Home  |  Archive  |  AJA @ Nature  |  Online Submission  |  News & Events  |  Subscribe  |  APFA  |  Society  |  Links  |  Contact Us  |  中文版

Cr(V) involvement in the toxicity pathway of testicular damage

M. L. Pereira1, T. M. Santos2, R. Pires das Neves1, F. G. Costa3, J. Pedrosa de Jesus2

1Department of Biology; 2Department of Chemistry, University of Aveiro, 3810-193, Aveiro, Portugal
3 Histology and Anatomic Pathology Section, Faculty of Veterinary Medicine, Technical University of Lisbon, Portugal

Asian J Androl  2002 Jun; 4:  153-155             

Keywords: Cr(V); blood-testis barrier; integrity; spermatogenesis; horseradish peroxidase

Aim: The functional integrity of the blood-testis barrier (BTB) in male mice exposed to Cr(V) was studied in order to clarify the mechanism underlying testicular injury. Methods: Adult male mice were subcutaneously injected repeated doses of 8.02 mmol (0.5 mL) of Cr/mouse.day for 5 days. Animals receiving a similar volume of bis(hydroxyethyl)-aminotris(hydroxymethyl)methane buffer (BT) were used as controls. The animals were sacrificed on day 6 and small fragments of seminiferous tubules, approximately 8-10 mm length, were incised and sutured at both ends. They were exposed in vitro to horseradish peroxidase-containing culture medium for 10 minutes. Tissues were then fixed and processed for ultrastructural studies. Results: Controls and Cr(V)-treated group resulted in the uptake of the tracer by Sertoli cells. However, the major finding consisted in the permeability of the BTB only in the Cr(V)-group, as evidenced by the presence of the tracer within the junctions between the neighbouring Sertoli cells. Conclusion: The BTB is disrupted in mice submitted to Cr(V). The permeability of the BTB is a crucial feature to be investigated for the understanding of lesions within the seminiferous tubule.

1 Introduction

Chromium(VI) is a well-recognized environmental pollutant that may bring about adverse effects on both humans and animals [1-2]. Additionally, there are potential hazards for industrial workers [3-4]. Cr(V), generated as a metabolic intermediate from the intracellular reduction of Cr(VI), is one of the major candidates for the carcinogenic forms of the chromium compounds [5-7]. Recently, it was indicated that Cr(VI) also produce undesirable effects on male reproductive physiology [8-11]. We have just demonstrated that a Cr(V) complex, [CrV-BT]2-, was able to induce testicular lesions in the mouse [12]. To our knowledge, no other references are so far available in the literature concerning the effects of Cr(V) on male reproduction.

The present work describes for the first time the effects of the new Cr(V) compound on the functional integrity of the blood-testis barrier (BTB), which plays a significant role on spermatogenesis.

2 Materials and methods

2.1 Animals

Experiments were conducted in 60 days old male Charles River mice (30 g) kept under laboratory conditions for small rodents (12h light/12h dark cycle; 60 % humidity; 222). Food and water were available ad libitum. Twenty animals were randomly divided in two groups, the Cr(V)-treated and the controls of 10 animals each.

2.2 Preparation of chromium(V) compound

[CrV-BT]2- [BT is bis(hydroxyethyl)-aminotris(hydroxymethyl)methane buffer] was obtained in situ in aqueous solution, by full ligand substitution (30 min) from the precursor complex Na[CrVO(ehba)2], sodium bis(2-ethyl-2-hydroxybutanoato)oxochromate(V) [6,13].

2.3 Treatment of animals

Mice of the Treated group were subcutaneously administered 8.02 mmol (0.5 mL) of Cr/mouse.day for 5 days [14]. The Control group was injected with a similar volume of a physiological solution (BT) for the same period of time. Mice were sacrificed on day 6 under ether anaesthesia and the testis was removed.

2.4 In vitro incubation with horseradish peroxidase

The seminiferous tubules were isolated from the control and the treated mice and around sixty small fragments of the tubules (each about 8-10 cm in length) were incised and sutured at both ends with a cotton thread. Tubules were then incubated in vitro for 10 min at 37 with Eagle's medium (Biomrieux) containing 5 mg/mL of horseradish peroxidase (HRP Type II; Sigma) supplemented with 10 % fetal calf serum (GIBCO) as described [15].

2.5 Ultrastructural studies

The tubular fragments were then fixed with 2.5 % glutaraldehyde in 0.1 M caccodilate buffer and placed for 45 min in 10 mL of Tris buffer solution with 5 mg of diaminobenzidine and 0.1 mL of 1 % H2O2. Tissues were postfixed in 2 % buffered osmium tetroxide, dehydrated and embedded in Epon 812. Thin sections prepared with a diamond knife were double stained with uranyl acetate and lead citrate. At least 2 or 3 sections were made in each tubule. Observations were conducted using a Hitachi transmission electron microscope.

3 Results

The seminiferous epithelium fragments of the Cr(V)-injected mice, in particular the Sertoli cells, were able to uptake the HRP added to the culture medium. The uptake of this macromolecule was accomplished by small vesicles, which moved into the intercellular space where the releasing of the tracer took place. HRP also permeated the intercellular junctions between the neighbouring Sertoli cells, reaching the adlumenal compartment of the seminiferous tubules (Figure 1). The intercellular spaces around the Sertoli cells were widened, and the tight contacts with the neighbouring cells were disrupted in all the Cr(V)-treated animals. In the controls, the Sertoli cells were also able to uptake the tracer, but the BTB was not damaged (inset).

Figure 1. Ultrastructural aspect of the intercellular junctions between Sertoli cells in Cr(V)-injected mouse filled with the tracer (arrows); S -Sertoli cell; V -Vacuoles; inset -The impenetrable inter-Sertoli cell tight junctions of a control mouse (arrows). Bar = 1 mm.

4 Discussion

This work was conducted to shed light on the possible mechanisms of Cr(V)-induced testis injury and demonstrated for the first time that the BTB is the potential target site. Consequently, exfoliation of immature germ cells within the lumen of the seminiferous tubules, as described in our previous paper [12], may result from the failure of the BTB integrity. It is well known that Sertoli cells provide, select and/or transport from the systemic circulation the required molecules for germ cell development. Thus, substances from the interstitial tissue must pass through or between Sertoli cells in order to reach the adlumenal compartment of the seminiferous tubules. However, the presence of tight junctions between these somatic cells, prevent most substances from reaching the adluminal compartment, unless they pass through the Sertoli cell cytoplasm [16,17]. These findings support the conclusion that Cr(V) is implicated in the bio-toxic pathway of testis injury, by disrupting the BTB.

Other investigations have already demonstrated damages on male reproductive physiology induced by Cr(VI) compounds [8-11]. Comparative studies between Cr(VI) and Cr(V) effects on mice testis are in progress in our lab in order to obtain a better understanding of the mecha-nisms underlying chromium in vivo toxic effects.


This study was supported in part by grants from the Research Centre on Inorganic Chemistry and Materials, from Aveiro University (Portugal).


[1] IARC. Monographs on the evaluation of carcinogenic risks to humans: chromium, nickel and welding. International Agency for Research on Cancer (Lyon) 1990; 49: Chap. 3.
[2] Dayan AD, Paine AJ. Mechanisms of chromium toxicity, carcinogenicity and allergenicity: review of the literature from 1985 to 2000. Hum Exp Toxicol 2001; 20: 439-51.
[3] De Flora S. Threshold mechanisms and site specificity in chromium(VI) carcinogenesis. Carcinogenesis 2000; 21: 533-41.
[4] Gibb HJ, Lees PS, Pinsky PF, Rooney BC. Lung cancer among workers in chromium chemical production. Am J Ind Med 2000; 38: 115-26.
[5] Dillon CT, Lay PA, Bonin AM, Cholewa M, Legge GJ, Collins TJ et al. Permeability, cytotoxicity, and genotoxicity of chromium (V) and chromium (VI) complexes in V79 Chinese hamster lung cells. Chem Res Toxicol 1998; 11: 119-29.
[6] Codd R, Dillon CT, Levina A, Lay PA. Studies on the genotoxicity of chromium: from the test tube to the cell. Coord Chem Rev 2001: 216-17, 537-82.
[7] Liu KJ, Shi X. In vivo reduction of chromium(VI) and its related free radical generation. Mol Cell Biochem 2001; 222: 41-7.
[8] Sutherland JE, Zhitkovich A, Kluz T, Costa M. Rats retain chromium in testis following chronic ingestion of drinking water containing hexavalent chromium. Biol Trace Elem Res 2000; 74: 41-53.
[9] Chowdhuri DK, Narayan R, Saxena DK. Effect of lead and chromium on nuclei acid and protein synthesis during sperm-zona binding in mice. Toxicol in vitro 2001; 15: 605-13.
[10] Li H, Chen Q, Li S, Yao W, Li L, Shi X, et al. Effect of Cr(VI) exposure on sperm quality: human and animal studies. Ann Occu Hyg 2001; 45: 505-11.
[11] Veeramachaneni DN, Palmer JS, Amann RP. Long-term effects on male reproduction of early exposure to common chemical contaminants in drinking water. Hum Reprod 2001; 16: 979-87.
[12] Pereira ML, Pires das Neves R, Santos TM, Pedrosa de Jesus J. Morphological alterations of male reproductive organs in mice exposed to Cr(V). Reprod Toxicol (accepted).
[13] Fonkeng BS, Moghaddas S, Bose RN. Electron paramagnetic resonance, kinetics of formation and decomposition studies of (bis(hydroxyethyl)amino-tris(hydroxymethyl) methane)oxochromate(V): a model Cr(V) complex for DNA damage studies. J Inorg Biochem 1998; 72: 163-71.
[14] Solis-Heredia MJ, Quintanilla-Vega B, Sierra-Santoyo A, Hernandez JM, Brambila E, Cebrian ME, et al. Chromium increases pancreatic metallothionein in the rat. Toxicol 2000; 142: 111-7.
[15] Pereira ML. Studies on the permeability of the blood-testis barrier in stainless-steel-administered mice. Cell Biol Internat 1995; 7: 619-24.
[16] Grootegoed JA, Siep M, Baarends WM. Molecular and cellular mechanisms in spermatogenesis. Baillire's
Clin Endocrinol Metabol 2000; 14: 331-43.
[17] Creasy DM, Foster PM. Male reproductive system. In: Wanda M, Hascek-Hock C, Rousseaux C, Wallig M, editors. Handbook of Toxicologic Pathology. London: Academic Press; 2002. p 785-845.


Correspondence to: Dr. M. L. Pereira, Department of Biology, University of Aveiro, 3810-193, Aveiro, Portugal.
Fax: +351-234-426 408
E-mail: lpereira@bio.ua.pt
Received 2002-03-05      Accepted 2002-05-10