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Quantitative changes in the testicular structure in mice exposed to low doses of cadmium
Environmental Toxicology and Pharmacology 23 (2007) 96–101
Quantitative changes in the testicular structure in mice exposed to low doses of cadmium Alfonso Blanco a , Rosario Moyano b , Joaqu´ın Vivo a , Rafaela Flores-Acu˜na a , Ana Molina b , Carmen Blanco a , Eduardo Ag¨uera a , Jos´e G. Monterde a,∗ a
Department of Comparative Anatomy and Pathological Anatomy, Veterinary Faculty, University of C´ordoba, 14071 C´ordoba, Spain b Department of Pharmacology and Toxicology, Veterinary Faculty, University of Cordoba, 14071 C´ ordoba, Spain Received 28 March 2006; received in revised form 25 July 2006; accepted 31 July 2006 Available online 4 August 2006
Abstract The purpose of this study was to quantify the consequences of a long-term exposure to low doses of cadmium on the testicular structure. Sexually mature male mice were orally exposed to cadmium (0.015 g/1 of CdCl2 in drinking water) for 1, 3, 6 and 12 months and then sacrificed; cadmium withdrawal was also considered in two groups raised with cadmium for 3 and 6 months, and without cadmium for 3 and 6 months before sacrifice, respectively. Morphometrical and stereological estimations were applied to quantify the structural constituents of the testes. The morphological parameters (testicular mass and size) were significantly decreased at 6 and 12 months of cadmium exposure. Interstitium was the testicular constituent most sensitive to cadmium so that significant decreases in the volume fractions of interstitium and Leydig cells were recorded as from 3 months of cadmium exposure. Cadmium-exposed seminipherous tubules showed increased diameters and lumens together with decreased tubular densities and epithelial percentages. © 2006 Elsevier B.V. All rights reserved. Keywords: Cadmium; Testes; Testicular structure; Stereology; Interstitum; Leydig cell; Seminal epithelium; Seminipherous tubules
1. Introduction Cadmium is a well-known heavy metal widely used in industry and agricultural products. Tobacco smoke is another important source of cadmium exposure, so it has been ranked among the 10 most toxic compounds for human health (Hirano and Suzuki, 1996). In recent years, there has been increasing concern about this metal as an environmental pollutant mainly derived from its extremely long biological half life, so long-term past exposures could still result in direct toxic effects from the residual metal (Benoff et al., 2000). Cadmium accumulates primarily in the kidney and in the liver where it is bound to metallothionein and is thereby detoxified, at least temporarily. Swiergosz-Kowalewska (2001) reviewed the toxicological effect of cadmium on vital organs after acute and chronic exposures highlighting the incidence of ∗ Correspondence to: Departamento de Anatom´ıa y Anatom´ıa Patol´ ogica Comparadas, Edificio de Sanidad Animal, Campus de Rabanales, 14071 C´ordoba, Spain. Tel.: +34 957218675; fax: +34 957218847. E-mail address: [email protected] (J.G. Monterde).
1382-6689/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2006.07.008
nephropaties, hepatic necrosis, pulmonary emphysema, osteoporosis and its well-known carcinogenic effect. The adverse consequences on reproductive organs of exposure to this heavy metal have been widely considered. After acute exposure, cadmium-induced testicular damage is found at interstitial and tubular levels; permeability changes in the capillary endothelium, which derive in oedemas, haemorrhages or necrosis, seem to be clearly implicated in the histopathological mechanism of these lesions (Biswas et al., 2001; Laskey et al., 1984). Although it is well known that long-term cadmium exposure has carcinogenic effects on the male reproductive organ (Koyama et al., 2002; Waalkes, 2003) and causes a diminution of reproductive capacities (Saygi et al., 1991; Selypes et al., 1992), few studies have quantified the morphological consequences of subclinic and chronic cadmium exposures. The purpose of this article was to histomorphometrically evaluate whether a long-term exposure to low doses of cadmium causes modifications in testicular morphology and structure, and the possible reversibility of the testicular changes was also considered.
A. Blanco et al. / Environmental Toxicology and Pharmacology 23 (2007) 96–101
97
2. Materials and methods
2.2. Quantitative study
Forty-eight 12-week-old sexually mature male mice (Mus musculus, Swiss OF-1) were acquired from the University of Granada. Mice were divided randomly into four groups, which were controlled during 1 month (n = 10), 3 months (n = 10), 6 months (n = 14) and 12 months (n = 14), respectively. Mice were housed under light (12-h light:12-h dark cycles) and temperature (22 ± 2 ◦ C) controls with an ad libitum access to food and tap water. Six animals from each group (Cd-exposed) received cadmium orally (CdCl2, Acros Organics, New Jersey, USA) in drinking water (0.015 g/1 CdCl2). The dose was equivalent to a cadmium concentration in drinking water of 1.5 ppm and it was decided by taking into account the non-observable effect levels (NOEL = 1 ppm) and the lowest observable effect levels (LOEL = 5 ppm) for cadmium chloride in drinking water determined by Laskey et al. (1980) for male rat reproductive toxicity under chronic exposure. Also, this dissolution was calculated so that the sum of individual doses ingested per animal over 12 months was 7.8 mg/kg; 10-fold the oral LD50 dose of 63–88 mg/kg. The possible reversibility of the response of testicular structure to cadmium exposure was also considered in four mice (Cd-withdrawal) of specimens slaughtered at 6 and 12 months; cadmium was withdrawn 3 and 6 months before slaughter (respectively), so that cadmium was administered only for the first 3 and 6 months, respectively. Four animals from each group (control) were raised without any cadmium addition to the drinking water. Animals were killed by cervical dislocation and both testes were dissected out. For morphological estimations, one testis was weighed, measured and fixed in 10% formaldehyde; the other testis was frozen for analytical cadmium determinations.
For the structural quantifications, the fixed testis was cut into top, middle and bottom sections. Each portion was then histologically processed, dehydrated in a graded series of ethanol, immersed in xylol and embedded in paraffin wax. The first section (4 m thick) of each block was stained with haematoxylin and eosin and used for the stereological study. The slides were analysed by a trinocular microscope connected by a colour video camera to a computer equipped with a frame grabber board. For the selection of microscopic images, each section was sampled in a systematic manner and digitalised under ×4 (NA = 0.10), ×10 (NA = 0.25) and ×100 (NA = 1.25) magnifications. An average of 20 microscopic fields per slide and objective lens were captured in each specimen. The microscopic images were processed using the Visilog 5® software. The tissue volume fraction (also named volume density and noted Vv ), expresses the relative volume of a structure per unit volume; its estimation was obtained by using an integral test system composed of regularly spaced test points superimposed on each microscopic image (Fig. 1A). The calculation was: Vv (tiss/tes) = P(tiss)/P(tes), where Vv (tiss/tes) is the tissue volume fraction and P(a tiss) and P(tes) are the total number of points hitting the tissue and the testis, respectively. The study also measured the nuclear volume fraction of the Leydig cells by using the same integral test system of regularly spaced points on high magnifications of the interstitial tissue images. The tubular diameter minimum was measured by tracing the boundary of the seminiferous tubule cross sections with the cursor of the analysis system previously calibrated in the images at low magnifications. The same tubular profiles were used to calculate the length density of testicular tubules (relative length of a structure per unit volume) which can be calculated by counting the profiles intersected within a delimited test area (Fig. 1B) by the formula: est Lv (tub/tes) = 2 × Q/A; where est Lv (tub/tes) is the relative length of seminiferous tubules per unit of testicular volume; Q and A are the total of the profiles of seminal tubules and the total of the delimited test area, respectively.
2.1. Analysis of cadmium content in testes Frozen testes samples of approximately 0.5 g of tissue were allowed to thaw and were digested twice with 6 ml of HNO3 and 1 ml of H2 O2 by an analytical microwave digestion system and applied to a graphyte tube atomizer for detection of cadmium by atomic absorption spectrophotometer (Varian Spectra A-600, Varian, Inc., USA).
2.3. Statistical analysis Data were analysed using the General Lineal Model (GLM) included in the Statistica software (Statistica, Version 6. Statsoft inc) to determine the effects of cadmium at each group of time exposure and the overall effects of cadmium,
Fig. 1. Photomicrographs of sections of cadmium-exposed testes for 1, 3, 6 and 12 months (A–D). Note the decrease in the testicular tissues and the increase in the lumens. The tests system superimposed in (A) and (B) was used in the estimation of tissues volume fraction and tubular length density, respectively. Scale bar = 50 m.
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time and the cadmium per time interaction. The Fisher LSD post hoc test was used to perform multiple comparisons between groups. Results are expressed as the mean ± standard deviation and p < 0.05 was considered to be significant.
3. Results Fig. 2 shows the levels of cadmium concentration in the testes of mice at the different times of cadmium exposure. Whereas no cadmium was detected in the controls, a time-dependent increase in the cadmium concentration was observed in the testes of exposed animals so that differences were highly significant from the control and from 3 months of exposure. The figures recorded for cadmium withdrawn at 6 and 12 months reflected a significant drop in cadmium levels. Results for the macroscopic features of the testes are shown in Table 1. Whereas no significant differences were found in the body weight between the control and cadmium-exposed animals, the testicular mass (testicular weight and testicular/live weight percentage) and testicular dimensions (maximum diameter) were clearly decreased in the cadmium-exposed animals with respect to the control; those differences reached high significant levels at 6 and 12 months of cadmium exposure. Despite the small size recorded in the testes of exposed animal, no other differences in the external appearance were observed between the dosed and control subjects. Regarding the tissue constituents of the testis, the different tissue volume fractions were clearly affected by the cadmium exposure, so that, from 3 months of administration, the Vv (int/tes) (% of testicular volume occupied by interstitium) was significantly
Fig. 2. Concentrations of cadmium in the testes at the different times of cadmium exposure.
decreased in the cadmium-exposed groups with respect to the control. Moreover, the data concerning the Vv (Leydig/int) (% of interstitium occupied by the Leydig cells population) showed a remarkable decrease in cadmium-dosed groups as compared to the control (Fig. 3). The data for the Vv (epth/tes) (% of testicular volume occupied by seminal epithelium) also decreased in the groups exposed to the cadmium but from 6 months of dosage. The Vv (lumen/tes) parameter reflected a highly significant increase in the percentage of testicular volume occupied
Table 1 Macroscopic testicular data (mean ± S.D.) for control, cadmium-exposed (Cd-E) and cadmium withdrawn (Cd-W) animals at the different time of recordings Cadmium exposition
Time
Significance
1 month
3 months
6 months
12 months
Cd
T
Cd × T
Live weight (g) Control Cd-E Cd-W
31.70 ± 1.62 33.85 ± 2.37
36.42 ± 1.11 37.68 ± 1.86
46.18 ± 2.66 47.27 ± 3.10 45.18 ± 3.63
46.63 ± 1.25 48.12 ± 2.97 45.40 ± 0.67
ns
ns
ns
Testicular weight (mg) Control Cd-E Cd-W
106.6 ± 6.8 107.3 ± 16.7
127.1 ± 13.4 128.0 ± 12.6
152.0 ± 13.3 124.0 ± 17.0c** 131.3 ± 12.9c*
169.0 ± 4.8 131.8 ± 8.8c*** 145.4 ± 14.9c*
***
*
**
Testicular/live weight (%) Control Cd-E Cd-W
0.34 ± 0.04 0.32 ± 0.04
0.35 ± 0.04 0.34 ± 0.02
0.34 ± 0.01 0.26 ± 0.04c*** 0.29 ± 0.05c*
0.36 ± 0.01 0.27 ± 0.01c*** 0.32 ± 0.03
***
ns
*
Diameter maximum (mm) Control Cd-E Cd-W
8.42 ± 0.24 8.47 ± 0.48
9.03 ± 0.11 9.15 ± 0.33
9.63 ± 0.36 8.76 ± 0.3c*** 9.12 ± 0.20c*
9.95 ± 0.32 9.05 ± 0.24c*** 9.21 ± 0.41c**
***
ns
*
Diameter minimum (mm) Control Cd-E Cd-W
5.40 ± 0.04 5.31 ± 0.43
5.63 ± 0.21 5.55 ± 0.43
5.90 ± 0.04 5.70 ± 0.42 5.60 ± 0.38
5.85 ± 0.18 5.51 ± 0.21 5.59 ± 0.22
ns
ns
ns
Overall significant effects of cadmium (Cd), time (T) and cadmium by time (Cd × T) interaction are also shown. ns: not significant; *, **, ***: p < 0.05, p < 0.01 and p < 0.001, respectively. c*, c**, c*** different from the control with p < 0.05, p < 0.01 and p < 0.001, respectively.
A. Blanco et al. / Environmental Toxicology and Pharmacology 23 (2007) 96–101
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Fig. 3. Histograms for percentages of testicular tissue constituent of interstitium, Vv (int/tes); seminal epithelium, Vv (epth/tes) and lumen, Vv (lumen/tes), as well as for nuclear volume fraction of Leydig cells, Vv (Leydig/int), at the different times of cadmium exposure. Overall significance effects of cadmium, time and the cadmium by time interaction are also shown. Asterisks (*, **, ***) denote different from the control with p < 0.05, <0.01, <0.001, respectively.
by the tubular lumens, especially from 6 months of cadmium administration (Fig. 1). The parameters of seminipherous tubules showed that the cadmium administration caused significant increases in the tubular diameter, together with a conspicuous decrease in the density of the tubular length from 6 months of cadmium exposure (Fig. 4), which means that cadmium caused a significant decrease in the relative length of seminiferous tubules per unit of testicular volume; taking into account that the size testicular parameters also decreased in the cadmium-exposed animals, it could be deduced that the total length of the seminal epithelium was clearly decreased as a consequence of cadmium exposure. In the groups where the cadmium was withdrawn, data were closer to the control for all parameters so that only the minimum tubular diameter and Vv (Leidig/int) percentages showed significant differences with respect to the control. 4. Discussion Studies of the effects of cadmium have demonstrated that the testis is more sensitive to cadmium than other important organs, and that low doses with no detectable effect on general health
can interfere with the testis function (Foote, 1999; Selypes et al., 1992; Swiergosz-Kowalewska, 2001). The results of this study showed that the morphology and structure of the testes were clearly altered by a prolonged exposure (from 6 months on) to a low dose of cadmium administered in drinking water, whereas the physical appearance and body weight of the animals remained unaltered. The results showed that the cadmium administered was accumulated within the testes of exposed animals and reached conspicuous concentrations even taking into account the low doses given, whereas no testis cadmium content was observed in the control animals. These results are consistent with the data of previous works that have reported the high affinity of cadmium for concentrating in the testes in cases of chronic exposure (Bench et al., 1999; Teiichiro et al., 2002). Nevertheless, the consequences on testicular morphology and structure of the cadmium’s ability to concentrate in the testes are more controversial. Although no changes in testicular weight have been reported in some subchronic cadmium intoxications (Foote, 1999; Teiichiro et al., 2002), our data showed a marked testicular hypoplasia in the cadmium-administered mice from 6 months of exposure when the testes/bodyweight ratio was 25% smaller in the exposed animals with respect to the con-
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exposure so that the interstitium percentage was significantly decreased compared to the control from 3 months of cadmium administration. Moreover, the percentages of Leydig cells in the interstitium were also markedly decreased in the exposed group, which suggests that this cell population may be especially sensitive to long-term cadmium toxicity as has been reported in acute cadmium intoxication (Laskey et al., 1984; Selypes et al., 1992; Waalkes, 2003; Yang et al., 2003). However, in our study, no images of haemorrhages or necrosis were seen in the interstitium testicular, only mild morphological differences, such as some oedemas and degenerative signs of Leydig cells (nuclear shrinkage and hyperchromasia) were observed between the treated and control images. Parameters of seminiferous tubules in the cadmium-exposed animals showed significant differences at 6 and 12 months compared to the control; dilatation of cross section diameters and increased lumens together with a decreased percentage of the volume fraction of the seminal epithelium suggested that the cadmium administration caused a notable loss of spermatogenic elements. These morphometric data are in line with the previously reported cadmium-induced impairment of spermatogenesis (Foote, 1999; Hew et al., 1993; Teiichiro et al., 2002). The cadmium withdrawal was reflected in a diminution of the cadmium testicular levels and a recovery of morphological and structural data although it was not possible to assure that, as has been reported, the carcinogenetic effect of cadmium could have done unreserved damage to the testicles. Taken together, our results reflected that prolonged exposure to low doses of cadmium caused important impairments in testicular morphology and structure mainly derived from the cadmium’s ability to concentrate in the testes, so even at low doses prolonged exposure to cadmium ends up reaching testicular levels thus causing testicular damage. Acknowledgement
Fig. 4. Histograms for tubular minimum diameter and length density of testicular tubules, Lv (tub/tes), at the different times of cadmium exposure. Overall significance effects of cadmium, time and the cadmium per time interaction are also shown. Asterisks (*, **, ***) denote different from the control with p < 0.05, <0.01, <0.001, respectively.
trol, whereas no differences were observed at shorter exposure times. Apart from the diversity of the administration, duration of treatment, age, along with other factors that can obviously affect the results, previous works in acute cadmium exposure have reported reductions in testicular weight in correlation with the cadmium dosage (Laskey et al., 1984), which have been attributed to the necrotic and degenerative cadmium-induced changes (Biswas et al., 2001; Shen and Sangiah, 1995). Our results highlight the significant influence of the time of exposure to cadmium on the morphological changes and alert about the risk of cadmium’s ability to accumulate in testes. Data concerning testicular structure showed that the interstitium was the tissue most highly affected by the cadmium
Our thanks to Prof. Joaqu´ın Reyes L´opez (University of C´ordoba, Spain) for his help and advice in the statistic analysis. References Bench, G., Corzett, M.H., Martinelli, R., Balhorn, R., 1999. Cadmium concentrations in the testes, sperm, and spermatids of mice subjected to long-term cadmium chloride exposure. Cytometry 35 (1), 30–36. Benoff, S., Jacob, A., Hurley, I.R., 2000. Male infertility and environmental exposure to lead and cadmium. Hum. Reprod. Update 6 (2), 107– 121. Biswas, N.M., Sen, G.R., Chattopadhyay, A., Choudhury, G.R., Sarkar, M., 2001. Effect of atenolol on cadmium-induced testicular toxicity in male rats. Reprod. Toxicol. 15 (6), 699–704. Foote, R.H., 1999. Cadmium affects testes and semen of rabbits exposed before and after puberty. Reprod. Toxicol. 13 (4), 269–277. Hew, K.W., Ericson, W.A., Welsh, M.J., 1993. A single low cadmium dose causes failure of spermiation in the rat. Toxicol. Appl. Pharmacol. 121 (1), 15–21. Hirano, S., Suzuki, K.T., 1996. Exposure, metabolism, and toxicity of rare earths and related compounds. Environ. Health Perspect. 104 (Suppl. 1), 85–95. Koyama, H., Kitoh, H., Satoh, M., Tohyama, C., 2002. Low dose exposure to cadmium and its health effects. (1). Genotoxicity and carcinogenicity. Nippon Eiseigaku Zasshi 57 (3), 547–555.
A. Blanco et al. / Environmental Toxicology and Pharmacology 23 (2007) 96–101 Laskey, J.W., Rehnberg, G.L., Cahill, D.F., Pieirzak-Flis, Z., 1980. Chronic ingestion of cadmium and/or tritium. II. Effects on growth, development and reproductive function. Environ. Res. 22, 466–475. Laskey, J.W., Rehnberg, G.L., Laws, S.C., Hein, J.F., 1984. Reproductive effects of low acute doses of cadmium chloride in adult male rats. Toxicol. Appl. Pharmacol. 73 (2), 250–255. Saygi, S., Deniz, G., Kutsal, O., Vural, N., 1991. Chronic effects of cadmium on kidney, liver, testis, and fertility of male rats. Biol. Trace Elem. Res. 31 (3), 209–214. Selypes, A., Serenyi, P., Boldog, I., Bokros, F., Takacs, S., 1992. Acute and “long-term” genotoxic effects of CdCl2 on testes of mice. J Toxicol. Environ. Health 36 (4), 401–409.
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Shen, Y., Sangiah, S., 1995. Na+, K(+)-ATPase, glutathione, and hydroxyl free radicals in cadmium chloride-induced testicular toxicity in mice. Arch. Environ. Contam. Toxicol. 29 (2), 174–179. Swiergosz-Kowalewska, R., 2001. Cadmium distribution and toxicity in tissues of small rodents. Microsc. Res. Tech. 55 (3), 208–222. Teiichiro, A., Hirmichi, I., keisuke, M., Kunihiro, H., Makoto, H., 2002. Cadmium-induced testicular damage in a rat model of subchronic intoxication. Reprod. Med. Biol. 1, 59–63. Waalkes, M.P., 2003. Cadmium carcinogenesis. Mutat. Res. 533 (1–2), 107–120. Yang, J.M., Arnush, M., Chen, Q.Y., Wu, X.D., Pang, B., Jiang, X.Z., 2003. Cadmium-induced damage to primary cultures of rat Leydig cells. Reprod. Toxicol. 17 (5), 553–560.
Quantitative changes in the testicular structure in mice exposed to low doses of cadmium Alfonso Blanco a , Rosario Moyano b , Joaqu´ın Vivo a , Rafaela Flores-Acu˜na a , Ana Molina b , Carmen Blanco a , Eduardo Ag¨uera a , Jos´e G. Monterde a,∗ a
Department of Comparative Anatomy and Pathological Anatomy, Veterinary Faculty, University of C´ordoba, 14071 C´ordoba, Spain b Department of Pharmacology and Toxicology, Veterinary Faculty, University of Cordoba, 14071 C´ ordoba, Spain Received 28 March 2006; received in revised form 25 July 2006; accepted 31 July 2006 Available online 4 August 2006
Abstract The purpose of this study was to quantify the consequences of a long-term exposure to low doses of cadmium on the testicular structure. Sexually mature male mice were orally exposed to cadmium (0.015 g/1 of CdCl2 in drinking water) for 1, 3, 6 and 12 months and then sacrificed; cadmium withdrawal was also considered in two groups raised with cadmium for 3 and 6 months, and without cadmium for 3 and 6 months before sacrifice, respectively. Morphometrical and stereological estimations were applied to quantify the structural constituents of the testes. The morphological parameters (testicular mass and size) were significantly decreased at 6 and 12 months of cadmium exposure. Interstitium was the testicular constituent most sensitive to cadmium so that significant decreases in the volume fractions of interstitium and Leydig cells were recorded as from 3 months of cadmium exposure. Cadmium-exposed seminipherous tubules showed increased diameters and lumens together with decreased tubular densities and epithelial percentages. © 2006 Elsevier B.V. All rights reserved. Keywords: Cadmium; Testes; Testicular structure; Stereology; Interstitum; Leydig cell; Seminal epithelium; Seminipherous tubules
1. Introduction Cadmium is a well-known heavy metal widely used in industry and agricultural products. Tobacco smoke is another important source of cadmium exposure, so it has been ranked among the 10 most toxic compounds for human health (Hirano and Suzuki, 1996). In recent years, there has been increasing concern about this metal as an environmental pollutant mainly derived from its extremely long biological half life, so long-term past exposures could still result in direct toxic effects from the residual metal (Benoff et al., 2000). Cadmium accumulates primarily in the kidney and in the liver where it is bound to metallothionein and is thereby detoxified, at least temporarily. Swiergosz-Kowalewska (2001) reviewed the toxicological effect of cadmium on vital organs after acute and chronic exposures highlighting the incidence of ∗ Correspondence to: Departamento de Anatom´ıa y Anatom´ıa Patol´ ogica Comparadas, Edificio de Sanidad Animal, Campus de Rabanales, 14071 C´ordoba, Spain. Tel.: +34 957218675; fax: +34 957218847. E-mail address: [email protected] (J.G. Monterde).
1382-6689/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2006.07.008
nephropaties, hepatic necrosis, pulmonary emphysema, osteoporosis and its well-known carcinogenic effect. The adverse consequences on reproductive organs of exposure to this heavy metal have been widely considered. After acute exposure, cadmium-induced testicular damage is found at interstitial and tubular levels; permeability changes in the capillary endothelium, which derive in oedemas, haemorrhages or necrosis, seem to be clearly implicated in the histopathological mechanism of these lesions (Biswas et al., 2001; Laskey et al., 1984). Although it is well known that long-term cadmium exposure has carcinogenic effects on the male reproductive organ (Koyama et al., 2002; Waalkes, 2003) and causes a diminution of reproductive capacities (Saygi et al., 1991; Selypes et al., 1992), few studies have quantified the morphological consequences of subclinic and chronic cadmium exposures. The purpose of this article was to histomorphometrically evaluate whether a long-term exposure to low doses of cadmium causes modifications in testicular morphology and structure, and the possible reversibility of the testicular changes was also considered.
A. Blanco et al. / Environmental Toxicology and Pharmacology 23 (2007) 96–101
97
2. Materials and methods
2.2. Quantitative study
Forty-eight 12-week-old sexually mature male mice (Mus musculus, Swiss OF-1) were acquired from the University of Granada. Mice were divided randomly into four groups, which were controlled during 1 month (n = 10), 3 months (n = 10), 6 months (n = 14) and 12 months (n = 14), respectively. Mice were housed under light (12-h light:12-h dark cycles) and temperature (22 ± 2 ◦ C) controls with an ad libitum access to food and tap water. Six animals from each group (Cd-exposed) received cadmium orally (CdCl2, Acros Organics, New Jersey, USA) in drinking water (0.015 g/1 CdCl2). The dose was equivalent to a cadmium concentration in drinking water of 1.5 ppm and it was decided by taking into account the non-observable effect levels (NOEL = 1 ppm) and the lowest observable effect levels (LOEL = 5 ppm) for cadmium chloride in drinking water determined by Laskey et al. (1980) for male rat reproductive toxicity under chronic exposure. Also, this dissolution was calculated so that the sum of individual doses ingested per animal over 12 months was 7.8 mg/kg; 10-fold the oral LD50 dose of 63–88 mg/kg. The possible reversibility of the response of testicular structure to cadmium exposure was also considered in four mice (Cd-withdrawal) of specimens slaughtered at 6 and 12 months; cadmium was withdrawn 3 and 6 months before slaughter (respectively), so that cadmium was administered only for the first 3 and 6 months, respectively. Four animals from each group (control) were raised without any cadmium addition to the drinking water. Animals were killed by cervical dislocation and both testes were dissected out. For morphological estimations, one testis was weighed, measured and fixed in 10% formaldehyde; the other testis was frozen for analytical cadmium determinations.
For the structural quantifications, the fixed testis was cut into top, middle and bottom sections. Each portion was then histologically processed, dehydrated in a graded series of ethanol, immersed in xylol and embedded in paraffin wax. The first section (4 m thick) of each block was stained with haematoxylin and eosin and used for the stereological study. The slides were analysed by a trinocular microscope connected by a colour video camera to a computer equipped with a frame grabber board. For the selection of microscopic images, each section was sampled in a systematic manner and digitalised under ×4 (NA = 0.10), ×10 (NA = 0.25) and ×100 (NA = 1.25) magnifications. An average of 20 microscopic fields per slide and objective lens were captured in each specimen. The microscopic images were processed using the Visilog 5® software. The tissue volume fraction (also named volume density and noted Vv ), expresses the relative volume of a structure per unit volume; its estimation was obtained by using an integral test system composed of regularly spaced test points superimposed on each microscopic image (Fig. 1A). The calculation was: Vv (tiss/tes) = P(tiss)/P(tes), where Vv (tiss/tes) is the tissue volume fraction and P(a tiss) and P(tes) are the total number of points hitting the tissue and the testis, respectively. The study also measured the nuclear volume fraction of the Leydig cells by using the same integral test system of regularly spaced points on high magnifications of the interstitial tissue images. The tubular diameter minimum was measured by tracing the boundary of the seminiferous tubule cross sections with the cursor of the analysis system previously calibrated in the images at low magnifications. The same tubular profiles were used to calculate the length density of testicular tubules (relative length of a structure per unit volume) which can be calculated by counting the profiles intersected within a delimited test area (Fig. 1B) by the formula: est Lv (tub/tes) = 2 × Q/A; where est Lv (tub/tes) is the relative length of seminiferous tubules per unit of testicular volume; Q and A are the total of the profiles of seminal tubules and the total of the delimited test area, respectively.
2.1. Analysis of cadmium content in testes Frozen testes samples of approximately 0.5 g of tissue were allowed to thaw and were digested twice with 6 ml of HNO3 and 1 ml of H2 O2 by an analytical microwave digestion system and applied to a graphyte tube atomizer for detection of cadmium by atomic absorption spectrophotometer (Varian Spectra A-600, Varian, Inc., USA).
2.3. Statistical analysis Data were analysed using the General Lineal Model (GLM) included in the Statistica software (Statistica, Version 6. Statsoft inc) to determine the effects of cadmium at each group of time exposure and the overall effects of cadmium,
Fig. 1. Photomicrographs of sections of cadmium-exposed testes for 1, 3, 6 and 12 months (A–D). Note the decrease in the testicular tissues and the increase in the lumens. The tests system superimposed in (A) and (B) was used in the estimation of tissues volume fraction and tubular length density, respectively. Scale bar = 50 m.
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A. Blanco et al. / Environmental Toxicology and Pharmacology 23 (2007) 96–101
time and the cadmium per time interaction. The Fisher LSD post hoc test was used to perform multiple comparisons between groups. Results are expressed as the mean ± standard deviation and p < 0.05 was considered to be significant.
3. Results Fig. 2 shows the levels of cadmium concentration in the testes of mice at the different times of cadmium exposure. Whereas no cadmium was detected in the controls, a time-dependent increase in the cadmium concentration was observed in the testes of exposed animals so that differences were highly significant from the control and from 3 months of exposure. The figures recorded for cadmium withdrawn at 6 and 12 months reflected a significant drop in cadmium levels. Results for the macroscopic features of the testes are shown in Table 1. Whereas no significant differences were found in the body weight between the control and cadmium-exposed animals, the testicular mass (testicular weight and testicular/live weight percentage) and testicular dimensions (maximum diameter) were clearly decreased in the cadmium-exposed animals with respect to the control; those differences reached high significant levels at 6 and 12 months of cadmium exposure. Despite the small size recorded in the testes of exposed animal, no other differences in the external appearance were observed between the dosed and control subjects. Regarding the tissue constituents of the testis, the different tissue volume fractions were clearly affected by the cadmium exposure, so that, from 3 months of administration, the Vv (int/tes) (% of testicular volume occupied by interstitium) was significantly
Fig. 2. Concentrations of cadmium in the testes at the different times of cadmium exposure.
decreased in the cadmium-exposed groups with respect to the control. Moreover, the data concerning the Vv (Leydig/int) (% of interstitium occupied by the Leydig cells population) showed a remarkable decrease in cadmium-dosed groups as compared to the control (Fig. 3). The data for the Vv (epth/tes) (% of testicular volume occupied by seminal epithelium) also decreased in the groups exposed to the cadmium but from 6 months of dosage. The Vv (lumen/tes) parameter reflected a highly significant increase in the percentage of testicular volume occupied
Table 1 Macroscopic testicular data (mean ± S.D.) for control, cadmium-exposed (Cd-E) and cadmium withdrawn (Cd-W) animals at the different time of recordings Cadmium exposition
Time
Significance
1 month
3 months
6 months
12 months
Cd
T
Cd × T
Live weight (g) Control Cd-E Cd-W
31.70 ± 1.62 33.85 ± 2.37
36.42 ± 1.11 37.68 ± 1.86
46.18 ± 2.66 47.27 ± 3.10 45.18 ± 3.63
46.63 ± 1.25 48.12 ± 2.97 45.40 ± 0.67
ns
ns
ns
Testicular weight (mg) Control Cd-E Cd-W
106.6 ± 6.8 107.3 ± 16.7
127.1 ± 13.4 128.0 ± 12.6
152.0 ± 13.3 124.0 ± 17.0c** 131.3 ± 12.9c*
169.0 ± 4.8 131.8 ± 8.8c*** 145.4 ± 14.9c*
***
*
**
Testicular/live weight (%) Control Cd-E Cd-W
0.34 ± 0.04 0.32 ± 0.04
0.35 ± 0.04 0.34 ± 0.02
0.34 ± 0.01 0.26 ± 0.04c*** 0.29 ± 0.05c*
0.36 ± 0.01 0.27 ± 0.01c*** 0.32 ± 0.03
***
ns
*
Diameter maximum (mm) Control Cd-E Cd-W
8.42 ± 0.24 8.47 ± 0.48
9.03 ± 0.11 9.15 ± 0.33
9.63 ± 0.36 8.76 ± 0.3c*** 9.12 ± 0.20c*
9.95 ± 0.32 9.05 ± 0.24c*** 9.21 ± 0.41c**
***
ns
*
Diameter minimum (mm) Control Cd-E Cd-W
5.40 ± 0.04 5.31 ± 0.43
5.63 ± 0.21 5.55 ± 0.43
5.90 ± 0.04 5.70 ± 0.42 5.60 ± 0.38
5.85 ± 0.18 5.51 ± 0.21 5.59 ± 0.22
ns
ns
ns
Overall significant effects of cadmium (Cd), time (T) and cadmium by time (Cd × T) interaction are also shown. ns: not significant; *, **, ***: p < 0.05, p < 0.01 and p < 0.001, respectively. c*, c**, c*** different from the control with p < 0.05, p < 0.01 and p < 0.001, respectively.
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Fig. 3. Histograms for percentages of testicular tissue constituent of interstitium, Vv (int/tes); seminal epithelium, Vv (epth/tes) and lumen, Vv (lumen/tes), as well as for nuclear volume fraction of Leydig cells, Vv (Leydig/int), at the different times of cadmium exposure. Overall significance effects of cadmium, time and the cadmium by time interaction are also shown. Asterisks (*, **, ***) denote different from the control with p < 0.05, <0.01, <0.001, respectively.
by the tubular lumens, especially from 6 months of cadmium administration (Fig. 1). The parameters of seminipherous tubules showed that the cadmium administration caused significant increases in the tubular diameter, together with a conspicuous decrease in the density of the tubular length from 6 months of cadmium exposure (Fig. 4), which means that cadmium caused a significant decrease in the relative length of seminiferous tubules per unit of testicular volume; taking into account that the size testicular parameters also decreased in the cadmium-exposed animals, it could be deduced that the total length of the seminal epithelium was clearly decreased as a consequence of cadmium exposure. In the groups where the cadmium was withdrawn, data were closer to the control for all parameters so that only the minimum tubular diameter and Vv (Leidig/int) percentages showed significant differences with respect to the control. 4. Discussion Studies of the effects of cadmium have demonstrated that the testis is more sensitive to cadmium than other important organs, and that low doses with no detectable effect on general health
can interfere with the testis function (Foote, 1999; Selypes et al., 1992; Swiergosz-Kowalewska, 2001). The results of this study showed that the morphology and structure of the testes were clearly altered by a prolonged exposure (from 6 months on) to a low dose of cadmium administered in drinking water, whereas the physical appearance and body weight of the animals remained unaltered. The results showed that the cadmium administered was accumulated within the testes of exposed animals and reached conspicuous concentrations even taking into account the low doses given, whereas no testis cadmium content was observed in the control animals. These results are consistent with the data of previous works that have reported the high affinity of cadmium for concentrating in the testes in cases of chronic exposure (Bench et al., 1999; Teiichiro et al., 2002). Nevertheless, the consequences on testicular morphology and structure of the cadmium’s ability to concentrate in the testes are more controversial. Although no changes in testicular weight have been reported in some subchronic cadmium intoxications (Foote, 1999; Teiichiro et al., 2002), our data showed a marked testicular hypoplasia in the cadmium-administered mice from 6 months of exposure when the testes/bodyweight ratio was 25% smaller in the exposed animals with respect to the con-
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exposure so that the interstitium percentage was significantly decreased compared to the control from 3 months of cadmium administration. Moreover, the percentages of Leydig cells in the interstitium were also markedly decreased in the exposed group, which suggests that this cell population may be especially sensitive to long-term cadmium toxicity as has been reported in acute cadmium intoxication (Laskey et al., 1984; Selypes et al., 1992; Waalkes, 2003; Yang et al., 2003). However, in our study, no images of haemorrhages or necrosis were seen in the interstitium testicular, only mild morphological differences, such as some oedemas and degenerative signs of Leydig cells (nuclear shrinkage and hyperchromasia) were observed between the treated and control images. Parameters of seminiferous tubules in the cadmium-exposed animals showed significant differences at 6 and 12 months compared to the control; dilatation of cross section diameters and increased lumens together with a decreased percentage of the volume fraction of the seminal epithelium suggested that the cadmium administration caused a notable loss of spermatogenic elements. These morphometric data are in line with the previously reported cadmium-induced impairment of spermatogenesis (Foote, 1999; Hew et al., 1993; Teiichiro et al., 2002). The cadmium withdrawal was reflected in a diminution of the cadmium testicular levels and a recovery of morphological and structural data although it was not possible to assure that, as has been reported, the carcinogenetic effect of cadmium could have done unreserved damage to the testicles. Taken together, our results reflected that prolonged exposure to low doses of cadmium caused important impairments in testicular morphology and structure mainly derived from the cadmium’s ability to concentrate in the testes, so even at low doses prolonged exposure to cadmium ends up reaching testicular levels thus causing testicular damage. Acknowledgement
Fig. 4. Histograms for tubular minimum diameter and length density of testicular tubules, Lv (tub/tes), at the different times of cadmium exposure. Overall significance effects of cadmium, time and the cadmium per time interaction are also shown. Asterisks (*, **, ***) denote different from the control with p < 0.05, <0.01, <0.001, respectively.
trol, whereas no differences were observed at shorter exposure times. Apart from the diversity of the administration, duration of treatment, age, along with other factors that can obviously affect the results, previous works in acute cadmium exposure have reported reductions in testicular weight in correlation with the cadmium dosage (Laskey et al., 1984), which have been attributed to the necrotic and degenerative cadmium-induced changes (Biswas et al., 2001; Shen and Sangiah, 1995). Our results highlight the significant influence of the time of exposure to cadmium on the morphological changes and alert about the risk of cadmium’s ability to accumulate in testes. Data concerning testicular structure showed that the interstitium was the tissue most highly affected by the cadmium
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