Volume 13, Issue 8 (9-2015)                   IJRM 2015, 13(8): 489-494 | Back to browse issues page

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Farhadi M, Mohseni Kouchesfahani H, Shockravi A, Foroozanfar M, Parivar K. The adverse effects of the methoxsalen and ultraviolent A radiation on spermatogenesis in mice. IJRM. 2015; 13 (8) :489-494
URL: http://journals.ssu.ac.ir/ijrmnew/article-1-674-en.html
1- Department of Mcrobiology, Karaj Branch, Islamic Azad University, Karaj, Iran , monafarhadi@yahoo.com
2- Department of Biology, Kharazmi University, Tehran, Iran
3- Faculty of Chemistry, Kharazmi University, Tehran, Iran
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Introduction
 
Psoralens are a class of three rings, heterocyclic and planar compounds that occur naturally as secondary metabolites in various plant species (1, 2, 3, 4) Methoxsalen (8-methoxypsoralen) together with ultraviolet A (UVA) long-wave therapy (PUVA) is known to be a human carcinogen based on sufficient evidence of carcinogenicity from studies in humans. In photo chemotherapy methoxsalen and UVA (320-400 nm) are used for treatment of some epidermal proliferative disorders such as psoriasis and vitiligo (5, 6). Psoralens are known to be toxic to a wide range of organisms (7). In the last three decades researchers were able to show that psoralens inhibit epidermal cell proliferation after UV light irradiation (6).
It has been shown that methoxsalene has toxic effects on reproductive and developmental processes (8). Studies by Diawara et al. (1997) showed that, oral administration of methoxsalen to male and female rats reduces birth rate significantly. Also, 200 to 400 mg/Kg dosage of methoxsalen could reduce animal weight and could change the histology of the liver, testes and adrenal (9). In 2013 it was shown that methoxsalen decreased mean level of estrogen and progesterone significantly and it significantly reduced the number and diameter of corpus luteum and number of growing follicles (10). Ruta graveolens (RG) is one of the species of herbal medicinal families that use for male contraception (11, 12). Long term administration of RG can decrease the weight of genital organs, sperm motility and sexual behavior in rat (13). RG plus 8-methoxy psoralen (sodab) caused weight gain in testicle and epydidim (14). These findings showed that psoralen could affect the male and female reproductive systems. Despite of the long term application of furocoumarines (methoxsalen, xanthotoxin, bergapten, isopimpinellin) in traditional medicine, the teratogenicity of methoxsalen alone or together with UV radiation is not clear yet. This study was applied to investigate the probable teratogenicity of methoxsalen on unmated male mature Balb/C mice.
 
Materials and methods
 
In an experimental study in Kharazmi University we used Methoxsalen with formula of 9-methoxy-7H-furo [3, 2-g] [1]-benzopyran-7-one that was purchased from Sigma Aldrich (Germany) and Tween 80 was used as solvent. The UVA source consisted of a L40/79k (Osram) lamp emitting 7.632×10 (w/cm) at 50 cm distance. The UVA dose which causes erythema is 0.046 J/cm2. Male Balb/C mice were obtain from Pasture Institute (Experimental Animal Keeping Center, Tehran, Iran) and were grown in the animal room at temperature of 22-23°C, 50-55% humidity and lighting cycle of 12-h light/12-h dark. The animals were randomly divided in three experimental, sham and control groups which all were unmated mature Balb/C mice (65 days old), 6 in each group. The LD50 standard was determined 160 mg/kg body weight and the UVA dose which causes erythema was applied (0.046 g/cm). A sub-lethal dose of 80 mg/kg methoxsalen solution was injected intrapritoneally to mature mice and after one hour the mice were exposed to UVA radiation for 20 minutes. Experimental group I received methoxsalen only; experimental group II received methoxsalen with UVA and PUVA, while experimental group III were under UVA only. Injections were applied for two consecutive weeks. The animals of experimental groups (I, II, III) were sacrificed 48 hours after the last injection, and their testes were removed and fixed in Bouins’s fixative and embedding with paraffin. Then serial cross sections (5 µm thickness) were prepared and slides were stained with H & E solutions for morphological and histological studies (15). The following observations were recorded: initial and final male body weights, testes weights, Tunica albuginea diameter, and number of spermatogonia and histological investigation of the testes. Morphometric measurements were applied by light microscopy. Tubule diameters of 100 nearly round seminiferous tubules were measured for each testis using an ocular micrometer at 250X. Diameter averaged for each rat for statistical analysis (16). All animal-related protocols were approved by Ethical Committee at Kharazmi University, Tehran, Iran.
 
Statistical analysis
In all experiments, data were analyzed using SPSS (Statistical Package for the Social Sciences, SPSS Inc., Chicago, IL, USA) software, and one way ANOVA statistical method. For each variable, means were calculated at the 5% level using LSD test.
 
Results
 
The results showed that body weight reduced significantly in all experimental groups compared to the control group. Relative testes weight and the thickness of tunica albuginea, was significantly increased (Figure 1) in experimental groups I and II, while the testis volume and its length and diameter did not show significant changes (Table I). The number of type A and B spermatogonia, primary spermatocytes, spermatids and sperms were significantly reduced in all experimental groups, compared to control group (Table II). In addition, PUVA experience (experimental group I) and methoxsalen alone expeience (experimental group II) showed shrinkage of seminiferous tubules, release of primary spermatocytes and sperms into the lumen, and disorganized seminiferous tubules (Figure 2).

Table I. Results of the effects of methoxsalen on the testes (mean±SE)




Table II. Results of the effects of methoxsalen on the number of spermatogenic cells (mean±SE)




Figure 1. Photomicrograph of tunica albuginea of testis. Control (left), experimental: methoxsalen with UVA (right). The increase of tunica albuginea diameter (2500X)