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Environmental pollutant Aroclor 1242 (PCB) disrupts reproduction in adult male rhesus monkeys (Macaca mulatta)
ARTICLE IN PRESS . Environmental Research 93 (2003) 272–278
Environmental pollutant Aroclor 1242 (PCB) disrupts reproduction in adult male rhesus monkeys (Macaca mulatta)$ Syed Uzair Ahmad, Saeed Tariq, Samina Jalali, and M. Maqbool Ahmad Department of Biological Sciences, Quaid-I-Azam University, Islamabad, Pakistan Received 15 September 2002; received in revised form 6 May 2003; accepted 23 May 2003
Abstract Adult male rhesus monkeys (Macaca mulatta) were given oral treatment of either Aroclor 1242 or vehicle (corn oil and glycerol) at a dose of 200 mg/kg body wt/day for 6 months to investigate the effects of the pollutant on plasma testosterone and the morphology of testes and accessory glands. Aroclor 1242 treatment significantly decreased testicular size and testosterone levels in plasma and adversely affected spermatogenic activity by disrupting epithelial organization. All components of the germinal epithelium were greatly reduced. The spermatogonia were either hypertrophied or had shrunken vesiculated cytoplasm with distorted mitochondria and nuclear pyknosis. Changes were milder in the Sertoli cells, where nuclear infoldings were reduced. Characteristic features of treated Leydig cells were the presence of electron-dense and electron-opaque zones, appearing as plaques, cell membrane abnormalities, and high variability in nuclear shape and heterochromatin distribution. All the Aroclor 1242-treated accessory glands contained more connective tissue than their vehicle-treated counterparts. The epithelium contained many layers of irregularly shaped necrotic cells possessing stereocilia in the epididymides, either hypochromic and hypertrophied or hyperchromic and hypotrophied cells in the prostate and shrunken cuboidal cells with elongated nuclei in the seminal vesicles. In conclusion, Aroclor 1242 treatment causes severe structural alterations on gonads and accessory organs in adult male rhesus monkeys, and these effects could be mediated through both estrogen and Ah receptors. r 2003 Elsevier Inc. All rights reserved. Keywords: Aroclor 1242; Macaca mulatta; Testis; Accessory organs; Ultrastructure
1. Introduction Polychlorinated biphenyls (PCBs) are persistent organic pollutants that have contaminated all parts of the globe. PCBs are mixtures of congeners, each of which consists of a biphenyl nucleus with chlorine at any one or all of the 10 available sites, of which the ortho, meta, and para positions are important in determining the properties of the congener. Aroclors are commercial mixtures of PCBs which were manufactured in the $ This work was funded by Quaid-I-Azam University and the World Health Organization. Experiments were conducted in accordance with national and institutional guidelines for animal welfare, and due consideration was taken to alleviate distress and discomfort in the animals. Corresponding author. Department of Biological Sciences, Quaid-iAzam University, Islamabad, Pakistan. E-mail address: [email protected], uzair [email protected] (S.U. Ahmad). URL: www.qau.pak.edu.
0013-9351/03/$ - see front matter r 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0013-9351(03)00110-5
United States. They were usually given a four-digit number, of which the first two digits refer to the number of carbon atoms attached to the biphenyl ring and the last two indicate the percentage (by weight) of chlorine (Nessel and Gallo, 1994). The constituent congeners of Aroclor 1242 are 2,20 4,40 -tetra- and 2,20 4,40 5,50 -hexa chlorinated biphenyls. This PCB mixture is of toxicological interest because of its estrogenic properties. In primates, besides damaging hematological and lymphoreticular systems (Hori et al., 1982; Tryphonas et al., 1986), the blood chemistry (Bell et al., 1994), and the histology of various body organs (Allen et al., 1974; Arnold et al., 1997; Becker et al., 1979; Hori et al., 1982; McNulty, 1985; Tryphonas et al., 1986), PCBs are also known to adversely affect the menstrual cycle, conception (Allen et al., 1980; Arnold et al., 1995; Bryce et al., 2000) and infant size (Kuratsune et al., 1972) and to cause developmental problems (Allen et al., 1974; Barsotti et al., 1976; Truelove et al., 1982). Noting the heretofore meager focus on PCB toxicity to male
ARTICLE IN PRESS S.U. Ahmad et al. / Environmental Research 93 (2003) 272–278
reproduction, we investigated the effects of Aroclor 1242 on the testes and accessory sex glands in adult rhesus monkeys.
2. Materials and methods Eight adult (5–7-year-old) male rhesus monkeys (Macaca mulatta) were purchased from local suppliers. The ages of animals were estimated using dentition formula. The animals were housed in individual stainless steel cages under standard colony conditions (temperature: 2472 C, 12:12 light/dark cycle) and quarantined for 2 months. Groups of animals (n ¼ 4) were given oral treatment of either Aroclor 1242 (Electrical Grade; Lot KB05-612; Monsanto Company, St. Louis, MO, USA) or vehicle (corn oil and glycerol) at a dose of 200 mg/kg/ day for a period of 6 months in self-ingesting gelatin capsules. Body weight and testicular size were noted, and blood samples (3 mL 2) were collected weekly by venipuncture under ketamine sedation. Following humane sacrifice (euthanasia with sodium pentobarbital), the testes and accessory glands (epididymes, seminal vesicles, and prostate) were removed, cleaned, and weighed. The tissues were fixed in 2% glutaraldehyde, postfixed in 1% osmium tetroxide (Sigma Chemical Co., St. Louis, MO, USA) and embedded in epoxy resin media (LX-112; LADD Research Industries Inc., Williston, VT, USA) using standard procedures. Semithin sections were made with glass knives on LKB ultratome, stained with 1% toluidine blue, and studied using a Nikon Optiphot light microscope. The ultrathin sections were transferred to copper grids and contrasted sequentially with uranyl acetate and lead citrate. Observations were made on a JEOL SX 100 transmission electron microscope. Plasma testosterone concentrations were determined by radioimmunoassay.
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Testicular diameter (Cl)
3. Results Aroclor 1242 treatment slightly reduced body weights and significantly decreased (Po0:05) testicular diameters (Fig. 1). The mean testosterone levels of Aroclor 1242-treated animals declined during the treatment period (Fig. 2); however, this decline was only statistically (Po0:05) significant for two of the treated monkeys. Administration of Aroclor 1242 for 6 months caused remarkable changes in the histological and ultrastructural features of the testes. The germinal epithelium population was drastically reduced. Necrotic Myeoid cells were egressing into the intertubular connective tissue through frequent discontinuities in the basal lamina. Collagen deposition thickened the basement membrane (Fig. 3a). The internal and external lamellae frequently separated widely and split into several layers (Fig. 3c). The spermatogonia either were hypertrophied with vesiculated cytoplasm (Fig. 3a and b) or contained numerous clear areas in the cytoplasm. Cell membranes were disrupted; mitochondrial cristae appeared distorted; and the endoplasmic reticulum was largely absent (Fig. 3d). The nuclei lost their round or oval shape and were pyknotic or had increased heterochromatic portions (Fig. 3d). Shrinkage was also evident in the spermatocyte cytoplasm, which possessed nuclei with condensed chromatin and nucleoli (Fig. 3e). However, the degree of chromatin condensation was lower than in the vehicle-treated spermatocytes. The process of spermiogenesis was characterized by shrinkage of the cytoplasm and thickening of cell membranes of round spermatids, distortion of elongated spermatids (Fig. 3f), and the absence of stages of acrosomal formation. Though a few sperms were found, these were abnormal, showing
A 1242
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7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Weeks
Fig. 1. Mean testicular diameters of Aroclor 1242-treated and control animals.
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Testosterone fmoles/l
2500
2000
1500
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0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 weeks Control Aroclor 1242
Fig. 2. Mean plasma testosterone levels in Aroclor 1242-treated and control animals.
shrunken heads and a total absence or degeneration of tails. The paucity of spermatogic epithelical cells made the Sertoli cells highly conspicuous. Many tubular lumens contained only extensions of Sertoli cells. The cytoplasm of the Sertoli cells contained numerous vesicles with a dense core (Fig. 3a and b). Mitochondria, endoplasmic reticulum, Golgi apparatus, and fat droplets were conspicuous. Sertoli cell nuclear shapes varied greatly, the infoldings of the nuclei were also reduced and heterochromatin was increased (Fig. 3d). The Leydig cells exhibited nuclear and cytoplasmic degeneration. Nuclei in many cells became elongated or sickle-shaped with pyknosis. The heterochromatin was scattered all over the nucleoplasm, with only a peripheral ring evident at the nuclear membrane (Fig. 3g). The cytoplasms of these cells developed zones of electron-dense and electron-opaque regions that appeared as plaques with no evidence of organelles. In some places the Leydig cells also contained fat droplets and were vacuolated. The cell membranes had threadlike extensions. Such cells contained very little cytoplasm (Fig. 3h). All accessory glands of Aroclor 1242-treated animals showed adversely affected morphology (Fig. 4b, d and f) as compared to controls (Figs. 4a, 4c and 4e). The epididymal lumens lacked spermatozoa. The epithelium contained irregular-shaped necrotic cells, with brush borders oriented toward the lumen (Fig. 4b). In the prostate, the epithelium contained a single layer of cuboidal cells, of which a few were hypertrophied and hypochromic without nuclear contents (Fig. 4d). In the seminal vesicles there were irregular mucosal folds lined by tightly packed columnar cells that became narrow to a great extent, and the basally located nuclei also assumed an elongated shape (Fig. 4f). Fibrosis was a feature in all treated animals.
4. Discussion Adult male rhesus monkeys were administered Aroclor 1242 orally at a dose of 200 mg (5 ppm)/kg/day for 6 months. This dietary level of a PCB congener (Aroclor 1248) for 6 months is reported to cause overt signs of toxicity in rhesus monkeys (Barsotti et al., 1976) and has been known to produce similar clinical toxicity symptoms in cynomolgus monkeys when administered as Aroclor 1254 (Arnold et al., 1990). In the present study, testosterone levels in Aroclor 1242-treated monkeys generally declined during the treatment period, which was accompanied by a number of light microscopic and ultrastructural abnormalities in the Leydig cells. In rodents, Aroclor 1242 has been found to decrease testosterone levels. In one study, lactating female rats receiving daily subcutaneous injections of 80 or 8 mg of Aroclor 1242 in corn oil showed significant reductions in serum testosterone levels and lowered LH-stimulated testosterone production per testis and per Leydig cell in offspring than the corn oil-treated controls (Kim et al., 2001). Aroclor 1242 has been reported to possess estrogenic properties by a number of investigators. Uterotropic activity in immature or ovariectomized adult female rodents has been accepted to reflect standard estrogenic activity (Hansen, 1998), while in male animals disruption of androgen production is considered to be an estrogenic response (Loomis and Thomas, 2000). Administration of Aroclor 1242 to immature female Sprague–Dawley rats significantly increased uterine weights and cell proliferation in the uterus. However, in the same females coadministration of Aroclor 1242 and congener 3,30 ,4,40 TCB resulted in a decrease in uterine weight (Krishan and Safe, 1993). Furthermore, Aroclor 1242 is also known to enhance the responsiveness of cultured anterior pituitary cells to GnRH in a
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Fig. 3. (a and b) Light micrographs of Aroclor 1242-treated monkey testes showing the detachment of myeoid cells from the basement membrane and their accumulation in the interstitium, a hypertrophied Ap-type spermatogonia, Sertoli cells with vesiculated cytoplasm, and much smaller nuclei ( 40). (Ap, A pale-type spermatogonium; B, B-type spermatogonium; BM, basement membrane; L, Leydig cell; M, myeoid cell; S, Sertoli cell; V, vesiculations). (c–h) Electron micrographs of Aroclor 1242-treated monkey testes. (c) Tunica propria with a wider gap between the lamellae and disassembly of collagenous fibers ( 5000). (d) A spermatogonium with a highly pyknotic nucleus, distorted mitochondria, and shrinkage of the cytoplasm ( 8000). (e) A spermatocyte having empty space within the cytoplasm, a low degree of chromatin condensation, and a prominent nucleolus ( 8000). (f) Distortion in the shape of elongated spermatids, with thickened cell membrane ( 4000). (g) Leydig cells showing nuclear shape abnormalities and cytoplasmic disorganization ( 3000). (h) Thread-like projections of the cytoplasm, which is very scanty ( 5000). (CF, collagen fibers; CM, cell membrane; EL, external lamella; EP, empty patches; IL, internal lamella; M, Myeoid cells; Mi, mitochondria; N, nucleus; Nu, nucleolus; S, Sertoli cell; Sg, Spermatogonium; St, spermatid).
manner comparable to that observed in estradiol-treated pituitary cultures (Jansen et al., 1993). These findings suggest that predicting the estrogenic effects of a complex mixture of PCBs requires the determination
of the net effects of all estrogenic and antiestrogenic congeners present in the mixture (Battershill, 1994). The present study demonstrates that oral administration of Aroclor 1242 to adult male rhesus monkeys
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Fig. 4. Photomicrographs of accessory glands of vehicle- and Aroclor 1242-treated animals. (a) Vehicle-treated epididymis showing principal cells and the basal cells in the epithelium surrounded by connective tissue. (b) Aroclor 1242-treated epididymis showing that the luminal margins of the epithelium posses a brush border (arrow) and that the thickness has increased. (c) A portion of vehicle-treated prostate gland. (d) Aroclor 1242treated prostate gland containing hypochromic and hyperchromic cells in the epithelium. A few of them are abnormally large. (e) Vehicle-treated seminal vesicles exhibiting normal features of the gland. (f) Aroclor 1242-treated seminal vesicle showing disorganized mucosal folds and epithelial degeneration. (BB, brush border; CT, connective tissue; E, epithelium; S, sperms).
causes severe structural alterations in the testes and accessory sex organs. This supports the hypothesis that PCBs may cause aspermia in monkeys (Nessel and Gallo, 1994) and decreased sperm motility and concentration and reduced semen quality in humans (Bush et al., 1986; Rozati et al., 2000). The structural damage caused by Aroclor 1242 in rhesus monkeys was similar to that of Aroclor 1254; however, the severity of damage was greater in the Aroclor 1254-treated testes, where most of the components of the germinal epithelium were lost (Ahmad et al., 2001). Various studies provide clues for possible mechanisms of PCB toxicity at cellular and molecular levels. It has been found that the PCB congener 2,20 ,5,50 -tetrachlorobiphenyl induces apoptosis in human neuronal cells. The capability of this congener to induce apoptosis is associated with the proteolytic cleavage of specific target proteins, such as poly(ADP-ribose) polymerase (PARP) and beta-catenin proteins, suggesting the possible involvement of caspases in the process (Hwang et al., 2001). Ultrastructural observations of myogenic cells demonstrate that Aroclor 1254 prevents the accumulation of contractile filaments while inducing hypertrophy of the smooth endoplasmic reticulum and causing the appearance of membrane-filled autophagosomes. Aroclor 1254 inhibits the fusion of L6 myoblasts into multinucleated myotubes and increases creatine kinase activity dose-dependently, with no effect on cell density. This may explain the observations that the offspring of PCB-exposed mothers (both in humans and rodents) have reduced body mass, since PCBs have the capability of effecting differentiation of both
myogenic cell lines and primary myogenic cell cultures (Coletti et al., 2001). The neuroendocrine system plays a very important role in the control of reproduction. The effects of PCBs on neurochemistry may contribute to reproductive toxicity. Evidence for the toxic effects of PCBs on neurochemistry has been provided by a recent report in which Aroclor 1254 exposure at a dose of 1 mg/gm of body wt in the diet of Atlantic croaker (Micropogonias undulatus) for 30 days caused a significant decrease in GnRH content in the preoptic– anterior hypothalamic area, pituitary GnRH receptor concentrations, and hypothalamic 5-hydroxytryptamine (5-HT) concentrations, thus inhibiting hypothalamic tryptophan hydroxylase (TPH), which is a rate-limiting enzyme in 5-HT synthesis. This demonstrates that TPH is one of the targets of PCB neurotoxicity and a decrease in 5-HT availability in PCB-exposed croakers results in disruption of the stimulatory 5-HT/ GnRH pathway controlling LH secretion (Khan and Thomas, 2001). In the present study, Aroclor 1242 treatment also caused damage to the histology of accessory sex organs. In rats, Aroclor 1254 at doses of 10 and 25 mg/kg/day caused severe physiological alterations, significant reductions in the number of sperm stored in the cauda epididymis, and decreased accessory gland weights (Gray et al., 1993). In another study, the PCB congener hexachlorobiphenyl (6-CB) failed to reduce serum testosterone levels or its synthesis in vitro in mice without affecting the accessory gland weights, demonstrating that the reductions in seminal vesicle weight are not a consequence of reduced serum levels of
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testosterone (Johansson, 1987). It has also been suggested that the effects of PCBs on the accessory sex glands involve a number of mechanisms such as increased testosterone turnover (Orberg and Lundberg, 1974), abnormal concentrations of extra gonadal hormones (Brain, 1972; Bryne et al., 1998; Christian and Davis, 1971; Dadoune, 1985), inhibition of such enzymes as 5-a-reductase, and induction of hepatic enzyme systems such as cytochrome p 450 (Orberg and Lundberg, 1974; Dieringer et al., 1979).
Acknowledgments The authors are grateful to Dr. Larry Hansen, University of Illinois, Urbana, IL, USA for providing Aroclor 1242 for this study.
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McNulty, W.P., 1985. Toxicity and fetotoxicity of TCDD, TCDF and PCB isomers in Rhesus macaques (Macaca mulatta). Environ. Health Perspect. 60, 71–88. Nessel, C.S., Gallo, M.A., 1994. Dioxins and related compounds. In: Lippmann, M. (Ed.), Environmental Toxicants: Human Exposure and their Health Effects. Van Nostrand-Reinhold, New York, pp. 163–182. Orberg, J., Lundberg, C., 1974. Some effects of DDT and PCB on the hormonal system of the male mouse. Environ. Physiol. Biochem. 6, 116–221.
Rozati, R., Reddy, P.P., Reddanna, P., Mujtaba, R., 2000. Xenoesterogens and male infertility: myth or reality? Asian J. Androl. 2, 263–269. Truelove, J., Grant, D., Mes, J., Tryphonas, H., Tryphonas, L., Zawidzka, Z., 1982. Polychlorinated biphenyl toxicity in the pregnant cynomolgus monkey: a pilot study. Contam. Toxicol. 11, 583–588. Tryphonas, L., Arnold, D.L., Zawidzka, Z., Mes, J., Charbonneau, S., Wong, J., 1986. A pilot study in adult rhesus monkey (M. mulatta) treated with Aroclor 1254 for two years. Toxicol. Pathol. 14, 1–10.
Environmental pollutant Aroclor 1242 (PCB) disrupts reproduction in adult male rhesus monkeys (Macaca mulatta)$ Syed Uzair Ahmad, Saeed Tariq, Samina Jalali, and M. Maqbool Ahmad Department of Biological Sciences, Quaid-I-Azam University, Islamabad, Pakistan Received 15 September 2002; received in revised form 6 May 2003; accepted 23 May 2003
Abstract Adult male rhesus monkeys (Macaca mulatta) were given oral treatment of either Aroclor 1242 or vehicle (corn oil and glycerol) at a dose of 200 mg/kg body wt/day for 6 months to investigate the effects of the pollutant on plasma testosterone and the morphology of testes and accessory glands. Aroclor 1242 treatment significantly decreased testicular size and testosterone levels in plasma and adversely affected spermatogenic activity by disrupting epithelial organization. All components of the germinal epithelium were greatly reduced. The spermatogonia were either hypertrophied or had shrunken vesiculated cytoplasm with distorted mitochondria and nuclear pyknosis. Changes were milder in the Sertoli cells, where nuclear infoldings were reduced. Characteristic features of treated Leydig cells were the presence of electron-dense and electron-opaque zones, appearing as plaques, cell membrane abnormalities, and high variability in nuclear shape and heterochromatin distribution. All the Aroclor 1242-treated accessory glands contained more connective tissue than their vehicle-treated counterparts. The epithelium contained many layers of irregularly shaped necrotic cells possessing stereocilia in the epididymides, either hypochromic and hypertrophied or hyperchromic and hypotrophied cells in the prostate and shrunken cuboidal cells with elongated nuclei in the seminal vesicles. In conclusion, Aroclor 1242 treatment causes severe structural alterations on gonads and accessory organs in adult male rhesus monkeys, and these effects could be mediated through both estrogen and Ah receptors. r 2003 Elsevier Inc. All rights reserved. Keywords: Aroclor 1242; Macaca mulatta; Testis; Accessory organs; Ultrastructure
1. Introduction Polychlorinated biphenyls (PCBs) are persistent organic pollutants that have contaminated all parts of the globe. PCBs are mixtures of congeners, each of which consists of a biphenyl nucleus with chlorine at any one or all of the 10 available sites, of which the ortho, meta, and para positions are important in determining the properties of the congener. Aroclors are commercial mixtures of PCBs which were manufactured in the $ This work was funded by Quaid-I-Azam University and the World Health Organization. Experiments were conducted in accordance with national and institutional guidelines for animal welfare, and due consideration was taken to alleviate distress and discomfort in the animals. Corresponding author. Department of Biological Sciences, Quaid-iAzam University, Islamabad, Pakistan. E-mail address: [email protected], uzair [email protected] (S.U. Ahmad). URL: www.qau.pak.edu.
0013-9351/03/$ - see front matter r 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0013-9351(03)00110-5
United States. They were usually given a four-digit number, of which the first two digits refer to the number of carbon atoms attached to the biphenyl ring and the last two indicate the percentage (by weight) of chlorine (Nessel and Gallo, 1994). The constituent congeners of Aroclor 1242 are 2,20 4,40 -tetra- and 2,20 4,40 5,50 -hexa chlorinated biphenyls. This PCB mixture is of toxicological interest because of its estrogenic properties. In primates, besides damaging hematological and lymphoreticular systems (Hori et al., 1982; Tryphonas et al., 1986), the blood chemistry (Bell et al., 1994), and the histology of various body organs (Allen et al., 1974; Arnold et al., 1997; Becker et al., 1979; Hori et al., 1982; McNulty, 1985; Tryphonas et al., 1986), PCBs are also known to adversely affect the menstrual cycle, conception (Allen et al., 1980; Arnold et al., 1995; Bryce et al., 2000) and infant size (Kuratsune et al., 1972) and to cause developmental problems (Allen et al., 1974; Barsotti et al., 1976; Truelove et al., 1982). Noting the heretofore meager focus on PCB toxicity to male
ARTICLE IN PRESS S.U. Ahmad et al. / Environmental Research 93 (2003) 272–278
reproduction, we investigated the effects of Aroclor 1242 on the testes and accessory sex glands in adult rhesus monkeys.
2. Materials and methods Eight adult (5–7-year-old) male rhesus monkeys (Macaca mulatta) were purchased from local suppliers. The ages of animals were estimated using dentition formula. The animals were housed in individual stainless steel cages under standard colony conditions (temperature: 2472 C, 12:12 light/dark cycle) and quarantined for 2 months. Groups of animals (n ¼ 4) were given oral treatment of either Aroclor 1242 (Electrical Grade; Lot KB05-612; Monsanto Company, St. Louis, MO, USA) or vehicle (corn oil and glycerol) at a dose of 200 mg/kg/ day for a period of 6 months in self-ingesting gelatin capsules. Body weight and testicular size were noted, and blood samples (3 mL 2) were collected weekly by venipuncture under ketamine sedation. Following humane sacrifice (euthanasia with sodium pentobarbital), the testes and accessory glands (epididymes, seminal vesicles, and prostate) were removed, cleaned, and weighed. The tissues were fixed in 2% glutaraldehyde, postfixed in 1% osmium tetroxide (Sigma Chemical Co., St. Louis, MO, USA) and embedded in epoxy resin media (LX-112; LADD Research Industries Inc., Williston, VT, USA) using standard procedures. Semithin sections were made with glass knives on LKB ultratome, stained with 1% toluidine blue, and studied using a Nikon Optiphot light microscope. The ultrathin sections were transferred to copper grids and contrasted sequentially with uranyl acetate and lead citrate. Observations were made on a JEOL SX 100 transmission electron microscope. Plasma testosterone concentrations were determined by radioimmunoassay.
10
Testicular diameter (Cl)
3. Results Aroclor 1242 treatment slightly reduced body weights and significantly decreased (Po0:05) testicular diameters (Fig. 1). The mean testosterone levels of Aroclor 1242-treated animals declined during the treatment period (Fig. 2); however, this decline was only statistically (Po0:05) significant for two of the treated monkeys. Administration of Aroclor 1242 for 6 months caused remarkable changes in the histological and ultrastructural features of the testes. The germinal epithelium population was drastically reduced. Necrotic Myeoid cells were egressing into the intertubular connective tissue through frequent discontinuities in the basal lamina. Collagen deposition thickened the basement membrane (Fig. 3a). The internal and external lamellae frequently separated widely and split into several layers (Fig. 3c). The spermatogonia either were hypertrophied with vesiculated cytoplasm (Fig. 3a and b) or contained numerous clear areas in the cytoplasm. Cell membranes were disrupted; mitochondrial cristae appeared distorted; and the endoplasmic reticulum was largely absent (Fig. 3d). The nuclei lost their round or oval shape and were pyknotic or had increased heterochromatic portions (Fig. 3d). Shrinkage was also evident in the spermatocyte cytoplasm, which possessed nuclei with condensed chromatin and nucleoli (Fig. 3e). However, the degree of chromatin condensation was lower than in the vehicle-treated spermatocytes. The process of spermiogenesis was characterized by shrinkage of the cytoplasm and thickening of cell membranes of round spermatids, distortion of elongated spermatids (Fig. 3f), and the absence of stages of acrosomal formation. Though a few sperms were found, these were abnormal, showing
A 1242
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Fig. 1. Mean testicular diameters of Aroclor 1242-treated and control animals.
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Fig. 2. Mean plasma testosterone levels in Aroclor 1242-treated and control animals.
shrunken heads and a total absence or degeneration of tails. The paucity of spermatogic epithelical cells made the Sertoli cells highly conspicuous. Many tubular lumens contained only extensions of Sertoli cells. The cytoplasm of the Sertoli cells contained numerous vesicles with a dense core (Fig. 3a and b). Mitochondria, endoplasmic reticulum, Golgi apparatus, and fat droplets were conspicuous. Sertoli cell nuclear shapes varied greatly, the infoldings of the nuclei were also reduced and heterochromatin was increased (Fig. 3d). The Leydig cells exhibited nuclear and cytoplasmic degeneration. Nuclei in many cells became elongated or sickle-shaped with pyknosis. The heterochromatin was scattered all over the nucleoplasm, with only a peripheral ring evident at the nuclear membrane (Fig. 3g). The cytoplasms of these cells developed zones of electron-dense and electron-opaque regions that appeared as plaques with no evidence of organelles. In some places the Leydig cells also contained fat droplets and were vacuolated. The cell membranes had threadlike extensions. Such cells contained very little cytoplasm (Fig. 3h). All accessory glands of Aroclor 1242-treated animals showed adversely affected morphology (Fig. 4b, d and f) as compared to controls (Figs. 4a, 4c and 4e). The epididymal lumens lacked spermatozoa. The epithelium contained irregular-shaped necrotic cells, with brush borders oriented toward the lumen (Fig. 4b). In the prostate, the epithelium contained a single layer of cuboidal cells, of which a few were hypertrophied and hypochromic without nuclear contents (Fig. 4d). In the seminal vesicles there were irregular mucosal folds lined by tightly packed columnar cells that became narrow to a great extent, and the basally located nuclei also assumed an elongated shape (Fig. 4f). Fibrosis was a feature in all treated animals.
4. Discussion Adult male rhesus monkeys were administered Aroclor 1242 orally at a dose of 200 mg (5 ppm)/kg/day for 6 months. This dietary level of a PCB congener (Aroclor 1248) for 6 months is reported to cause overt signs of toxicity in rhesus monkeys (Barsotti et al., 1976) and has been known to produce similar clinical toxicity symptoms in cynomolgus monkeys when administered as Aroclor 1254 (Arnold et al., 1990). In the present study, testosterone levels in Aroclor 1242-treated monkeys generally declined during the treatment period, which was accompanied by a number of light microscopic and ultrastructural abnormalities in the Leydig cells. In rodents, Aroclor 1242 has been found to decrease testosterone levels. In one study, lactating female rats receiving daily subcutaneous injections of 80 or 8 mg of Aroclor 1242 in corn oil showed significant reductions in serum testosterone levels and lowered LH-stimulated testosterone production per testis and per Leydig cell in offspring than the corn oil-treated controls (Kim et al., 2001). Aroclor 1242 has been reported to possess estrogenic properties by a number of investigators. Uterotropic activity in immature or ovariectomized adult female rodents has been accepted to reflect standard estrogenic activity (Hansen, 1998), while in male animals disruption of androgen production is considered to be an estrogenic response (Loomis and Thomas, 2000). Administration of Aroclor 1242 to immature female Sprague–Dawley rats significantly increased uterine weights and cell proliferation in the uterus. However, in the same females coadministration of Aroclor 1242 and congener 3,30 ,4,40 TCB resulted in a decrease in uterine weight (Krishan and Safe, 1993). Furthermore, Aroclor 1242 is also known to enhance the responsiveness of cultured anterior pituitary cells to GnRH in a
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Fig. 3. (a and b) Light micrographs of Aroclor 1242-treated monkey testes showing the detachment of myeoid cells from the basement membrane and their accumulation in the interstitium, a hypertrophied Ap-type spermatogonia, Sertoli cells with vesiculated cytoplasm, and much smaller nuclei ( 40). (Ap, A pale-type spermatogonium; B, B-type spermatogonium; BM, basement membrane; L, Leydig cell; M, myeoid cell; S, Sertoli cell; V, vesiculations). (c–h) Electron micrographs of Aroclor 1242-treated monkey testes. (c) Tunica propria with a wider gap between the lamellae and disassembly of collagenous fibers ( 5000). (d) A spermatogonium with a highly pyknotic nucleus, distorted mitochondria, and shrinkage of the cytoplasm ( 8000). (e) A spermatocyte having empty space within the cytoplasm, a low degree of chromatin condensation, and a prominent nucleolus ( 8000). (f) Distortion in the shape of elongated spermatids, with thickened cell membrane ( 4000). (g) Leydig cells showing nuclear shape abnormalities and cytoplasmic disorganization ( 3000). (h) Thread-like projections of the cytoplasm, which is very scanty ( 5000). (CF, collagen fibers; CM, cell membrane; EL, external lamella; EP, empty patches; IL, internal lamella; M, Myeoid cells; Mi, mitochondria; N, nucleus; Nu, nucleolus; S, Sertoli cell; Sg, Spermatogonium; St, spermatid).
manner comparable to that observed in estradiol-treated pituitary cultures (Jansen et al., 1993). These findings suggest that predicting the estrogenic effects of a complex mixture of PCBs requires the determination
of the net effects of all estrogenic and antiestrogenic congeners present in the mixture (Battershill, 1994). The present study demonstrates that oral administration of Aroclor 1242 to adult male rhesus monkeys
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Fig. 4. Photomicrographs of accessory glands of vehicle- and Aroclor 1242-treated animals. (a) Vehicle-treated epididymis showing principal cells and the basal cells in the epithelium surrounded by connective tissue. (b) Aroclor 1242-treated epididymis showing that the luminal margins of the epithelium posses a brush border (arrow) and that the thickness has increased. (c) A portion of vehicle-treated prostate gland. (d) Aroclor 1242treated prostate gland containing hypochromic and hyperchromic cells in the epithelium. A few of them are abnormally large. (e) Vehicle-treated seminal vesicles exhibiting normal features of the gland. (f) Aroclor 1242-treated seminal vesicle showing disorganized mucosal folds and epithelial degeneration. (BB, brush border; CT, connective tissue; E, epithelium; S, sperms).
causes severe structural alterations in the testes and accessory sex organs. This supports the hypothesis that PCBs may cause aspermia in monkeys (Nessel and Gallo, 1994) and decreased sperm motility and concentration and reduced semen quality in humans (Bush et al., 1986; Rozati et al., 2000). The structural damage caused by Aroclor 1242 in rhesus monkeys was similar to that of Aroclor 1254; however, the severity of damage was greater in the Aroclor 1254-treated testes, where most of the components of the germinal epithelium were lost (Ahmad et al., 2001). Various studies provide clues for possible mechanisms of PCB toxicity at cellular and molecular levels. It has been found that the PCB congener 2,20 ,5,50 -tetrachlorobiphenyl induces apoptosis in human neuronal cells. The capability of this congener to induce apoptosis is associated with the proteolytic cleavage of specific target proteins, such as poly(ADP-ribose) polymerase (PARP) and beta-catenin proteins, suggesting the possible involvement of caspases in the process (Hwang et al., 2001). Ultrastructural observations of myogenic cells demonstrate that Aroclor 1254 prevents the accumulation of contractile filaments while inducing hypertrophy of the smooth endoplasmic reticulum and causing the appearance of membrane-filled autophagosomes. Aroclor 1254 inhibits the fusion of L6 myoblasts into multinucleated myotubes and increases creatine kinase activity dose-dependently, with no effect on cell density. This may explain the observations that the offspring of PCB-exposed mothers (both in humans and rodents) have reduced body mass, since PCBs have the capability of effecting differentiation of both
myogenic cell lines and primary myogenic cell cultures (Coletti et al., 2001). The neuroendocrine system plays a very important role in the control of reproduction. The effects of PCBs on neurochemistry may contribute to reproductive toxicity. Evidence for the toxic effects of PCBs on neurochemistry has been provided by a recent report in which Aroclor 1254 exposure at a dose of 1 mg/gm of body wt in the diet of Atlantic croaker (Micropogonias undulatus) for 30 days caused a significant decrease in GnRH content in the preoptic– anterior hypothalamic area, pituitary GnRH receptor concentrations, and hypothalamic 5-hydroxytryptamine (5-HT) concentrations, thus inhibiting hypothalamic tryptophan hydroxylase (TPH), which is a rate-limiting enzyme in 5-HT synthesis. This demonstrates that TPH is one of the targets of PCB neurotoxicity and a decrease in 5-HT availability in PCB-exposed croakers results in disruption of the stimulatory 5-HT/ GnRH pathway controlling LH secretion (Khan and Thomas, 2001). In the present study, Aroclor 1242 treatment also caused damage to the histology of accessory sex organs. In rats, Aroclor 1254 at doses of 10 and 25 mg/kg/day caused severe physiological alterations, significant reductions in the number of sperm stored in the cauda epididymis, and decreased accessory gland weights (Gray et al., 1993). In another study, the PCB congener hexachlorobiphenyl (6-CB) failed to reduce serum testosterone levels or its synthesis in vitro in mice without affecting the accessory gland weights, demonstrating that the reductions in seminal vesicle weight are not a consequence of reduced serum levels of
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testosterone (Johansson, 1987). It has also been suggested that the effects of PCBs on the accessory sex glands involve a number of mechanisms such as increased testosterone turnover (Orberg and Lundberg, 1974), abnormal concentrations of extra gonadal hormones (Brain, 1972; Bryne et al., 1998; Christian and Davis, 1971; Dadoune, 1985), inhibition of such enzymes as 5-a-reductase, and induction of hepatic enzyme systems such as cytochrome p 450 (Orberg and Lundberg, 1974; Dieringer et al., 1979).
Acknowledgments The authors are grateful to Dr. Larry Hansen, University of Illinois, Urbana, IL, USA for providing Aroclor 1242 for this study.
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