1 Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600113, 2 Department of Biochemistry, Central Leather Research Institute, Chennai 600025 and 3 Department of Animal Science, Bharathidasan University, Tiruchirappalli 620024, India.4 Present address: Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, USA.
5 To whom correspondence should be addressed. E-mail: aruldhasmm{at}yahoo.com
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Abstract |
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Key words: chromium/multinucleate spermatids/oxidative stress/testicular toxicity/testis ultrastructure
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Introduction |
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Hexavalent chromium (CrVI), used in more than 50 industries, is an important heavy metal pollutant (Barceloux, 1999). Several systemic toxicities of CrVI have been demonstrated in experimental animals in vivo and in vitro (Bagchi et al., 2002
; Levina et al., 2003
). However, reproductive toxicity of chromium has been underplayed since the report of Bonde (1993)
, which stated that low-level exposure to CrVI might not be a major hazard affecting spermatogenesis in stainless steel welders. Even in a recent review, Bonde (2002)
emphasized the need for additional data to recognize the reproductive toxicity of chromium. Nevertheless, a number of investigations using laboratory animals have pointed out testicular toxicity of CrVI (Behari et al., 1978
; Ernst, 1990
; Saxena et al., 1990
; Zahid et al., 1990
; Murthy et al., 1991
; Ernst and Bonde, 1992
; Chowdhury and Mitra, 1995
; Sutherland et al., 2000
). Two recent reports also correlated chronic occupational exposure to CrVI to abnormal semen quality in men (Li et al., 2001
; Danadevi et al., 2003
), though the amount and type of CrVI used by Li et al. were questioned (Duffus, 2002
).
Uptake of CrVI by the testis and its subsequent reduction to trivalent chromium (CrIII) are well known (Sipowicz et al., 1997; Sutherland et al., 2000
). Recently we reported accumulation of uni- and multinucleate germ cells in the epididymal lumen of the monkeys treated with CrVI causing ductal obstruction (Aruldhas et al., 2004
). We attributed this to the disruption of spermatogenesis and testicular histoarchitecture and this was tested in the present study.
In an attempt to understand the mechanism underlying the testicular toxicity of chromium, we also studied the status of antioxidants and pro-oxidants in the testes of these animals. The involvement of oxidative stress in heavy metal-induced cellular toxicity is known (Sugiyama, 1994). Maintenance of a critical balance between pro-oxidants and antioxidants is a physiological feature of a cell, to sustain its survival (Aitken, 1989
; Yu, 1994
). Production of free radicals/reactive oxygen species (ROS) by sperm (Iwasaki and Gagnon, 1992
) and the adverse effect of excess ROS and peroxidation on sperm motility and viability were also reported (de Lamirande and Gagnon, 1992
; Kim and Parthasarathy, 1998
). The testis, epididymis, sperm and seminal plasma contain high activities of antioxidant enzymes, which protect sperm against the deleterious effects of ROS (Kobayashi et al., 1991
; Zini et al., 1993
; Potts et al., 2000
). Increased concentrations of ROS and subnormal antioxidant status have been recorded in the seminal plasma of infertile men (Pasqualotto et al., 2000
). In the light of the above background information, it is hypothesized that chronic exposure to chromium would disrupt spermatogenesis and testicular histoarchitecture by inducing oxidative stress. Bonde and Ernst (1992)
considered only the nasal route of human exposure to CrVI in the welding industry, whereas workers in tanneries are subjected to direct exposure to chromium. A number of people who live around such industries suffer indirectly through contamination of drinking water due to percolation of untreated or incompletely treated effluents into the ground. There are several such large-scale leather industries in India and other developing countries, where pollution control laws may not be implemented effectively. Therefore, it would be pertinent to revisit the testicular toxicity of CrVI to man, adopting a protocol simulating oral exposure and using an animal model closer to the human, in order to understand and overcome the imminent problem of chromium-induced infertility/subfertility in such men.
In the earlier experimental studies, rabbits, rats and mice have been used as animal models to test the testicular toxicity of CrVI (Behari et al., 1978; Ernst, 1990
; Saxena et al., 1990
; Zahid et al., 1990
; Murthy et al., 1991
; Chowdhury and Mitra, 1995
; Sutherland et al., 2000
). Fine details of spermatogenesis, the relative duration of postnatal development and the onset of sexual maturation differ between rodents and primates (de Kretser and Kerr, 1994
). Taking into consideration all the above facts, we tested the hypothesis in a non-human primate model, Macaca radiata subjected to chronic exposure to chromium through drinking water.
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Materials and methods |
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Animals
Adult male monkeys weighing 78 kg (68 years old as calculated by the dental formula), trapped by the Department of Forests and Wild Life, Government of Tamil Nadu State, for creating public nuisance, and kept in captivity, were procured with the permission of the Chief Wildlife Warden, Chennai, India. Animals were kept under quarantine, screened for infectious diseases and acclimatized to the animal house for 2 months before the experiments were initiated. The monkeys were maintained in cages (60 x 60 x 80 cm) individually, under natural temperature (28 ± 2°C), and light and dark schedules (12 ± 1 h) in a well-ventilated animal quarter. All animals were fed ad libitum with pellet diet (Brooke Bond India, Kolkatta, India), rice cooked with lentils, vegetables such as potatoes, carrots and beetroot, and seasonally available fresh fruits such as banana and guava.
Experimental design
The experimental design was the same as reported earlier (Aruldhas et al., 2004). Briefly, adult male bonnet monkeys (Macaca radiata) were divided into four groups, each consisting of six animals. Animals belonging to groups I, II and III were provided with drinking water containing CrVI (potassium dichromate) at concentrations of 100, 200 or 400 p.p.m. respectively, ad libitum for 180 days. Group IV consisted of control animals, which were provided with drinking water without chromium. Potassium dichromate was selected as it is highly soluble in water and is used in many industries. The concentrations of chromium were selected on the basis of a doseresponse study with reference to reproductive toxicity, as judged by decline in sperm characteristics. Chromium at a concentration of 10 mg/l has been considered as safe for humans (Barceloux, 1999
). Therefore, we tested increasing concentrations of potassium dichromate, starting a little above the safe level, i.e. 12.5, 25, 50, 100, 200, 400 and 800 mg/l (p.p.m.) for reproductive toxicity. The doses (100, 200, 400 mg/l) that impaired spermatogenesis (sperm count decreased by 5070% of the control value of 350 to 375 x 106/ml) by 6 months with the least systemic toxicity were selected for the study. At the end of the experiments, testes from three monkeys in each of the experimental groups and the control group were removed surgically under deep sodium pentathol anaesthesia (28 mg/kg body weight, i.p. injection). The testes of remaining three monkeys in each group were removed after withdrawing CrVI treatment for a period of 180 days. Immediately after removing, the testes were weighed and used for histological and biochemical analyses. After removing the testes, the wound was closed using nylon stitches; antibiotics were applied every day and stitches were removed on the eighth day.
Tissue processing for light microscopy and transmission electron microscopy (TEM)
Tissues were not subjected to perfusion fixation for TEM, as samples from the same monkeys were also used for light microscopic and biochemical studies. The entire right testis was immersion-fixed in 2.5% glutaraldehyde in cacodylate buffer (Hess and Moore, 1993) immediately after removal. Thin slices of the testis were again fixed in the same fixative to ensure proper fixation. The tissues were postfixed in 1% osmium tetroxide and embedded in thin viscosity resin (Spurrs mix; Sigma, St Louis, MO, USA). Semithin sections (1µm) were obtained with an Ultratome (Reichert Jung, Vienna, Austria) and stained in toluidine blue O (TBO) for light microscopic observations. The diameter of the seminiferous tubules (20 randomly selected tubules from each animal) was measured using a calibrated ocular micrometer (Erma, Tokyo, Japan). Ultrathin sections were cut with an ultramicrotome (Leica Microsystems, Nussloch, GmbH Nussloch, Germany), stained with uranyl acetate and lead citrate, and observed in a Phillips 201-C (Amsterdam, Holland) TEM. Image analysis and processing were done using Axivision image analysis software (Carl Zeiss, Jena, Germany). Tissues from the recovery group of monkeys were subjected to light microscopic analysis only.
Biochemical analysis
Blood collection and chromium analysis
Blood samples were collected by vein puncture before starting, every month during and at the end of experimentation, and plasma was separated and used for estimation of chromium in an atomic absorption spectrophotometer (Perkin-Elmer Life and Analytical Sciences, Shelton, CT, USA), following the protocol of the manufacturer. All glassware used for chromium analysis was washed thoroughly in deionized and double-distilled water, after overnight soaking in nitric acid.
Antioxidant enzymes
Tissue preparation. Immediately after removal, the left testis from each monkey was washed in ice-cold saline and a 10% homogenate was prepared in 0.1 M TrisHCl buffer (pH 7.4). To 1.0 ml of 1:10 diluted tissue extract, 0.25 ml absolute ethanol and 0.15 ml chloroform were added and the mixture was placed in a mechanical shaker for 15 min, centrifuged at 3000 g for 10 min at 4°C and the supernatant was used for the assay of antioxidant enzymes.
Superoxide dismutase (SOD) (EC 1.15.11). SOD activity was estimated according to the method of Marklund and Marklund (1974). The percentage inhibition of pyrogallol auto-oxidation at 470 nm was converted to units of enzyme activity. The amount of enzyme required to cause 50% inhibition of pyrogallol auto-oxidation was considered as 1 unit of enzyme activity.
Catalase (EC No. 1.15.1.6). Catalase activity was quantified colorimetrically (Sinha, 1972) based on the intensity of the green colour developed due to the conversion of dichromate into chromic acetate. The amount of enzyme that uses 1 µM of H2O2 per minute is equivalent to 1 unit.
Glutathione peroxidase (GPx) (EC 1.11.1.9). GPx activity was determined colorimetrically by estimating the amount of reduced glutathione (GSH) oxidized per minute, using GSH as the standard (Rortruck et al., 1973).
Glutathione reductase (GR) (EC 1.6.4.2). GR activity was assayed colorimetrically by following the rate of reduction of oxidized glutathione (Stall and Vegel, 1969).
Glutathione-S-transferase (GST) (EC 2.5.1.18). GST activity was determined by estimating the amount of enzyme that catalysed the conjugation of a known amount of 1-chloro-2,4 dibenzene with GSH, as described by Habig et al. (1973).
Glucose-6-phosphate dehydrogenase (G-6-PDH) (EC 1.1.1.49). G-6-PDH activity was assayed spectrophotometrically by following the rate of oxidation of glucose-6-phosphate to 6-phosphogluconate, according to Beutler (1983).
-Glutamyl transpeptidase (
-GT) (EC 2.3.2.2).
-GT activity was estimated colorimetrically by quantifying the amount of p-nitroaniline released from
-glutamyl-p-nitroanilide in a given time, using the method of Orlowski and Meister (1965)
with p-nitroaniline as the standard.
Non-enzymic antioxidants
Reduced glutathione (GSH). GSH concentration was quantified colorimetrically by measuring the product of the reaction between GSH and DTNB (Moren et al., 1979).
Vitamin C. Vitamin C was estimated colorimetrically using ascorbate as the standard (Omaye et al., 1971).
Vitamin E. Vitamin E content was quantified by the colorimetric method of Desai (1984), using
-tocopherol as the standard.
Vitamin A. Vitamin A content was quantified by the colorimetric method of Bayfield and Cole (1974), using
-carotene as the standard.
Reactive oxygen species
Hydrogen peroxide (H2O2). H2O2 production was assessed spectrophotometrically (Holland and Storey, 1981) by estimating the oxidation product of ferrocytochrome.
Hydroxyl radical. The production of hydroxyl radical was quantified by the colorimetric method described by Puntarulo and Cederbaum (1988).
Statistics
Data were subjected to one-way analysis of variance, and whenever the F value was significant, the data were analysed by Duncans multiple comparison test to find the within-group significance at the P < 0.05 level.
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Results |
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Organ weight
The absolute weights of the testis did not show any appreciable change due to CrVI treatment. However, there was a statistically significant decrease in the relative weight of the testes of monkeys that were exposed to CrVI The weight of the testes in monkeys belonging to the withdrawal group showed a trend of recovery to control level (Table I). (Author please re-check this.
Histopathology
Light microscopic observations
Control testes. The seminiferous tubules in the testes of control monkeys had the typical organization (Figure 1a), with different generations of germ cells associated with Sertoli cells (Figure 1b). The Leydig cells also had their characteristic organization (Figure 1a).
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Testes of chromium-treated monkeys. The seminiferous tubules of the chromium-treated monkeys were disorganized, with decreased diameter (0.61 ± 0.08, 0.52 ± 0.07 and 0.97 ± 0.11 mm for the 100, 200 and 400 p.p.m. groups, respectively, versus the control value of 1.32 ± 0.13 mm). Depletion of germ cells and hyperplasia of Leydig cells (Figure 1ce) were the common features in all the experimental groups. Spermatids were the most affected germ cells in most of the tubules and were totally absent in a few tubules. Most of the germ cells in the adluminal compartment were prematurely released into the lumen, resulting in Sertoli cell fibrosis. Even those spermatids that adhered to the epithelium had vacuoles around them, indicating that they were detaching from the Sertoli cell (Figure 2a). Of the round and elongating spermatids, the former appeared to be the earlier target for chromium as the round spermatids were missing or being lost in many of those tubules which should possess two generations of spermatids, whereas the elongating spermatids were present (Figure 2a).
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In several tubules, which were in the process of losing round spermatids, the adluminal compartment contained multinucleate giant cells (Figure 1e). The organization of the nuclei of the multinucleate giant cells suggested they were spermatids (Figure 2b), and in a few such giant cells the nuclei were abnormally shaped, with marginalized chromatin, creating a hollow in the centre (Figure 2c). In rare cases, the cytoplasm of the multinucleate spermatids appeared vacuolated and the nuclei were pycnotic (Figure 2d).The lumen was invariably filled with prematurely released germ cells and cell debris (Figures 1c, e and 2b, d).
In several postzygotene spermatocytes, the chromatin appeared fragmented and discontinuous (Figure 3a), whereas in those with chromosomes in the metaphase plate the spindle fibres were not discernible (Figure 3b). Cells resembling macrophages were noticed in the adluminal compartment (Figure 3a). In most of the prematurely released germ cells, the chromatin was either intact or marginalized; in a few instances hydropic swelling and necrosis were also noticed (Figure 3c). Depending upon the extent of the loss of germ cells, the seminiferous epithelium reflected prominence of Sertoli cells and a few such tubules reflected the Sertoli cell-only syndrome (Figure 3d), but closer examination revealed the presence of spermatogonia in the basal compartment Accumulation of electron-dense granules in the basal and perinuclear cytoplasm of Sertoli cells was a common manifestation of chromium toxicity (Figure 3c, d).
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In the recovery group of monkeys (i.e. 6 months after the withdrawal of chromium exposure) the diameter (100 p.p.m., 1.18 ± 0.12 mm; 200 p.p.m., 1.22 ± 0.08 mm; 400 p.p.m., 1.25 ± 0.14 mm) and the histological organization of the seminiferous tubules were comparable to those of control monkeys. Spermatogenesis recovered fully, though a few prematurely released germ cells were still noticed in the lumen (Figure 4a, b).
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TEM observations
Control testes. Low-power electron micrographs of the testes of control monkeys revealed closely packed Sertoli cells contacting the basement membrane. The spermatogonia and spermatocytes up to preleptotene stage were present in the basal compartment, whereas spermatocytes beyond preleptotene stage and spermatids were present in the adluminal compartment of Sertoli cells. The cell types reflected their characteristic ultrastructural organization (Figure 5a).
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Testes of chromium-treated monkeys: elongating spermatids. Sertoli cells in which sperm heads were embedded developed vacuoles around the latter and had electron-dense granules around the posterior aspect of the sperm nucleus. The cytoplasm of the differentiating midpiece was also vacuolated (Figure 5b). Other important manifestations in spermatids of the monkeys exposed to chromium included: (i) chromatin becoming heterogeneous and granular; (ii) the appearance of a dense, plaque-like acrosomal cap; (iii) association of the nuclear cap with microtubule-like structures; (iv) the presence of a large vacuole at the junction between the acrosomal cap and the manchette microtubules; (v) vacuoles in the developing midpiece (Figure 5c); (vi) formation of the manchette microtubules into a continuous patch (Figure 5d); and (vii) the midpiece lagging in pace behind that of the head (Figure 5e).
Round spermatids. In the 400 p.p.m. chromium-treated group the round spermatids were prematurely released from the Sertoli cells, with little pathological change (Figure 6a), whereas in the 100 and 200 p.p.m. groups the round spermatids developed vacuoles in the cytoplasm, particularly in the Golgi area (Figure 6b). The mitochondria were swollen, with collapsed cristae (Figure 6c).
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Spermatocytes and spermatogonia. In spermatocytes at the advanced pachytene stage, the chromatin appeared fragmented and the mitochondria underwent hydropic swelling accompanied by collapse of cristae (Figure 6d). A few spermatocytes showed necrotic changes with thoroughly damaged chromatin (Figure 7a). However, the pale and dark spermatogonia and preleptotene spermatocytes manifested little pathological change (Figure 7b,c), though mitochondria were slightly damaged in the pale spermatogonia (Figure 7d).
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Sertoli cells. Sertoli cells of CrVI-treated monkeys accumulated large electron-dense bodies in the basal and perinuclear cytoplasm, and at a higher magnification these bodies appeared to be pale, transitional and dark lysosomes (Figure 8a). In certain rare cases, deformed sperm heads were seen in a vacuole in the Sertoli cell cytoplasm (Figure 8a). The structure of inter-Sertoli cell junctions (Figure 8b) and the Sertoli cellgerm cell junctions appeared to be affected (Figures 6a and 8b).
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Multinucleate giant cells and their origin. Ultrastructural analysis revealed the multinucleate giant cells to be spermatids possessing two or more nuclei in a common cytoplasm. The presence of an acrosome in the nuclear cap in some of these cells suggested the initiation of spermiogenesis, though in respect of the different constituent cells this was not uniform (Figure 9a). Occasionally, the nuclear envelope in the binucleate spermatid was indented at varying degrees, and the mitochondria that were destined to develop into the mitochondrial sheath accumulated at this pole of the nucleus (Figure 9b). The formation of bi- and multinucleate spermatids appeared to be due to incomplete cytokinesis (Figure 9c), as a result of which two nuclei shared a common nuclear cap with a common Golgi apparatus lying on top of it (Figure 9d).
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Macrophages in the seminiferous epithelium. The occurrence of macrophages in the seminiferous epithelium was confirmed in the TEM study, and such macrophages were seen to align close to sperm heads that detached from the Sertoli cell (Figure 10a). In some cases, damaged macrophages containing germ cell fragments were found within the Sertoli cell cytoplasm (Figure 10b).
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Biochemical analysis
Antioxidant enzymes
Chromium treatment for 6 months led to a dose-dependent decrease in the specific activities of testicular SOD, catalase, GPx, GR and G-6-PDH, with the maximum decrease in monkeys exposed to 400 p.p.m. chromium (Figures 11 and 12). The specific activity of -GT (Figure 11) decreased in the testes of monkeys exposed to 400 p.p.m. chromium alone. Unlike the other enzymes, GST activity (Figure 12) increased in the testes of chromium-treated monkeys in all groups.
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Non-enzymatic antioxidants
The concentration of reduced glutathione (Figure 12) increased in the testes of monkeys exposed to chromium, whereas the concentration of antioxidant vitamins A, C and E (Figure 13) decreased.
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Free radicals
The concentrations of H2O2 and OH increased significantly in the testes of monkeys exposed to chromium, in a dose-dependent manner; the maximum concentration was recorded in monkeys exposed to 400 p.p.m. chromium (Figure 14).
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Discussion |
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CrVI as a genotoxic agent
The histopathological changes observed in the testis of chromium-treated monkeys suggest the cytotoxic effect of the metal. The impact of chromium on meiotic germ cells (spermatocytes) is interesting because the chromosomes appeared to undergo breakage in two ways, subsequent to pairing in the zygotene stage. In most instances, chromosomes of the pachytene spermatocytes underwent fragmentation and clumping, leading to karyolysis and necrosis. In a few cells, which had developed the metaphase plate, the chromosomes failed to proceed towards the poles and underwent fragmentation to form dot-like chromatoid bodies.
Chromium is known to induce chromosomal aberrations such as single-strand breaks and 8-oxo-guanosine substitutions (Sugiyama et al., 1993; Wise et al., 1993
; Qi et al., 2000
). Direct interaction of reactive chromium intermediates with DNA, leading to DNAchromium binding, DNADNA crosslinks and DNAprotein crosslinks are known (Misra et al., 1994
). Interaction of chromium with GSH results predominantly in chromiumDNA binding and strand breaks (Aiyar et al., 1991
). Therefore, the increased concentration of H2O2, OH and GSH observed in the present study may favour the operation of such a mechanism in the testes of monkeys exposed to CrVI, resulting in damage to the chromosomes/chromatin of meiotic/postmeiotic germ cells.
The data on antioxidants and free radicals in the present study indicate free radical toxicity in the testis of chromium-treated monkeys, as there was a significant increase in the concentration of H2O2 and OH and subnormal activity of most of the antioxidant enzymes tested, accompanied by decreased concentrations of the antioxidant vitamins A, C and E. Thus, chronic exposure to chromium appears to result in oxidative stress in the testis due to poor scavenging of free radicals The histopathological changes in the testis suggest that spermatocytes and spermatids are susceptible to chromium toxicity, whereas spermatogonia are resistant to the hostile environment of increased free radicals in the tissue. GSH is a major cellular reductant, and plays a pivotal role against the nefarious actions of hydroxyl radicals (Pal et al., 1993). The increased concentration of GSH observed in the testis of monkeys exposed to chromium probably acted as a protector of spermatogonia. The mechanism underlying such a cell-specific effect of GSH may be another area of future research for a better understanding of the mechanism of the testicular toxicity of chromium.
GT couples
-glutamyl moiety to a suitable amino acid acceptor for transport into the cell and makes it suitable for the intracellular synthesis of GSH (Markey et al., 1998
).
GT also mediates the cleaving of the dipeptidyl cysteinyl glycine, which provides cells with cysteine, a rate-limiting factor for the synthesis of GSH (Pal et al., 1993
). GR converts oxidized glutathione into GSH (Sen, 1997
). Therefore, the reduced activity of
GT (specifically in 400 p.p.m. chromium-treated monkeys) and GR (in all groups) observed in the present study suggests decreased degradation as a possible mechanism underlying increased concentration of GSH in chromium-treated monkeys. Presumably, this is a defence mechanism against chromium-induced free radical toxicity. Increased activity of GST in all groups and
GT in monkeys exposed to 50 p.p.m. CrVI (data not shown) also indicates an attempt by the testis to overcome the toxic effect of chromium. GST catalyses the conjugation of the electrophilic xenobiotics to the -SH group of glutathione, and thus increases their water solubility to facilitate excretion (Sen, 1997
). Therefore, the enhanced activity of testicular GST observed in chromium-treated monkeys may be a pointer to augmented detoxification of xenobiotic compounds.
Thus, it is clear that testicular tissue tends to overcome the adverse effects of the free radical toxicity induced by chronic exposure to chromium. Spermatogonia and preleptotene spermatocytes appear to be the preferred cell types for such protective measures in the testis, which help the revival of spermatogenesis after the withdrawal of chromium treatment.
Reversibility of the toxic effect of chromium on the testis was vividly reflected in the normal organ weight and histoarchitecture of the testis and normal sperm counts (data not shown here) in monkeys that were kept free of chromium exposure for 6 months. It is well established that it takes about 10 weeks for a spermatozoon to complete testicular and post-testicular maturation and to be ready for ejaculation in monkeys (Amann et al., 1976; Shrape, 1994
). In fact, normozoospermia was achieved in experimental monkeys in the present study by the end of 3 months after the withdrawal of chromium treatment (Aruldhas et al., 2000
).
In Wistar rats exposed to chromium water for 30 days, concurrent supplementation with vitamin C or vitamins C and E protected the testis from the adverse effects of chromium, as evident from the normal histoarchitecture and antioxidant enzymes (Subramanian, 2001). Vitamin A and E are also essential for the maintenance of normal spermatogenesis (Huang and Hembree, 1979
). Vitamin E deficiency was reported to induce spermatogenic arrest (Bensoussan et al., 1998
). Therefore, decreased concentrations of vitamins A, E and C in the testes of chromium-treated monkeys might also have contributed to the premature release of germ cells into the lumen. This proposal was ascertained by the finding of the maintenance of normozoospermia in monkeys supplemented with any one of these vitamins while concurrently treated with chromium (data not shown).
Multinucleate giant cells
The multinucleate spermatids appearing in the seminiferous epithelium of chromium-treated monkeys, irrespective of the dose, may explain another mechanism of chromium toxicity, independent of its genotoxic effect. It is also important to note that the formation of multinucleated giant cells and the degenerative features described in the present study are common in animals exposed to a wide range of toxic compounds (Russel et al., 1990). This may be due to disruption of the cytoplasmic bridges connecting germ cell clones or failure of cytokinesis (Stanley and Akbarsha, 1992). The binucleate spermatids with the cap of the two nuclei remaining fused and the incomplete cytokinesis in a binucleate spermatid, observed in the present study, are clear indications of failure of cytokinesis as the probable mechanism underlying the generation of multinucleate spermatids in the context of chromium toxicity.
Premature loss of germ cells and Sertoli cell toxicity
The results of the present study clearly point to the germ cells present in the adluminal compartment of the seminiferous epithelium as the principal target of chromium toxicity in the testis. The manifestations are clearly pathological and the affected cells have become non-viable or dead and hence do not go through the process of spermatogenesis and/or spermiogenesis. Such cells are lost from any epithelium through either necrosis or apoptosis (Levin et al., 1999). The seminiferous epithelium responds to such a situation with premature release of the affected cells, which reach the epididymal duct for further processing and removal (Agnes and Akbarsha, 2001
; Aruldhas et al., 2004
). The TEM observations in the present study suggest occasional necrosis in the testis of chromium-treated monkeys. Though DNA labelling and in situ staining by the TUNEL method are the most reliable techniques for the confirmation of apoptosis, marginalization of nuclear chromatin is a morphological indication of apoptosis (Flickinger et al., 1999
), the mode of cell death caused by chromium (Blankenship et al., 1994
).
ROS produced during the reduction of CrVI was shown to be responsible for initiation of p53-induced apoptosis (Ye et al., 1999). Data on enzymatic and non-enzymatic antioxidants and free radicals clearly establish the development of oxidative stress in the testis of CrVI-treated monkeys. Therefore, it may be logical to propose that ROS-mediated apoptosis may occur in the testis of CrVI-treated monkeys. This could be another mechanism in the system towards maintenance of a viable epithelium to provide scope for revival of spermatogenesis, apart from the preservation of spermatogonia.
The participation of Sertoli cells in the process of premature loss of germ cells is indispensable. The germ cells in the adluminal compartment are held in position by the characteristic inter-Sertoli cell junctions and the Sertoli cellgerm cell junctions (Bardin et al. 1994). These junctional complexes are maintained by cytoskeletal elements and the ectoplasmic specialization of the Sertoli cell cytoplasm (Bardin et al., 1994). Once a germ cell has arrived at the adluminal compartment, it is released only at the time of spermiation in the normal course (de Kretser and Kerr, 1994
). During toxic manifestations, as in the present case, the Sertoli cells may release the germ cells prematurely, and this would necessarily involve alterations in the junctional complexes and the cytoskeletal framework of the Sertoli cells (de Kretser and Kerr, 1994
). When such a response to chromium toxicity is imminent, the Sertoli cell itself becoming a target of the toxicant cannot be ruled out. The changes occurring in the mitochondria of the Sertoli cell and the accumulation of pale, transitional and dark lysosomes in the basal aspect of the Sertoli cell cytoplasm, as observed in the present study, may be reflections of such consequences of chromium toxicity.
One of the important histopathological observations in the present study is that the germ cells of the adluminal compartment alone are irrevocably affected, whereas those in the basal compartment remained unaffected. It is an established fact that nutritive and/or regulatory substances reach germ cells of the adluminal compartment through the Sertoli cell (Bardin et al., 1994), and chromium may also take the same route. Germ cells at different stages of development require specific nutrients, such as lactate, pyruvate, fatty acids and regulatory peptides (de Kretser and Kerr, 1994
). Chromium toxicity probably affects the supply of such specific nutrients and/or regulatory substances required by meiotic/postmeiotic germ cells, resulting in their premature death.
Macrophages in the seminiferous epithelium
Because of the thick tunic around the seminiferous tubules and the consequent absence of a direct blood supply (Setchell et al., 1994), macrophages cannot enter the seminiferous tubules under normal circumstances (Russell et al., 1990
). Thus, the present study is unique in reporting macrophages in the seminiferous epithelium of CrVI-treated monkeys. Chromium treatment has probably damaged the bloodtestis barrier (Pereira et al., 2002
), paving the way for the entry of macrophages into the seminiferous tubules. It is an established fact that Sertoli cells phagocytose the residual bodies and the other cell debris under physiological conditions as well as in certain pathological states (Russell et al., 1990
). The presence of degenerating spermatozoa in vacuoles in the Sertoli cell cytoplasm of chromium-treated monkeys indicates the operation of such a mechanism of cleaning cell debris in the testes of these animals. The presence of macrophages inside the seminiferous tubules may enhance cleansing the damaged cellular materials from the tubules along with the apical portions of the Sertoli cells, as the latter could not assimilate such large volumes of degenerating cells, a situation which can attract macrophages (Russell et al., 1991
).
Thus, from the present study on the testis and our earlier findings on the epididymis of monkeys subjected to chronic chromium exposure (Aruldhas et al., 2004), and in the light of recent reports of poor reproductive health in men experiencing occupational exposure to chromium (Li et al., 2001
; Danadevi et al., 2003
), it may be concluded that occupational and/or environmental exposure to CrVI can affect the male reproductive health of primates. Our results support the hypothesis that chromium-induced changes in the histoarchitecture of the testis are due to increased ROS, leading to oxidative stress in the organ, and that these changes can be prevented by supplementation with antioxidants and are reversible. Since antioxidant supplementation has been advocated for infertile men (Irvine, 1996
), it becomes likely that the management of reproductive health of men who have occupational exposure to chromium may benefit from supplementation of antioxidants.
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Acknowledgements |
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Submitted on December 6, 2004; resubmitted on May 5, 2005; accepted on May 20, 2005.
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