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From the Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore, India.
| Correspondence to: Dr Muralidhara, Department of Biochemistry and Nutrition, CFTRI, Mysore 570 020, India (e-mail: mura16{at}yahoo.com). |
| Received for publication April 30, 2004; accepted for publication July 7, 2004. |
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Abstract |
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Key words: Nickel chloride, oxidative damage, apoptosis, abnormal sperms, dominant lethal mutations
In mammalian cells, induction of DNA single-strand breaks, DNA protein cross-links, sister chromatid exchanges, and chromosomal aberrations has been demonstrated with various nickel salts (Kawanishi et al, 1989; Torreilles and Gurein, 1990; Kasprzak, 1991). Since nickel ions bind weakly to purified DNA in vitro, indirect mechanisms such as generation of ROS have been previously proposed to explain nickel-induced DNA damage (Patierno and Costa, 1985; Zhong et al, 1990; Huang et al, 1993). Further, nickel salts are shown to enhance LPO in various somatic tissues, such as blood, muscle, liver, and kidney of rats (Athar et al, 1987; Kasprzak and Hernandez, 1989; Misra et al, 1990; Stinson et al, 1992; Chen et al, 1998).
Effects of nickel compounds on reproduction in rodent models are well documented (Pandey et al, 1999; Das and Dasgupta, 2000). Although bioaccumulation of nickel in testis is well demonstrated, the exact mechanisms of nickel-induced male reproductive toxic effects are not clear (Kakela et al, 1999; Obone et al, 1999). Further, the involvement of oxidative stress (OS) mechanisms in nickel-induced testicular toxicity has not been unequivocally demonstrated. In addition, studies on the relationship between induction of LPO and DNA damage in testis in vivo and susceptibility of epididymal sperm (ES) to nickel-induced OS are totally lacking. More importantly, to our knowledge, the ability of nickel salts to induce apoptosis (programmed cell death) and DNA damage in testis, implications on sperm morphology and development, and their possible genotoxic effects have not been previously addressed.
Accordingly, in the present study, we investigated the propensity of nickel to induce OS in testis and ES of mice following administration of single or multiple sublethal doses. Using similar doses, induction of DNA damage in testis or ES and apoptosis (in terms of DNA fragmentation) in testis was also investigated. Furthermore, nickel-induced genotoxic effects were ascertained by examining their effects on caudal sperms (counts and head abnormalities) and their ability to induce male-mediated dominant lethal (DL) mutations. Our results suggest that OS induced by multiple low doses of nickel chloride in subcellular fractions (mitochondrial or microsomal) of testis is associated with significant DNA damage and apoptosis. Further, nickel chloride has the propensity to cause stage-specific genotoxic effects measured in terms of a higher percentage of sperm head abnormalities and DL-type mutations discernible only during specific posttreatment weeks.
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Materials and Methods |
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Animals and Care
Adult mice (10 weeks old) of CFT-Swiss strain of both sexes were randomly
drawn from the stock colony of our animal house facility and were held in
groups of 5 in polypropylene cages under standard conditions. During a week of
acclimatization and throughout the studies, they were maintained on commercial
mouse pellets (Gold Mohur, supplied by Lipton India Pvt Ltd, India) ad libitum
and had free access to water. The cages were kept on racks built of slotted
angles and were housed in a controlled atmosphere with a temperature of
25°C ± 5°C and a mean relative humidity of 50% ± 5%.
Animal Treatment
To assess the induction of OS in testis, groups of male mice were initially
administered (intraperitoneally) nickel chloride (dissolved in distilled
water) at single dosages (highest dose, 5 µmol/100 g of body weight). For
all other investigations, a multiple exposure regimen (0.625, 1.25, 2.5, and
5.0 µmol/100 g of body weight for either 3 or 5 consecutive days) was used.
These dosages were based on our earlier study in which the toxicity profile of
nickel chloride was computed (Doreswamy et
al, 2001). Mice were killed 24 hours after the last dose
(biochemical studies, DNA damage, and fragmentation experiments) and at 1, 2,
3, and 5 weeks following the beginning of the treatment (sperm head
abnormality study). Body weights were recorded on the days of treatment and
autopsy. Immediately after euthanatizing, testes and epididymis were excised
and weights were recorded. One testis and its correspondent epididymis were
fixed in Bouin fixative and processed for histopathological examination.
Induction of LPO in Testis and ES
Induction of OS was ascertained by measuring the extent of LPO in
testicular homogenates, microsomes, mitochondrial fractions, and ES of adult
mice at 24 hours after the last dose in both single- and multiple-exposure
regimens. For homogenate preparation, testes were decapsulated, weighed, and
homogenized with a glass Teflon grinder at 4°C in phosphate buffer, pH
7.4. Microsomes were prepared following the method of Shimoji and Aniya
(1994) with minor
modifications, and mitochondrial fractions were prepared as per the method
described by Trounce et al
(1996). ES suspensions were
prepared as per the procedure described earlier
(Muralidhara and Narasimhamurthy,
1991). LPO was quantified by measuring the formation of
thiobarbituric acid reactive substances (TBARS) by following the method of
Ohkawa et al (1979) using
1,1,3,3-tetramethoxy propane as the standard.
Measurement of Antioxidant Enzyme Activity
Testis samples were homogenized in 50 mM phosphate buffer (pH 7.4) and
sonicated over ice. The activities of enzymes (catalase, glutathione
peroxidase, and glutathione S-transferase) were determined in 1000 x
g supernatants of testis homogenates. Catalase (EC 1.11.1.6) activity
was assayed by the method of Aebi
(1984), and the activity was
expressed as micromoles of H2O2 consumed per minute per
milligram of protein. The activity of glutathione peroxidase (EC 1.11.1.9) was
determined using the tbHP as the substrate according to the method described
by Flohe and Gunzler (1984),
and the activity was expressed as nanomoles of NADPH oxidized per minute per
milligram of protein (e340 = 6.22 mM/cm). Glutathione S-transferase (EC
2.5.1.18) was assayed at 340 nm by measuring the rate of enzyme catalyzed
conjugation of reduced glutathione with 1-chloro-2-4-dinitro benzene according
to the method of Guthenberg et al
(1985). Enzyme activity was
expressed as nanomoles of s-2,4-dinitrophenyl glutathione formed per minute
per milligram of protein. Protein level was determined using bovine serum
albumin as the standard as per the method of Lowry et al
(1951).
Quantification of DNA Damage
The fluorimetric analysis of DNA unwinding (FADU) assay was performed
according to the procedure described by Birnboim
(1990). Testicular cell
suspensions were prepared as described previously from our laboratory
(Rajeshkumar et al, 2002).
Final cell pellet was suspended in a known volume of KRBS. An aliquot of
testicular cell suspension (approximately 1 x 106 cells) or
ES (approximately 2 x 106) was transferred into test tubes
and cells lysed for 10 minutes. The assay was performed in triplicate. The pH
was increased by adding, successively and carefully, the alkaline solutions to
allow DNA unwinding. Following neutralization, the percentage of
double-stranded DNA (ds-DNA) formed was detected by measuring the fluorescence
of samples after addition of ethidium bromide. Measurements were performed in
a Shimadzu F-2000 fluorescence spectrophotometer with 520 and 575 as
excitation and emission wave-lengths, respectively. The percentage of ds-DNA
that remained after the unwinding process was calculated by determining the
following ratio: (unwound DNA fluorescence - denatured DNA
fluorescence)/(native DNA fluorescence - denatured DNA fluorescence).
Agarose Gel Electrophoresis of Testicular DNA
DNA from testis of both control and nickel-treated mice was isolated
following the method of Hughes and Gorospe
(1991). Briefly, testes were
decapsulated and teased in lysis buffer, and following the addition of diethyl
pyrocarbonate, the samples were digested with proteinase K. After repeated
extraction with phenol-chloroform, the DNA in the supernatant was precipitated
by adding 3 M sodium acetate and an equal volume of ice cold absolute alcohol.
The samples were kept at -20°C for DNA precipitation. The DNA was pelleted
and washed with 70% alcohol. The pellet was dried and dissolved in Tris EDTA
buffer (pH 8.0). The DNA was quantified spectrophotometrically, and 5 µg of
DNA was loaded onto a 1.8% agarose gel and electrophoresed at 50 V in Tris
borate EDTA buffer. The gel was stained with ethidium bromide and visualized
on a UV transilluminator.
ES Counts and Sperm Head Abnormalities
Following administration of multiple doses of nickel chloride, male mice
were killed by cervical dislocation at 1, 2, 3, and 5 weeks after treatment.
Fresh cauda epididymis held in 0.9% NaCl was processed according to the method
described previously (Muralidhara and
Narasimhamurthy, 1991) to determine the sperm counts. Aliquots of
sperm suspensions were stained in 1% eosin Y, and the smears were examined for
abnormal sperms (a minimum of 1000 sperms per animal) and expressed as the
percentage of abnormal sperms based on the original method
(Wyrobeck and Bruce,
1978).
DL Mutation Response
For this study, 10 adult males were administered (intraperitoneally) nickel
chloride (2.5 µmol/100 g of body weight) for 5 consecutive days and mated
with untreated (1:1) virgin females each week sequentially for 5 weeks.
Successful mating was ascertained by the presence of vaginal plugs, and all
the pregnant females were humanely killed 16 to 17 days after detection of
plugs and assessed for the degree of postimplantation embryolethality as
described previously (Muralidhara and
Narasimhamurthy, 1996). Males that were administered
(intraperitoneally) a single dose of cyclophosphamide (100 mg/100 g of body
weight) and mated with virgin females for 5 consecutive weeks served as the
positive control.
Statistical Analyses
Data on both LPO and DNA damage were analyzed using Student's t
test, and P < .05 was set as the minimum level of statistical
significance. However, data on ES count, sperm head abnormalities, and 3
variables of DL assay (pregnancy, total implantations, and postimplantation
deaths) were analyzed using a nonparametric test, the Mann-Whitney U
test (Snedecor and Cochran,
1980).
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Results |
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Induction of LPO in Testicular Homogenates, Microsomes, Mitochondrial Membranes, and ES
Following single doses, only a marginal increase (15%) in the endogenous
TBARS (an index of in vivo LPO) levels was evident in the testis at 24 hours
after exposure at the highest dosage of 5 µmol/100 g of body weight only
(data not shown). However, there were significant increases in the TBARS
levels in whole homogenates and mitochondrial and microsomal fractions
following multiple exposure regimens
(Figure 1) measured at 24 hours
after the last dose. Although the degree of LPO induction was marginal at the
lowest dose (1.25 µmol), a nearly 15% to 30% increase was evident at the
higher doses (2.5 and 5 µmol/100 g of body weight). However, the
mitochondrial fractions showed a dose-related increase in LPO induction, which
ranged from a 20% to 50% increase over the control levels. Further, nickel
treatment resulted in a relatively higher degree of LPO induction (25% to 60%)
in the microsomal fractions at similar doses
(Figure 1). Interestingly,
there was a 10% to 20% increase in the LPO in the ES during the first week
with nickel treatment (Figure
1).
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Antioxidant Enzymes in Testis
Alterations in the activities of antioxidant enzymes measured in testis
following nickel treatment are presented in
Figure 2. Dose-related
elevations were evident in the activities of all antioxidant enzymes. A
significantly enhanced activity of glutathione peroxidase was evident at the
highest dosage (5.0 µmol). The activities of glutathione S-transferase were
elevated significantly (15% to 26%) at all the doses. Although the increase
was marginal at the lowest dose, the catalase activities were enhanced
significantly (10% to 25%) at the higher doses.
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DNA Damage in Testis and ES
The effect of multiple doses of nickel chloride on DNA damage in testis and
ES quantified at 24 hours after the last dose is presented in
Table 2. The formation of
strand breaks (as measured by the FADU assay) that led to a decreased
percentage of ds-DNA was evident only at higher doses (2.5 and 5 µmol/100 g
of body weight). In the testis, the background DNA damage was 17%. However,
treatment with nickel chloride resulted in increased dose-related DNA damage.
A similar trend was evident in ES, and only a marginal increase in DNA damage
was observed at the highest dose.
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Agarose gel electrophoresis of genomic DNA extracted from both control and nickel-administered mice as visualized by ethidium bromide fluorescence is shown in Figure 3. As evident from the figure, the control samples showed only a minimal degree of DNA damage (lane A). However, nickel treatment induced significant apoptosis, since typical ladder patterns of DNA fragmentation were discernible at all doses. Although the laddering of DNA appeared to be weak at the lower doses (0.625 and 1.25 µmol/100 g of body weight; lanes B and C), it was marked at the higher doses (2.5 and 5 µmol/100 g of body weight; lanes D and E).
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ES Counts and Incidence of Sperm Head Abnormalities
The sperm caudal counts among nickel-treated mice were on par with those of
controls at all sampling periods (data not shown). Data on the frequency of
sperms with head abnormalities determined during various posttreatment weeks
are presented in Table 3. The
spontaneous incidence of the percentage of abnormal sperms among controls
ranged from 1.4 to 2.0 and was within the background incidence reported
previously for the CFT-Swiss mice from our laboratory. However, nickel
treatment induced a significant increase in the percentage of abnormal sperms
only during the first 3 weeks and the frequency normalized thereafter. Nickel
treatment induced a 3- to 4-fold increase during weeks 1, 2, and 3. The major
head abnormalities consisted of amorphous heads, balloon heads, and big heads
and hammerheads as described originally by Wyrobeck and Bruce
(1978).
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Assessment of DL Mutation Response
Administration of nickel chloride at 2.5 µmol/100 g of body weight per
day on 5 consecutive days seemed to marginally affect the mating efficiency of
males only during the first 3 weeks. The percentage of induced pregnancies
during weeks 1, 2, and 3 were 75%, 55%, and 75%, respectively. During weeks 4
and 5, it was comparable to those of negative controls (80% to 90%). However,
no significant difference was found with regard to the total number of
implantations among pregnancies that resulted from nickel-treated males during
all the 5 weeks (Table 4). The
total implantations per litter in the nickel group ranged from 10.9 to 11.4.
However, there was a marked decrease in the number of live implantations among
the nickel group during weeks 1, 2, and 3. Further, there was no decrease in
the number of live implantations in the nickel-treated group during weeks 4
and 5. The number of live implantations per litter during weeks 1, 2, and 3
was 9.0, 8.2, and 9.00, respectively. As a consequence, the mean incidence of
dead implantations per litter during these weeks increased to 1.9, 3.2, and
2.2, respectively, which was statistically significant. The dead implantations
per litter expressed as a percentage of total implantations
(Table 5) showed a significant
increase (approximately 17%, 28%, and 20%) compared with the controls
(<10%). For comparison, data on the size of increase in the incidence of
dead implantations following treatment of males with a single dose of
cyclophosphamide (a positive DL mutagen) was also obtained. The
cyclophosphamide-treated males induced nearly a seven-fold increase in the
dead implantations per litter during the first 2 weeks and a fourfold increase
during the third week.
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Discussion |
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Although much evidence indicates the involvement of OS mechanisms in nickel-mediated toxic effects in somatic organs (Sunderman et al, 1985; Athar et al, 1987; Sunderman, 1989; Misra et al, 1990; Stinson et al, 1992; Chen et al 1998), studies on the propensity of nickel to induce OS in testis are scarce. More importantly, data on the consequences of DNA damage on sperm morphology and development and their possible genotoxic implications are nonexistent. In the present study, low, single doses of nickel failed to induce any notable increase in LPO in both testicular homogenates and ES. However, significant induction was evident with multiple sublethal doses, clearly indicating the importance of repeated exposures. Although the degree of induction was relatively low in homogenates, significant increases were evident in both mitochondrial and microsomal fractions, suggesting the higher susceptibility of these membranes to nickel intoxication.
In the present study, we also observed moderate elevations in the activities of few antioxidant enzymes (glutathione peroxidase, glutathione S-transferase, and catalase) in testis, suggesting the induction of OS. Previously, we reported significant increases in protein carbonyls and iron levels in rat testis following administration of sublethal multiple doses of nickel chloride (Doreswamy et al, 2001). Further, we found no evidence of pathological lesions (necrosis) in testis due to nickel treatment (data not shown). However, nickel induced a significant degree of apoptosis in testis, which was assessed biochemically (in terms of DNA laddering). Although this is the first report, to our knowledge, on nickel-induced apoptosis in testis, other authors have documented similar cell death in testis of experimental animals following exposure to cadmium (Xu et al, 1996), cyclophosphamide (Cai et al, 1997), and various other apoptogenic agents (Blanco-Rodriguez and Martinez-Garcia, 1998). Further studies are essential to understand the mechanisms related to nickel-induced apoptotic cell death in testis and its implications.
In the present study, nickel induced significant DNA damage only at higher doses in testis. Our data are consistent with earlier reports on nickel chloride–induced DNA strand breaks in rat liver (Stinson et al, 1992) and in vitro cell models (Patierno and Costa, 1985; Kawanishi et al, 1989). Although the mechanism of nickel-induced DNA damage is not clear, it may be caused by the induction of Fenton-generated hydroxyl radicals as suggested earlier in somatic cells (Misra et al, 1990; Stinson et al, 1992). Other authors (Athar et al, 1987) have hypothesized that nickel-induced accumulation of iron in hepatic tissue may be directly responsible for the oxidative damage to macromolecules. Although speculative, similar mechanisms may also be operating in germ cells, since we also noted significant increases in iron in the rat testis following nickel intoxication (Doreswamy et al, 2001).
The susceptibility of epididymal spermatozoa to OS induced by nickel is evident, since a significant increase in LPO was observed following multiple exposures. Although sperms produced in the testis are reasonably well protected by the microenvironment of Sertoli cells, they are less protected against the oxidant environment in epididymis, owing to the slow transit time and prolonged storage (Cummins et al, 1994). In the current study, the sperm counts were in the normal range, suggesting the absence of any detrimental effect on total production of sperms. However, other authors have reported reduced sperm counts in experimental animals (Obone et al, 1999; Pandey et al, 1999) administered nickel sulfate at higher doses and for a longer time. Since higher doses of nickel tend to induce severe pathological lesions and have a direct effect on spermatogenesis, we chose to experiment with very low doses for 3 or 5 days.
Interestingly, nickel-induced OS in testis was associated with significant genotoxic effects measured in terms of production of abnormal sperms and DL mutations. The higher frequency of abnormal sperms during weeks 1, 2, and 3 clearly suggests a stage-specific effect on the development of sperms and also indicates the higher susceptibility of postmeiotic germ cells to nickel intoxication. These data are consistent with earlier observations that many germ cell mutagens induce genotoxic effect by producing a higher incidence of abnormal sperms in mice (Ehling, 1977). Furthermore, in our DL assay, administration of nickel (2.5 µmol/100 g of body weight per day for 5 days) failed to induce any detrimental effect on the incidence of implantations. However, a marked increase of dead implantations among pregnancies arising from nickel-treated males clearly suggested a specific induction of DL-type mutations. The peak DL response observed in weeks 2 and 3 can probably be interpreted as a direct effect on the spermatids that appear to be the most susceptible stage to nickel exposure.
In conclusion, multiple doses of nickel exposure produced moderate OS in testis of mice, which was apparently associated with apoptotic cell death and DNA damage in testis and ES. The genotoxic effects (ie, increased frequency of ES with abnormal heads and higher percentage of DL-type mutations) can be interpreted as a specific effect on spermatozoa and spermatids (early or late), which can play a significant role in the development of male infertility. Further, these data taken together suggest that nickel-induced testicular dysfunction at lower sublethal doses is wholly or partly mediated through oxidative damage to macromolecules, including damage to DNA.
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Footnotes |
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