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From the Departments of * Pharmacology and
Therapeutics and Obstetrics and Gynecology,
McGill University, Montréal, Québec, Canada
| Correspondence to: Dr Bernard Robaire, Department of Pharmacology and Therapeutics, 3655 Promenade Sir-William-Osler, Montréal, Québec, Canada H3G 1Y6 (e-mail: bernard.robaire{at}mcgill.ca). |
| Received for publication December 22, 2006; accepted for publication March 14, 2007. |
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Abstract |
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Key words: Anticancer drugs, spermatogenesis, male germ cell apoptosis, adverse effects, developmental toxicity
All 4 of the drugs in the CHOP cocktail have been reported to be sterilizing as single agents. Previous studies from our laboratories using a rat model have shown that chronic cyclophosphamide treatment significantly increases DNA single- and double-strand breaks and cross-links in spermatozoa and elevates preimplantation and postimplantation embryo loss and fetal growth retardation and malformations (Trasler et al, 1985, 1986, 1987; Codrington et al, 2004). A number of studies have shown that male rats exposed to chronic treatment with doxorubicin have significant decreases in the number of spermatozoa in the cauda epididymides, sperm motility, the percentage of progressive sperm, and sperm velocity (Kato et al, 2001); a decrease in the numbers of implantations and live embryos and an increase in pre-implantation loss have also been observed. Adachi et al (2000) reported a suppression of body weight gain and decreased testis weight in rats treated for 2 weeks with doxorubicin (1 mg/kg/wk); histologic examination revealed that the number of spermatogonia was dramatically decreased. Kinkead et al (1992) and Stanley and Akbarsha (1994) showed that vincristine treatment of rats resulted in Leydig cells that were necrotic and swollen, with severe damage to the steroidogenic machinery. Chronic vincristine treatment of male mice resulted in decreases in testis weight and sperm count, effects on sperm morphology, and increases in DNA damage (Dobrzynska et al, 2005). However, the impact of exposure to a drug combination may be different than that of each alone. The goal of this study was to determine the consequences of exposure to the combination of drugs used in the CHOP chemotherapeutic regimen on male reproduction, fertility, and progeny outcome using the rat model.
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Materials and Methods |
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One week after arrival males were randomly divided into 4 treatment groups, each composed of 8 rats. Rats were treated with the combination chemotherapy most often used for NHL, which involves the administration of CHOP. Cyclophosphamide (Sigma-Aldrich, Oakville, Canada), doxorubicin and vincristine (LKT Laboratories, St Paul, Minn), and prednisone (Sigma-Aldrich) were administered for 1 week out of 3, in 4 cycles. The doses of the drugs administered to rats were analogous to human doses after adjustment for the differences in surface area to weight ratio. Thus, in rats, the 1x dose regimen was 27 mg/kg cyclophosphamide, 1.8 mg/kg doxorubicin, and 0.05 mg/kg vincristine, given intraperitoneally (1 injection per cycle on the first day of the first week of each cycle), and 1.47 mg/kg prednisone by gavage (5 times per week during the first week of each cycle) (Figure 1). The rats received 1/3x, 2/3x, or 1x, with 1x representing the analogous human dose; control rats received saline. Males were weighed every second day. During the 10th week each male was mated with 2 females in pro-estrus; the females were euthanized and pregnancy outcome was assessed, as described below, 20 days later. Males were anaesthetized, and the left testes and epididymides, seminal vesicles, and ventral prostates were removed, trimmed of fat, and weighed in week 11 of the treatment protocol. The epididymides were divided into caput-corpus and cauda regions. The caput-corpus epididymides and testes were frozen in liquid nitrogen for the determination of spermatozoal head counts. Blood samples were collected in plastic syringes and transferred to plastic tubes for the blood cell counts. The right testes and epididymides were perfuse-fixed through the abdominal aortas with Bouin fluid (BDH Inc, Toronto, Canada) for 15 minutes.
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Enzyme-Linked Immunosorbent Assay Measurement of Testosterone
Serum testosterone concentrations were evaluated with a testosterone
enzyme-linked immunosorbent assay (ELISA) kit (IBL Immuno-Biological
Laboratories, Hamburg, Germany) following the manufacturer's
recommendations.
Spermatozoal Head Counts
The frozen testis and caput-corpus epididymidis from each of the 8 rats per
group were homogenized (Polytron PTA7, setting 5; Brinkman Instruments,
Westbury, NY), for two 15-second periods separated by a 30-second interval in
5 mL of 0.9% NaCl, 0.1% thimerosal, and 0.5% Triton X-100. To assess tissue
content of spermatozoa, the heads of spermatozoa were counted
hemocytometrically (Robb et al,
1978).
Testicular Histology
The perfuse-fixed testes were cut in 2 parts, placed in Bouin fluid
overnight, and processed for routine paraffin embedding. The testes were cut
into 5-µm sections. Three serial sections per testis were mounted on
slides, deparaffinized, rehydrated, and stained with periodic acid Schiff
(Sigma-Aldrich) following the manufacturer's recommendations. Sections of the
seminiferous tubules at all stages were examined by light microscopy; tubule
diameters were assessed in each testis (
200 random determinations per
testis) using a previously calibrated micrometer eyepiece.
In Situ Detection of Apoptosis
Sperm DNA fragmentation was evaluated with the terminal deoxynucleotidyl
transferase (TdT)–mediated dUTP-biotin nick end labeling (TUNEL) assay
(Gavrieli et al, 1992), which
detects apoptotic cells (ApopTag In Situ Apoptosis Detection Kit;
CHEMICON International Inc, Temecula, Calif). Testes embedded in paraffin were
cut into 5-µm sections. After deparaffinization and rehydration, tissue
sections were incubated with proteinase K (20 µg/mL) for 15 minutes at room
temperature, washed in distilled water, treated with 3.0% hydrogen peroxide in
phosphate-buffered saline (PBS) for 5 minutes at room temperature to quench
endogenous peroxidase activity, and washed twice with water for 5 minutes
each. Sections were treated with equilibration buffer and then incubated with
working-strength TdT enzyme in a humidified chamber for 1 hour at 37°C.
Following this incubation, slides were immersed for 10 minutes at room
temperature in working-strength stop/wash buffer and washed in 3 changes of
PBS for 1 minute each. Antidigoxigenin conjugate was applied to the slides for
30 minutes at room temperature. After incubation, the slides were washed in 4
changes of PBS for 2 minutes per wash at room temperature. To determine the
optimal staining time, color development was monitored by looking at the
slides under the microscope. Specimens were counterstained, rehydrated, and
mounted following the recommendations of the manufacturer. At least 200
tubules per slide were randomly examined with the light microscope.
Acridine Orange Assay for Sperm DNA Denaturation
To measure the susceptibility of sperm nuclear DNA to denaturation in situ,
the acridine orange sperm chromatin structure assay (AO, SCSA®) was
completed as described (Stanley and
Akbarsha, 1994; Evenson et al,
2002). Sperm samples were thawed and sonicated on ice to remove
the tails. A 200-µL aliquot of spermatozoa in PBS was mixed with 400 µL
of denaturation buffer containing 0.08 N HCl, 0.15 M NaCl, and 0.1% Triton
X-100 (pH 1.4) and incubated for 30 seconds at room temperature to denature
uncondensed sperm DNA. After 30 seconds spermatozoa were stained with 1.2 mL
of AO staining solution (0.2 M N2HPO4, 0.1 M citric acid
buffer, 1 mM EDTA, 0.15 M NaCl [pH 6.0], and 5–10 µg of AO).
Stained spermatozoa were analyzed using a FACSCAN flow cytometer (BD Biosciences, San Jose, Calif) fitted with an argon ion laser (488-nm line excitation). Fluorescence of individual cells was measured at wavelengths of green (515- to 530-nm band pass filter) and red (630- to 650-nm long pass filter) in the peak mode. A total of 10 000 sperm were analyzed for each sample. The data were processed using WinList Software (Verity Software House; Verity Software, Topsham, Me). The AO parameters obtained from this analysis included DNA fragmentation index (DFI), the standard deviation of DFI, and mean DFI ± SD.
Analysis of Pregnancy Outcome
The vaginas of all mated females were flushed with saline the morning after
mating to assess the presence of spermatozoa. On day 20 of gestation (day 0 =
morning of the day when sperm were found in the vagina), females were
euthanized with CO2 asphyxiation, decapitated, and sectioned by
caesarean incision. The ovaries were removed and the numbers of corpora lutea
counted. The uteri were removed and opened, and the numbers of implantations,
resorption moles, and live fetuses were recorded. Preimplantation loss per
litter was determined as the number of corpora lutea minus the number of
implantation sites divided by the number of corpora lutea for each female.
Postimplantation loss was calculated as the number of implantation sites minus
the number of live fetuses divided by the total number of implantations. Each
fetus was examined for any external malformations, weighed, sexed, killed by
hypothermia, and fixed in ethanol. Fetuses weighing less than 75% of the mean
weight for each group were considered as growth-retarded fetuses
(Kirk and Lyon, 1984).
Statistical Evaluation
Data were analyzed by 1-way analysis of variance with Dunnet's multiple
range test. Data are presented as means ± SEM. The level of
significance was P < .05.
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Results |
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The weights of sex accessory tissues and concentrations of serum testosterone are presented in Figure 3. There was a significant decrease in ventral prostate weights in the 1x CHOP treatment group (Figure 3A). In contrast, CHOP treatment had no effect on seminal vesicle weights (Figure 3B) or on serum testosterone concentrations (Figure 3C). 1x CHOP treatment resulted in significant decreases in the weights of the testes and epididymides (Figure 4A and B), which were accompanied by decreases in testicular and epididymal spermatozoal head counts (Figure 4C and D). Interestingly, the total numbers of spermatozoa per testis were also decreased in the rats exposed to lower doses of CHOP (1/3x and 2/3x).
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Effects of CHOP Treatment on Testicular Histology
CHOP treatment–induced disturbances in the histology of the
seminiferous tubules varied from small vacuoles in the epithelium to total
loss of germ cells (Figure 5).
Staging could not be evaluated due to the dramatic damage to tubules. Among
the 5 animals analyzed for each treatment group, the greatest damage occurred
in the 1x CHOP–treated group. Although the extent of damage was
variable, a mean of 10% of the seminiferous tubules were heavily damaged; the
range was 3% to 23%. Although various tubules contained round spermatids with
the acrosomic head caps of spermatogenesis stage VI, some did not have the
second generation of round or elongated spermatids
(Figure 5C). In some tubules,
germ cells were completely absent (Figure
5D); in others, the number of germ cells was greatly decreased
(Figure 5E). Seminiferous
tubule diameters were significantly reduced in the 1x CHOP treatment
group compared with controls (Figure
5F); this effect was more pronounced in the periphery of the
testis compared with central tubules (data not shown).
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AO SCSA for Sperm DNA Denaturation
Results from the AO SCSA clearly showed a significant increase in the
percentage of cells with abnormal DNA denaturation in the rats treated with
1x CHOP (Figure 7). The
mean DFI, representing the mean fluorescence observed in the population, was
higher in 1x CHOP–treated rats compared with controls
(Figure 7A), as was the SD of
DFI, a reflection of the width of the sample population
(Figure 7B). The percent DFI,
an assessment of the percent of cells outside the main population, was
elevated more than fivefold in the rats exposed to the highest dose of CHOP
relative to controls (Figure
7C).
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Discussion |
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We found that the CHOP drug regimen produced oral mucositis, also called stomatitis, in most of the rats; this is the most debilitating side effect of this cancer therapy seen in patients. Stomatitis is a common dose-limiting toxicity of cancer therapy (Sonis and Clark, 1991; Peterson, 1999) and occurs in approximately 40% of patients treated with chemotherapy (Sonis and Clark, 1991). Jones et al (2006) reported that approximately 15% of patients treated with CHOP chemotherapy develop severe oral or gastrointestinal complications during the treatment.
Alterations in the light microscopic appearance of the seminiferous tubules were observed mostly at the periphery of the testis sections. The vasculature in the rat testis has been described to be denser in the periphery (Chubb and Desjardins, 1982), suggesting that the drugs may be more concentrated and have a greater effect in this region of the testis. The general decrease in seminiferous tubule diameters observed after 1x CHOP treatment supports the observation that the number of germ cells per tubule is reduced. Previous studies showed that antitumor drugs such as cisplatin, etoposide, and cyclophosphamide induce apoptosis in male germ calls (Huddart et al, 1995; Chresta et al, 1996; Cai et al, 1997). High-dose CHOP treatment did induce a significant increase in apoptosis, which would contribute to the decrease in sperm count.
The AO SCSA was used to investigate if CHOP treatment alters DNA chromatin structure in spermatozoa. AO fluoresces green when bound to condensed or double-stranded DNA and red when intercalated into denatured DNA. Only sperm derived from the cauda epididymides from the 1x CHOP–treated males had higher mean DFI, SD of DFI, and percent DFI values than control rats, indicating an effect on DNA integrity. Previous studies have shown that there is a significant inverse correlation between SCSA parameters and fertility (Evenson et al, 1980; Ballachey et al, 1987; Huddart et al, 1995); the SD of DFI was more strongly correlated with infertility than percent DFI (Evenson and Wixon, 2005).
Our data suggested that AO parameters correlate with the number of DNA strand breaks as measured by the TUNEL assay (Evenson et al, 2002; Delbes et al, 2007).
The effects of CHOP treatment on both sperm count and sperm chromatin quality/DNA susceptibility to low pH denaturation suggest that fertility may be affected. Indeed, the fertility of 2/3x and 1x CHOP–treated male rats was decreased significantly. In addition, we observed dramatic increases in both pre-implantation and postimplantation loss among the progeny sired by 1x CHOP–treated rats. Interestingly, postimplantation loss was also higher among the progeny sired by males exposed to 2/3x CHOP; in the absence of effects on AO assay parameters of sperm quality, the only other parameter affected in this treatment group was the spermatozoal count per testis. These data suggest that early embryo failure is a sensitive measure of male genome integrity.
In men, the spontaneous abortion rate is twofold higher if >30% of the sperm have fragmented DNA, as measured by SCSA (Evenson and Wixon, 2005). Previous animal studies have shown that sperm with fragmented DNA are capable of fertilization (Marchetti et al, 2004); the chromosomal aberrations in the zygotes that were produced were highly predictive of subsequent abnormal embryonic development. Ahmadi and Ng (1999) noted that epididymal mouse sperm exposed to different doses of radiation fertilized mouse eggs with equal efficiency; however, the incidences of pre-implantation and postimplantation loss increased dramatically in a dose-response fashion. Kinkead et al (1992) reported a significant increase in pre-implantation loss among the progeny of male rats exposed to cis-platinum. In previous studies from our laboratories, we demonstrated that male germ cells exposed to chronic low doses of cyclophosphamide throughout spermatogenesis had more DNA damage, as assessed by the comet assay, and transmitted this damage to the conceptus (Harrouk et al, 2000; Codrington et al, 2004). DNA repair does not occur during the later stages of spermiogenesis (Codrington et al, 2004; Olsen et al, 2005); higher pre-implantation and early postimplantation losses most likely represent a failure on the part of the conceptus to respond to the genotoxic stress introduced by a damaged male genome.
In conclusion, CHOP treatment affected male reproductive tissue weights, testicular histology, and sperm counts. The impact of CHOP exposure on male germ cells was translated into increases in pre-implantation and postimplantation loss with a resulting decrease in the number of live fetuses per litter. While early embryo loss clearly represents a sensitive measure of the adverse effects of chemotherapeutics on spermatozoal quality, new end points are needed to elucidate how drug exposures impact male germ cell genomic structure and function to further our understanding of the mechanisms underlying male-mediated developmental toxicity.
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Acknowledgments |
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Footnotes |
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