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Aromatherapy is increasingly popular alternative therapy but scientific research in this field are rare. In this experimental study, harmful effects of formaldehyde (FA) inhalation on sperm concentration, sperm quality, serum testosterone levels and the rat testes were investigated. In addition, the possible protective effects of rose oil against to these harmful effects were evaluated. For this purpose, 21 albino-Wistar rats were used. The rats in group I were used as control group. When the rats of group II were exposed FA (10 ppm/ 1hour) for 35 days, the rats of group III inhalated rose oil (1ml/1 hour) with FA. At the end of the experiment, when the epididymal tissues were taken for sperm analyzing, testes were removed for histological examination. In addition, testosterone levels were determined from the blood samples taken from animals. While the testosterone levels, the epididymal sperm concentration, and the progressive sperm motility significantly decreased, the abnormal sperm rate significantly increased in the group II when compared to group I. In the group III, it was determined that the testosterone levels and the epididymal sperm concentration significantly increased, the abnormal sperm rate significantly decreased when compared to the group II. The progressive sperm motility increased but not significantly. When the rats in the group II compared with the control group, it was determined that the diameter of tubuls and the number of Leydig cells decreased and Leydig cells with damaged nucleus increased. It was also seen atrophic changes in the tubuls. In the group III, it was determined that the histological changes of testes occured via FA exposure were improved. It can be expressed that serious damages occured via formaldehyde exposure in reproductive system and that the rose oil had protective effects against these damages.
Keywords: Formaldehyde, rat, rose oil, sperm, testes.
Formaldehyde (FA) is a pungent, irritant, and colorless gas. It is a member of aldehyde family and found in nature in domestic air, cigarette smoke, and the polluted atmosphere of cities due to the incomplete combustion of organics, photochemical smog, and release from FA containing products. FA has a strong tendency to combine with protein, DNA, RNA which leads to harmful effects [Heck and Casanova, 1999; Usanmaz et al., 2002]. Use of FA in anatomy, histology, and pathology laboratories has been increasing. In addition, FA is used in production of industrial and cosmetic products. Therefore, exposure to FA by inhalation is every time possible [Khanzadeh et al., 1994; Cohen et al., 1998].
FA has been shown to have harmful effects on the central nervous, respiratory, and digestive systems [Sarsilmaz et al., 2007; Kriebel et al, 2001]. It also affects the reproductive system and causes infertility [Collins et al., 2001; Taskinen H et al., 1994]. Various studies have showed that FA causes testicular damage and decreases the testosterone levels as well as sperm concentration and quality [Majumder and Kumar, 1995; Odeigah, 1997; SarsÄ±lmaz and Ã-zen, 2000].
Aromatherapy is the therapeutic use of essential oils. Essential oils are defined as volatile parts of aromatic plants extracted by steam distillation or expression. Rose oil, which is often used in aromatherapy, is extracted from rose flowers by steam distillation. Nearly 4000 kg of rose are used to produce 1 kg of rose oil. Rose oil is a limpid, light yellow, and volatile oil. Contents of five major constituents of the oil are: citronellol, geraniol, nerol, linalool, and phenylethyl alcohol [Ergen et al., 2003; KürkçüoÄŸlu and BaÅŸer, 2003].
In traditional medicine, rose oil is used for infections, anxiety, skin care, stomach and chest aches, and menstrual and digestive disorders [AltÄ±ntaÅŸ, 2007; Basim and Basim, 2003; Boskabady et al., 2006]. Recent experimental studies have revealed that rose oil has antibacterial [Basim and Basim, 2003], anti-HIV [Mahmood et al., 1996], anxiolytic [Almeida, 2004; Bradley 2007], anti-inflammatory, analgesic, hypnotic, anti-spasmodic, antitussive [Boskabady et al., 2006], and antioxidant [Wei and Shibamoto, 2007] effects. In addition, rose oil improves learning and memory [Köse et al., 2007].
Therefore, the present study aimed to examine the toxic effects of formaldehyde inhalation on sperm concentration, sperm quality, serum testosterone levels and the testes and to investigate the protective effects of rose oil against these toxic effects
Clinical findings: In the groups that were exposed to FA, after the 10th day of the study, the hair color started to fade. This was more marked in the animals that were exposed to FA only than in the animals that were exposed to both rose oil and FA. In addition, the animals in the FA only groups displayed frequent blinking, dyspnea, increased nasal cleaning, sneezing, and excessive licking associated with irritation due to FA exposure.
Biochemical findings: In the comparisons of the FA exposed rats with the controls, the serum testosterone levels of the FA exposed groups were significantly lower than those of the controls (p<0.05). Moreover, in the group that inhaled FA and rose oil, the serum testosterone levels significantly increased compared to the levels of the rats exposed to FA only (p<0.05) (Fig. 1).
Spermiogram results: After the right epididymis of each rat was prepared by a special hemocytometric method, it was evaluated under a microscope with Neaubauer glass, and thus, the total sperm counts were determined. Sperm motility and abnormal sperm counts were determined using the samples from the left cauda of epididymis, which were evaluated via a microscope with a heating plate.
The epidydimal sperm counts and sperm motility of the rats that were exposed to FA significantly decreased compared to the control group (p<0.001). In addition, the sperm counts of the rats in this group also increased (p<0.001).
The epidydimal sperm count of the group that was exposed to rose oil and FA significantly increased compared to that of the group that exposed to FA only and the abnormal sperm count improved. Moreover, there was an increase in the sperm motility of this group. However, the difference between the rose oil and FA exposed group and only FA exposed group for sperm motility was not statistically significant (p>0.05) (Table 1).
Histological results: In the light microscopic evaluation, the testes of the control group were normal. When the rats exposed to FA were compared with the control group, it was determined that the number of Leydig cells decreased in the FA only exposed group (p<0.001). The count of Leydig cells in the group that was exposed to rose oil and FA increased compared to that in only FA exposed group (p<0.001) (Fig. 2).
The count of Leydig cells with damaged nucleus in the rats exposed to FA only decreased compared with that of the control group (p<0,001). In the comparison of the rose oil and FA exposed and only FA exposed groups, it was determined that the count of the Leydig cells with damaged nucleus increased in only FA exposed group (p<0,001) (Figs. 3, 4, and 5).
The diameters of tubules in the rats exposed to FA only decreased when compared with the control group (p<0.001). In the group that was exposed to rose oil and FA, the diameters of the tubules increased compared with the rats exposed to FA only; however, the difference was not statistically significant (p>0.05) (Figure 6).
Formaldehyde has been shown to present negative effects on the respiratory, digestive, and nervous systems, skin, and eyes and have mutagenic and carcinogenic characteristics [Sarsilmaz et al., 2007; Kriebel et al, 2001; Marsh et al, 2007]. In addition, it has negative effects on the reproductive system. In the experimental studies to date, FA applied systemically or externally was shown to inflict changes in the morphology of the testes as well as in the spermogenetic cells [Collins et al., 2001; Taskinen H et al., 1994; Majumder and Kumar, 1995; Odeigah, 1997; SarsÄ±lmaz and Ã-zen, 2000].
The testes are one of the organs affected by FA. SarsÄ±lmaz and Ozen  applied FA on rats and showed the damages caused by FA in the Leydig cells and their nuclei. Ã-zen et al  applied FA in the subchronic stage for 91 days at the doses of 5-10 ppm and determined significant reductions in the tubular diameters (p<0.001). Similarly, Golalipour et al  used inhaled form of FA for 18 days in their subjects for 4 days a week, 2 and 4 hours a day, and 2 days a week, 2 hours daily and they reported significant reduction in the tubule diameter and epithelial thickness. The findings of our study are compatible with the findings of the studies above. In our study, FA was applied for 35 days and one hour a day at a dose of 10 ppm and it was found that the count of Leydig cells decreased and the rate of the damages in the nuclei of these cells increased, while the diameters of the seminipherous tubule reduced.
The spermium analyses of the epididymal tissues, there were decreases in the total spermotozoa counts and sperm motility of the animals that were exposed to FA only. However, the count of abnormal sperms increased. In other experimental studies, intraperitoneally-applied FA was shown to have negative effects on the sperm count and motility [Majumder and Kumar, 1995; Zhou et al., 2006; Tang et al., 2003]. Damage to the seminipherous tubule, in which the sperms develop, negatively affected the sperm count; i.e. the sperm count reduced. Zhou et al  and Tang et al  have suggested that intraperitoneal FA causes atrophy and degeneration in the seminipherous tubule, which leads to reductions in the sperm counts. Likewise, significant reduction was reported in the sperm motility as a result of damage to the Leydig cells. Henkel et al  have determined a direct correlation between the sperm motility and testosterone expressed by the Leydig cells. In the study by Tang et al  on rats, increased abnormal sperm counts were reported. In the same vein, Odeigah  determined anomalies of the sperm head in the rats that were applied intraperitoneal FA. The findings of the sperm analyses in our study are compatible with the findings of earlier studies and supportive of histological findings as well.
The serum levels of testosterone, which has an important role in the reproductive functions, are also negatively affected by FA exposure. In the study by Chowdhury et al  and Zhou et al , intraperitoneally applied FA led to significant reductions in the serum testosterone levels. Similarly, with the inhalation form of FA, Ã-zen et al  reported a significant reduction in the testosterone levels. All of these studies emphasized that the damage in the Leydig cells caused the decreases in the testosterone levels [Chowdhury et al., 1992; Zhou et al., 2006; Ã-zen et al., 2005]. In our study, the serum testosterone levels of the rats that were exposed to FA only significantly reduced due to the damage in the Leydig cells. Various studies have attributed these effects to the oxidative damage caused by FA [ZararsÄ±z et al., 2004; Ã-zen et al, 2008]. Thus, the changes in the Leydig cells, their nuclei, tubule diameters, epidydimal sperm analysis findings, and testosterone levels determined in our study may be explained by the oxidative damage caused by FA.
Rose oil, which is clear and light yellow in color and characteristically fragrant, primarily contains acyclic terpenic substances and 40-50% of the oil consists of citronellol, and 20% geraniol. Geraniol produces the characteristic aroma of the rose [Ergen et al., 2003; KürkçüoÄŸlu and BaÅŸer, 2003]. Rose oil is used in cosmetics as well as in aromatherapy as an alternative treatment. Its medical effects have also been shown in various experimental studies [Basim and Basim, 2003; Mahmood et al., 1996; Wei and Shibamoto, 2007].
The experimental studies to date have determined the antioxidant effects of rose oil. Wei et al and Shibamoto , based on the results of aldehyde/carboxylic acid test that is used in showing long-term antioxidant activity, have reported that rose oil presents an antioxidant activity almost at a 100% compared to the Î±-tokoferol, which was taken as a reference. In the same study, 1-diphenyl-2-picrylhydrazil (DPPH) free radical cleansing and malonaldehyde/gas chromatography (MA/GC) tests revealed 70% antioxidant activity of rose oil. Î±-tokoferol used as a reference in these tests showed nearly 90% antioxidant activity [Wei and Shibamoto, 2007]. Likewise, in the studies using thiobarbituric acid (TBA) test, citronellol, nerol, and geraniol, the constituents of rose oil have been shown to inhibit MA formation at various levels [Roberto and Maratta, 2000].
The Leydig cell counts of the animals exposed to rose oil and FA increased compared to those of the animals exposed to FA only (p<0.001) and the number of damaged nuclei of the Leydig cells in the rose oil and FA exposed group significantly reduced (p<0.001). Seminipherous tubule diameters of this group also increased, but the difference was not significant (p>0.05). In some of the earlier studies, the disorders of the testes due to oxidative damage were reported to improve with antioxidant therapy [Türk et al., 2007; Ã-zen et al., 2008]. Türk et al , in their study evaluating the testes exposed to cycloporin A, used likopen to prevent oxidative damage and histological changes and reported successful findings. According to the results of these studies, prevention of oxidative damage in the testes by antioxidants have yielded improvements in histopathological changes. In our study, the significant improvement in the histopathological changes in the testes of the rats exposed to rose oil and FA compared to the rats exposed to FA only may be attributed to the antioxidant effect of rose oil.
The increase in the sperm count and motility and reduction in the abnormal sperm count may also be associated with the antioxidant effect of rose oil. This characteristic of rose oil may have indirectly led to improvements in the Leydig cells and seminipherous tubules, sperm counts, motility, and reductions in the abnormal sperm counts.
Total testosterone levels of the group exposed to rose oil and FA was significantly higher than those of the FA exposed group (p<0.05). This might also be due to the antioxidant effect of rose oil. Recent studies have reported a direct correlation between Leydig cells and testosterone levels [Henkel et al., 2005; Joensenet al., 2008]. In our study, increase in the testosterone releasing Leydig cell counts and reduction in the nucleic damage may be responsible for increased testosterone levels.
In conclusion, the decreases in the Leydig cell counts and tubular diameters increase in the nucleic damage, reduction in the sperm count and motility, and decreased serum testosterone levels indicate the negative effects of FA inhalation on the productive system. Rose oil, with its antioxidant characteristics, can be used to improve some of these negative effects.
MATERIALS AND METHODS
Adult male Wistar rats (weighing 310-320 g, n=21) comprised the study material. All procedures were approved by the Institutional Animal Care and Use Committee of the Medical School, FÄ±rat University, Turkey. The animals were divided into three groups. The rats in Group I (n=7) were used as the controls. While the rats in Group II were exposed to FA (10 ppm/1 hour--formalin, Sigma-Aldrich Formaldehyde 37% solution, Deisenhofen, Germany) for 35 days, the rats in Group III inhaled rose oil (1ml/1 hour--Gülbirlik, Isparta, Turkey) along with FA. At the end of the experiment, the epididymis tissues were taken for sperm analysis and testes were removed for histological examination; testosterone levels were determined using the blood samples of the animals.
Histological studies: The testicular tissue specimens were fixed in Bouin's solution. Tissue specimens were embedded in paraffin wax and sectioned (5 µm). For light microscopic evaluation, paraffin sections were stained with hematoxylin-eosin (H&E) and Mason tricrom and examined with an Olympus BH2 light microscope.
In this investigation, the diameters of seminipherous tubule, the number of Leydig cells, and the number of Leydig cells with damaged nucleus (pyknosis, karyolysis, karyorrhexis) were determined.
Determination of testosterone levels: Plasma was stored at -20°C for analysis. The plasma testosterone level was assayed using Coat-a-Count Radioimmunoassay kit (Active Testosterone RIA DSL-4000, Diagnostic System Laboratories Inc, Texas, USA) and expressed as ng/mL.
Determination of epididymal sperm concentration: The epididymal sperm concentration was determined with a hemocytometer (Improved Neubauer, Weber, UK) using a modification of the hemoctometric method described by Turk et al  and Sönmez et al. . Briefly, the right epididymis was finely minced using anatomical scissors in 1mL of physiological saline (NaCl, 0.9%) in a Petri dish. It was completely squashed with tweezers for 2 min. Then, it was incubated at room temperature for 5 min to provide the migration of all spermatozoa from epididymal tissue to the fluid. After incubation, the epididymal tissue-fluid mixture was filtered via a strainer to separate the supernatant from tissue particles. The supernatant fluid was drawn into the capillary tube up to 0.5 line of the pipette designed for counting red blood cells. The solution containing 5 g sodium bicarbonate, 1mL formalin (35%, v/v) and 25 mg eosin per 100 mL distilled water were pulled up to 101 lines of the pipette. Approximately 10 mL of the diluted sperm suspension was transferred to counting chambers of hemocytometer and allowed to stand for 5 min. The sperm cells in both chambers were counted with the help of light microscope at the magnification of 200X.
Determination of epididymal sperm motility: The percentage of progressive sperm motility was evaluated using a light microscope with heater table as described by Sönmez et al . For this process, a slide was placed on microscope and allowed to warm to a temperature of 35â-¦C on a heating table. Several droplets of Tris buffer solution [Tris (hydroxymethyl) aminomethane 3.63 g, glucose 0.50 g, citric acid 1.99 g, and distilled water 100 mL] were dropped on the slide and a very small droplet of fluid obtained from the left cauda of epididymis with a pipette was added on this solution and mixed with a cover-slip. The percentage of progressive sperm motility was visually evaluated using a score ranging from 0 to 100% under magnification.
Determination of percentage of abnormal spermatozoa: Percentage of abnormal spermatozoa was determined by the method described by Türk et al.30 To determine the percentage of morphologically abnormal spermatozoa, the slides stained with eosin-nigrosin (1.67 g eosin, 10 g nigrosin, and 2.9 g sodium citrate per 100 ml distilled water) were prepared. After preparation, the slides were viewed under a light microscope at 400 magnification. For each animal, 300 sperm cells were examined on each slide.
Statistical analysis: All the statistical analyses were undertaken with the statistical software package SPSS, version 12.00 (SPSS, Chicago, IL, USA). For all group evaluations, Kruskall Wallis test was used. For intergroup comparisons, Mann-Whitney U test was used. The level of significance was set at p<0.05. Quantitative data are expressed as means ± standard deviations (SD) and shown in figures.
This work was supported by The Firat University Research Foundation (FÜBAP-1286).
Declaration of Interest: The authors report no con¬‚icts of interest. The authors alone are responsible for the content and writing of the paper.