Abnormalities in the male reproductive system after exposure to diesel and biodiesel blend

Abstract

Altering the fuel source from petroleum-based ultralow sulfur diesel to biodiesel and its blends is considered by many to be a sustainable choice for controlling exposures to particulate material. As the exhaust of biodiesel/diesel blends is composed of a combination of combustion products of polycyclic aromatic hydrocarbons and fatty acid methyl esters, we hypothesize that 50% biodiesel/diesel blend (BD50) exposure could induce harmful outcomes because of its ability to trigger oxidative damage. Here, adverse effects were compared in murine male reproductive organs after pharyngeal aspiration with particles generated by engine fueled with BD50 or neat petroleum diesel (D100). When compared with D100, exposure to BD50 significantly altered sperm integrity, including concentration, motility, and morphological abnormalities, as well as increasing testosterone levels in testes during the time course postexposure. Serum level of luteinizing hormone was significantly depleted only after BD50 exposure. Moreover, we observed that exposure to BD50 significantly increased sperm DNA fragmentation and the upregulation of inflammatory cytokines in the serum and testes on Day 7 postexposure when compared with D100. Histological evaluation of testes sections from BD50 exposure indicated more noticeable interstitial edema, degenerating spermatocytes, and dystrophic seminiferous tubules with arrested spermatogenesis. Significant differences in the level of oxidative stress assessed by accumulation of lipid peroxidation products and depletion of glutathione were detected on exposure to respirable BD50 and D100. Taken together, these results indicate that exposure of mice to inhalable BD50 caused more pronounced adverse effects on male reproductive function than diesel.

Keywords: DNA fragmentation; biodiesel particles; male reproduction; oxidative stress; pulmonary exposure; sperm quality.

Fig. 1

The degree of oxidative stress evaluated by HNE-His adduct (A) and GSH (B) in the testes of C57BL6 mice on Days 7 and 28 after repeated exposure to D100 or BD50 exhaust particles. Mice were exposed via pharyngeal aspiration (15 µg/mouse/day of total carbon, twice a week for 2 weeks). Black columns: 7 days after last repeated exposure to BD50 or D100 exhaust particulate; and clear columns: 28 days after last repeated exposure to BD50 or D100 exhaust particulate. Data are presented as percent of control. Means ± SE (n = 10 mice per group). Significantly different from *controls (P < 0.05) and ^αD100 exhaust particulate-exposed group (P < 0.05).

Fig. 2

Concentration (A) and motility (B) of epididymal sperm from C57BL6 mice on days 7 and 28 after repeated exposure to D100 or BD50 combustion exhaust particles. Mice were exposed to exhaust particles via pharyngeal aspiration (15 µg/mouse/day of total carbon, twice a week for 2 weeks). Black columns: 7 days after last repeated exposure to BD50 or D100 exhaust particulate; and clear columns: 28 days after last repeated exposure to BD50 or D100 exhaust particulate. Means ± SE (n = 10 mice per group). Significantly different from *controls (P < 0.05) and ^αD100 exhaust particulate-exposed group (P < 0.05).

Fig. 3

Morphological abnormalities of epididymal sperm from C57BL6 mice on Days 7 and 28 postrepeated exposure to D100 or BD50 combustion exhaust particles. (A) Day 7 and (B) Day 28 postexposure to combustion exhaust particles. Mice were exposed to exhaust particles via pharyngeal aspiration (15 µg/mouse/day of total carbon, twice a week for 2 weeks). Black columns: control mice; gray columns: mice exposed to D100 exhaust particulate; and clear columns: mice exposed to BD50 exhaust. Data presented as percent of abnormal cells population when compared with total number of sperm. Means ± SE (n = 10 mice per group). Significantly different from *controls (P < 0.05) and ^αD100 exhaust particulate-exposed group (P < 0.05).

Fig. 4

Testicular testosterone (A) and serum LH (B) levels in C57BL6 mice on Days 7 and 28 after repeated exposure to D100 or BD50 exhaust particulate. Mice were exposed to exhaust particles via pharyngeal aspiration (15 µg/mouse/day of total carbon, twice a week for 2 weeks). Black columns: 7 days after last repeated exposure to BD50 or D100 exhaust particulate; and clear columns: 28 days after last repeated exposure to BD50 or D100 exhaust particulate. Data presented as percent of control. Means ± SE (n = 10 mice per group). Significantly different from *controls (P < 0.05) and ^αD100 exhaust particulate-exposed group (P < 0.05).

Fig. 5

Light micrographs of H&E stained sections from testes of mice 28 days after pharyngeal aspiration of D100 or BD50 (cumulative dose 60 mg per mouse): (A) control mice, (B) mice exposed to D100, and (C) mice exposed to BD50. Sections of D100 group showed interstitial edema and occasional dystrophic seminiferous tubules with arrested spermatogenesis and the presence of degenerating spermatocytes (arrows). These histologic changes were more prominent in BD50 group, where dystrophic seminiferous tubules were clustering, especially in subcapsular areas (arrows).

Fig. 6

DNA damage of epididymal sperm from C57BL6 mice on Days 7 and 28 postrepeated exposure to D100 or BD50 exhaust particles. Mice were exposed to exhaust particles via pharyngeal aspiration (15 µg/ mouse/day of total carbon, twice a week for 2 weeks). Black columns: 7 days after last repeated exposure to BD50 or D100 exhaust particulate; and clear columns: 28 days after last repeated exposure to BD50 or D100 exhaust particulate. Data presented as percent of control. Means ± SE (n = 10 mice per group). Significantly different from *controls (P < 0.05) and ^αD100 exhaust particulate-exposed group (P < 0.05).

Fig. 7

A schematic representation of several biological effects associated with inflammation, oxidative stress, and male infertility. This figure illustrates how enhanced oxidative stress can cause changes at different levels in the male reproductive system, leading to infertility. The precise mechanisms that are modulated/effected on BD50 particulate exposure in male mice are highlighted in red colored font.