Reproductive toxicity of 2,5‐hexanedione in male rats

Abstract

Background, Aims and Method:  2,5‐Hexanedione is an industrial solvent which causes peripheral neuropathy. In the current study, the effect of 2,5‐hexanedione on testicular histology of adult rats as well as sperm concentration, motility, and morphology were studied by administering 100 mg, 200 mg, 400 mg/kg per day for 12 weeks.

Results:  No sperm motility was observed in the 200 mg and 400 mg/kg per day treatment groups and significantly reduced motility was observed in the 100 mg/kg per day group. The morphology were also significantly reduced in the 200 mg and 400 mg/kg per day groups compared to the control group, but the sperm concentration was significantly reduced only in the 400 mg/kg per day group. Histological examination of the testes in the 400 mg/kg per day group revealed that two‐thirds of the testes had Sertoli cell only syndrome, whereas in the 200 mg/kg per day group half of the testes showed maturation arrest and sperm as well as spermatids were observed in 83% of the testes.

Conclusions:  In conclusion, we have shown that 2,5‐hexanedione severely affected sperm motility even at low doses, whereas high doses adversely affected all the sperm parameters as well as causing testicular injury. (Reprod Med Biol 2004; 3: 59–62)

INTRODUCTION

THE COMMONLY USED industrial solvents n‐hexane and methyl n‐butyl ketone produce the toxic metabolite 2,5‐hexanedione (2,5‐HD) during oxidation. Exposure to 2,5‐HD or its precursors results in a slowly progressive peripheral polyneuropathy ¹ , ² , ³ and testicular injury. ⁴ , ⁵ , ⁶ 2,5‐Hexanedione toxicity targets the Sertoli cells that produce semineferous tubular fluid and whose function is to nurture the germ cells. ⁷ Blanchard et al. showed that after 2,5‐HD‐induced testicular atrophy, exogenous administration of stem cell factor promoted recovery of spermatogenesis. ⁸

The chemical basis of the injury involves reaction of 2,5‐HD with protein amines, such as the epsilon‐amine of lysine; to form pyrroles that further react to form microtubule protein (tubulin) cross‐linking which leads to altered transport and deficient formation of semineferous fluid. Subsequently, germ cells are compromised leading to apoptosis of germ cells which continues even after cessation of the toxicity. Some studies have shown less than 1% of the semineferous tubules have germ cells reaching the spermatogonia stage 12 weeks after exposure. ⁴ , ⁷ , ⁹

Recently, deterioration in human sperm quality over the last four decades, has been observed by several researchers ¹⁰ , ¹¹ , ¹² , ¹³ , ¹⁴ , ¹⁵ and because the deterioration occurred over a short period of time, environmental rather than genetic factors have been suggested as the causative agents. ¹⁶ , ¹⁷ 2,5‐Hexanedione is one of the important environmental toxicants, ¹ , ¹⁶ but there are only a few studies on the effect of 2,5‐HD on important semen parameters such as motility, morphology and concentration. Nagano et al. exposed rats to 2,5‐HD 200 mg/kg for 3 weeks and 300 mg/kg for 5 weeks after which they analyzed the semen and found multinucleated giant spermatids. ¹⁸

In all the aforementioned studies, neither spermatid were found in the testes nor mature sperm in the epididiymides. However, Horimoto et al. treated males rats with 100 or 250 mg/kg per day for 28 days and then analyzed epididymal semen. ¹⁹ They found that sperm cells were present in normal concentrations and there was normal morphology in all mice. The only abnormal sperm parameter was reduced sperm motility.

The aim of the present study was to attempt to placate previous conflicting results on the effect of 2,5‐HD on principal sperm parameters in the 2,5‐HD treated rats.

MATERIALS AND METHODS

MALE WISTAR RATS, approximately 10 weeks old, were used in the current study. The health status of the rats were checked and then acclimated to the laboratory environment for 2 weeks before the experiment. The room temperature and relative humidity were set at 24°C and 54%, respectively. The light was controlled to provide a 12 h light cycle and a 12 h dark cycle. All the animals were kept in stainless‐steel cages and given pelleted commercial laboratory animal food and water throughout the study.

Male rats were randomly allocated to three treatment groups. Each group had six rats and each group was given 2,5‐HD subcutaneously at dose levels of 100, 200 and 400 mg/kg per day for 5 days per week (no injection given on the other 2 days i.e., resting days) for 12 weeks. The control group had 10 rats which were given only food and water throughout the experiment. The concentration of the doses were based on the most recent bodyweights. The dose levels and period were selected as most likely to cause toxicity based on preliminary studies on both testicular and nerve toxicity. ¹⁹

Evaluation of sperm motility and morphology

On day 90 of 2,5‐HD treatment, all the rats were anesthetized with diethyl ether, weighed and killed. The testis and epididymides were collected for analysis of sperm motility and count. The right‐sided epididymis was excised and placed in a pre‐warmed Petri dish containing 1 mL of calcium and magnesium free Hank's solution at 37°C. The tissue was minced with scaples for approximately 60 s and then placed in a 37°C incubator for approximately 20 min prior to determining the sperm motility. The suspension was stirred and one drop was placed on to a pre‐warmed slide and a coverslip added. A minimum of 10 microscopic fields were observed for each slide at 400× magnification using a standard optical microscope and the percentage of motile sperm were counted. For the morphological examination, another drop of the sperm suspension was added to another slide and then a thin smear was made and stained with Papanicolau and the morphology of 200 sperm cells was assessed for each slide. ²⁰

The left‐sided epididymis and testis were frozen immediately until evaluation. After thawing at room temperature, the whole epididymis and the testis specimens were homogenized separately in 1 mL of a solution of 0.9% NaCl containing 0.01 mL Triton X‐100. The testis and epididymis homogenates were each diluted with 1.5 mL of the same solution and spermatozoa and spermatids were counted 400× in a Neubauer hemocytometer (Erma, Tokyo, Japan) and three counts per sample were averaged. ²⁰

Histologic examination

Histologic examination of the testis was performed after fixing the right‐sided testis in a formalin solution. Six‐micron thick paraffin sections were stained with hematoxylin and eosin and examined by light microscopy.

Data were analyzed using the Levene's test for equality of variances. If P > 0.05, the variances were considered equal and the Student's t‐test was calculated and P < 0.05 was considered as significant. If P < 0.05, then the variances were considered unequal and a Wilcoxon signed‐ranked test was also performed and P < 0.05 was considered significant.

RESULTS

SPERM PARAMETERS ARE given in Table 1. No motile epididymal spermatozoa were found in the 200 and 400 mg/kg per day groups and sperm motility (25.0%) was significantly lower in the 100 mg/kg per day group than the control group (71.7%) (P < 0.05). Forward progressive motion was also significantly decreased in the 200 mg and 400 mg/kg per day group compared to the control group (P < 0.05). The epididymal sperm concentration in the 400 mg/kg per day group was significantly lower than the control group and there were tendencies of decreased epididymal sperm concentrations in both the 200 mg and 100 mg/kg per day group but the decrease was insignificant. The sperm morphologies in the 200 mg and 400 mg/kg per day groups were significantly lower than the control group (P < 0.05). The spermatid counts were also significantly decreased in the 200 mg and 400 mg/kg per day (P < 0.05).

Table 1.

Sperm parameters of male rats treated with 2,5‐hexanedione

Sperm parameters	Control	Exposed dose (mg/kg per day)
		100	200	400
Sperm count per epididymis (×106)	194.0 ± 63.9	109.2 ± 69.2	96.7 ± 68.3	28.5 ± 36.5*
Motility (%)	71.7 ± 17.3	25.0 ± 23.4*	0*	0*
Normal morphology (%)	88.0 ± 6.8	65.2 ± 25.5	58.2 ± 36.0*	51.1 ± 28.6*
Spermatid count per testis (×106)	87.1 ± 31.2	81.7 ± 52.4	24.4 ± 13.2*	18.5 ± 11.9*

^* Significantly different from control value at P < 0.05.

Table 2 shows that in the 400 mg/kg per day group, the histological examination of the testes revealed that two‐thirds of the rats showed Sertoli cell only syndrome (SCOS) with 100% destruction of the semineferous tubules and no germ cells were found. Leydig cell hypertrophy and occasional giant cells were seen in the tubules. Only two rats showed normal histology with all germ cells present. In the 200 mg/kg per day group, one rat showed SCOS with no germ cells, whereas 50% of the rats showed maturation arrest with very few visible spermatid, while two rats had normal histology. In the 100 mg group, one‐third of the rats showed mild semineferous tubule destruction (<10%) with normal concentrations of spermatid and the remaining two‐thirds had normal semineferous tubules. In the control group, all the rats showed normal histology.

Table 2.

Histology of the testis of male rats treated with 2,5‐hexanedione

Histology	Control	Exposed dose (mg/kg per day)
		100	200	400
Normal	100% (10/10)	83% (5/6)	33% (2/6)	33% (2/6)
Sertoli cell only syndrome	0	0	1	4
Maturation arrest	0	1	3	0

DISCUSSION

IN THE PRESENT study, subcutaneous administration of 2,5‐HD to male rats resulted in significant adverse effects in all the principal sperm parameters including complete loss of sperm motility in the 200 mg and 400 mg/kg per day and significant reduction in progressive motility and concentrations in both groups (P < 0.05). Even at a low dose of 100 mg/kg per day, both the motility and progressive motilities were significantly reduced compared to the control group and the concentration of sperm was reduced although insignificantly. The histology of these testes showed abnormal changes consisting of SCOS in two‐thirds of the testes in the 400 mg/kg per day and maturation arrest in half of the testes in the 200 mg/kg per day, whereas one‐third of the testes in each group showed normal histology. Nonetheless, despite the fact that one‐third of the testes in each treatment group showed normal histology with all the stages of germ cell maturation, yet absolutely no sperm motility was detected in the 200 mg and 400 mg/kg per day groups and was significantly reduced even in the 100 mg/kg per day group. Apparently sperm motility was the most sensitive parameter for detecting 2,5‐HD toxicity as sperm motility was severely affected even in the one‐third histologically normal testes in both the 200 mg and 400 mg/kg per day groups after 2,5‐HD treatment. We do not understand why the histologies of one‐third of the testes in both the 200 mg and 400 mg/kg per day and 83% in the 100 mg/kg per day were completely unaffected despite 12 weeks of 2,5‐HD exposure although, at the same time, the motility, concentration and morphology were significantly adversely affected. As our study is ongoing, we hope to obtain an answer when we have a large sample.

In a similar experiment, Horimoto et al. treated male rats with 100 mg and 250 mg/kg per day of 2,5‐HD, and revealed that only sperm motion was significantly reduced to 70 and 56%, respectively, but the sperm count and morphology remained normal. ¹⁹ Their findings differ from ours because we found absolutely no sperm motility in both 200 mg and 400 mg/kg per day and merely 25% motility in the 100 mg/kg per day group. Unfortunately they did not observe the histology of their specimens, as their observation would have probably explained the discrepancy between our data and theirs. Another study by Nagano et al. administered 200 mg/kg per day and during histology the finding of abnormal spermatid was only mentioned. ¹⁸ However, they did neither sperm/spermatid count nor histology of the testes which would have made comparison with our data interesting.

Also unlike our study, in human infertile men exposed to environmental solvents ¹⁴ , ¹⁵ , ¹⁶ , ²¹ , ²² , ²³ , ²⁴ the sperm motility was not as severely affected as in our study.

The histological findings in the few previous studies ⁵ , ⁶ , ⁸ , ⁹ carried out on the effect of 2,5‐HD on the testes revealed that in 2,5‐HD induced testicular injury, less than 1% of the semineferous tubules had germ cells reaching the spermatogonia stage 12 weeks after exposure. Apoptosis of spermatids occurred first at approximately 5–6 weeks followed by spermatocytes and spermatogonia between 6 and 12 weeks and no spermatid/mature sperm were seen in their studies, a finding which disagrees with both our study and Horimoto et al.’s. ¹⁹ We found spermatid in one‐third of the testis in the 400 mg/kg per day and 83% of the testis in the 200 mg/kg per day group, although, their concentrations were significantly decreased (P < 0.05). However, in the 100 mg/kg per day group, the concentration was normal indicating that spermatogenesis was severely affected at high doses of 200 mg/kg per day and 400 mg/kg per day only.

In conclusion, 2,5‐HD intoxication resulted in poor sperm characteristics and testicular injury.