Subchronic cyanide toxicity on male reproductive system of albino rat

Abstract

Sexually mature albino rats were orally treated with sodium cyanide, NaCN (0, 0.64, 1.2 and 3.2 mg kg⁻¹ BW) for 90 days. After 90 days of treatment, the rats were euthanized and male reproductive functions were assessed by histopathology, sperm head counts, sperm motility, sperm morphology and hormonal assay. Only a higher dose (3.2 mg kg⁻¹ BW) of NaCN caused significant changes in body and reproductive organs weight, sperm motility, sperm count, sperm abnormality and in the levels of luteinizing hormone (LH), follicular stimulating hormone (FSH) and testosterone, whereas the group treated with 1.2 mg kg⁻¹ BW showed significant changes in testis and prostate weight, sperm motility, sperm count, LH and testosterone levels. In contrast, insignificant changes were observed in body weight gain, reproductive organs weight, sperm parameters and hormonal levels in the rats treated with the lowest (0.64 mg kg⁻¹ BW) dose of NaCN. Histopathologically, NaCN caused atrophy, degeneration in testis, increased number of clearing cells, vacuolation in epididymis and decreased secretion, and desquamations of glandular epithelium in the prostate were observed only at higher (1.2 and 3.2 mg kg⁻¹ BW) dose levels compared to control. Whereas no changes in histology were observed in the rats treated with 0.64 mg kg⁻¹ BW. Our results suggest that a high (3.2 mg kg⁻¹ BW) dose of NaCN can exert reproductive toxicity in male Wistar albino rats.

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Introduction

Cyanide is known as one of the most rapidly acting poisons.¹ It is extensively used in industrial processes, such as electroplating, plastic, chemical synthesis and in mining operations through the milling of high grade ores and heap leaching of low grade ores throughout the world.^2–4 From all these industrial usages of cyanide, it has been released into the environment.⁵ The estimated free cyanide in industrial effluent was more than 14 million kg per year.⁶ Consequently, the discharge of such cyanide contaminated industrial effluent may lead to environmental pollution.⁷ By this means of cyanide contamination, non-target animals like humans are exposed to cyanide. Furthermore, animals and humans are exposed to cyanide by the consumption of many plants, including bitter almond, cassava, apricot etc., which, as a source of carbohydrates, contain cyanogenic glycosides that can release cyanide after metabolism.^8–10

Apart from these two above-mentioned routes of cyanide exposure, animals and humans are exposed to cyanide by cigarette smoke and smoke from industries containing the free cyanide, and by certain drugs, such as laetrile and nitroprusside also release cyanide ions after metabolism.^8,11–13 Rao et al.¹⁴ reported that globally, 26 000 people are exposed to cyanogens every day; these compounds release toxic levels of cyanide ions in the body. By all these sources, it has created complex problems in modern society and in the environment.^1,15,16

In fact, only free cyanide (i.e., the sum of molecular hydrogen cyanide, HCN, and the cyanide anion, CN⁻) is considered to be a biologically meaningful expression of cyanide toxicity, regardless of its origin.³ The toxicity results by inhibiting the enzyme, cytochrome c oxidase in the electron transport system of mitochondria and disrupting the aerobic production of adenosine triphosphate (ATP) and leads to anaerobic respiration.^17–20 However, with the help of another mitochondrial enzyme rhodanase, lower doses of cyanide can get detoxified from the body without causing any harm. The enzyme rhodanase helps to metabolize most of the absorbed cyanide to a cytotoxic metabolite, which is known as thiocyanate (SCN⁻). Consequently, Bradbury et al.¹⁰ found higher level of thiocyanate (SCN⁻) in school children consuming more cassava at the time of cassava harvest. But, SCN⁻ has specific antithyroid properties and its bioconcentration has been implicated as a possible etiologic factor in the alteration of thyroid function and development of goitre in humans and rats.^21,22

Studies demonstrated that cyanide affect spermatogenesis via the hypothalamic–pituitary–gonadal axis in male rainbow trout.²³ Kamalu²⁴ has observed that cyanide can reduce the spermatogenic cycle, testicular germ cell sloughing, degeneration and occasional abnormal cells in dogs. Whereas, several studies demonstrated that the maternal consumption of cyanogenic plants lead to the fetal malformations in pigs, horses, sheep, cattle and humans.^8,25 However, studies pertaining to the cyanide toxicity on the male reproductive system of Wistar albino rats were very rare. Two decades back in 1993, National Toxicology Program (NTP)²⁶ evaluated NaCN male reproductive toxicity in a F344/N rat strain and found that up to 100 ppm (4.5 mg kg⁻¹ BW) of cyanide can cause mild (insignificant) alteration in the male reproductive system. However, several other studies demonstrated that cyanide (dose lower than the 4.5 g kg⁻¹ BW) can induce hepatotoxicity, renal toxicity, neurotoxicity, oxidative stress in functionally different tissue.^15,20,27,28 Therefore, based on the literature review, the present study was executed to examine the hypothesis; subchronic exposure of cyanide may affect the male reproductive system in Wister albino rats.

Materials and methods

Chemicals

Sodium cyanide of 95% purity was procured from Loba Chemie Pvt. Ltd, Mumbai, India. Doses were freshly prepared by dissolving NaCN in double distilled water using a standard volumetric flask.

Animals

Sexually mature (90 days old) male Wistar albino rats weighing about 180–190 g were utilized for the present study. Animals were maintained at the animal care facility in the Department of Zoology, Karnatak University, Dharwad, in plastic cages, fed a standard laboratory ration and watered ad libitum, and exposed to a 12 h light/dark cycle, under a controlled temperature (23 ± 2 °C) and air humidity of 65 ± 5%. All animals were acclimatized for one week before the initiation of experiments and handled in accordance with the CPCSEA guidelines for the care and use of laboratory animals.

Experimental design

After the period of acclimation, animals were randomly divided into four groups of seven animals each and treated with respective doses.

Group I – Control animals (received the vehicle only).

Group II – 0.64 mg kg⁻¹ BW cyanide (this dose equals to 1/10^th of LD₅₀).

Group III – 1.2 mg kg⁻¹ BW cyanide (this dose equals to 1/5^th of LD₅₀).

Group IV – 3.2 mg kg⁻¹ BW cyanide (this dose equals to 1/2^th of LD₅₀).

The selected LD₅₀ value of NaCN was based on available literature.²⁹ The treatment was given in the morning (between 09:00 and 10:00 h) to non-fasted rats for 90 days. The dose volume equals to 1 mL/100 g BW and was treated through oral gavage.

Clinical signs

Clinical signs and behavioral changes were observed daily in all groups for attraction to feed and water, activity or depression, responsiveness to tapping at the cage wall.

Body and reproductive organs weights

After 90 days treatment, all animals were sacrificed under light ether anaesthesia and the final body weight was measured on the electric balance. The weight of reproductive organs, including testis, epididymis and prostate of respective groups were recorded after sacrificing the animals.

Sperm motility

The epididymis was collected as quickly as possible and placed in clean petri plates. The cauda epididymis was cut into several pieces and then incubated in 3 mL prewarmed phosphate buffer saline (PBS) solution at 37 °C for 10 min to allow the sperm to release from the cauda epididymis. The sperm suspensions were then evaluated for sperm motility, sperm head count and sperm morphology. The sperm suspension was pipetted several times; one drop of the suspension was placed on a slide, covered by a 22 × 22 mm coverslip. At least 10 microscopic fields were observed at 400× magnification using a phase-contrast microscope (Olympus BX51, Tokyo, Japan). The sperm were categorized on the basis of their motility as “motile” or “immotile.” The results were recorded as the percentage of sperm motility.

Epididymal sperm count

The sperm head count was determined with a hemocytometer. A sample of 0.5 mL of the sperm suspension was diluted with 9.5 mL of sperm diluting solution [5 g NaHCO₃, 1 mL formalin (35%) and 25 mg eosin per 100 mL distilled water]. Then, 10 μL of diluted sperm suspension was transferred to each counting chamber and was allowed to stand for 5 min, and the concentration of epididymal sperm was evaluated as millions of sperm cells per mL of suspension under 400× magnification using a phase contrast microscope (Olympus CH20i), calculated according to the formula:

Sperm count = Total number of sperm in 5 squares × 50 000 × 100 (Sperms mL⁻¹).

Epididymal sperm morphology

For sperm morphology, one drop of the suspension was smeared on glass slides, then air dried and stained with 1% Eosin Y. The morphological abnormalities of sperms were evaluated, from a total of two hundred sperm per animal using the criteria of Nahas et al.³⁰ and Mori et al.,³¹ and the results were recorded as the percentage of abnormal sperm.

Hormone assays

Blood samples were collected in dry glass centrifuge tubes by cardiac puncture technique under sodium pentobarbital anaesthesia (40 mg kg⁻¹). The blood was then allowed to stand for 10 min at room temperature to clot and centrifuged at 3000 rpm for 5 min at 4 °C. The serum was then collected into separate vials and subsequently subjected to the assessment of LH, FSH and T levels determined by Fully Automated Bidirectionally Interfaced Chemi Luminescent Immuno Assay.

Histopathology

For histopathological examination, the testis, epididymis and prostate gland tissues were dissected and the tissue samples were fixed in Bouin's fluid for 24 h, processed using a graded alcohol series, and embedded in paraffin wax. The paraffin blocks were cut to 5 μm thick using a semi-automated microtome (LeicaRM 2255) and sections were stained with hematoxylin and eosin (H&E) for light microscopic examination. The sections were evaluated for histopathological lesions in the testis, epididymis and prostate (Table 3) on the basis of arbitrary scores (−/+). For each slide in every case, at least 10 fields were randomly selected for such scoring and then a cumulative figure was obtained for each treatment group and photographed using a phase contrast microscope (Olympus BX51, Tokyo, Japan) with an attached photograph machine (ProgResC3, Jenoptic-Germany).

Statistical analysis

Data were analyzed using SPSS 16.0 for Windows and expressed as mean ± SEM. The significance was performed using one-way ANOVA followed by Tukey's post-doc or student's t-test. All statistical analysis was conducted at the significance level of P < 0.05.

Results

Clinical evaluations, body and reproductive organ weights

Death was not observed in all the treated and control groups throughout the experiment. There were clinical signs of toxicity observed in the behavioral activity; roaming and arrogant posture was found only in highest dose of cyanide (data not shown). Body weight gain and absolute weight of reproductive organs did not significantly change (P > 0.05) at 0.64 and 1.2 mg kg⁻¹ BW, except the testis and prostate weight at 1.2 mg kg⁻¹ BW. But the body weight gain and absolute weight of the testis, epididymis and prostate gland significantly decreased (P < 0.05) in rats treated with 3.2 mg kg⁻¹ BW of NaCN and prostate weight at 1.2 mg kg⁻¹ BW compared to the control group (Table 1).

Table 1 Effect of sodium cyanide on body and reproductive organ weight

Groups	Body weight gain (g)	Organ weight
		Testis (g)	Epididymis (g)	Prostate (g)
The values (mean ± SE) (n = 7) bearing dissimilar letters in column differ significantly (P < 0.05).
Control	96.42 ± 6.1^a	1.52 ± 0.09^a	0.39 ± 0.2^a	0.52 ± 0.09^a
0.64 mg kg^−1 BW	86.42 ± 5.08^a	1.44 ± 0.03^a	0.35 ± 0.1^a	0.47 ± 0.2^a
1.2 mg kg^−1 BW	86.53 ± 2.6^a	1.30 ± 1.2^b	0.33 ± 0.1^a	0.44 ± 0.2^b
3.2 mg kg^−1 BW	82.77 ± 3.24^b	1.38 ± 0.05^b	0.29 ± 0.2^b	0.40 ± 0.1^b

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Sperm motility, sperm head counts and morphology

There was no significant (P > 0.05) difference observed in the sperm motility, sperm head count and sperm morphological abnormality in the second group of rats treated with 0.64 mg kg⁻¹ BW. The third group treated with 1.2 mg kg⁻¹ BW showed significant (P < 0.05) changes in the sperm motility and sperm count but insignificant (P > 0.05) changes in the sperm morphological abnormality compared to control. However, in the fourth group of rats treated with 3.2 mg kg⁻¹ BW, NaCN showed significant (P < 0.05) changes in all sperm parameters, including motility, count and abnormality compared to those in the control group (Table 2).

Table 2 Subchronic effect of sodium cyanide on sperm motility, sperm morphology and abnormal sperm morphology in rats

Groups	Parameters
	Sperm count (×10^6 mL^−1)	Sperm motility (%)	Sperm abnormality (%)
The values (mean ± SE) (n = 7) bearing dissimilar letters in the column differ significantly (P < 0.05).
Control	292 ± 3.7^a	84.42 ± 2.7^a	8.67 ± 0.46^a
0.64 mg kg^−1 BW	281 ± 4.7^a	80.71 ± 3.83^a	8.73 ± 1.33^a
1.2 mg kg^−1 BW	264 ± 8.6^b	71.28 ± 2.59^b	10.23 ± 2.91^a
3.2 mg kg^−1 BW	256 ± 8.4^b	69.07 ± 5.21^b	13.25 ± 2.62^b

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Hormone concentration

There were no significant (P > 0.05) differences observed in the serum level of LH, FSH and T in the second group of rats treated with 0.64 mg kg⁻¹ BW NaCN. The third group of rats treated with 1.2 mg kg⁻¹ BW showed significant (P < 0.05) changes in serum LH and testosterone level and insignificant changes were observed in the FSH level. However, in the fourth group, significant changes were observed in the serum FSH (P < 0.05) and LH and testosterone (P < 0.01) levels compared to those in the control group (Fig. 1).

Fig. 1 Effect of NaCN on LH (A), FSH (B) and t (C) after subchronic exposure. The rats were exposed to different doses of sodium cyanide for 90 days. Bars represent mean ± SEM. the asterisks above the bar denote significantly different from compared to control (*P < 0.05, **P < 0.01).

Gross histopathology

Testis. In the control group, normal testis histology with regular seminiferous tubules and spermatogenic cell lines with abundant spermatids in the seminiferous tubules were observed (Fig. 2A). The second group treated with 0.64 mg kg⁻¹ BW showed no changes in the histoarchitecture of testis compared to that of the control (Fig. 2B). The third and fourth group of rats treated with 1.2 and 3.2 mg kg⁻¹ BW of NaCN, respectively, showed histological alteration, including atrophy, degenerated seminiferous tubules with cell debris in the lumina. In addition to this, there was a thin population of spermatogenic cells, spermatocytes, spermatids and spermatozoa in the tubules (Fig. 2C, D). All these changes are more prominent in the fourth group of rats compared to the third group (Table 3).

Fig. 2 H&E stained paraffin sections of testis; (A) shows normal histoarchitecture of testis, seminiferous tubules (St), interstitial tissue (*); (b) 0.64 mg kg⁻¹ BW NaCN dosed testis sections shows normal histoarchitecture of testis, seminiferous tubules (St), interstitial tissue (*); (C) 1.2 mg kg⁻¹ BW NaCN dosed testis sections shows vacuoles (v) in the germinal epithelial layers; (D) 3.2 mg kg⁻¹ BW NaCN dosed testis sections shows vacuoles (v) in the germinal epithelial layers, atrophy (A) and degenerated seminiferous tubule (200×).

Table 3 Histopathological changes in the testis, epididymis and prostate of experimental rats, based on scoring severity of injury in both organs

Groups	Testicular injury		Epididymis injury
	Score average (range)	Severity	Score average (range)	Severity
Scoring was done as follows: none (−), mild (+), moderate (++) and severe (+++).
Control	−	Normal	−	Normal
0.64 mg kg^−1 BW	−	Normal	−	Normal
1.2 mg kg^−1 BW	+	Mild	+	Mild
3.2 mg kg^−1 BW	++	Moderate	++	Moderate

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Epididymis. In the control group, the epididymal histology with luminal cell lines with an abundant number of sperm was observed (Fig. 3A). In the second (0.64 mg kg⁻¹ BW) group, observable difference was not seen compared to the control (Fig. 3B). In the third and fourth groups, increase in the number of clearing cells with vacuolation in the laminar cell layer was observed. In addition to this, sperm density was observed to be low compared to the control (Fig. 3C, D). These alterations were moderate in the fourth group and mild in the third group compared to those in the control group (Table 3).

Fig. 3 H&E stained paraffin sections of epididymis; (A) shows normal histoarchitecture of epididymis tubules with bulk of sperm (Sp); (B) 0.64 mg kg⁻¹ BW NaCN dosed epididymis shows normal histoarchitecture as seen in control group; (C) 1.2 mg kg⁻¹ BW NaCN dosed epididymis low density of sperm; (D) 3.2 mg kg⁻¹ BW NaCN dosed epididymis shows low sperm density and constricted at center, vacuoles (v) in the germinal epithelial cell lining (200×).

Prostate gland. In the control group, normal prostate histology with normal luminal cell lines with an abundant amount of prostate secretion was observed (Fig. 4A). The second group of rats treated with 0.64 mg kg⁻¹ BW showed normal histoarchitecture as the control prostate (Fig. 4B). However, the third and fourth groups treated with 1.2 and 3.2 mg kg⁻¹ BW, respectively, show decreased prostate secretion in the lumen (Fig. 4C, D); desquamations of glandular epithelium were only observed at 3.2 mg kg⁻¹ BW (Fig. 4D).

Fig. 4 H&E stained paraffin sections of prostate gland; (A) shows normal histoarchitecture of prostate lumen with bulk of prostate secretion (Ps); (B) 0.64 mg kg⁻¹ BW NaCN dosed rat prostate shows normal histoarchitecture; (C) 1.2 mg kg⁻¹ BW NaCN dosed rat prostate shows low secretion (arrow head); (D) 3.2 mg kg⁻¹ BW NaCN dosed rat prostate shows low secretion, desquamation of glandular epithelium (arrow) (200×).

Discussion

The gold mines and cyanide using industries brought with them not only development, employment and wealth, but also the most overwhelming changes in nature, such as pollution, negative health impacts and ecological destruction.³² Clark and Hothem³³ have reported that the industrial effluent and metal processing pound contained free cyanide along with metallocyanide complex concentrations ranging from 0.3 to 216 ppm and 200 to 300 ppm respectively. Thus, in the present study, the first two chosen doses (0.64 and 1.2 mg kg⁻¹ BW) are considered to be environmentally relevant.³⁴ However, the labourers working in these industries have every chance of cyanide exposure. Furthermore, occupational and dietary exposure to cyanide occurs by the large scale cassava processing and ingestion of cassava based food products.^35,36 In view of this, the subchronic (90 days) effect of cyanide (NaCN) toxicity at sublethal doses (0.64, 1.2 and 3.2 mg kg⁻¹ BW) were evaluated on the male reproductive system of the albino rat.

This study was strongly supported by NTP study on NaCN.²⁶ However, the differences were the use of a different rat strain and different selected dose levels. Wister strain rats were used in the present study, while the F344/N strain was used in the NTP study. The higher dose (3.2 mg kg⁻¹ BW) tested in the present study was lower than the dose (100 ppm/4.5 mg kg⁻¹ BW) used in the NTP study. We evaluated hormonal levels as they play a very important role in substantiation in male reproductive toxicity assessment. The results of the present study reveal that Wister strain male albino rats were more susceptible to cyanide ions and male reproductive organs may have low cyanide detoxification capacity. These findings agree with Kimani et al.,²⁰ as these findings demonstrate that the cyanide detoxification mechanism varies from species to species and tissue to tissue.

The lower dose (0.64 mg kg⁻¹ BW) in the present study induced no significant (P > 0.05) alteration in body weight gain or reproductive organs weight. However, the third group of rats treated with 1.2 mg kg⁻¹ BW showed significant reduction only in the prostate weight compared to control (Table 1). While the fourth group treated with 3.2 mg kg⁻¹ BW showed significant (P < 0.05) changes in body weight gain and reproductive organ weights compared to control. And there were some behavioural changes, including posture, activity, roaming and arrogance observed in the higher dose treated group, but no mortality was found throughout the experiment. On the other hand, diet consumption was comparably the same in all the treated groups compared to the control group. The histopathological changes observed in the testis, epididymis and prostate gland may be attributed to the reduction of organ weight following the NaCN treatment. These finding are consistent with previous studies that show cyanide causing poor body weight gain.^20,37

Results from the current study showed that lower doses (0.64 mg kg⁻¹ BW) of cyanide may not be a potential cause of the male reproductive toxicity in albino rats. This may be due to the existing detoxifying mechanism, which involves the mitochondrial enzyme rhodanase. Rhodanase can catalyze the reaction between CN⁻ and thiosulfate to produce SCN⁻.²⁰ In which, it transfers a sulfur atom to CN⁻.³⁸ And SCN⁻ is approximately 120 times less toxic than CN⁻ and is excreted over several days.^39,40 In turn, cyanide can rapidly detoxify and enable animals to ingest sublethal doses of cyanide over extended periods without harm. However, the liver is highly sensitive to cyanide and high doses of cyanide are beyond the detoxification capacity of the rhodanase system in the liver.^14,41

The hormonal secretion from the pituitary facilitates the normal spermatogenesis by the paracrine and autocrine regulation of various components in the testis. Testosterone is the most important hormone involved in the regulation of the alterations observed in the testis of male rat, which is regulated by the pituitary-secreted gonadotropin hormones. Testosterone plays a key role in the regulation of spermatogenesis, together with gonadotropins. Its secretion from the Leydig cells is dependent on the secretion of LH from the pituitary gland.⁴² In the present study, the significant reduction in serum LH levels of the third (P < 0.05) and fourth (P < 0.01) group rats is a sign towards the possible effect of NaCN on the hypothalamus (Fig. 1B). This may be attributed to the dysfunction of Leydig cells, which will lead to the decrease in the synthesis of testosterone in the testis (Fig. 1C). The FSH level significantly declined in the fourth group treated with 3.2 mg kg⁻¹ BW (Fig. 1A). However, FSH was a very important hormone required for the proliferation of Sertoli cells. Moreover, the same was evidenced by histopathological changes observed in the testis of the rats treated with higher doses (1.2 and 3.2 mg kg⁻¹ BW) (Fig. 2C, D). These findings were in consistence with the earlier reports.^43,44 Further, Sylvia et al.²³ demonstrated that subchronic cyanide treatment affects the spermatogenesis cycle through the hypothalamic–pituitary–gonadal axis in the male rainbow trout.

In the present study, it was observed that the rats treated with 1.2 and 3.2 mg kg⁻¹ BW caused histopathological changes in testis, including atrophy and degenerated seminiferous tubules with cell debris in the lumina (Fig. 2C, D). Ultimately, these changes have affected the spermatogonial cells, primary spermatid, secondary spermatid and spermatozoa. These histological changes in the testis caused by NaCN led to low sperm counts and increased sperm abnormality (Table 2). Observed histopathological changes in the testis and alteration in sperm parameters may be attributed to oxidative stress and anaerobic cellular respiration.^45,46 Studies have demonstrated that cyanide induces oxidative stress in the functionally different tissues of rats.⁴⁷ Another possible cause for decreased sperm count, sperm motility and increased abnormal sperm in higher dose treated rats was decreased availability of androgens (Fig. 1B, C). Sperms leaving the testis are not physiologically mature, and such maturation occurs during their epididymal transit; after maturation, they become motile.⁴⁸ The changes in the epididymis essentially involve the addition and modification of proteins by principal cells and the removal of existing proteins by clearing cells.⁴⁹ These changes in the epididymal create an appropriate environment for spermatozoa to become mature.⁵⁰ It implies that any histological changes in the epididymis may affect the maturation of the sperm. The notable histopathological changes include an increased number of clearing cells and vacuolation in the epithelial cell layer of the epididymis at higher dose. (Fig. 3A, B). Sperm motility is a very important feature as it provides fertilizing capacity. Any negative impact on motility would seriously affect the fertilizing ability of sperms.⁵¹ Studies demonstrate that cyanide can cause depletion in ATP synthesis in the spermatozoa of crab by inhabiting the enzyme necessary for ATP synthesis.⁵² ATP plays a crucial role in the forward movement of sperm.⁵³ Thus, reduced sperm motility on exposure to NaCN may be due to changes in the ATP pool. However, the secretion of the prostate is required to activate the sperms to fertilize the ovum; the prostate in turn requires androgen for differentiation, development, and maintenance of epithelial cells.^54,55 Decreased prostate secretion in the rats treated with 1.2 and 3.2 mg kg⁻¹ BW (Fig. 4C, D) may be due to the decreased serum testosterone level (Fig. 1C). Desquamation of tubuloalveolar glandular epithelial cells of prostate in the rats treated with 3.2 mg kg⁻¹ BW (Fig. 4D) could be attributed to either the decreased serum testosterone level or may be oxidative stress induced by NaCN in the current study.

From the results, we conclude that subchronic exposure to low doses of cyanide may produce mild effects; however, the high dose (3.2 mg kg⁻¹ BW) tested in the present study, induces an adverse effect on the male reproductive system in Wister strain albino rats. However, humans are more sensitive to the cyanide ions, subchronic exposure to the lower dose of cyanide may lead to infertility in males. Therefore, care has to be taken while using cyanide in industries.

Acknowledgements

The first author is thankful to Karnatak University, Dharwad for awarding University Research Fellowship (KU/SC/URS/2011–2012/20045) and University Grant commission for Research Fellowship in Science for Meritorious Student [F.4-1/2006(BRS)/7-102/2007(BRS] and the authors are also thankful to the Department of Zoology, Karnatak University, Dharwad, Karnataka, India to carry the present work.

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