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Keywords:

  • benzo(a)pyrene;
  • DNA strand breaks;
  • DNA adducts;
  • TUNEL assay

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

The objective of this study was to assess whether subchronic exposure to benzo(a)pyrene (BaP) via oral ingestion alter endpoints of the reproductive system of mice. Hsd: ICR (CD1) 10-week-old males (n = 8) were randomly assigned to the exposure group and control group. Mice were administered BaP for 30 and 60 days by daily gavage at doses of 1, 10, 50, and 100 mg/kg body weight per day. At the end of the experiments, mice were anesthetized and reproductive organs, including testes, seminal vesicles, prostate, and cauda epididymis, were removed and examined. Spermatozoa quality and DNA strand breaks were assessed—1 and 10 mg/kg/day of BaP for 30 and 60 days did not significantly induce altered morphology or weights of testes, prostate, seminal vesicle, and epididymis, and spermatozoa quality of mice; 100 mg/kg/day of BaP for 60 days decreased weights of testes, seminal vesicle, and cauda epididymis. BaP exposure also significantly decreased motility, normal head morphology, vitality, and concentration of mature spermatozoa. In addition, BaP exposure induced a significant increase in DNA strand breaks. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 1–8, 2015.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Benzo(a)pyrene (BaP) is a semivolatile, lipophilic compound that belongs to the polycyclic aromatic hydrocarbon family. This compound can be formed as a result of incomplete combustion from industrial processes, smoking tobacco, charring of grilled and broiled foods, and exhaust from diesel and gasoline engines. Inhalation and oral ingestion are two major routes of BaP exposure. After exposure, BaP can be metabolized by the cytochrome P-450 family to form reactive epoxides, that is, 7,8-epoxide, BaP-7,8-dihydrodiol 9,10-epoxide, BaP-9, and quinones (Godschalk et al., 2003). In addition, the active intermediates can undergo redox cycling and generated excessive reactive oxygen species (ROS). ROS are known to alter transcription and cell signaling and damage cellular membranes resulting in apoptosis. In the last stage of BaP biotransformation, BaP epoxides can then be conjugated with glutathione and yield a product that is easily excreted into urine (Penning, 2004).

Because of the blood–testis barrier of testes, spermatozoa are more protected from chemical insults than other cell types. However, animal studies have demonstrated that BaP, and/or its metabolites, via oral, inhalation, and intravenous exposure can cross the blood and testis barrier and could reach testicular tissues of mice (Ramesh et al., 2004a). BaP and some of its metabolites were substantially present in the male gonad after 8 h (oral) or 4 h (inhalation) postexposure (Ramesh et al., 2001). BaP exposure can induce apoptosis in germ cells, interfere with spermatogenesis cycles, and lead to decreased spermatozoa quality (Ramesh et al., 2004b, 2008; Mohamed et al., 2010).

The objective of this study was to assess whether subchronic exposure to BaP via oral administration alters endpoints of the reproductive system by examining spermatozoa quality, including concentration, motility, vitality, morphology, and DNA strand breaks. Histological assessment was conducted to assess the effects of BaP on testicular tissues. The exposure periods (30 and 60 days) encompassed two sperm cycles in the mouse species used in this study.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Animal Experimental Design and Exposure Protocol

Hsd: ICR (CD1) 10-week-old males, weighing 30–40 g, were used (Harlan Laboratories). Mice were caged individually with a 12-h light/dark cycle. Rodent chow and water were supplied ad libitum. Each mouse was weighed every other day and was examined daily for behavioral and clinical symptoms. Upon arrival, mice were acclimated for 2 days before BaP exposure began. Mice (eight per group) were randomly assigned to the exposure group and control group. Mice were administered BaP for 30 and 60 days by daily gavage at doses of 1, 10, 50, and 100 mg/kg of body weight (BW) per day. The inclusion of BaP concentrations were chosen based on other studies (Revel et al., 2001; Mohamed el et al., 2010). Control animals (n = 8) received the same weight-based volume of vehicle solution (coil oil). On day 30 and day 60, mice were anesthetized using intraperitoneal injection of sodium pentobarbital (50 mg/mL) to the surgical plane, confirmed by the toe-pinch method. Reproductive tissues were removed with testes, seminal vesicles, prostate, and cauda epididymis placed on a petridish and weighed. Testes were placed in 10% formalin for histological preparation, or placed in freezing media-RPMI 1640 (Cellgro), 1× Pen/Strep (Cellgro), 5% glycerol (Sigma) on ice until snap frozen at −80°C. All procedures were performed in accordance with protocols approved by the Old Dominion University's Institutional Animal Care and Use Committee.

Spermatozoa Collection and Analysis

A consistent length of each vas deferens, 2 cm, was flushed with 80 μL of 0.5% bovine serum albumin (fraction V, MP Biomedical) in RPMI+ 1× Pen/St using a dulled 25-gauge needle to recover spermatozoa. Spermatozoa quality, including concentration, vitality, motility, and morphology, was assessed. Fifty microliters of spermatozoa suspension was applied to a Makler chamber to determine concentration and motility according to manufacturer's instructions. Spermatozoa concentrations were expressed as the sum of 10 squares on the Makler ×106/mL, and the percentage of motility was determined by counting both motile and immotile spermatozoa area. For vitality analysis, at least 100 spermatozoa per sample were assessed from an eosin stained preparation. For morphology assessment, two slide smears were prepared for each sample. At least 100 spermatozoa were classified as either normal, abnormal head, or curly tail. Each analysis was conducted in duplicate.

Histological Evaluation of Testes

The testes were fixed in 10% formalin in a 1% phosphate buffer at room temperature for 24–48 h. Testes were then dehydrated using ethanol and xylene. After dehydration, the testes were embedded in paraffin wax. Then, serial sections (8 μm) were cut from the middle of each testis using a microtome. These sections were placed in a warm DNA water bath at 47°C and transferred to charged microscope slides. Sections were dried overnight onto glass slides at 35°C and deposited in a storage box at room temperature until processed for histology. For staining and morphometric evaluation, sections were deparaffinized and rehydrated using graded ethanol (2× 100%, 2× 95%, and 1× 70%). Slides were stained with hematoxylin and eosin. Images were acquired using an Olympus CX41 microscope with a DP72 camera and CellSens Standard software.

Detection of DNA Strand Breaks

Terminal deoxy-nucleotidyl transferase-mediated digoxigenin-deoxyuridine triphosphate nick end labeling (TUNEL) assay was performed to detect DNA strand break in spermatozoa and testicular tissues according to manufacturer specifications (Roche, In Situ Cell Death Detection, Fluorescein). DNA fragmentation was identified by labeling the free 3′-OH terminal with fluorescein-labeled nucleotides using terminal deoxynucleotidyle transferase (TdT). For spermatozoa, 200 μL of spermatozoa of each mouse collected from the previous step was washed twice with 1× phosphate-buffered saline (PBS)/1% human serum albumin (HSA) by centrifugation at 300 RCF for 10 min. The final concentration was adjusted to 30 × 106/mL. Spermatozoa were fixed to the slides, permeabilized, and 10K units/mL of DNase I (Sigma) was added to positive control samples. Samples and positive controls were incubated with label and enzyme solutions, whereas negative controls were incubated with label solution only. Slides were washed as before, and 3 μL of VectaShield H-100 was added to the slides followed by a coverslip. A minimum of 100 spermatozoa were counted as either positive (green) or negative (absence), at 1000× under oil immersion, using a Nikon Eclipse 80i fluorescent/brightfield microscope with the X-Cite series 120 and FITC filter. Spermatozoa samples from all of the studied mice were analyzed using the same setting at the same time. DAN fragmentation percentages of each B[a]-treated mice group were averaged.

For testicular tissues, testes were fixed in formalin for 24–48 h and then embedded in paraffin. Sections were sliced in 6 μm ribbons on a Leica microtome. Individual sections were mounted on heavy teflon coating (HTC)-coated slides. The tissue sections were washed with 1% HSA (Irvine Scientific) in 1× PBS (10×, Fisher) and permeabilized using 0.1% (v/v) Triton X-100 (Fisher), 0.1% (w/v) sodium citrate in 1× PBS, and incubated at room temperature (20–25°C) for 10 min. Appropriate volumes of the In Situ Cell Death Detection Kit vials 1 & 2 (Roche) were mixed and applied to tissues. Slides were incubated for 60 min at 32.5°C, in the dark, with humidity, and were monitored for wetness by adding 1% HSA in 1× PBS as needed. In a dimly lit or dark room, wells were washed twice with 1% HSA in 1× PBS, and an antiquench/mounting solution (VectaShield H-1000) was applied. Then, slides were analyzed immediately using a Fluorescent Microscope (Nikon Eclipse 80i) with a Cool Snap EZ camera and NIS Elements BR3.2 software. A minimum of 20 fields of view at 1000× magnification were randomly selected for analysis of cells that contain DNA strand breaks for percent with breaks and the intensity of fluorescing cells.

Statistical Analysis

All data were tested for normality and variance before statistical analysis took place. Data are presented as mean ± standard deviation (SD). Multivariate Analysis of Variances and LSD post hoc tests were used to assess statistically significant differences in spermatozoa quality, reproductive organ weight, and DNA integrity among the exposed groups and the control group.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Table 1 summarizes the weights of the total body and reproductive organs of mice at the end of the experimental periods. Total BW remained stable for each exposure group. Testes, seminal vesicles, and cauda epididymis weights decreased as exposure increased, whereas the prostate weight remained stable. Exposure to 100 mg/kg/day BaP for 60 days resulted in a significant decrease in testes weights (P = 0.02), seminal vesicle (P = 0.03), and cauda epididymis (P = 0.03).

Table 1. Body weight and reproductive organs of mice exposed to BaP for 30 and 60 days versus the control
 100 mg/kg/day50 mg/kg/day10 mg/kg/day1 mg/kg/dayControl
 30 days60 days30 days60 days30 days60 days30 days60 days30 days60 days
  1. Value for each measurement expressed as mean (SD) (n = 8).

  2. a

    Significantly different than control: P < 0.05.

Body weight (kg)0.035 (0.002)0.034 (0.002)0.034 (0.001)0.038 (0.002)0.037 (0.002)0.036 (0.002)0.037 (0.002)0.036 (0.002)0.036 (0.002)0.036 (0.002)
Testes (g)0.251 (0.056)0.190 (0.049)a0.284 (0.039)0.225 (0.058)0.261 (0.033)0.248 (0.052)0.269 (0.045)0.302 (0.015)0.280 (0.071)0.292 (0.050)
Prostate (g)0.083 (0.009)0.087 (0.021)0.084 (0.021)0.106 (0.011)0.093 (0.011)0.098 (0.021)0.080 (0.017)0.109 (0.025)0.074 (0.025)0.101 (0.016)
Seminal vesicle (g)0.203 (0.050)0.153 (0.087)a0.213 (0.065)0.223 (0.049)0.227 (0.037)0.299 (0.047)0.276 (0.078)0.316 (0.078)0.254 (0.055)0.265 (0.040)
Cauda epididymis (g)0.041 (0.005)0.035 (0.007)a0.038 (0.009)0.040 (0.007)0.038 (0.005)0.042 (0.003)0.043 (0.004)0.042 (0.005)0.051 (0.010)0.048 (0.005)

Table 2 tabulates spermatozoa quality of mice exposed to BaP for 30 and 60 days. For the 30-day exposure, mice exposed to 1, 10, 50, and 100 mg/kg/day of BaP generally had decreased motility and vitality compared with the control; but there was no significant difference among the groups. For the 60-day exposure, mice exposed to 10, 50, and 100 mg/kg/day of BaP induced decreased spermatozoa concentrations, which were significantly lower than the control group's measurements. In addition, 100 mg/kg/day of BaP induced a significant decrease in motility and vitality compared with the control (P = 0.033 and 0.003, respectively). Furthermore, 50 and 100 mg/kg/day of BaP significantly altered spermatozoa morphology. In the control group, 65.7% spermatozoa had normal head morphology, whereas mice exposed to 50 and 100 mg/kg/day had 56.3 and 51.5% spermatozoa with normal head morphology (P = 0.03 and 0.02), respectively.

Table 2. Assessment of spermatozoa collected from the vas deferens after exposure to BaP for 30 days and 60 days versus the control
 100 mg/kg/day50 mg/kg/day10 mg/kg/day1 mg/kg/dayControl
 30 days60 days30 days60 days30 days60 days30 days60 days30 days60 days
  1. All values are mean (SD).

  2. a

    Significantly different than control: P < 0.05.

Concentration (×106/mL)3.9 (2.5)3.1 (2.7)3.5 (2.3)3.0 (2.4)3.3 (3.6)5.7 (4.0)3.2 (2.2)4.33 (3.0)4.75 (2.9)5.35 (2.3)
Motility (%)56.5 (15.6)49.8 (12.8)a40.8 (12.6)60.7 (18.5)56.3 (19.1)55.6 (12.5)67.5 (20.4)50.9 (3.9)67.0 (28.0)68.7 (8.7)
Vitality (%)65.9 (14.7)91.6 (5.5)a49.5 (12.1)93.8 (3.9)62.9 (18.2)95.9 (1.6)80.1 (18.6)94.5 (0.8)73.6 (28.0)97.7 (2.0)
Morphology (%)          
Normal head67.0 (9.9)51.1 (11.5)a67.7 (7.3)56.3 (8.3)a72.4 (4.6)61.4 (6.8)76.5 (5.6)58.8 (7.1)69.5 (6.8)71.1 (7.9)
Abnormal head12.0 (7.7)34.3 (10.7)a14.2 (6.5)29.4 (5.8)a12.2 (6.0)22.7 (5.5)11.0 (5.3)27.2 (5.5)9.5 (4.1)16.6 (8.2)
Curly tail21.0 (6.4)14.3 (5.5)18.2 (5.9)16.5 (6.2)15.4 (9.4)15.9 (5.8)12.6 (12.5)16.1 (5.6)21.0 (5.2)12.3 (4.5)

Figure 1(A–C) demonstrates abnormal head and curly tails of mature spermatozoa observed in this study. Morphology slides [Fig. 1(A–C)] illustrate the appearance of normal spermatozoa, with a hooked head and straight tail, spermatozoa with an abnormal head, and spermatozoa with a curly tail.

image

Figure 1. Morphology of spermatozoa from (A) the control group with normal morphology; (B) Spermatozoa from mice exposed to 50 mg/kg BaP for 30 days [arrow indicates an abnormal spermatozoa head]; (C) spermatozoa from mice exposed to 50 mg/kg BaP for 30 days [arrow indicates an abnormal spermatozoa curled tail]. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Figure 2 shows the histological preparations from the testes of control and BaP-exposed mice. The testes of control mice were found to have normal morphology. Mice espoused to at or less than 10 mg/kg/day showed no tangible difference from the control. In the BaP-treated mice exposed to 50 and 100 mg/kg/day, however, testicular morphology, including seminiferous tubule and seminiferous epithelium, lost increasing degrees of integrity as exposure increased and in many areas some basement of the cellular membrane did not remain intact.

image

Figure 2. Histological evaluation on seminiferous epithelium of mice exposed to BaP from 10 mg/kg/day to 100 mg/kg/day for 30 days, and the control. (A) Control, 100× showing seminiferous tubule integrity, organized seminiferous epithelium, normal luminal space, and numbers of maturing spermatozoa; (B) 10 mg/kg/day, 100× showing seminiferous tubule integrity, tubule integrity, organized seminiferous epithelium, normal luminal space, and numbers of maturing spermatozoa; (C) 50 mg/kg/day, 100×, seminiferous tubule integrity beginning to decline, seminiferous epithelium although still highly organized, showing a loss of fidelity among the Sertoli cells; (D) 100 mg/kg/day, 400× with noticeable variation in the diameter of the seminiferous tubules and reduced integrity, decrease of the seminiferous epithelium, and a decrease in the luminal volume of maturing spermatozoa, increasing variance in the seminiferous tubule size, and organization with a pronounced and uneven Sertoli cell maintenance of the seminiferous epithelium. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

The study demonstrated that BaP doses less than 10 mg/kg/day for 30 days did slightly alter spermatozoa quality, whereas BaP doses greater than 50 mg/kg/day for 30 and 60 days induced a detrimental impact on spermatozoa morphology by significantly increasing the percent of abnormal heads shape. Only BaP at 100 mg/kg/day for 60 days significantly decreased motility and vitality of spermatozoa. Our results were in agreement with those from Ramesh et al., reporting that BaP reduced motility and mean density of spermatozoa in rats exposed to 75 μg/m3 for 60 days via inhalation (Ramesh et al., 2008).

Unmetabolized BaP was observed to enter reproductive organs that lead to the presence of reactive metabolites in reproductive tissues (Ramesh et al., 2001). That outcome indicates that BaP could have an effect on gamete production and maturation of spermatozoa via the entire cycle of spermatogenesis. Using the In Situ Death Detection Kit, spermatogenic cells, that is, spermatocytes, spermatids, in seminiferous tubules of testes were in a state of ongoing apoptosis (Fig. 3). The findings suggested that decreased spermatozoa quality could initiate before spermatozoa have become mature. In addition, another possibility was that BaP and its metabolites could directly attack mature spermatozoa when they progress to, and are stored in the epididymis until ejaculation.

image

Figure 3. TUNEL assay used to evaluate DNA strand breaks in testicular tissues, including spermtogenic cells, early and mature spermatozoa; (A) control, 400×, no fluorescence indicates no DNA strand breaks in the cells in the testicular tissue; (B) 100 mg/kg/day for for 60 days, 400×, detected fluorescence indicates DNA strand breaks in spermtogenic cells and spermtozoa. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Unpaired, accumulated DNA strand breaks in mature spermatozoa were detected in mice exposed to BaP (Fig. 4). Percent of DNA strand breaks positively correlated with BaP exposure doses. During the 30-day exposure cycle, mice exposed to 50 mg/kg/day started experiencing a statistically significant increase in DNA strand breaks. During the 60-day exposure cycle, mice had a significantly higher percent of DNA strand breaks than the control at 10 mg/kg and >10 mg/kg. Such DNA lesions may arise at varying stages during spermatogenesis and spermiogenesis. The susceptibility at the various stages to BaP depends on the ability of the spermatogenic cells to remove the damage by DNA repair. At 1 mg/kg/day for 30 days, spermatogenic cells were seemingly capable of repairing the lesions, because mean percent of DNA stand break was at the baseline percent occurring in the control mice. However, as the exposure duration increased to 60 days, spermatogenic cells, that is, primary and secondary spermatocytes, and spermatids, failed to repair enough of the lesions and led to accumulated DNA lesions in mature spermatozoa, or the mature sperm may have been damaged in situ at a late stage of maturation, that is, in the epididymis. Spermatozoa DNA lesions may occur in mature spermatozoa or before spermatozoa reach the mature stage, such as spermatids, where the activity of nucleotide excision repair (NER) and the base excision repair (BER) mechanisms were limited or none existed at all (Ozturk and Demir, 2011). Our study showed that protective mechanisms did not seem to sufficiently protect the spermatogonial cell DNA upon exposure to BaP. In postmeiotic stages, because spermatids lack DNA repair, induced DNA damage may not be removed until after fertilization. The persistence of DNA damage could increase the vulnerability of germ cells to mutation (Adler, 1996).

image

Figure 4. DNA strand breaks of mature spermatozoa exposed to BaP with doses from 1 mg/kg/day to 100 mg/kg/day for 30 and 60 days. DNA strand breaks expressed as % positive detected using the TUNEL assay (mean ± SD and n = 8). *P < 0.05 versus the control.

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BaP at 100 mg/kg/day for 60 days caused a decrease in testis weight. The result was in agreement with Ramesh et al. who observed that exposure to inhaled 75 mg BaP/m3 for 60 days induced a decrease in weights of testis and testicular volume. Both the oral ingestion and inhalation exposure routes had cause similar effects on testis of mice and rats. Spermatogenic compartments of the testis were founded to significantly decrease as compared with the controls. There was a 23% reduction in total weight of interstitium per paired testis versus a 35% reduction in tubular weight of mice exposed to 100 mg/kg/day for 60 days. The luminal space became less regular and the number of maturing spermatozoa was less abundant. Seminiferous tubules of BaP-treated mice were not uniform, irregular most notably at the basement membrane, and appear to lose the apical connections with the neighboring cells (Fig. 2). There was evidence of a reduction in spermatogenetic support and testicular mass. Similar harmful effects in the reproductive organs in mice and rats have been reported in other studies (Blazak et al., 1985; Ramesh et al., 2008). Our results support the observation that BaP exposure targeted the reproductive organs, e.g. testes, cauda epididymis, and seminal vesicle, and altering their microenvironment that supports the integrity of the cycle of spermatogenesis.

The observations of the testicular morphometric data was in agreement with the direct measurement of spermatozoa quality, showing a reduction of spermatozoa concentration, because the number of spermatozoa transported into the epididymides post spermiation decreased. Regression in the epididymal epithelium, especially the caput epididymal region, may decrease spermatozoa maturation and survival. Weights of cauda epididymis and seminal vesicle from mice exposed to BaP had a 27% reduction as compared to the controls. It is likely that less degenerated spermatozoa became present in the caudal epididymis and vas deferens (Jones, 2004). Beside the change in the microenvironment of reproductive organs, BaP was reported to impair testicular endocrine and exocrine function, which consequently lead to a decrease in stored spermatozoa and motility (Ramesh et al., 2008).

Histological assessment showed that seminiferous tubular cells, Sertoli cells and Leydig cells, in the seminiferous epithelium, were regressing. Using a cell death kit, Sertoli cells and Leydig cells in mice exposed to BaP were found to undergo apoptosis (Figs. 5 and 6). That suggests that BaP could possibly alter normal steoidogenic and spermatogenic function. The reduction of cellular function in Sertoli cells could decrease structural and nutritional support for the development of spermatogenic cells within the tubules through the stages of spermatogenesis. In addition, the secretory function of Sertoli cells could be adversely impacted in their ability to secrete certain substances, that is, testosterone, androgen binding protein, and estradiol to activate and stimulate spermatogenesis (Hazra et al., 2013). BaP was observed to incorporate into the Leydig lipogenic tissue (Ramesh et al., 2001). The apoptosis of Leydig cells could affect the development of spermatogenic cells by reducing steroidial hormone biosynthesis and release, of testosterone (Shima et al., 2013). In addition, it could lead to a reduction in the function of androgen receptors to stimulate spermatogenesis.

image

Figure 5. Leydig cell adjacent to the seminiferous tubules in the testicle from mice exposed to BaP for 60 days. (A) The control; (B) mice exposed to 10 mg/kg/day; (C) mice exposed to 50 mg/kg/day for 60 days; fluorescence indicated apoptosis of Leygid cells; (D) mice exposed to 100 mg/kg/day. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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image

Figure 6. Sertoli cells from mice exposed to 100 mg/kg/day of BaP for 60 days. Fluorescence indicates apoptosis of Sertoli cells detected using a Cell Death Kit. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Taken together, the findings of the study suggest that subchronic exposure of mice via oral administration to BaP could be a reproductive toxin that insults the testes, cauda epididymis, and seminal vesicle. In addition, oral subchronic exposure to BaP could compromise spermatozoa quality. Such insult of BaP could occur during spermatogenesis or to mature spermatozoa in the epididymis.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES