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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2016 Jul 28;97(4):310–316. doi: 10.1111/iep.12193

Long‐term effects of perinatal exposure to low doses of cadmium on the prostate of adult male rats

Viviane P Santana 1, Évila S Salles 2, Deborah E Correa 2, Bianca F Gonçalves 1, Silvana G Campos 3, Luiz A Justulin 1, Antonio F Godinho 1, Wellerson R Scarano 1,✉
PMCID: PMC5061764  PMID: 27469444

Summary

Developmental toxicity caused by environmental exposure to heavy metals during the perinatal period has raised questions about offspring health. Cadmium (Cd) is an endocrine‐disrupting chemical with the potential to interfere with morphogenesis and susceptibility to diseases in reproductive organs. Taking into account that in the rat prostate morphogenesis occurs during the perinatal period, and that pregnant females absorb and retain more dietary Cd than their non‐pregnant counterparts, it is important to understand the effects of perinatal Cd exposure on the adult rat prostate. Therefore this study investigated the effects of gestational and lactational Cd exposure on adult offspring rat prostate histopathology. Pregnant rats (n = 20) were divided into two groups: Control (treated with aqueous solution of sodium acetate 10 mg/l) and treated (treated with aqueous solution of cadmium acetate 10 mg/l) administered in the drinking water. After weaning, male offspring from different litters (n = 10) received food and water ‘ad libitum’. The animals were euthanized at postnatal day 90 (PND90), the ventral prostates (VPs) were removed, weighed and examined histopathologically. Blood was collected for the measurement of testosterone (T) levels. Immunohistochemistry for androgen receptor (AR) and Ki67, and a TUNEL assay were performed. There were no differences in T levels, cell proliferation and apoptosis indexes, or AR immunostaining between the experimental groups. Stromal inflammatory foci and multifocal inflammation increased significantly in the treated group. These changes were associated with inflammatory reactive epithelial atypia and stromal fibrillar rearrangement. In conclusion, VP was permanently affected by perinatal Cd exposition, with increased incidence of inflammatory disorders with ageing.

Keywords: cadmium, perinatal exposure, prostate, rat

Studies suggesting that cadmium has carcinogenic potential in human have appeared with increasing frequency in recent decades, and this has led to increased interest in its toxicological potential. Recently, experimental evidence has suggested that exposure to low concentrations of cadmium from the environment can induce DNA damage and mutation, which decreases genetic stability by inhibiting the repair of endogenous and exogenous DNA lesions which leads to an increased likelihood of mutations and therefore the initiation of cancer (Filipic et al. 2006).

The liver and the kidneys are the organs most affected by the toxicity of cadmium (Satarug et al. 2003). However, recent studies indicate that chronic exposure to low doses of cadmium (10 mg/l) may cause neurobehavioral problems in animals (Leret et al. 2003). In occupationally exposed workers, cadmium can hinder visual motor function, promotes emotional balance changes and undermines concentration, even when no kidney damage is discovered (Viaene et al. 2000).

Cadmium exposure was considered as a factor in the aetiology of testicular and prostate cancer (Waalkes & Rehm 1994). The prostate epithelial dysplasia induced in rats by cadmium (80 ppm) over 18 months showed increase in proliferative activity and high expression of bcl‐2, as described in human prostate epithelial cells (Martin et al. 2001) and in pubertal rats (Lacorte et al. 2011). The rat is used as the main experimental model for the studies of human prostate cancer (Pollard et al. 2001; Morrissey et al. 2002) because morphological similarities were observed between human prostatic intra‐epithelial neoplasia and the dysplastic disorders observed in rodents (Bosland 1999; Martin et al. 2001).

The male reproductive system can also be particularly affected by cadmium because a heavy metal can function as an endocrine disrupter, modifying the reproductive function and interfering with the development of reproductive organs and, consequently, the quality of semen (Oldereid et al. 1993; Telisman et al. 2000).

Cigarette smoking during pregnancy causes an accumulation of cadmium in the foetus, due to tobacco contamination by environmental cadmium, decreasing weight at birth (Sikorski et al. 1988). Considering the fact that toxic effects may result from in utero exposure to cadmium, as cadmium can be transferred through the foetal blood and through breast milk (Barlow & Sullivan 1982; Bhattacharya 1983; Ali et al. 1986; Anderson et al. 1997; Mokhtar et al. 2002), cigarette smoking during pregnancy is a source of exposure of constant concern.

Exposure at an early stage of one's life causes more concern, because this metal is retained to a greater extent in the young than in adults and may therefore cause more serious consequences. In this sense, experimental studies that mimic the situation of perinatal exposure are important because they can give a greater understanding of the effects of cadmium on the development and consequences on the lives of the individuals who are exposed in the uterus.

The aim of this study was to investigate the effects of cadmium on the prostate of adult rats exposed to low doses of cadmium in the gestational and lactational periods.

Materials and methods

Animals

Adult female (80 days of age, n = 20) and male (90 days of age, n = 10) Wistar rats were obtained from the Central Biotherium of São Paulo State University and housed in polypropylene cages (43 × 30 × 15 cm) with laboratory‐grade pine shavings as bedding. Rats were maintained under controlled temperature (23 + 1°C) and lighting conditions (12:12 h photoperiod). Balanced rat chow (Nestlé Purina PetCare, St. Louis, MO, USA) and filtered tap water were provided ad libitum. For mating, three female rats were housed with one male, during the dark portion of the light–dark cycle, and the day that sperm cells were detected in the vaginal smear was considered gestation day 0. The gravid females were randomly assigned between the experimental groups alternately and housed individually in cages.

Ethical approval

The experimental protocol was approved by the Ethics Committee for Animal Use (Protocol: 214‐CEUA/2010) from Institute of Biosciences of Botucatu‐UNESP following Brazilian laws.

Treatment

Pregnant rats from the treated group (T, n = 10) received cadmium acetate (CdAc) (Sigma®, St. Louis, MO, USA) at 10 mg/l, in drinking water ad libitum, for all gestation and lactational periods [until postnatal day 21(PND 21)]. Pregnant control rats (C, n = 10) received sodium acetate (NaAc) at the same molarity as CdAc, in drinking water ad libitum, following the same experimental protocol. The cadmium dose in the present study was chosen based on the literature data and referenced as low exposure dose (Antonio et al. 2002; Leret et al. 2003; Ronco et al. 2011) and mimics that observed in humans due to environmental exposure (Alvarez et al. 2004; Lacorte et al. 2011).

At birth, the litter size was standardized to eight pups per litter. In the litters with more than eight pups, the extra animals were excluded randomly. Litters with less than six pups were not considered for the experiment. If necessary, females from other litters in the same experimental group were moved to balance the litter size. Males and females chosen and ignored in this study were selected randomly. Males from different litters were not moved to balance the litter size, to preserve the litter independence. For experiments, one pup from each mother (control and treated) was separated from their mother at PND21 and maintained in groups (two males per cage), receiving food and water ad libitum, until PND90.

On PND 90, the male rats were weighed and then lightly anaesthetized in a saturated CO2 chamber and killed by decapitation. The experimental animals (control and treated groups) were killed the following morning. Blood was collected for the hormone assays; the ventral prostate (VP) was removed and weighed and its distal segment was fragmented for the analyses.

Serum testosterone assays

Blood samples from the ruptured cervical vessels were collected in a tube (additive free) at the time of death (between 9:00 and 11:30 am). The serum was separated by centrifugation (∼1,000×g, 15 min, 4°C) and stored at −20°C until hormonal determination. Testosterone levels were determined by double‐antibody radioimmunoassay, using the Testosterone MAIA® kit (Biochem Immunosystem, Milan, Italy), at the Neuroendocrinology Laboratory, Dental School of Ribeirão Preto, University of São Paulo – USP. All the samples were checked in the same assay, to avoid interassay errors. The lowest detection limit was 0.064 ng/ml, with a 4% intra‐assay variation.

Histopathological analysis

Ventral prostates from the control and treated groups (n = 10 per group) were removed, and the fragments selected from the distal segment of the gland were fixed by immersion in methacarn solution (Puchtler et al. 1970) for 3 h. Fixed tissue samples were dehydrated in a graded ethanol series and embedded in Paraplast Plus (Sigma®). Histological sections (5 μm) were stained haematoxylin–eosin (H&E) for general studies and with Gömöri′s reticulin staining to assess the collagen and reticular fibres. Histopathological analyses were performed on Zeiss photomicroscopy, and the microscopic fields were digitized using the software AxioVision® (Carl Zeiss, Oberkochen, DEU), version 4.8. The histopathological classification of prostate lesions presented here was performed according to the study of Bernoulli et al. (2008), and the level is focal inflammation, multifocal inflammation (larger or equal to three microscopic fields per histological section) was recorded, reactive epithelial atypia and atypical hyperplasia. The slides were coded for analysis ‘blind’. The analyses were performed by one of the authors (WRS) and confirmed by an experienced veterinarian pathologist.

Morphometric‐stereological analysis

Using an imaging analysis system (AxioVision®, version 4.8 for Windows™ software), histological sections stained by H&E were studied. Random H&E images of 100 histological fields per experimental group were captured and analysed by the stereological method, such that the histological fragments of all animals were evaluated equally (10 per animal). Stereological analyses were obtained by Weibel's multipurpose graticulate, with 120 points and 60 test lines (Weibel 1963) to compare the relative proportion among the prostate components (epithelium, stroma and lumen) in the experimental groups.

Immunohistochemistry

AR (clone SC‐816; Santa Cruz Biotechnology® Inc., Santa Cruz, CA, USA) and Ki67 (clone ab16667; Abcam® Inc., Cambridge, MA, USA) primary antibodies were used for IHC. For the immunohistochemical technique, histological sections (5 μm) were dewaxed and then rehydrated in graded alcohol and distilled water. Antigenic retrieval was realized in Tris–EDTA (pH 9.0) at high temperature (100°C) for 30 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol for 45 min, followed by a quick rinse in distilled water and phosphate‐buffered saline (PBS), and then incubated in 3% bovine serum in PBS for 1 h, to block protein–protein non‐specific binding. Sections were incubated with the primary antibody at 4°C overnight. After incubation with the primary antibody, sections were incubated for 2 h with the secondary antibody HRP (IgG goat‐anti rabbit, ab97051; Abcam® Inc). Chromogen colour development was accomplished with 3,3‐diaminobenzidine tetrahydrochloride (DAB; Sigma‐Aldrich Co®). The slides were counterstained with Harris’ haematoxylin. For negative control, the primary antibody was replaced with the corresponding normal isotype serum.

TUNEL assay

Ventral prostate sections from experimental animals (n = 10 per group) were digested by proteinase K. The sections were processed according to the instructions of the kit apoptosis (FragEL™ DNA Fragmentation Detection Kit, Colorimetric – TdT Enzyme; Merck Millipore's Calbiochem® Darmstadt, DEU), which is based on the TUNEL assay. The staining was revealed with DAB, and the sections were counterstained with methyl green.

Proliferation and apoptosis indexes’ determination

The cell proliferation (Ki‐67‐positive cells) and cell death (TUNEL positive) indexes in VP were determined by counting all nuclei of at least 100 microscopic fields/group at × 40 objective lens (n = 10 animals/group; 10 microscopic fields/animal). The proportion of epithelial proliferating and apoptotic cells was determined by dividing the number of stained cells by the number of cells analysed (approximately 2000 cells per VP) and finally the value multiplied by 100 for each animal in the different experimental groups.

AR semiquantitative analysis

At least five histological sections were analysed per group, and Androgen Receptor (AR)‐positive nuclei were randomly selected by section (100 nuclei/group). The images of the selected nuclei were cut in a regular and constant rectangular form (always in central part of the nucleus, in view of the homogeneity of nuclear staining/reactivity for AR) and those ‘rectangles’ were submitted to optical densitometry analysis by scion (Scion Corporation, Frederick, MD, USA) Image for Windows Software® as described in the study by Fossato da Silva et al. (2012).

Statistics

For the comparison of the results between the experimental groups, nonparametric Mann–Whitney U‐test was used. The stereological data were expressed as median followed by quartiles [Q1–Q3], while the other data were expressed as mean ± SEM. For the incidence of lesions, the results are expressed in percentage and experimental groups were compared by the chi‐square test. Differences were considered significant when P ≤ 0.05. After weaning, data from the body weight, hormonal analysis and prostate parameters were performed using 1 to 2 male/litter (selected randomly), with the litter as the unit of measure into the experimental groups.

Results

There was no change in body and VP weight between the experimental groups in PND90 (Table 1). Similarly, serum testosterone levels did not differ between them (Table 1). Stereological analysis showed no significant differences in the relative proportion of tissue components in VP between the experimental groups at PND90 (Table 2).

Table 1.

Quantitative data from experimental groups at PND90

Control Treated
Biological parameter (n = 10)
Body weight (g) 375.5 ± 22.0 388.2 ± 29.1
Ventral prostate (g) 0.324 ± 0.039 0.351 ± 0.041
Serum testosterone (ng/ml) 0.84 ± 0.24 1.01 ± 0.34
Histopathological change incidence (n = 10)
Focal inflammation 1/10 (10%) 6/10 (60%)a
Multifocal inflammation 0/10 (0%) 4/10 (40%)a
Reactive epithelial atypia 0/10 (0%) 5/10 (50%)a
Atypical hyperplasia 1/10 (10%) 3/10 (30%)
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Results are expressed by mean ± SD in biological parameters. Statistical test: Mann–Whitney U‐test, P ≤ 0.05. Histopathological changes: values are expressed by the occurrence of VP lesions/number of animals evaluated, followed by the percentage of incidence and compared by chi‐square test.

a

P ≤ 0.05.

Table 2.

Stereology of prostatic tissue compartments from experimental groups at PND90

Groups Epithelium (%) Stroma (%) Lumen (%)
Control (n = 10) 13.99 [11.31–19.64] 37.20 [31.70–46.73] 44.64 [40.63–52.23]
Treated (n = 10) 13.39 [10.71–17.11] 39.29 [27.98–46.88] 46.13 [39.29–56.85]
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Results are expressed in percentage: median [Q1–Q3]. Statistical test: Mann–Whitney U‐test, P ≤ 0.05.

In general, the VP of adult rats is composed of a group of glandular acini with simple columnar epithelium, surrounded by a stroma composed of a tenuous layer of vascular connective tissue, surrounded by smooth muscle cells (Figure 1a).

Figure 1.

Figure 1

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Histological sections from VP of the experimental animals from control (a, c, e and g) and treated (b, d, f and, h) stained by haematoxylin–eosin (a and b); picrosirius (c and d); and reticulin (e and f). Sections submitted to immunohistochemistry to AR (g and h). Abbreviations: ep, epithelium; st, stroma; lu, lumen; arrows in c and d indicate collagen fibres; arrows in e and f indicate reticular fibres; arrows in g and h (details) indicate positive reactivity to AR; asterisk in b indicates a reactive atypia and in h (detail) shows negative reactivity to AR in atrophic epithelium.

Inflammatory foci in the stromal compartment were present in 60% of the treated animals and multifocal inflammation was observed in 40% of animals, whereas no significant inflammatory changes were observed in control animals (Figure 1a,b and Table 1). Chronic inflammation with predominance of mononuclear cells, such as lymphocytes, was present in these animals (Figure 1b). The presence of inflammatory reactive epithelial atypia adjacent to the inflammatory focus was observed in 50% of treated animals (Table 1) and was identified by the presence of stratification and epithelial dysplasia (Figure 1b).

Furthermore, changes in the distribution and compaction of collagen and reticular fibres were observed in the treated group, mainly in regions with inflammation (Figure 1e,f). There was an apparent increase in the collagen fibre layer between the acini (Figure 1c,d).

No changes were observed in the AR immunoreactivity pattern in the prostate of experimental animals (Figures 1g,h and 2). However, in some regions the immunoreactivity was apparently less intense in treated animals than in control animals, and in atrophic acini the reactivity was practically absent (Figure 1h). Epithelial cell proliferation and apoptosis indexes were not different between experimental groups at PND90 (Figures 3 and 4).

Figure 2.

Figure 2

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Semiquantitative analysis (IOD, integrated optical density) of the nuclear epithelial cells (n = 100 nuclei/group) AR immunoreactivity from VP of the experimental groups. Values are expressed as mean ± SEM.

Figure 3.

Figure 3

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Mean ± SEM of the cell proliferation index in VP of the experimental animals in PND90 (n = 10).

Figure 4.

Figure 4

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Mean ± SEM of the apoptosis index in VP of the experimental groups in PND90 (n = 10).

Discussion

Several studies have shown that environmental endocrine disruptors may affect prostatic development causing changes in the prostate of adult animals exposed during perinatal life (Cowin et al. 2008; Scarano et al. 2009). These changes included inflammatory processes and epithelial adaptive and preneoplastic lesions that emerge from an altered environment during prostate development.

The prostate develops from the pelvic part of the urogenital sinus (UGS), a tube derived from the endoderm of posterior primitive intestine (Timms et al. 1994). The UGS is morphologically indistinguishable in males and females until gestation days 17–18 in rats and mice, and around weeks 10–12 in humans. From this period, prostatic morphogenesis begins an androgen‐dependent process (Marker et al. 2003) that continues until puberty in rats (Vilamaior et al. 2006).

The effects of cadmium on the prostate have been discussed in several studies, varying doses and routes of administration (Martin et al. 2001; Arriazu et al. 2005; Lacorte et al. 2011). The bioavailability of heavy metal molecules and their permanency in a determined tissue, as for example the prostate, enhance the specific organ toxicity. The present results do not contemplate the measurement of Cd in prostate tissue but, as a comparison, in a recent study Lacorte et al. (2013) evaluated the Cd levels in prostate of Wistar rats that received aqueous solution containing 15 ppm of CdAc and found an increase of four times in Cd levels in ventral and dorsolateral prostate compared with control animals. So, it is possible to consider that the observed effects in the prostate of animals receiving Cd is due to the presence of metal in the organ.

Martin et al. (2001) found dysplastic changes in the rat ventral prostate lobe in animals treated for 18 months (80 ppm) from PND60. Moreover, Alvarez et al. (2004), using rats exposed to cadmium from PND21 (15 ppm) for 3 months, found the presence of dysplastic lesions in the ventral lobe of the prostate. Arriazu et al. (2005) started their experiment with rats at PND30, using a dose of 60 ppm for up to 24 months. They described dysplastic epithelial changes, confirming the possible role of cadmium in prostate tumorigenesis. Experimental protocols applying oral route to cadmium exposition have the advantage of being physiologically more relevant.

Lacorte et al. (2011) observed an increase in cell proliferation and in androgen receptor expression after 30 days of treatment in postpubertal rats, at the same dose used in this experiment. Our data showed that the rats exposed during prostate organogenesis, at a low dose of cadmium acetate (10 mg/l), did not show a difference in cell proliferation and the apoptosis indexes or in androgen receptor immunoreactivity compared with the control. Androgen receptor expression is essential to normal prostate morphogenesis, and its altered expression can cause abnormalities in prostate growth and differentiation (Cunha et al. 1986; Marker et al. 2003). Our results, as well as the data described by Lacorte et al. (2011), did not show a reduction in the prostate weight after cadmium treatment. This aspect reinforces that the treated animals were not affected in their ability to respond to androgens; or, if they were, they probably recovered these properties such that changes may be adaptive and/or transitional, which could explain the normality of these parameters in adulthood.

Many reports have proposed that adverse intrauterine and neonatal environments could interfere in initial development and, sometimes, delay the development of organs like the prostate (Pinho et al. 2014). However, although some structural and molecular aspects could be recovered in the long term, some consequences from this adverse environment could not (Peixoto et al. 2015).

In this sense, changes were observed, such as inflammatory focus associated with or not associated with inflammatory reactive atypia and atypical hyperplasia as described by Bernoulli et al. (2008). Previous reports from our group and others, using some EDs, have shown that the perinatal period is an essential time to induce the silent permanent changes that can modify genetic and epigenetic patterns and increase the susceptibility to diseases in adulthood (Ho et al. 2006; Cowin et al. 2008; Scarano et al. 2009; Peixoto et al. 2015).

In theory, the histopathological findings of this study are not directly related to the changes in the hypothalamic–pituitary–gonadal axis, as the serum testosterone levels were normal. Thus, they may be related to metabolic stress caused by cadmium, which causes the release of chemotactic mediators of inflammation and alters the structure of epithelial cells resulting in adaptive or permanent changes, important to adaptive and carcinogenic processes (Arriazu et al. 2005; Cowin et al. 2008).

Stromal rearrangement in the inflammation areas has been previously reported by Scarano et al. (2009), including increases in the activity of metalloproteinase 9 (MMP9) in rats treated with BPD (di‐n‐butyl‐phthalate) during the prenatal period. Lacorte et al. (2011) reported a decrease in the amount of collagen fibres in the prostate gland of animals treated with cadmium after puberty. Our results showed an increase in thickness of collagen bundles between the prostatic acini, which at first might seem contradictory. However, it is important to emphasize the treatment period used in this protocol. During foetal and neonatal periods, the prostate morphogenesis begins, and this time is essential to establishing the budding and branching of acini coordinated by mesenchyme. Changes in the pattern of development during this stage could determine the alterations in the epithelial–stromal interaction in adult glands (Timms et al. 1994; Marker et al. 2003; Thomson 2008).

The results of this study are valuable because they showed that the exposure to cadmium in low doses during prostate morphogenesis can increase the incidence of histopathological findings in adulthood, mainly those associated with inflammation, without, however, showing significant biological changes in systemic parameters such as hormone levels, or in the prostate, such as the weight of the gland or cell proliferation.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by the São Paulo State Research Foundation (FAPESP, Scholarship number 2010/05979‐7) and Foundation for the Development of UNESP (FUNDUNESP). The authors are grateful to José Eduardo Bozano for the excellent technical assistance and ProPe/UNESP for financing the English review.

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