Chronic Water-Pipe Smoke Exposure Induces Injurious Effects to Reproductive System in Male Mice

Abstract

There is a global increase in the popularity of water-pipe tobacco smoking including in Europe and North America. Nevertheless, little is known about the male reproductive effects of water-pipe smoke (WPS), especially after long-term exposure. Here, we assessed effects of WPS exposure (30 min/day) in male mice for 6 months. Control mice were exposed to air-only for the same period of time. Twenty-four hours after the last exposure, testicular histopathology, and markers of inflammation and oxidative stress, and the tyrosine-protein kinase vascular endothelial growth factor receptor 1 (VEGFR1) were assessed in testicular homogenates. Moreover, plasma testosterone, estradiol, and luteinizing hormone (LH) concentrations were also measured. Chronic WPS exposure induced a significant decrease of testosterone and estradiol, and a slight but significant increase of LH. Glutathione reductase, catalase, and ascorbic acid were significantly decreased following WPS exposure. Plasma concentration of leptin was significantly decreased by WPS exposure, whereas that of tumor necrosis factor α and interleukin 6 was significantly increased. Histopathological analysis of the testes revealed the presence of a marked reduction in the diameter of the seminiferous tubules with reduced spermatogenesis. Transmission electron microscopy examination showed irregular thickening and wrinkling of the basement membranes with abnormal shapes and structures of the spermatozoa. VEGFR1 was overexpressed in the testis of the mice exposed to WPS and was not detected in the control. The urine concentration of cotinine, the predominant metabolite of nicotine, was significantly increased in the WPS-exposed group compared with the control group. We conclude that chronic exposure to WPS induces damaging effects to the reproductive system in male mice. If this can be confirmed in humans, it would be an additional concern to an already serious public health problem, especially with the increased use of WPS use all over the world, especially in young adults.

Keywords: mice; reproductive hormones; testes; tobacco; water-pipe smoke.

Figure 1

The plasma concentrations of testosterone, luteinizing hormone (LH) and estradiol following air exposure (AE) of control mice (open columns), and water-pipe smoke (WPS) exposure for 30 min/day for 6 months (filled columns). Each column and vertical bar represent the mean ± SEM (n = 10–11 mice in each group).

Figure 2

Ascorbic acid concentration and catalase, glutathione reductase and superoxide dismutase (SOD) in testicular homogenates following air exposure (AE) of control mice (open columns), and water-pipe smoke (WPS) exposure for 30 min/day for 6 months (filled columns). Each column and vertical bar represent the mean ± SEM (n = 10–11 mice in each group). P < 0.05 was considered significant.

Figure 3

The total protein and cholesterol concentrations, and the activity of alkaline phosphatase in testicular homogenates following air exposure (AE) of control mice (open columns), and water-pipe smoke (WPS) exposure for 30 min/day for 6 months (filled columns). Each column and vertical bar represent the mean ± SEM (n = 10–11 mice in each group). P < 0.05 was considered significant.

Figure 4

The concentrations of leptin, tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in plasma following air exposure (AE) of control mice (open columns), and water-pipe smoke (WPS) exposure for 30 min/day for 6 months (filled columns). Each vertical column and bar represent the mean ± SEM (n = 10–11 mice in each group). P < 0.05 was considered significant.

Figure 5

Representative photomicrographs of a Hematoxylin-Eosin stained sections of testicular tissue obtained following air exposure (AE) of control mice (A,B), and water-pipe smoke (WPS) exposure (C and D) for 30 min/day for 6 months. Control mice showed normal spermatogenesis (A,B), whereas those from mice exposed to WPS showed signs of damage (A,B). The tubule diameter and the Johnson's score were both significantly decreased by the exposure to WPS (p < 0.05).

Figure 6

Transmission electron microscopy testicular tissue photomicrographs from a representative control mouse (exposed to air only, A) and a representative mouse exposed to water-pipe smoke (WPS, B) for 30 min/day for 6 consecutive months. The photomicrograph in the control group (A) showed normal basement membranes (single arrows) and normal structure of the spermatozoa (double arrows). The electron micrograph of the WPS group (B) showed irregular thickening and wrinkling of the basement membranes (single arrow) with abnormal shapes and structures of the spermatozoa (double arrows).

Figure 7

Representative images for the expression of the vascular endothelial growth factor receptor 1 (VEGFR1) in testicular tissues in control mice exposed to air, and mice exposed daily for 30 min to water-pipe smoke (WPS) for 6 months. VEGFR1 was overexpressed in the testes of the mice treated with WPS, and was not detected in the control not treated mice. The scale bar equals 50 μm.

Figure 8

The concentrations of cotinine in plasma (A) and urine (B) following air exposure (AE) of control mice (open columns), and mice exposed to water-pipe smoke (WPS) for 30 min/day for 6 months (filled columns). Each vertical column and bar represent the mean ± SEM (n = 6 mice in each group). p < 0.05 was considered significant.