Our previous studies have demonstrated that naturally occurring peptide, Nisin possess antibacterial activity and did not interfere with rabbit vaginal mucosa. In this study, the reproductive toxicity of the Nisin in male rats was evaluated. Rats were fed orally with Nisin (10, 25, and 50 mg/kg/day) for 13 weeks. No treatment related mortality was observed. The body weight gain, food consumption and serum biochemical parameters were at par with the control group. Histomorphology of the selected reproductive (testis, epididymis, ventral prostate, and seminal vesicle) and nonreproductive (liver and kidney) tissues was observed to be normal. There was no treatment-related increase or decrease in the expression of testis-specific genes (c-Kit, GATA-1, and HILS-1) and the activity levels of epididymal
Microbicides are now recognized as the most promising prevention technologies to combat the current pandemic of sexually transmitted infections (STIs) including AIDS [
A group of compounds exhibiting the above properties are naturally occurring antimicrobial peptides (AMPs). One of the well-characterized AMPs is Nisin, a 34 amino acid cationic, amphiphilic peptide belonging to the lantibiotic family [
The present study was designed to evaluate (1) the cumulative toxicity of Nisin in male rats, determine its metabolic tolerance, and also ascertain its risk/benefit profile and (2) determine the effect of Nisin on general health and reproductive performance in male rats.
Nisin (Sigma St. Louis, USA) was purified as described earlier [
Mature, 70-day-old male (mean body weight
Adult male rats were randomly divided into six groups: three controls and three treated, consisting of 10 animals per group. Nisin was administered by oral gavage, once daily at 10, 25, and 50 mg doses per kg body weight for 13 consecutive weeks. Since the parameters studied did not differ among the three groups, the results obtained with 50 mg dose are discussed in this study.
Body weights, clinical and behavioral changes of control and treated rats (10, 25 and 50 mgs/kg/day) were recorded daily during the treatment period. Control animals received only the vehicle (0.9% saline). On each data point the mean body weights of all the three treatment groups was plotted. On day 91, five animals from each group were sacrificed. Prior to autopsy, 3-4 mL of blood was collected into heparinized tubes by cardiac puncture for hematological and serum biochemical analysis. The vital nonreproductive (liver and kidney) and reproductive (testis, epididymis, ventral prostate, and seminal vesicles) tissues were excised, cleared of fat, connective tissues, and weighed to the nearest milligram. All organs were stored at −80°C until processed for histological and biochemical analysis.
The remaining five rats from each group were cohabited with proven fertile female rats in the proestrus-estrus transition phase. The mated females rats (
The tissues such as testis, epididymis, ventral prostate, seminal vesicle, liver, and kidney were isolated from five rats each of the control and treated groups. A portion of each tissue was fixed in Bouin’s solution. Paraffin sections of 5
After removal of tunica albuginea, the testis was minced and homogenized in 10 mL of NaCl containing 0.5% Triton-X100 and centrifuged at 600 ×g for 1 min. The number of homogenization-resistant spermatids was counted in a hemocytometer. Daily sperm production (DSP) was derived by dividing the total number of homogenization-resistant spermatids per testis by 6.1 days (the number of days of a seminiferous cycle in which the spermatids are present). Also, transit time in days through each of these regions was calculated by dividing the epididymal sperm number by DSP. To assess the percentage of morphologically abnormal sperm, the vas deferens was rinsed with 0.5 mL of physiological saline (0.9% NaCl) to obtain a sperm suspension. Aliquots of sperm suspension were stained with 2% eosin. Two hundred spermatozoa per animal were analyzed microscopically at 400x magnification and spermatozoa with abnormal heads and/or abnormal tails were scored.
Epididymal sperm count of the control and treated animals was determined by the method as described in the WHO manual [
The hematological parameters were analyzed using a multiparameter, automated analyzer (Swelab instruments, Stockholm, Sweden), which was standardized for rat blood [
Biochemical analysis of blood plasma samples was performed spectrophotometrically (Shimadzu-UV 160, Japan). The levels of total proteins, albumin, creatinine, uric acid, blood urea nitrogen, total cholesterol, glucose, phosphorus, sodium, potassium, chloride, calcium, alanine aminotransferase (AlAT) (EC: 2.6.11), and aspartate aminotransferase (AAT) (EC: 2.6.1.2) [
Extracts of each of the accessory reproductive organs were prepared by homogenizing preweighed tissues in distilled water. Protein levels were determined by Folin-Ciocalteu method [
After mating studies, the control and treated male rats were autopsied, testes collected aseptically and RNA was isolated using Qiagen RNA extraction kit and Trizol reagent [
Sequences of oligonucleotide primers used for PCR.
Gene | Product size (bp) | Primer sequences | Annealing temperatures |
---|---|---|---|
C-Kit | 403 | F: 5′-ACC CAC AGG TGT CCA ATT ATT C-3′ | 60°C |
GATA-1 | 242 | F: 5′-TGT GTG AAC TGT GGA GCA ACG GC-3′ | 62°C |
HILS1 | 218 | F: 5′-TGG AGT ATC TAG CAC CTG GAG T-3′ | 62°C |
Cyclophilin-A | 465 | F: 5′-AAG TTC CAA AGA CAG CAG AAA ACT-3′ | 59°C |
The total reaction mixtures (50
To determine the fertility and reproductive performance, Nisin-treated male rats were cohabited with proven fertile females. The mated female rats were monitored for duration of gestation and weight gain in the proestrus-oestrus transition phase during the first week of the treatment. At the end of gestation the litter size, number of dead fetuses, weight of pups, and their growth were recorded.
To examine the reproductive ability of male mice born to Nisin-treated parental males, F1 males were mated with normal healthy female mice. Total number of pups born per mother and sex ratio were recorded.
Repeated measures of body weight gain were determined by the analysis of covariance (ANCOVA) between the data points of the control and treated groups. Food consumption, hematological and serum biochemical profiles were evaluated by one-way Analysis of Variance (ANOVA) [
The toxicological profile of Nisin was evaluated by monitoring its effects on general health and behavior, weight changes, hematology, serum biochemical parameters, functional efficiency of different tissues, and reproductive performance. Repeated oral administration of Nisin (10, 25 and 50 mg/kg) for 13 consecutive weeks revealed no adverse effects in treated rats. Mortality did not occur, and all the animals were clinically healthy at the end of the study. Mean body weight gain and final mean body weight of the rats did not differ during the treatment period and were similar to the controls (Figure
Weight changes (mg) in various reproductive organs of male rats exposed orally with Nisin (50 mg/kg/day) for 90 days. Each value is the mean ± S.D. of five observations. No significant difference (
Testis | Epididymis | Ventral prostate | Seminal vesicle | |
---|---|---|---|---|
Control | 1710 ± 263 | 901 ± 134 | 320 ± 36 | 738 ± 65 |
Treated | 1805 ± 318 | 879 ± 92 | 333 ± 29 | 711 ± 54 |
Body weight changes in control and Nisin-treated rats. Each data point is the mean of all the three treatment groups per week. No significant difference was observed in the body weights between control and Nisin-treated groups as determined by analysis of covariance (ANCOVA) between the data points of the control and treated groups.
Histological evaluation of testis, epididymis, ventral prostate, and seminal vesicle revealed no gross changes in the morphology of testicular parenchyma, diameter of the seminiferous tubules/lumen, and thickness of the epididymal epithelium (Figure
Effect of oral exposure of male rats with Nisin (50 mg/kg/day) for 90 days on daily sperm production, sperm number in cauda epididymis, sperm transit rate and sperm morphology was determined as described in Section
Daily sperm production | Sperm number | Sperm transit rate | Abnormal sperm (%) | |
---|---|---|---|---|
Control | 40.32 ± 3.42 | 286 ± 37 | 6.34 ± 1.97 | 1.22 ± 0.13 |
Treated | 40.63 ± 4.14 | 280 ± 30 | 6.22 ± 2.13 | 0.99 ± 0.10 |
Effect of oral administration of Nisin (50 mg/kg/day) for 90 days on structural organization of testis (a, b) and epididymis (c, d) in control (a, c) and treated (b, d) rats. No treatment related abnormalities were observed. S: Epididymal sperm, L: Testicular lumen, and ET: Epididymal tubule.
The values of hematological parameters (total RBC count, haemoglobin concentration, and haematocrit) and white blood cells (lymphocytes, neutrophils, eosinophils, and basophils) were within the normal limits, and no biologically significant differences were observed between the control and treated groups. Analysis of serum biochemical parameters also did not reveal any treatment-related changes (Table
Effect of oral exposure of Nisin (50 mg/kg/day) on hematological profiles in male rats. Each value is the mean ± S.D. of five observations. No significant difference was observed between the control and treated groups.
Parameter | Control | Treated |
---|---|---|
RBC (×106/mm3) | 4212 ± 192 | 4298 ± 236 |
Hematocrit (%) | 36.88 ± 3.14 | 35.64 ± 2.11 |
Hemoglobin (g/dL) | 11.99 ± 1.34 | 12.07 ± 0.91 |
WBC (×103/mm3) | 5008 ± 491 | 5063 ± 388 |
Neutrophils (%) | 23.94 ± 1.00 | 24.01 ± 1.68 |
Monocytes (%) | 2.63 ± 0.98 | 2.71 ± 0.34 |
Lmphocytes (%) | 4.86 ± 0.27 | 4.77 ± 0.30 |
Eosinophils (%) | 2.31 ± 0.37 | 2.68 ± 0.30 |
Effect of oral exposure of Nisin (50 mg/kg/day) on serum biochemical profiles in male rats. Each value is the mean ± S.D. of five observations. No significant difference was observed between both the groups.
Parameter | Control | Treated |
---|---|---|
Total protein (g %) | 5.17 ± 0.40 | 5.34 ± 0.63 |
Albumin (gm %) | 1.21 ± 0.19 | 1.30 ± 0.28 |
Blood urea nitrogen (mg %) | 12.17 ± 0.94 | 13.01 ± 1.86 |
Creatinine (mg %) | 0.63 ± 0.10 | 0.71 ± 0.05 |
Glucose (mg %) | 87.00 ± 4.14 | 90.32 ± 5.06 |
Uric acid (mg %) | 2.11 ± 0.27 | 2.32 ± 0.30 |
Calcium (mg %) | 7.87 ± 0.91 | 8.01 ± 1.07 |
Phosphorus (mg %) | 3.07 ± 0.20 | 3.17 ± 0.19 |
Sodium (meq/L) | 123.03 ± 12.11 | 121.18 ± 9.34 |
Potassium (meq/L) | 3.11 ± 0.23 | 3.03 ± 0.18 |
Chloride (meq/L) | 81.94 ± 7.03 | 83.15 ± 7.07 |
Alanine amino transferase ( | 108.04 ± 3.53 | 1.16.23 ± 1.94 |
Asparate amino transferase ( | 77.01.94 ± 9.03 | 76.32 ± 7.14 |
Total cholesterol (mg %) | 99.04 ± 2.87 | 97.61 ± 2.58 |
The concentrations of lactic acid, fructose and the activity levels of
Effect of oral exposure of Nisin (50 mg/kg/day) on various biochemical parameters of reproductive tissues in male rats. Each value is the mean ± S.D. of six observations. No significant difference was observed between the two groups.
Epididymis | Seminal vesicle | Ventral prostate | Liver | Kidney | ||||
Lactic acid (mg/gm tissue) | Fructose (mg/gm tissue) | Alkaline phosphatase ( | AlAT | AAT | AlAT | AAT | ||
Control | 23.24 ± 1.18 | 2.17 ± 0.84 | 2.11 ± 0.76 | 30.34 ± 2.81 | 0.93 ± 0.07 | 0.67 ± 0.07 | 0.63 ± 0.05 | 0.51 ± 0.05 |
Treated | 24.19 ± 1.16 | 2.04 ± 0.71 | 2.81 ± 0.88 | 30.18 ± 3.00 | 0.89 ± 0.08 | 0.69 ± 0.06 | 0.62 ± 0.05 | 0.46 ± 0.05 |
Germ-cell-specific expression of different genes has been studied in the testis of control and Nisin-treated rats. The expression pattern of testicular germ-cell marker genes such as c-Kit (spermatogonial cells), GATA-1 (Sertoli cells), and HILSI (spermatids) was not altered in Nisin-treated animals and was found to be similar to the controls (Figure
(a) RT-PCR analysis of testicular germ-cell-specific marker genes in the control (a) and Nisin-treated (b) animals as described in Section
To assess the reproductive performance, rats treated with the highest dose of Nisin (50 mg/kg) were allowed to mate with proven fertile females. The mated female showed study increase in weight, and gestation period was also normal. No significant differences in litter size, weight, and general health of pups was noted with no perinatal or postnatal repercussions (Table
Effect of oral exposure of Nisin (50 mg/kg/day) on the evaluation of fetal parameters. Number of male rats is in parentheses. Values are mean ± S.D. of ten pregnant rats. No significant differences were observed between the control and treated rats.
Male Rats | Pregnant females | Litter size | Dead fetuses | Weight of pups (g) |
---|---|---|---|---|
Control (5) | 10 | 0 | ||
Treated (5) | 10 | 0 |
Nisin treatment did not induce any adverse effects on the subsequent fertility of the F1 progeny of both male and female offspring, born to the treated parental males when compared to the offspring born to the control animals. Total number of pups born per mother and sex ratio were not different between the two groups (data not shown).
In the present study, we have made an attempt to demonstrate the effect of Nisin on general health and reproductive performance of male rats. Nisin, a 34 amino acid naturally occurring peptide, has been shown to demonstrate antimicrobial and spermicidal properties [
Earlier reports by Hara and colleagues [
Subchronic exposure of rats to Nisin (10, 25, 50 mg/kg/day) for 13 weeks did not cause any increase and/or decrease in body and organ weights. Physical parameters such as weights of epididymis, ventral prostate and seminal vesicle, liver and kidney have been considered as gravimetric markers to assess metabolic efficiency of these organs [
Alkaline phosphatase (ALP) was analyzed as it plays an important role in the synthesis of fructose, an essential constituent of seminal plasma in ventral prostate [
Alanine aminotransferase (AlAT) and aspartate amino transferase (AAT) play an important role in the mobilization of amino acids into gluconeogenesis in the liver and kidney [
Since Nisin is known to be absorbed systemically and accumulate in vital organs in the body [
In order to determine the effect of Nisin on spermatogenesis at the molecular level, we studied the expression of a battery of cell- and stage-specific genes in the testes. Of the genes selected, c-Kit, GATA-1, HILS-1 genes play a key role in the differentiation of testicular germ cells. c-Kit a spermatogonial-cell-(SGC-) specific marker is required for germ-cell migration, proliferation, and survival [
The present data along with our previous studies suggest that Nisin does not affect the general health and reproductive performance of male rats. These observations are strongly supported by normal function of reproductive as well as nonreproductive tissues and unaltered expression of genes involved in spermatogenesis.
The authors are grateful to the Director for giving encouragement in carrying out this study. This work (NIRRH/32//2011) was supported by the Indian Council of Medical Research (ICMR), New Delhi.