Cr(VI) compounds have been declared potent occupational carcinogens by IARC 1. Although the first report on the development of lung cancer in the chrome ore industry appeared ca 80 years ago 2, several epidemiological studies among workers of almost one hundred different professional categories (chrome plating, stainless-steel welding, pigment and leather tanning) were published since 345. Numerous and different in vitro and/or in vivo studies have been undertaken either in animals or in humans and today it is known that there is a close relationship between workers health and the amounts of industrial pollution provoked by industries manufacturing chromium containing materials 167.

Cytotoxicity and carcinogenicity of Cr(VI) depend on intracellular reduction towards stable and inert Cr(III) compounds 89. During this process Cr(V) and Cr(IV) intermediate states have been gaining major relevance 101112. Cr(V) compounds appear as good candidates to elucidate the not yet fully understood mechanism of the intracellular trail of chromium 1112.

Cr(V) complexes possessing α-hydroxy-carboxylate moieties are believed to act as both structural and biomimetic models for a range of Cr(V) species generated in vivo from Cr(VI) in intracellular media 1112. The [Cr^V-BT]^2- complex, has a "design" which exploits an adequate co-ordination capacity to the Cr metal centre together with a strong chelate effect, and is stable under physiologically conditions over a rather long period of time, and therefore seems to be an adequate Cr(V) complex to be explored for in vivo studies 13.

Chromium in vivo studies in small rodents have dealt essentially with the effects of this element in the usual toxicological target organs. Recent works concern, for example, kidney and liver 1014, pancreas 15, lungs 6161718, brain 19, male reproductive organs 20, skin 21, or even blood 22. These studies have comprised both particulate chromium compounds, mainly related with lung pathology 16, together with soluble compounds containing chromium in all the available oxidation states, more adequate or available to reach other target organs.

In previous works we have undertaken studies on Cr(VI) and Cr(V) induced alterations on mouse liver 14, and spleen histology 23. Also, the functional properties of Sertoli cells from mice exposed to Cr(V) were investigated 24. The purpose of the present study was to assess the effects of [Cr^V-BT]^2- on the morphology of testis and epididymis of mice.

Bis(hydroxyethyl)aminotris(hydroxymethyl)methane, (BT) buffer (Fluka) was used and the pH of the solution was adjusted to 7.4 by addition of diluted HCl.

In the current study, all animals survived until the end of the dosage period. Additionally, no abnormal behaviour was observed, and at sacrifice, none of the animals evidenced any macroscopic lesions. Their body weight at the start and at the end of the experiment was 32 ± 3 g, and no loss or gain in body weight was noticed along the experiments. Also, no differences were noted between the organ weights of controls and Cr(V)-injected animals over the period of these experiments (testis weight = 267 ± 2 mg; epididymis = 96 ±1 mg). The seminiferous tubules in control mice demonstrated regular features (Figure 1A). However, degenerative histological changes were observed in the epithelium of seminiferous tubules of treated animals (Figure 1B). These changes were uniform within the testis, ranging from Sertoli cells to highly mature germ cells. Intraepithelial vacuoles were clearly observed. Abnormalities affecting nearly all stages of germ cell development were seen in the seminiferous tubules, being evident necrotic mass of germ cells. In addition, all the animals injected with [Cr^V-BT]^2- were affected equally.

The ultrastructural features of the basal as well as the upper compartments of the seminiferous tubules in control animals revealed regular morphology. However, [Cr^V-BT]^2- injected mice evidenced degenerative changes within the Sertoli cells and irregular features of spermatids, and displacement towards the lumen (Figures 2A–C). Necrotic and vacuolated germ cells were also present.

Epithelial cells of cauda epididymis from [Cr^V-BT]^2- treated animals also exhibited a large number of vacuoles (Figure 3). However, other portions like the boundary tissue, revealed apparently normal features.

Table 1 shows that [Cr^V-BT]^2- induced a slight reduction in sperm motility and density, although the differences when compared to controls were not statistically significant. Sperm acrosome integrity of mice treated with [Cr^V-BT]^2- was significantly lower than sperm acrosome integrity of the controls.

Cr(VI) is known to be toxic to animals and humans 3. However implementation of both environmental and occupational regulations have been established to decrease of the hazards caused by the different types of compounds of this element. Nevertheless their associated toxicity mechanism(s) still remain unclear. For this reason, a better knowledge of the action of chromium species in intermediate oxidation states, for example, that Cr(V), is relevant in the understanding the harmful effects of chromium.

Mainly due to the poor knowledge of the role of chromium in the intermediate oxidation state Cr(V), which is considered the first step in the potentially long and harmful Cr(VI) intracellular reduction 131112, in vivo experimental studies with [Cr^V-BT]^2- have been undertaken in the present work.

The results of this study indicate that Cr(V), in the form of [Cr^V-BT]^2-, is a male reproductive toxicant, causing several histological and ultrastructural changes in mice spermatogenesis, including alterations in both Sertoli and germ cells. The presence of strongly stained exfoliated germ cells in the lumen of seminiferous tubules of [Cr^V-BT]^2- treated animals demonstrates a degenerative process in this epithelium. As shown by the results of the present work, the exfoliation process is remarkable when compared with testis injuries reported for other toxicants 2027.

Cauda epididymis also presented some epithelial vacuolation. Similar results have been described for the epididymal epithelial cells of rats treated, for example, with lead acetate 28.

These results suggest that [Cr^V-BT]^2- beyond inducing alterations in testis and epididymis histology and ultrastructure also induces a reduction in sperm motility and density. Other authors have also described a reduction in epididymal sperm counts and sperm motility in rats treated with other chromium compounds, namely CrO₃ 20. Here, the reduction of acrosome sperm integrity in mice exposed to Cr(V) is a sign of sperm lost of fertilizing potential, reflecting its inability to penetrate the zona pellucida 29. Our previous studies have shown that Cr(V) affected the functional properties of Sertoli cells 24. These results show evidence that other components of testis are important toxicological targets of Cr(V) compounds.

Keeping in mind that the mammalian testis and epididymis provide a suitable and necessary environment for spermatogenesis, maturation, and storage of spermatozoa, the damaged induced by [Cr^V-BT]^2- compound appears of great importance in the understanding of the biological effects of chromium in regard to male reproductive function.

Further studies are now underway in our laboratories concerning other organs and other type of experiments, which combine different factors such as several doses, time intervals, and different chromium compounds. The aim is to obtain dose-response data and eventually to identify toxicity limits of toxicology for these types of compounds and finally to clarify the mechanisms of chromium induced toxicity.

BT – bis(hydroxyethyl)aminotris(hydroxymethyl)methane

ehba – sodium bis(2-ethyl-2-hydroxybutanoate)oxochromato(V)

CrO₃ – Chromic acid

The author(s) declare that they have no competing interests.

MLP, TMS, and JP conceived the study, and participated in its design and coordination and helped to draft the manuscript. TMS participated in the preparation of the Cr(V) compound [Cr^V-BT]^2-, and completed the review of literature. RPN, and HO carried out the experimental study with mice, namely the histological, and motility, acrosome integrity, and count of sperm cells under Cr(V) exposure, and performed the statistical analysis. MLP carried out the ultrastructural studies. All authors read, discussed, and approved the final manuscript.