different in this group, e.g., associated with high synthesis versus low catabolism. The increased HDL-c levels in obese rats exposed to cigarette smoke may be related to defects in VLDL-c removal . Obesity is strongly related to dyslipidemia, and this feature may be aggravated by the addition of another metabolic disruptor, such as exposure to cigarette smoke .
Plasma FFA levels are usually increased in obesity because the increased adipose tissue mass releases more FFA into the circulation and plasma FFA levels inhibit the antilipolytic action of insulin . Increased serum FFA levels may also be attributed to cigarette smoke , and this effect may lead the development of metabolic diseases, such as Diabetes mellitus and metabolic syndrome .
Tobacco smoke contains polycyclic aromatic hydrocarbons, aldehydes, nitrosamines  and reactive oxygen species (ROS) , all of which can induce genotoxicity and mutagenicity. Several studies have examined the studies of smoking-related DNA damage, and have shown that, in both passive and active smokers, tobacco induces oxidative stress and DNA adducts . Thavanati et al.  demonstrated that the strong associations among free radicals, DNA damage, and antioxidants are moderately correlated with cigarette smoking, age, and body mass index (BMI). These authors reported that BMI had a positive relationship with oxidative stress, but antioxidant levels did not vary with body mass index, corroborating with our results.
Lipid peroxidation (oxidative injury marker), represented by malonaldehyde (MDA), is significantly correlated with body mass index (BMI) and waist circumference . Our data shows that, regardless of cigarette smoke exposure, obesity tended to increase levels of MDA and decrease SOD activity, classic signs of biological oxidative stress that occurs because the first antioxidant barrier (SOD) does not remove adequate serum reactive oxygen species [31, 32]. However, Seiva et al.  showed that obese young male rats exposed to cigarette smoke presented increased SOD, GSH-Px and catalase enzymatic activities as a compensatory mechanism in response to cellular injury.
It is known that tobacco smoke contains various chemicals that induce primary DNA damage and mutations. Our data shows that nonobese rats exposed to similar levels of cigarette smoke presented no difference in tail moment compared to control rats , showing that the association between obesity and cigarette smoke exposure increased DNA damage levels. Moreover, it was also verified that obese rats exposed to tobacco cigarette smoke presented enhanced genotoxicity, confirmed by the significant increases in all of the comet assay parameters analyzed (Table 2). Among the lifestyle factors, smoking and obesity have important and synergistic effects on the health of an individual. DNA damage was observed to be significantly higher among smokers , and cigarette smoking is associated with carcinogenesis, heart disease, and premature death , whereas obesity leads to greater oxidative stress and contributes to diseases such as atherosclerosis, Diabetes mellitus and arterial hypertension  with a depletion of the total antioxidant levels .
In conclusion, this study showed that the association between obesity and cigarette smoke exposure exacerbated the genotoxicity, altered the lipid profile, impaired the antioxidant defenses and caused early glucose intolerance. Thus, the changes caused by cigarette smoke exposure can trigger the earlier onset of metabolic disorders associated with obesity, such as Diabetes mellitus and metabolic syndrome.
We are grateful to the Research Support Center (RSC) for its valuable contributions to the study design and statistical analysis.
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