ToxSci Advance Access originally published online on January 30, 2008
Toxicological Sciences 2008 103(1):137-148; doi:10.1093/toxsci/kfn020
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Motorcycle Exhaust Induces Reproductive Toxicity and Testicular Interleukin-6 in Male Rats
* Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC
Institute of Veterinary Pathobiology, National Chung-Hsing University, Taichung, Taiwan, ROC
Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taichung, Taiwan, ROC
Department of Anesthesiology
¶ Department of Obstetrics and Gynecology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC
1 To whom correspondence should be addressed at Institute of Toxicology, College of Medicine, National Taiwan University, 1 Jen Ai Road, Section 1, Taipei, Taiwan, ROC. Fax: +886-2-2314-0217. E-mail: thueng{at}ntu.edu.tw.
Received December 13, 2007; accepted January 15, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Motorcycle exhaust (ME) from two-stroke engines contains many toxicants and poses a potential health hazard. The major objectives of the present study were to investigate the male reproductive toxicity of ME and the underlying mechanisms of toxicity. Male Wistar rats were exposed to ME by inhalation 1 h each in the morning and afternoon, Monday through Friday. Exposures to 1:50 diluted ME for 4 weeks or to 1:10 diluted ME for 2 and 4 weeks showed concentration- and time-dependent decreases of testicular weight, spermatid number, and cauda epididymal sperm number. Subsequent studies were done using 4-week exposure to 1:10 diluted ME. ME caused histopathological changes including testicular spermatocytic necrosis and seminiferous tubule atrophy and cauda epididymal formation of clusters of pyknotic and necrotic sperm cells. ME-exposed male rats mated with untreated females showed decreases of male mating index and female fertility index and an increase of implantation site loss. ME decreased 7-ethoxycoumarin O-deethylase and superoxide dismutase activities but induced proinflammatory cytokine interleukin-6 (IL-6) messenger RNA (mRNA) in the testis. Male rats were exposed to ME with or without cotreatment with 50 mg/kg vitamin E orally for 4 weeks. ME decreased serum testosterone concentration. This effect was reversed by cotreatment with vitamin E. ME decreased testicular spermatid number and induced IL-6 mRNA and protein. These effects were also reversed by the vitamin E cotreatment. The present findings show that ME causes male reproductive effects and induces testicular IL-6 in rats by mechanisms involving induction of oxidative stress and inhibition of steroidogenesis.
Key Words: motorcycle exhaust; IL-6; testis; sperm; testosterone; oxidative stress; air pollution.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Motorcycles and scooters equipped with two-stroke and four-stroke engines are a popular means of transportation in many parts of the world. For instance, the total number of motorcycles exceeds 13 million in Taiwan, which has a population of 23 million. Emissions from the motorcycles are the predominant source of mobile pollutants in urban areas. Motorcycle exhaust (ME) from two-stroke engines poses a potential health hazard, because the gas phase of ME contains carcinogens benzene and 1,3-butadiene and pulmonary toxicant naphthalene (Jemma et al., 1995
) and the particulate phase contains carcinogenic polycyclic aromatic hydrocarbons (PAHs) benzo[a]pyrene, benz[a]anthracene, and chrysene (Ueng et al., 2000, 2005). In addition to motorcycles, the two-stroke engine is used in a variety of applications including outboard boats, snowmobiles, lawn mowers, and trimmers.
ME and the organic extracts of ME particulates (MEP) show biochemical, immunological, antiestrogenic, vascular, and genetic effects. ME inhalation exposure induced cytochrome P450 (CYP) 1A1 protein and monooxygenase activity in rat liver, kidney, and lung (Ueng et al., 1998). The exposure enhanced lipid peroxidation and increased proinflammatory cytokine interleukin (IL)-1
and fibroblast growth factor-9 messenger RNA (mRNA) expression in rat lung (Ueng et al., 2004b, 2005). Intratracheal administration of MEP extracts to mice increased airway inflammation and IL-4 in bronchoalveolar lavage fluid (Lee et al., 2004). Treatment of immature female rats with MEP extracts intraperitoneally suppressed 17β-estradiol-induced uterine weight and peroxidase activity (Ueng et al., 2004a). MEP extracts increased vasoconstriction in rat aorta under organ culture conditions (Tzeng et al., 2003). The extracts increased peroxide production, sister chromatid exchange, and 8-hydroxyguanosine formation in Chinese hamster lung V79 cells (Kuo et al., 1998). In these previous studies, oxidative stress has been suggested as a common mechanism of action for ME and MEP in vivo or in vitro.
The male reproductive systems of humans and animals are susceptible to the adverse effects of environmental chemicals. Extensive studies are available concerning the reproductive effects of individual compounds such as drugs, metals, organic solvents, or PAHs. Few studies are available concerning the effects of complex environmental mixtures, to which humans are exposed. Smoking is associated with decreases of sperm density, total count, and motility in men (Vine et al., 1996). Inhalation exposure of growing male rats to diesel exhaust (DE) containing 5.6 mg/m3 particulate matter 6 h daily and 5 days a week for 3 months reduced testicular sperm production and hyaluronidase activity, increased serum testosterone and estradiol concentrations, and decreased serum follicle-stimulating hormone and luteinizing hormone (Watanabe and Oonuki, 1999). Decreases of sperm concentration and luteinizing hormone receptor mRNA were found in the testes of male mice exposed to 3.0 mg/m3 DE particles 12 h daily for 6 months (Yoshida et al., 1999). In these DE studies, disruption of hormonal control of spermatogenesis was considered an underlying mechanism of toxicity. The roles of oxidative stress and other mechanisms were not investigated.
Oxidative stress plays an important etiological role in the development of toxicities and diseases including cancer (Aggarwal et al., 2006). IL-6, IL-1β, and cyclooxygenase-2 (COX-2) are critical inflammatory mediators of oxidative damages induced by toxicants and carcinogens. The antioxidant vitamin E protects cells and tissues against many chemically induced oxidative damages. Vitamin E also modulates the expressions of gene families involved in the uptake and degradation of tocopherols, lipid uptake, and atherosclerosis, modulation of extracelluar proteins, adhesion, and inflammation, and cell signaling and cell cycle regulation (Azzi et al., 2004).
ME reproductive effects have not been reported in the literature. Because more than tens of millions of people are exposed to ME on a regular basis, it is of environmental health importance to determine the reproductive hazard of ME exposure. Given that the chemical composition of ME is different from that of DE (Jemma et al., 1995), it is difficult to predict or assess ME reproductive toxicity based on the existing toxicity data of DE. Therefore it is necessary to conduct reproductive studies of ME. Cytokines are important regulators of the development and function of the testis. IL-6 and related proinflammatory cytokines have direct effects on testicular spermatogenic cell differentiation and steroidogenesis (Hedger and Meinhardt, 2003). Direct information regarding the effect of environmental chemicals on testicular IL-6 expression is not available. Many steroid-metabolizing P450 enzymes show 7-ethoxycoumarin O-deethylase activity (Ryan and Levin, 1999). Therefore, the O-deethylase activity can serve as a biochemical marker for the P450 enzymes. Effect of xenobiotics on the testicular O-deethylase activity has not been reported.
The major objectives of the present study were to determine the male reproductive toxicity of ME and to investigate the mechanism of toxicity. The present study was conducted using male rats exposed to 1:10 diluted ME by inhalation for 1 h each in the morning and afternoon daily on 5 days a week for 4 weeks, to provide more environmentally realistic conditions. The effects of ME on reproductive, histopathological, biochemical, and inflammatory parameters were determined using the testes and the epididymides. The effects on fertility and pregnancy outcome were determined using ME-exposed male rats mated with untreated females. Mechanistic studies were done to investigate the ability of vitamin E to block the effects of ME on serum testosterone level, sperm counts, and testicular IL-6. The present findings demonstrate that ME induces male reproductive toxicity, increases testicular IL-6 expression, and decreases serum testosterone in rats. The mechanisms of toxicity involve induction of oxidative stress and inhibition of steroidogenesis.
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MATERIALS AND METHODS |
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Animal treatment.
Male Wistar rats, 7 weeks old, were purchased from the Animal Center of the College of Medicine, National Taiwan University, Taipei, Taiwan. Before experiments began, rats were allowed 1-week acclimation period at the animal quarter with air conditioning and 12-h light/dark cycles. The animals were fed ad libitum a rodent laboratory chow purchased from Purina Mills, Inc., St Louis, MO. A 1992 Yamaha Cabin motorcycle with a two-stroke 50 c.c. engine and a variable venturi carburetor was used for the experiments. There were 11,450 miles on the motorcycle. A head-nose-only inhalation chamber purchased from Technical & Scientific Equipment GMBH, Bad Hamburg, Germany, was used for the inhalation studies. The inhalation chamber and exposure atmosphere conditions were described previously (Ueng et al., 2004b
, 2005). After preliminary exposure studies, the animals were exposed to ME from 9 to 10 A.M. and 4 to 5 P.M. daily, Monday through Friday, for 4 weeks. In antioxidant vitamin E studies, (+)--tocopherol (1000 IU/g, Sigma-Aldrich Corporation, St Louis, MO) was used. Vitamin E was dissolved in corn oil and administered orally to rats at 50 mg/kg/day once daily, Monday through Friday, for 4 weeks. Control rats were exposed to clean air with or without treatment with corn oil. Animals were killed after the last treatment by decapitation after light anesthesia with CO2. All animal care and experimental procedures were approved by the Institutional Care and Use Committee, College of Medicine, National Taiwan University.
Spermatid and sperm numbers.
The right testis was homogenized in phosphate buffered saline, pH 7.4 using a Teflon-pestle homogenizer. After incubation for 15 min at 37°C, the homogenate was mixed and diluted using phosphate buffered saline. The diluted sperm suspension was placed in a 60°C water bath for 1 min and counted for spermatid number on a hemocytometer using a phase-contrast microscope. The cauda epididymal sperm number was similarly determined.
Fertility and reproductive function.
At the end of ME treatment, each of 11 control and 12 ME-treated male rats was housed with two untreated virgin females overnight. A control male was housed with one female, due to the limited number of female rats available. Female was checked for the presence of plug the next morning or vaginal smear was collected and examined for the presence of sperm. The mating procedure was repeated daily for 2 weeks or until detection of plug or sperm in vaginal smear. The day of plug or sperm detection was considered day 0 of gestation. Females were sacrificed on day 20 and ovaries and uteri were collected. The numbers of corpora leutea, implantation sites, and live fetuses were determined. Preimplantation loss was determined by calculating the difference between the number of corpora leutea and the number of implantation sites. Postimplantation loss was determined by calculating the difference between the number of implantation sites and the number of live fetuses. Fetuses were blotted, weighed, sexed, and examined for external alterations. The male mating index was calculated as the ratio between the number of males with pregnant females and the number of males. The female fertility index was calculated as the ratio between the number of females presumed pregnant and the number of females cohabitated (Moore et al., 1995).
Histopathological evaluation.
The testis and the cauda epididymis were fixed in Bouin's buffered formalin solution for 1 week. Tissues were processed using standard histopathological techniques, sectioned at 2 µm, and stained using hematoxylin and eosin for light microscopy analysis (Opticphot-2, Nikon, Tokyo, Japan). Testicular and cauda epididymal histology was evaluated according to the criteria of Oakberg (1956) and Hess (1990) with slight modifications. The criteria for testicular score numbers and histopathological changes were (0), no observable effect; +: (1), minor changes, 1–10% of the seminiferous tubules with slight decrease and increase sloughing in sperm cells; ++: (2), mild changes, 10–20% of the seminiferous tubules showing atrophy with mild decrease in sperm cells, few necrotic sperm cells, and multinuclear giant cell formation; +++: (3), moderate changes, 21–50% of the seminiferous tubules showing atrophy with moderate decrease in sperm cells and increase of multinuclear giant cell formation; and ++++: (4), severe changes, above 50–100% of the seminiferous tubules showing atrophy with remarkable decrease or all loss of sperm cells and increase of multinuclear giant cell formation. The criteria for cauda epididymal score numbers and histopathological changes were –: (0), no observable effect; +: (1), minor changes, normal sperm concentration and 5–10 necrotic cells in the efferent ductuli; ++: (2), mild changes, slight decrease and 11–50 necrotic cells; +++: (3), moderate changes, moderate decrease and above 50 necrotic cells; and ++++: (4), severe changes, remarkable decrease in sperm concentration or azoospermia in the efferent ductuli.
Lipid peroxidation and glutathione content assays.
The testis and the epididymis homogenates were prepared and lipid peroxidation was determined by measuring the formation of malondialdehyde and related compounds at 532 nm using the thiobarbituric acid method (Ohkawa et al., 1979). Total glutathione contents of the testis and the epididymis homogenates were determined using the 5,5'-dithio-bis(2-nitrobenzoic acid) and glutathione reductase recycling method (Jollow et al., 1974).
Enzyme assays.
Microsomes and cytosols were prepared from the testis and the epididymis by differential centrifugation. Microsomal 7-ethoxycoumarin O-deethylase activity was determined by measuring the fluorescence of the product 7-hydroxycoumarin (Greenlee and Poland, 1978). Cytosolic glutathione S-transferase activity toward 1,2-dichloro-4-nitrobenzene was determined following the spectrophotometric method of Habig et al. (1974). Total superoxide dismutase activity was determined using the epinephrine method by measuring the capacity of the enzyme to inhibit auto-oxidation of adrenaline to adrenochrome (Misra and Fridovich, 1972). Catalase activity was determined by a spectrophotometric method measuring the disappearance of H2O2 at 240 nm (Pippenger et al., 1998). Glutathione peroxidase activity was measured using t-butyl hydroperoxide as a substrate following the oxidation of nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) (Flohë and Günzler, 1984). All the enzyme activities were determined under linear conditions with respect to protein concentration and reaction time. Protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.
Reverse transcription-polymerase chain reaction.
Total RNA was isolated from the testis and the cauda epididymis following the method of Chomczynski and Sacchi (1987). Complimentary DNA (cDNA) synthesis and PCR procedures were conducted as described previously (Wang et al., 2001). PCR primers for target genes and internal standard cyclophilin were synthesized according to the published sequences by Gibco/BRL, Life Technologies, Inc., Gaithersburg, MD. The sequences of forward and reverse primers of IL-6 (Han et al., 1999), IL-1β (Garcon et al., 2001), COX-2 (Ogata et al., 2004), and cyclophilin (Agardh et al., 2002) were described previously. The thermocycle conditions for each primer were: 60°C and 42 cycles for IL-6; 55°C and 34 cycles for IL-1β; 59°C and 30 cycles for COX-2; and 62°C and 35 cycles for cyclophilin. The cycle number for each primer was determined to keep signal amplification in the linear range. PCR products were separated on 2% agarose gels and stained with ethidium bromide. Intensity of PCR product was determined by scanning densitometry using an IS-1000 Digital Imaging System (Alpha Innotech Corporation, San Leandro, CA) and normalized against the intensity of internal control cyclophilin. Relative intensity of target gene PCR product from treated rats was calculated by dividing its intensity by the corresponding intensity from control rats.
Real-time reverse transcription–polymerase chain reaction.
To confirm the results of reverse transcription–polymerase chain reaction (RT-PCR), real-time RT-PCR was conducted. Total RNA was isolated from the testis using SV Total RNA Isolation System (Promega Corporation, Madison, WI). Five micrograms of total RNA was reverse transcribed and the resulting cDNA was used in subsequent real-time PCR reactions with fluorescence detection using an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA) as described previously (Ueng et al., 2005). Reaction was carried out in microAmp 96 well reaction plates, SYBR Green PCR Master Mix 2x, DNA polymerase, deoxy-nucleotide triphosphates with deoxy-uridine triphosphate, forward and reverse primers 0.15µM each (Invitrogen Corp., Carlsbad, CA), and 200 ng cDNA in a final volume of 25 µl. Amplification parameters were: denaturation at 94°C 10 min, followed by 45 cycles of 95°C, 15 s; 60°C, 60 s. PCR primers for IL-6, IL-1β, COX-2, and endogenous control β-actin were synthesized according to the published sequences (Peinnequin et al., 2004) by Gibco/BRL, Life Technologies, Inc. Samples were analyzed in triplicate. Quantitation of mRNA transcription was performed using a relative quantitation method with standard curves constructed from five log RNA concentrations of a specific gene or β-actin and their respective CT values. The input amount of a specific gene was calculated from its standard curve and normalized to the input amount of β-actin calculated from its standard curve. The relative difference between treatment and control groups was calculated from the ratio of the amount of specific gene to that of β-actin in each group.
IL-6 enzyme-linked immunosorbent assay.
The frozen testis was homogenized (1:4, wt/vol) in 20mM Tris–HCl buffer, pH 7.5, containing 2M NaCl, 0.1% Tween-80, 1mM phenylmethylsulfonyl fluoride, and 1mM ethylenediaminetetraacetic acid at 4°C. The homogenate was centrifuged at 19,000 x g for 30 min at 4°C. Supernatant was collected and used for enzyme-linked immunosorbent assay (ELISA) using a Quantikine Rat IL-Immunoassay kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). The minimum detectable dose of rat IL-6 was 21 pg/ml.
High-pressure liquid chromatography analysis of serum testosterone and metabolites.
Two hundred microliters of serum was extracted twice with 400 µl of ethyl acetate. The ethyl acetate extract was evaporated to dryness by nitrogen flow and then dissolved in 100 µl of methanol prior to high-pressure liquid chromatography (HPLC) analysis (Baltes et al., 1998). An Agilent HPLC system, a UV 1100 detector operating at 245 nm (Hewlett-Packard), and a Waters symmetry RP18 HPLC column (5 µm, 250 x 4.6 mm inner diameter, Waters, Milford, MA) were used. The column was eluted for 29 min with a gradient from 1% acetonitrile:33% methanol:64% water to 1% acetonitrile:43% methanol:56% water at a flow rate of 1.5 ml/min at 40°C.
Statistical analysis.
The statistical significance of difference between control and treated groups was evaluated by the use of analysis of variance followed by Student's t test. A p value < 0.05 was considered statistically significant.
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RESULTS |
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Initial studies were conducted to investigate the concentration- and time-dependent effects of ME. Inhalation exposure to 1:50 diluted ME for 4 weeks produced a 9% decrease of body weight and no marked effects on the relative testis and epididymis weights (Table 1, treatment A). The ME treatment resulted in a 16% decrease of spermatid number in the testis. Exposure to 1:10 diluted ME for 2 weeks had no effects on body weight or the relative testis and epididymis weights (treatment B). The 2-week ME treatment caused a 36% decrease of testicular spermatid number and a 66% decrease of cauda epididymal sperm number. Exposure to 1:10 diluted ME for 4 weeks decreased body weight by a 15% and reduced the relative testis weight by a 28% without affecting epididymis weight (treatment C). The treatment resulted in 84% and 75% decreases of testicular and cauda epididymal sperm counts, respectively. These results showed that the ME-mediated decreases of sperm counts were concentration- and time-dependent (treatment A vs. C and B vs. C). The decrease of testicular weight was selective, because ME had no effects on the relative weights of liver, kidney, lung, spleen, seminal vesicles, ventral prostate, adrenal, and thyroid (data not shown). The subsequent studies were done using 4-week exposure to 1:10 diluted ME.
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Microscopic examinations of the testes revealed that control rat testis was histologically normal, characterized by the different stages of spermatogenesis in the multiple seminiferous tubules (Fig. 1A) and the presence of the surrounding Leydig cells (Fig. 1B). In contrast, ME-exposed rat testis showed seminiferous tubule atrophy (Fig. 1C), and moderate to severe germ cell necrosis (Fig. 1D). ME also induced other lesions including absence of elongated spermatids, decrease of spermatocytes, and formation of multinuclear giant cells in the severely damaged seminiferous epithelium. Sertoli cells were still present (Fig. 1E). Control rat cauda epididymis showed abundant normal sperm in the efferent ductules (Figs. 2A and 2B). In contrast, ME-exposure produced either an almost absence of sperm or a mass cluster of pyknotic and necrotic sperm cells in the lumen of efferent ductules (Fig. 2C). Other epididymal lesions included the presence of necrotic spermatids in the lumen of efferent ductules (Fig. 2D). In all the testes examined, 85%, 10%, and 5% of control rats showed no, minor, and mild pathological changes, respectively, with no moderate nor severe ratings (Table 2). Five percent of ME-exposed rats showed respective minor and mild pathological changes, whereas 40% and 50% showed moderate and severe changes, respectively. ME exposure resulted in a marked increase of testicular histopathological score. In the cauda epididymides examined, 85%, 10%, and 5% of control rats showed no, minor, and mild pathological changes, respectively. In contrast, 5%, 15%, 10%, and 70% of ME-treated rats showed respective minor, mild, moderate, and severe changes. ME markedly increased cauda epididymal histopathological score.
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To further determine the reproductive effect of ME, a fertility study was done using control and ME-exposed male rats mated with untreated female rats. ME exposure caused 64% and 24% decreases in male mating index and female fertility index, respectively (Table 3). ME caused a 29% decrease in implantation site and a 226% increase in preimplantation loss. Litter size from the ME group was 28% smaller than the controls. ME exposure had no marked effects on postimplantation loss, fetal body weight, and fetal sex ratio. These fertility and gestation data were consistent with the sperm count and histopathology data that ME caused reproductive effects in male rats.
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Biochemical and cytokine studies were conducted to investigate the possible role of oxidative stress in ME reproductive toxicity. The results of biochemical study showed that ME exposure resulted in a trend toward decrease of glutathione content and increase of lipid peroxidation in the testis (Table 4). The decrease and increase were not significant. ME treatment caused a 78% decrease in cytochrome P450-dependent 7-ethoxycoumarin O-deethylase activity in testis microsomes. ME resulted in a 29% decrease of antioxidant enzyme superoxide dismutase activity and had no marked effects on glutathione S-transferase, catalase, and glutathione peroxidase activities in testis cytosol. In the epididymis, ME did not cause significant changes of the previously mentioned biochemical parameters. In cytokine study, the results of RT-PCR analysis showed that ME increased proinflammatory cytokine IL-6 mRNA in the testis, but not in the cauda epididymis (Table 4 and Fig. 3). These biochemical and cytokine data indicated that ME exposure resulted in a trend toward increase of oxidative stress in the testis. These data also indicated that the testis was possibly more susceptible than the epididymis to the oxidative damage associated with ME.
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To investigate the mechanistic role of oxidative stress in the reproductive effects, studies were conducted to determine the ability of vitamin E to protect against ME toxicity. Rats were exposed to 1:10 diluted ME with and without cotreatment with 50 mg/kg vitamin E orally for 4 weeks. Effects of these treatments on serum testosterone concentration, sperm count, and testicular IL-6 were determined. The results of HPLC analysis of serum samples showed that ME decreased the circulating testosterone concentration by a 67% and cotreatment with ME and vitamin E decreased serum testosterone by a 39%, compared with the controls (Fig. 4). Serum level of testosterone in the rats cotreated with ME and vitamin E was 85% higher than the level in rats exposed to ME. The treatments had no marked effects on serum 2β-hydroxytestosterone concentration (data not shown). Other hydroxylated metabolites such as 2
-, 6β-, 16-, and 16β-hydroxytestosterone were not detectable in the sera of control and ME-treated rats. ME produced a 52% decrease of spermatid number in the testis, relative to the controls (Fig. 5). ME and vitamin E cotreatment did not produce a significant decrease of testicular spermatid number, compared with the controls. The testicular spermatid number in rats cotreated with ME and vitamin E was 61% higher than the number in rats exposed to ME. ME resulted in a 76% decrease of sperm number in the cauda epididymis, relative to the controls. ME and vitamin E cotreatment caused a 64% decrease of cauda sperm number, compared with the controls. The cauda epidiymal sperm number in rats cotreated with ME and vitamin E was 50% higher than the sperm number in rats exposed to ME. These data showed that vitamin E reduced the ME-mediated decreases of sperm counts in the testis and the cauda epididymis.
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The results of RT-PCR analysis showed that ME exposure increased testicular IL-6 mRNA expression which was reduced by cotreatment with ME and vitamin E, determined by scanning densitometry (Fig. 6). These studies were extended to investigate the mRNA expression of inflammatory cytokine IL-1β and inflammatory enzyme COX-2. Testicular IL-1β and COX-2 mRNA were increased in ME-treated rats and the increases were reduced in rats cotreated with ME and vitamin E. Real-time RT-PCR studies were conducted to confirm the modulatory effects of ME exposure and cotreatment with vitamin E. The results showed that ME resulted in four-, three-, and twofold increases of IL-6, IL-1β, and COX-2 mRNA in the testis, respectively (Fig. 7). In contrast, cotreatment with ME and vitamin E reduced the ME-mediated increases of the cytokine and enzyme mRNA to levels which were not significantly different from their respective controls. An ELISA study was done to determine the effects of ME exposure and cotreatment with vitamin E on IL-6 protein level in the testis. The results showed that IL-6 protein in the ME-treated rat testis was threefold higher than the protein in controls (Fig. 8). Testicular IL-6 protein in rats cotreated with ME and vitamin E was reduced to a level similar to the controls. Treatment with 50 mg/kg vitamin E orally for 4 weeks had no effects on the testicular spermatid number or the cauda epididymal sperm number (data not shown). The results of RT-PCR analysis showed that vitamin E alone did not alter IL-6, IL-1β, and COX-2 mRNA levels in the testis (Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The present findings show that ME inhalation exposure causes male reproductive toxicity and induces testicular IL-6 expression in rats. To the best of our knowledge, this is the first report of IL-6 induction by environmental chemicals in the testis. The present findings may have significant health implications because a vast number of people are exposed to ME and an increase of IL-6 can produce adverse effects on the male reproductive function, inducing persistent testicular resistance to luteinizing hormone action, and suppression of Leydig cell steroidogenesis (Bornstein et al., 2004
). IL-6 protein levels in the seminal plasma of infertile and immunoinfertile men were higher than those of fertile men (Naz and Kaplan, 1994). It will be important to investigate the reproductive effects of ME exposure in humans in future studies.
The present study suggests that mechanisms of ME toxicity involve induction of oxidative stress and inhibition of steroidogenesis. This conclusion is supported by the following experimental results. ME induced the proinflammatory cytokine IL-6 and modulated the biochemical parameters in favor of oxidative stress in the testis. The ME-mediated decrease of sperm count and induction of testicular IL-6 were partly blocked by cotreatment with an antioxidant vitamin E. These experimental results support the idea that oxidative stress plays a mechanistic role. The partial protective effect of vitamin E suggests that there are other mechanisms involved, in addition to oxidative stress. ME inhalation exposure caused histopathological damages to the testis, the site for both sperm cell production and biosynthesis of testosterone. 7-Ethoxycoumarin is a substrate metabolized by a multiplicity of P450 enzymes (Ryan and Levin, 1999). ME decreased testicular 7-ethoxycoumarin O-deethylase activity (Table 4), which could be a reflection of decrease of the activity of steroidogenic P450 enzyme(s), a consequence of ME damage to testicular Leydig cells. Lastly, ME markedly decreased the circulating level of testosterone. These experimental results support that inhibition of steroidogenesis is a mechanism of ME toxicity.
The possible sequence of events following the repetitive inhalation exposure is that ME toxicants and their reactive oxygenated metabolites cross the blood–testis barrier to exert direct oxidative damages to the testicular cells to inhibit spermatogenesis and steroidogenesis (Fig. 10). Alternatively, ME oxidative stress can activate signaling pathways with transcription factors such as nuclear factor-kappa B and activator protein-1 to induce the expression of IL-6, IL-1β, and COX-2 in the testicular cells. IL-6 induction subsequently causes negative regulation of testicular spermatogenic and steroidogenic processes. The testicular cell types responsible for IL-6 induction may include macrophages, Sertoli cells, and Leydig cells (Fig. 10). Previous studies reported that treatment with lipopolysaccharide and interferon-
enhanced secretion of IL-6 by isolated rat testis macrophages (Kern et al., 1995). The bacterial endotoxin stimulated IL-6 secretion by enriched rat Sertoli cell preparations (Syed et al., 1993). Incubation of rat Leydig cell culture with IL-1β increased IL-6 release to the culture medium (Boockfor et al., 1994). Based on these previous studies, it is reasonable to assume that ME induces testicular IL-6 production via the autocrine and paracrine pathways.
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ME-exposed male rats mated with untreated females showed adverse effects on male fertility index, implantation, and fetus development (Table 3). An explanation for these adverse effects is that ME decreased testicular and epididymal sperm counts. Preliminary results from microscopic examinations indicated that ME also decreased the percentage of motile sperm in the vas deferens (data not shown). These decreases of sperm parameters suggest that ME has the ability to impair the production and function of sperm cells in male rats. The ability of ME to impair the integrity of the germ cells has not been investigated. It will be of interest to determine the effect of ME on spermatogenesis in male offspring, because male germ-line is capable of transmitting chemically induced developmental or genetic toxicity to the offspring. For example, Somers et al. (2004)
reported that when male mice exposed to ambient air at an urban-industrial site were mated to unexposed females, their offspring inherited a threefold more frequent germ-line mutation rate at repetitive DNA loci than those of control males mated to unexposed females. High efficiency air filtration of ambient air at the urban-industrial site reduced the heritable mutation rates in the mouse offspring, suggesting that the particulate phase, not the gas phase, was primarily responsible for the developmental toxicity. On the other hand, the gas phase of DE played a more predominant role than the particulate phase in DE reprotoxicity, because removal of most of the particles by filtration had no effects on the spermatogenesis and endocrine-disrupting effects (Watanabe and Oonuki, 1999). In ME, reproductive toxicants benzene, ethylbenzene, toluene, and xylene were detected in the gas phase (Jemma et al., 1995) and benzo[a]pyrene was detected in the particulate phase (Ueng et al., 2000, 2005). Additional studies are required to determine and compare reproductive effects of the gas phase and the particulate phase of ME.
ME and DE show similar reproductive effects but opposite effects on serum testosterone. Inhalation exposure of growing male rats to DE decreased daily sperm production but increased serum testosterone and estradiol concentrations. It was proposed that the DE-mediated increases of the steroids caused a negative feedback effect on gonadotropin-releasing hormone and depressed spermatogenesis in the testis (Watanabe and Oonuki, 1999). The present study shows that ME inhalation decreased both sperm production and serum testosterone. These similar and opposite effects of ME and DE would be expected because of their similarities and differences in the fuel mixture and the combustion temperature and pressure paradigms.
The male reproductive effects of ME are similar to the effects of benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in many aspects. Inhalation exposure of adult male rats to benzo[a]pyrene with carbon black carrier decreased plasma testosterone concentration and sperm progressive motility (Inyang et al., 2003). Treatment with TCDD decreased testicular weight and sperm content in rodents (Gray, 1998). Cotreatment with vitamin E protected against TCDD-induced oxidative stress in male rat testis (Latchoumycandane and Mathur, 2002). ME is also similar to benzo[a]pyrene and TCDD in estrogen disrupting and CYP inducing properties. Benzo[a]pyrene and TCDD are antiestrogens and potent CYP1A1 inducers (Safe, 2001). MEP extracts showed antiestrogenic effects in immature female rats and MCF-7 human breast cancer cells (Ueng et al., 2004a) and induced CYP1A1 expression in cultured human liver and lung cancer cells (Ueng et al., 2000, 2005). These similarities in reproductive toxicity, endocrine disruption, and enzyme induction suggest that ME shares some of the pathways through which benzo[a]pyrene and TCDD produce their toxicities and biological activities. ME might be regarded as a benzo[a]pyrene- or a TCDD-like complex environmental mixture.
In conclusion, the present study shows that repeated ME exposure decreases serum testosterone, induces testicular IL-6, and impairs spermatogenesis. Vitamin E blocks partly these effects suggesting that ME toxicity involves mechanisms dependent and independent of oxidative stress. Future studies are warranted to investigate the reason for the decrease of circulating testosterone and to explore the mechanistic role of endocrine disruption in ME reproductive toxicity.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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National Science Council, ROC grants (NSC94-2314-B-002-141 and NSC95-2314-B-002-256-MY3).
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ACKNOWLEDGMENTS |
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We thank the technical assistance of Mr Ping-Kun Chan, Ms Chung-Fan Wei, and Mr Xian-Yu Shuey. Conflict of interest: none declared.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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