Is rosuvastatin protective against on noise-induced oxidative stress in rat serum?

Noise, one of the main components of modern society, has become an important environmental problem. Noise is not only an irritating sound, but also a stress factor leading to serious health problems. In this study, we have investigated possible effects of rosuvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, thought to have an antioxidant effect, on noise-induced oxidative stress in the serum of rat models. Thirty-two male Wistar albino rats were used. In order to ease their adaptation, 2 weeks before the experiment, the rats were divided into four groups (with eight rats per each group): Noise exposure plus rosuvastatin usage, only noise exposure, only rosuvastatin usage and control. After the data had been collected, oxidant (Malondialdehyde, nitric oxide [NO], protein carbonyl [PC]) and antioxidant (superoxide dismutase [SOD], glutathione peroxidase [GSH-PX], catalase [CAT]) parameters were analyzed in the serum. Results indicated that SOD values were found to be significantly lower, while PC values in serum were remarkably higher in the group that was exposed to only noise. GSH-Px values in serum dramatically increased in the group on which only rosuvastatin was used. During noise exposure, the use of rosuvastatin caused significantly increased CAT values, whereas it resulted in reduced PC and NO values in serum. In conclusion, our data show that noise exposure leads to oxidative stress in rat serum; however, rosuvastatin therapy decreases the oxidative stress caused by noise exposure.

Introduction

Noise consists of undesirable sounds that have an adverse effect on people. Particularly in big cities, noise levels can be quite high. Noise pollution has become a serious problem due to the dramatic increase in the number of motor vehicles in towns and highways connecting them. Noise coming from technological and industrial advances, urbanization, swell in population, and growing number of entertainment centers is a serious environmental problem leading to numerous disturbances in human beings.[1]

It has been proved that exposure to noise may cause loss of hearing, changes in hormonal and immune systems, psychological, physical and behavioral disorders in humans. Besides, noise affects sleep, work performance and productivity, communication skills.[23] Continuous exposure to loud noise triggers oxidative stress and enhances production of free oxygen radicals, which can contribute to the tissue damage. Lipid peroxidation caused by free oxygen radicals and changes in protein and nucleic acid structure may lead to aging, neurodegenerative diseases and cancer.[456]

Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors used in order to reduce cholesterol rates. Statins also provide defending stroke and myocardial infarction with their pleiotropic effects.[7] Recent studies indicate that statins have neuroprotective, anti-inflammatory, anti-ischemic, and antioxidant effects.[89] Moreover, in particular rosuvastatin upregulates antioxidant defense mechanism and prevents tissue damage.[10]

In this study, we have investigated whether noise causes oxidative stress and the possible effect of rosuvastatin on oxidative stress in rat serum.

Methods

The investigation was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and approval has been received from Institutional Animal Ethics Committee at Fatih University.

Animals

Thirty-two male albino Wistar rats (210-230 g) were obtained from the laboratory animals department of the Medical Faculty of Fatih University. They were maintained at a temperature of (22°C ± 1°C) and relative humidity of (65-70%) on a 12 h light-dark cycle. Rats were fed ad libitum with commercially available rat chow and water. The animals were housed as eight animals per cage (50 cm × 35 cm × 20 cm).

Groups

Rats were allowed free access to tap water and laboratory for 2 weeks, as an adaptation, before the experiment and they were randomly divided into four groups (n = 8).

Group 1: Control — The rats were given 1 ml of distilled water via intragastric gavage every day for a period of 30 days.

Group 2: Rats were given 10 mg/kg of rosuvastatin (Crestor, Astra Zeneca) via intragastric gavage during 30 days.

Group 3: These rats were exposed to 100 dB(A) noise for 4 h/day during 20 days. Beginning 10 days prior the noise exposure, they were given 1 ml of distilled water via intragastric gavage every day for 30 days.

Group 4: These rats were exposed to 100 dB(A) noise for 4 h/day for 20 days. Beginning 10 days prior the experiment, they were given 10 mg/kg of rosuvastatin via intragastric gavage every day for 30 days.

Noise exposure

White noise was obtained by using the broadband noise CD (whitenoisemp3s.com/free-white-noise). It was intensified using a 40-W amplifier and octave band noise centered at 4 kHz was transmitted from the speakers placed 30 cm away from the cage [Figure 1]. The intensity of the noise was continuously measured by a sound level meter (sound level meter DT-8850, China), which is capable to measure 35-130 dB. The basal standard noise level and exposure noise level were adjusted to 45 dB(A) and 100 dB(A), respectively. The intensity of 100 dB(A) was chosen because it reflects the common noise level in industrial workplaces and entertainment centers.

Figure 1

Noise exposure system

Preparation of serum samples and biochemical study

At the end of the experimental period, animals were fasted for 12 h, anesthetized with ketamine (100 mg/kg) and xylazine (4 mg/kg). Blood samples were collected from inferior vena cava by exsanguinations. Blood samples were centrifuged at 4,000 rpm for 10 min, and the obtained serum samples were kept at −20°C for following biochemical investigations.

All preparation procedures were performed at +4°C. To assess the oxidative stress in the obtained serum, we used commercial chemicals supplied by Sigma (St. Louis, USA) for spectrophotometrical identification of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), nitric oxide (NO), protein carbonyl (PC) and malondialdehyde (MDA) levels.

Determination of superoxide dismutase activity

Total (Cu-Zn and Mn) (SOD, EC 1.15.1.1) activity was determined according to the method of Sun et al. with a slight modification by Durak et al.[1112] The principle of the method is based on the inhibition of nitroblue tetrazolium (NBT) reduction by the xanthine/XO system as a superoxide generator. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. Activity was expressed as units per milliliter.

Determination of catalase activity

Catalase activity was measured according to the method of Aebi.[13] The principle of the assay is based on the determination of the rate constant k (dimension: S−1, k) of H2O2 decomposition. By measuring the absorbance changes per min, the rate constant of the enzyme was determined. Activities were expressed ask (rate constant) per milliliter.

Determination of glutathione peroxidase activity

Glutathione peroxidase activity was measured by the method of Paglia and Valentine.[14] The enzymatic reaction in a tube-containing reduced from of nicotinamide adenine dinucleotide phosphate, reduced glutathione, sodium azide and glutathione reductase-was initiated by addition of H2O2 and the change in absorbance at 340 nm was monitored by a spectrophotometer. Enzyme activities were reported as units/liter.

Malondialdehyde determination

The MDA levels were determined by the method of Esterbauer.[15] This method based on the reaction with thiobarbituric acid (TBA) at 90-100°C. In the TBA test reaction, MDA or MDA like substances and TBA react together for the production of a pink pigment having an absorption maximum at 532 nm. The reaction was performed in pH 2-3 at 90°C for 15 min. After cooling, the absorbance was read at 532 nm. The results were expressed as micromol/liter.

Nitric oxide determination

As NO measurement is very difficult in biological specimens, serum nitrite and nitrate were estimated as an index of NO production according to the method of Cortas et al.[16] This method was based on the Griess reaction. Samples were initially deproteinized with Somogyi reagent. Total nitrite (nitrite + nitrate) was measured after conversion of nitrate to nitrite by copperized cadmium granules by a spectrophotometer at 545 nm. Results were expressed as micromole/liter.

Determination of protein carbonyl levels

The PC levels were determined spectrophotometrically according to the Method of Levine et al.[17] This method based on the reaction of the carbonyl group with 2, 4-dinitrophenylhydrazine to form 2, 4-dinitrophenylhydrazone. The results were given as nanomoles of carbonyl per milliliter.

Statistical analysis

Data were expressed as means ± standard deviation. Statistical analysis were carried out by analysis of variance (ANOVA), followed by appropriate post-hoc tests, including multiple comparison tests (least significant difference).

The Kruskal-Wallis one-way ANOVA by ranks was used for a simultaneous statistical test of the histopathologic score distribution in the groups. When the null hypothesis could be rejected, comparisons between controls and each other groups were made with the Mann-Whitney non-parametric test for independent samples. All analyses were made using the statistical package for the social sciences statistical software package and P < 0.05 were considered as significant.

Results

Serum oxidant parameter values

Serum PC levels were significantly higher in group III, compared to group I (P = 0.009). In group IV, PC levels were found to be lower than in group III and similar to group I (P = 0.004). Serum PC levels were also significantly lower in group II than in group IV (P = 0.002) [Figure 2].

Figure 2

Protein carbonyl, malondialdehyde and nitric oxide activities in serum of rat in each groups. *P < 0.009 versus all other groups, #P < 0.05 versus all other groups

Although there was no statistically significant distinction between the groups in serum MDA levels, MDA was found to be lower in group II and higher in group III, compared to group I. Similarly, the comparison between MDA levels in group III and group IV was not statistically significant, but MDA in group IV was lower and close to the levels of group I [Figure 2].

The levels of NO in group IV was statistically significantly low, compared to group I, II and III (P = 0.013, P = 0.007 and P = 0.000 respectively) [Figure 2].

Serum antioxidant parameter values

Although no significant difference was found between the groups in SOD values, compared to group I, the SOD value was reached borderline significance in group IV, and group III (P = 0.066). There was no important difference of SOD values between the other groups [Figure 3].

Figure 3

Superoxide dismutase, glutathione peroxidase and catalase activities in serum of rat for each groups. *P = 0.066 versus group I, **P < 0.024 versus all other groups. #P < 0.08 versus all other groups

Serum GSH-Px values were elevated in group II compared with group I, III, and IV (P = 0.015, P = 0.011, P = 0.024, respectively). There was no statistically meaningful difference compared to the other groups [Figure 3].

Serum CAT levels were increased in group IV, compared to the group I and II (P = 0.013 and P = 0.05 respectively). Although not statistically significant, the serum CAT levels were reached borderline significance in group IV, compared to group III (P = 0.08). Between the other groups, the serum CAT levels were not different in statistical terms [Figure 3].

Discussion

The world health organization declares noise as one of the most hazardous factors of the modern societies.[18] Noise attracts scientists as an environmental factor leading to oxidative stress. Study results have been shown that free radical molecules and consequence oxidative stress play a significant role in noise-induced tissue damage.[19] Zheng et al. have demonstrated in BALB/c mice that a 4-week exposure to 90 Db(A) noise leads to increase of 8-hidroxy-2’-deoxyguanozine, which is determinative for oxidative injury in urine.[3] In another study on long-Evans rats exposed to 120 dB(A) for 2 h, lipid peroxidation determined with TBA was found to be high 3 days after the exposure.[20]

In a study on New Zealand strain rabbits, after 1-h exposure to 100 dB(A) broadband noise, elevation in serum MDA values accompanied shifting of the hearing threshold in otoacoustic emission test.[21] Although our experiment revealed no statistically significant difference between the groups in the MDA values, it still showed that noise increases MDA values.

Free oxygen radicals may affect proteins as well as lipids. Oxidative stress leads to oxidative injury and increase in PC levels. There have been studies on many environmental factors, including noise, leading to oxidative stress and increasing PC levels.[2122] Similarly, we have shown that noise induces oxidative damage in proteins and increases PC levels.

There are antioxidant enzymes that prevent oxidative stress and neutralize the harmful effects of free radicals. Most important examples are SOD, GSH-Px and CAT. These enzymes function in the oxidative phosphorylation and neutralize the free oxygen radicals that form throughout electron transport in the electron transport chain.

Superoxide dismutase is the first enzyme to interact with free radicals. When superoxide levels are high, SOD becomes more active in order to inactivate the free radicals. The literature mentions decrease and increase in SOD enzyme activities during oxidative stress, which is paradoxical.[456] In our study, SOD values of rat serum exposed to noise was found to be borderline significance compared with the control group. The decrease in SOD activity together with increased lipid peroxidation can be explained with antioxidant enzymes being used up due to increasing in lipid peroxidation.

Hydrogen peroxide formed in the reaction catalyzed by SOD decomposes to water and oxygen by GSH-Px and CAT enzymes. Studies indicate that environmental stress factors affect these enzymes.[2223] There is conflicted evidence about GSH-Px and CAT values in studies conducted with noise. Some studies point out that GSH-Px and CAT values increase in acute or subacute noise exposure whereas decrease in chronic noise exposure, hence is still higher than normal levels overall.[42425] Yet, in other studies GSH-Px and CAT levels were found to be low in chronic noise exposure in Wistar strain male albino rats as well.[5626] In our study, noise exposure did not change serum GSH-Px and CAT values compared to the control group. No change in GSH-Px and CAT levels in spite of the low levels of SOD can be attributed to low production of hydrogen peroxide due to high SOD consumption.

Nitric oxide is a molecule that is always present in the human body. NO is formed from arginine as a result of the reaction catalyzed by NO synthase (NOS) in different tissues, such as brain, macrophage and vessel endotel. NO has radical characteristics as it contains unpaired electrons. However, compared to other radicals, it has significant physiological functions when it is low in concentration. Nevertheless, its excessive and uncontrolled synthesis is harmful to the cells.[2728]

Demirel et al. have found high levels of NO in the serum of Wistar rats exposed to noise 100 dB (A) in strength for a period of 20 days.[25] These changes are attributed to the oxidative stress which is caused by noise. However, in our study, noise exposure did not cause a notable statistical change in the serum, compared to the control group. NOS enzyme are known to be inhibited by glucocorticoids.[2728] This enzyme is present in vessel endothelial cells and the central nervous system. As shown in several studies, glucocorticoid level increases as a result of stress developed by various environmental factors including stress.[625] Although glucocorticoid levels have not been measured in our study, considering the findings in other studies, we can assume these levels to be high. For this reason, the lack of increase in NO levels can be explained by NOS inhibition by glucocorticoids. On the other hand, overproduction of superoxide radical causes it to unite with NO and leads to the formation of peroxynitrite. In our study, it can be assumed that increased superoxide resulting from SOD consumption reacts with NO, and that is why due to the consumption of NO its level does not grow.

Rosuvastatin is a new generation HMG-Co reductase inhibitor used in hypercholesterolemia treatment like other statins. Rosuvastatin research has also found pleiotrophic effects. The abovementioned studies describe the anti-inflammatory, neuroprotective, antioxidant, endothelial and blood-brain barrier repairing effects of statins.[78910] Rosuvastatin is more hydrophilic and shows more affinity to the liver compared to other statins.[2829]

Many experiments have been carried out to study antioxidant effects of rosuvastatin. In most of the animal studies, rosuvastatin was used in 10 mg/kg/day doses.[30] In our study investigating the effects of rosuvastatin on noise-induced oxidative stress, rosuvastatin was given in 10 mg/kg/day during 30 days.

When the antioxidant profile was analyzed in our study, the application of rosuvastatin singly did not affect SOD values significantly. In noise exposure, the use of rosuvastatin led to increase of SOD values, although not statistically significant, compared to the control group and the group exposed to noise only.

In our study, the use of rosuvastatin singly resulted in growth of GSH-Px levels, whereas when rats exposed to noise were treated with rosuvastatin, the serum GSH-Px levels did not show a significant change. There are only few studies on the impact of rosuvastatin on GSH-Px and they have conflicting evidence on this issue. In a study conducted by Gómez-García et al., it was observed that rosuvastatin treatment led to an increase in serum SOD and a decrease in the GSH-Px values in patients.[31] On the other hand, Shchukin et al. conducted a study in which rosuvastatin was given to individuals with atherosclerosis and hypercholesterolemia at a dosage of 10 mg/day for 12 weeks and proved that the low levels of GSH-Px increased significantly after rosuvastatin treatment.[32]

As rosuvastatin given singly does not affect serum CAT levels, rosuvastatin treatment combined with noise exposure was recognized to increase serum CAT levels in our study. Similar results were indicated in Schupp et al. study where they showed in human promyelocytic cells that rosuvastatin increased the messenger ribonucleic acid activity of CAT and SOD enzymes.[33]

Other studies on rosuvastatin declare that the antioxidant effect of rosuvastatin is determined by the reduced concentration of the superoxide radical as a result of the inhibition of asymmetric dimethylarginine (ADMA) and nicotinamide adenine dinucleotide phosphate-oxidase (NAD(P)H) oxidase.[34] Superoxide radicals are formed throughout the reaction in which NAD(P)H oxidase catalyses. ADMA triggers the formation of superoxide radicals. In our opinion, rosuvastatin inhibits the mentioned compounds, reducing the number of superoxide radicals, so that SOD would not be excessively consumed.

The activation of antioxidant enzymes such as SOD, GSH-Px and CAT in oxidative stress is a protective mechanism due to the activation of the antioxidant defense. The results of our study show that, in case of oxidative stress, rosuvastatin affects the antioxidant system by increasing SOD and CAT levels in the serum, but having no significant effect on GSH-Px levels. According to our results, in case of noise-induced oxidative stress, the use of rosuvastatin caused no change in serum GSH-Px, but a rise in CAT levels. This can be attributed to the split of hydrogen peroxide by CAT enzyme. However, it should be kept in mind that a higher dosage of rosuvastatin than what we used could be more effective on GSH-Px, because without oxidative stress rosuvastatin by itself has changed GSH-Px substantially.

There are conflicting findings on whether antioxidant effects of rosuvastatin depend on the dosage. Ajith et al. have shown that depending on the dosage, rosuvastatin has dramatically decreased lipid peroxidation in the rat brain and liver tissue.[9] In our study, it has been observed that given singly rosuvastatin has decreased MDA levels, although this decrease was not remarkable. With the use of rosuvastatin, MDA levels, which increase only during noise exposure, have dropped close to the level of the control group. Considering the results of the above mentioned study, it can be assumed that the decrease could be greater if rosuvastatin were given in a higher dose.

Not much has been done to investigate the effects of rosuvastatin on protein oxidation. Although the effects of rosuvastatin on PC had never been studied before, there are studies on nitrotyrosine, the marker of peroxynitrite, causing protein oxidative stress, which show that rosuvastatin reduces the increased levels of nitrotyrosine caused by the oxidative stress.[35] In our study, we have found that although rosuvastatin alone has caused no dramatic change in serum PC levels, these levels were lower than those of the control group. However, the serum PC levels, which rose drastically during noise exposure, have dropped to the level of the control group with the rosuvastatin use.

As mentioned above, NO is a molecule that has a dual effect both high and low levels of NO are dangerous. NOS system should be constantly balanced in order to obtain NO levels that have no undesirable effects. In several studies, it has been found that rosuvastatin has a regulating effect on the NOS enzyme of rats, apart from its lipid lowering effect.[3637] Similarly, in our study, while NO levels were not affected by rosuvastatin alone, they were remarkably low in case of rosuvastatin used together with noise.

Conclusion

As a result, our study has demonstrated that noise causes oxidative stress in rat serum, and this stress can decrease partially, if not completely, with use of rosuvastatin. Rosuvastatin achieves this effect by reducing the NO levels, decreasing lipid peroxidation and protein oxidation and by increasing GSH-Px. We believe that although this effect is not quite obvious in certain situations, it could be more prominent with a higher dose of rosuvastatin.