1 Introduction

Paracetamol is a non-prescription drug commonly used for antipyresis and analgesia^[1]. It contains a phenol ring, as does oestradiol, and an aceyl group as does progesterone^[2]. These structural features suggest that paracetamol may possess sex steroid agonist or antagonist activity. Indeed, uterine, ovarian and testicular atrophy has been reported in mice fed with high doses of paracetamol^[2]. Further, an inverse association between the use of paracetamol and ovarian cancer in women has been established^[3]. Collectively, these observations indicate a possible antigonadotrophic effect of paracetamol. Further, it is now known that paracetamol can inhibit nitric oxide generation^[4], which is essential for normal reproductive activity in the male rat^[5].

Paracetamol also has a mild inhibitory action on prostaglandin synthesis in peripheral tissues^[6]. Furthermore, prostaglandin synthesis inhibitors such as fenclozic acid^[7], aspirin^[8], sodium meclofenamate^[9], naproxen^[10] or indomethecin^[11] in high doses impair fertility of male rats, but not with polyphloretin phosphate^[12], a prostaglandin receptor blocking drug. These observations raise the possibility that long-term use of high doses of paracetamol may interrupt normal reproductive function due to its antigonadotrophic, and nitric oxide and prostaglandin synthesis blocking activity. This study was designed to investigate the effects of paracetamol on sexual behaviour and fertility in male rats.

2 Materials and methods

2.1 Materials and instruments

Paracetamol (State Pharmaceutical Corporation, Colombo, Sri Lanka), methyl cellulose (Griffin & George Ltd., London, UK), oestradiol benzoate and progesterone (Sigma chemical Co., St. Louis, MO, USA), ether (BDH, Poole, UK), haematoxylene, and eosin (Fluka Chemica, Buchs, Switzerland), enzyme assay kits for the measurement of serum glutamic oxaloacetic transaminase (EC 2.6.1.1, SGOT) and serum glutamic pyruvate transaminase (EC 2.6.1.2, SGPT) from Randox Laboratories Ltd., Antrium, UK. Pelleted rat food was obtained from Vet House Ltd., Colombo, Sri Lanka. The instruments used in the study were electronic balance (Chyo Balance Corporation, Tokyo, Japan), improved Neubauer haemocytometer (Fison Scientific Equipments, Loughborough, UK), microtome (Yamato Kohki Industrial Co., Tokyo, Japan), light microscope (Olympus Optical Co., Tokyo, Japan), spectrophotometer (Jasco V500, Jasco Corporation, Tokyo, Japan), Wifug Lab centrifuge (Eltex of Sweden Ltd., Bradford, UK), organ bath (Nutsume Seisakusho Co., Tokyo, Japan), pen recorder (Rikadenki Kogyo Co.,!Tokyo, Japan), isometric sensor (Model IM-20BG, Star Medicals, Tokyo, Japan) and SRI Stimulator (Scientific and Research Instrument Ltd., London, UK).

2.2 Paracetamol preparation

Paracetamol, 500 or 1000 mg, were suspended in 1 mL of 1% methyl cellulose for animal dosing.

2.3 Animals

Adult crossbred albino rats (males weighing 225-250 g and females weighing 220-240 g) were used. They were kept under standardised animal house conditions (temperature: 28-30; 12 h light/12 h dark, relative humidity 50-55%) with free access to food pellet and tap water.

2.4 Treatment and observation

Paracetamol and vehicle were administered orally by gastric intubation at 12.00 h daily for 30 consecutive days. Rats were observed twice daily (14.00 and 16.00 h) for signs of toxicity (salivation, rhinorrhoea, lachrymation, ptosis, squinted eyes, excessive gnawing and biting movements of jaw, wilting, convulsions, stupor, tremors, rapid rotational movement of head, neck and/or entire body around the spinal axis, yellowing of fur, pallor of lips, loss of hair, tail extension in a Straub-like reaction), postural changes, stress (erection of fur, exophthalmia) and non-sexual behaviour (such as cleaning of face, self grooming, climbing in the cage, rearings). Body weights were determined on days 1 and 30 of treatment. Food and water intake, consistency of faeces and colour of urine were noted.

2.5 Effects on sexual behaviour

Randomly selected male rats were treated with 500 mg/kg (n=6) or 1000 mg/kg (n=6) paracetamol daily for 30 consecutive days. The controls (n=6) were given 1 mL vehicle per day. On day 1 (2 h after treatment) and day 30, rats were individually caged with a 10 min adaptation period. Then a female that had been brought to oestrus by injecting subcutaneously 12 g of oestradiol benzoate in olive oil 56 h prior to pairing and 0.5 mg of progesterone in olive oil 8 h prior to pairing was placed into the cage^[13]. The following parameters of masculine sexual behaviour were monitored until ejaculation or 15 min after pairing; mount latency (the time from introduction of the receptive female to the first mount), intromission latency (the time from introduction of the receptive female to the first intromission), ejaculatory latency (the time from introduction of receptive female to ejaculation), number of mounts and number of intromissions. Using these measures the following parameters were calculated: % mount, % intromission, % ejaculation, copulatory efficiency (number of intromissions/number of mounts)100, intercopulatory interval (average time between intromissions) and intromission ratio number of intromissions/(number of mounts+number of intromissions).

2.6 Effects on fertility

Male rats were randomly assigned to two groups. One group (n=12) was orally treated with 1000 mg/kg paracetamol, and the other (n=5) with 1 mL of vehicle daily for 30 consecutive days. Libido, ejaculatory ability and fertility of these rats were assessed 7 days prior to treatment, on day 30 of treatment and 30 days following cessation of treatment. Each male was paired overnight with a pro-oestrous female (at 16.30-17.00 h). The pre-coital sexual behaviour of the paired rats was observed 1-2 h later. Vaginal smears of the females were taken in the following morning (08.00-08.30 h). The presence of spermatozoa was considered day 1 of pregnancy. If spermatozoa were present, their motility was noted (qualitatively) and the numbers were estimated in duplicate using an improved Neubauer haemocytometer and the gross morphology was observed microscopically (100 and 400).

At day 14 post-coitum, the mated females were subjected to laparotomy under ether anaesthesia and the number of conceptuses (both viable and dead) were counted to permit analysis of fertility. In addition, the colour, the number and the gross morphology of the corpora lutea in each ovary were recorded.

The following reproductive parameters were then calculated: index of libido=(number mated/number paired)100; quantal pregnancy=(number pregnant/number mated)100; fertility index=(number pregnant/number paired)100; pre-implantation loss=(number of corpora lutea-number of implantations)/number of corpora luteaݡ100; post-implantation loss=(total number of implants-number of viable implants)/total number of implantsݡ100.

2.7 Effect on ejaculated sperm density

To determine the earliest day of sperm count reduction, rats treated with 1000 mg/kg paracetamol (n=6) or 1 mL vehicle (n=4) were paired 1:1 with pro-oestrous females on days 3, 7 and 17 of treatment and vaginal sperm counts were estimated.

2.8 Effect on sperm number in urine

Twelve rats were treated with 1000 mg/kg of paracetamol and another 12 with 1 mL vehicle daily for 21 consecutive days. Rats were placed individually on wooden stands attached with polythene funnels and glass bottles to collect urine samples over the treatment period as described elsewhere^[14]. Urine collected over 24 h was removed, centrifuged at 1600g for 5 min and the precipitate was resuspended in normal saline. Ten L drops were examined in duplicate microscopically for the presence and the number of spermatozoa with an improved Neubauer haemocytometer.

2.9 Effect on hyperactivated sperm motility.

Hyperactivated motility was assessed as described by Yanagimachi^[15]. Briefly, spermatozoa from the cauda epididymis of etherised rats were extruded into BWW medium. BWW 200 L was placed at the centre of a glass petri dish and covered with paraffin oil; 100 L of the sperm suspension was then added to the BWW and mixed well. Finally, 100 L of either paracetamol1 mg/mL (n=5), 0.5 mg/mL (n=14) or 0.25 mg/mL (n=12)or BWW (n=5) was added to the sperm suspension and mixed thoroughly. The final concentration of spermatozoa was 3110⁶ mL. These preparations were incubated at 37 for 3 h in 5% CO₂ in air under relative humidity of 95%. Ten L of the sperm suspension was then transferred on to a glass slide and was examined microscopically (100), and the number of capacitated sperm (showing characteristic hyperactivated motility) were counted at least in 5 fields and expressed as a percentage.

2.10 Effects on muscle strength and muscle co-ordination

Male rats (n=22) were randomly divided into two groups. One group (n=11) was orally treated with 1000 mg/kg paracetamol and the other (n=11) with 1 mL of vehicle daily for 30 consecutive days. Rats were subjected to bar holding test (to evaluate muscle strength^[15]) and Bridge test (to evaluate muscle coordination^[15]) at 5-6 h after the last administration. In the former test the time taken (in s) for the rat to fall from the bar and in the latter test the latency to slide off (in s) were recorded.

2.11 Effect on liver function

From the above rats, about 2 mL of blood was collected from the tail under mild ether anaesthesia, allowed to clot (25-30 min) at room temperature (28-30) and subjected to 15 min centrifugation (at 3200g). Serum was collected and SGOT and SGPT were determined (within 1-2 h) using a Randox enzyme kit and a spectrophotometer. All readings were taken within 10 min after incubation.

2.12 Effects on sperm counts and motility

The spermatozoa from the right cauda epididymis of rats (used in 2.10) were extracted into isotonic saline and immediately examined at 100 magnification for gross morphology and motility and the number of motile spermatozoa were counted and expressed in percentage^[5]. The vasa deferentia, cauda epididymis and caput plus corpus epididymides were homogenised separately with a known volume of isotonic saline with a ground glass homogeniser. The number of spermatozoa present was determined in duplicate as described elsewhere^[16].

2.13 Effects on external morphology and accessory gland weights

Rats (used in 2.10) were killed with ether and the animals were weighed and necropsied. The gross external morphology of the liver, kidney, testes, excurrent reproductive ducts and accessory glands was noted. The length of the testes was measured using a pair of vernier calipers. Wet weights were recorded for the paired seminal vesicles plus coagulating glands (with glandular secretions), lateral prostates, left testis, vasa deferentia, cauda epididymis, caput plus corpus epididymis, testis, and liver and kidney. The weights were expressed as a percentage of body weight. The interstitial volume of the testes was measured as described by Sharpe^[17].

2.14 Effects on histopathology of liver, cauda epididymides and testes

Small pieces of liver, cauda epididymides and testes (of rats used in 2.10) were fixed in Bouin's fluid, mounted in paraffin, sectioned at 8 m and stained with haemotoxylin and eosin and were then examined microscopically (100 and 400).

2.15 Effects on contractility of epididymal tubules and vas deferens

Eighteen rats were anaesthetised with ether and their vasa deferentia (n=9) and epididymides (n=9) removed through a 4-5 cm midline lower abdominal incision. These were immediately placed in a 25 mL glass beaker containing fresh oxygenated (95 % O₂ and 5% CO₂) physiological salt solution of the following composition (mmol/L): Na⁺, 143; K⁺, 5.8; Ca²⁺, 2.6; Mg²⁺, 1.2; Cl^-, 128; H₂PO₄^-, 1.2; HCO₃^-, 25; SO₄^2-, 1.2; and glucose 11.1.

A small portion of the cauda epididymides (approximate length 25 mm) was uncoiled at the vas deferens end and freed from the surrounding connective tissue under a magnifying lens using a fine pair of forceps as described elsewhere^[13]. These portions of the cauda epididymides were suspended in a 50 mL organ bath at 371 under a resting tension of 0.25 g.

Following an equilibration period of 15-30 min three different concentrations of the paracetamol (2, 4 and 6 g/mL) were added cumulatively to the organ bath at 5 min intervals. Contractile responses were recorded isometrically with an isometric sensor and displayed on a pen recorder.

The isolated vasa deferentia (approximate length 35-40 mm) were also suspended in organ baths under 1.0 g resting tension and were allowed to equilibrate for 30-45 min. Nerve mediated contractions were elicited using platinum ring electrodes and an SRI stimulator (for 5 s at a frequency of 5 Hz with impulses of 0.5 ms and 90 V) as described elsewhere^[13].

2.16 Statistical analyses

3 Results

3.1 General health, body weight, side effects and stress

There were no treatment-related deaths. The paracetamol treated rats showed normal food and water intake and their body weight was not significantly reduced (control vs treatment: 266.18.7 g vs 270.44.7 g). The consistency and the colour of urine of paracetamol treated rats remained essentially similar to that of controls. Further, neither doses of paracetamol induced overt signs of toxicity or stress. The non-sexual behaviour and general alertness of the paracetamol-treated rats remained unaltered.

3.2 Sexual behaviour

The results are summarised in Table 1. At 2 h, the low dose had no effect on sexual behaviour whilst the higher dose significantly inhibited the number of mounts and intromissions, and prolonged the intercopulatory interval. In contrast, on day 30, both doses of paracetamol inhibited all of the parameters of sexual behaviour investigated except copulatory efficiency with the lower dose. Further, at day 30, the magnitude of changes in the sexual behaviour induced by the two doses of paracetamol was not significantly different.

Table 1.  Effect of oral treatment of paracetamol (500 or 1000 mgkg^-1d^-1) on the sexual behaviour of male rats (n=6, meanSEM; ranges in parantheses). ^bP<0.05, ^cP<0.01, compared with controls (Mann-Whitney U-test and G-test).

4 Discussion

This study has demonstrated that chronic paracetamol treatment impairs the sexual competence and fertility of male rats. This is a novel finding, which suggests that long term consumption of paracetamol may disrupt fertility. A four-week treatment period in male rats is considered sufficient for the evaluation of drug related effects on male reproduction^[18].

This antireproductive effect of paracetamol was not accompanied with overt signs of toxicity or stress. However, there was a significant SGOT elevation and moderate leucocyte infiltration of the hepatic vessels, suggesting a damaging effect on the liver. It has been indicated that paracetamol overdose causes liver necrosis and failure^[19]. The oral LD₅₀ of paracetamol in rats is 3.71 g/kg^[19]. Its therapeutic mechanism of action is still unclear, including whether paracetamol acts peripherally, centrally or both^[14].

In the sexual behaviour study, the number of rats mounting (except for the lower dose), intromitting or ejaculating was reduced. This is generally indicative of impairment of libido^[5]. Paradoxically, there was no  impairment of the libido index. Libido is androgen dependant^[20]. No reduction in weights of the accessory organs (except the epididymis) and an apparently normal morphology of Leydig cells suggest that paracetamol was not acting through suppression of the testosterone level. The depression in the epididymal weight is more likely to be due to low sperm numbers, which could result from an increased rate of sperm transport through it. Alternatively, the reduction in the caput and corpus epididymal weight may reflect its high sensitivity to testosterone^[21]. In sharp contrast, the antilibido action may have arisen from a direct inhibition of the sexual centre in the hypothalamus of brain^[22]; paracetamol passes the blood brain barrier^[1] and is claimed to provoke its antipyretic action through a selective effect on the hypothalamus^[23].

Paracetamol caused a considerable prolongation in the latencies of mounting, intromission or ejaculation, indicative of impairment of sexual arousability/motivation. An inverse relationship exists between latencies of these three sexual parameters and sexual arousability/motivation^[24]. In addition, paracetamol treatment resulted in a marked inhibition of mounting-and-intromission frequency and copulation efficiency (only with the higher dose) and intromission ratio. The former observation is suggestive of disruption in sexual vigour^[25] and the latter, of erectile dysfunction^[26]. Furthermore, the intercopulatory interval was prolonged even with acute paracetamol treatment indicating disturbed sexual performance^[24,25].

In the mating study, paracetamol inhibited fertility reversibly. Several mechanisms appear to be mediating this antifertility action. Paracetamol induced marked oligozoospermia but without teratozoospermia. In the rat, the number of spermatozoa in the ejaculate is up to 1000-fold higher than that required for maximum fertility^[27] and thus paracetamol induced oligozoospermia by itself cannot account for its antifertility action.

Paracetamol triggered apoptosis of spermatocytes and early spermatids, together with a reduction in testicular weight and interstitial volume, indicating a mild testicular toxicity. However, overall spermatogenesis remained uninhibited. Hence, the observed oligozoospermia cannot be attributed to decreased sperm production. Paracetamol failed to inhibit nerve mediated biphasic contraction^[13] of isolated vasa deferentia. Therefore, oligozoospermia is unlikely to be due to inhibition of orgasmic contraction of vas and cauda epididymis at ejaculation. Urine analysis and histopathological studies suggest that oligozoospermia is neither due to spermatorhoea nor to enhanced epididymal sperm resorption. Sperm granulomas or any other form of obstructions were not evident in the sperm tract and thus it is not a case of obstructive oligozoospermia^[28]. Paracetamol treatment caused a drastic drop in the sperm count of the caput and corpus epididymides, which could indicate accelerated sperm transport, thereby interrupting epididymal sperm maturation. In the rat, the immediate source of spermatozoa for ejaculation is the cauda epididymis^[16]. However, the sperm count in the cauda epididymis was not reduced. The principle cause of oligozoospermia is therefore unlikely to have resulted from reduced epididymal sperm counts. On the other hand, ejaculatory dysfunction through retrograde ejaculation^[29] and enhanced spontaneous ejaculations^[30] may play a contributory role in the production of oligozoospermia in this study. However, we have no evidence in favour or against such a claim.

Paracetamol treatment caused a reduction in the intromission ratio which indicates an impairment in erectile function^[26] possibly through its nitric oxide inhibiting activity^[4]. In addition, paracetamol largely reduced the number of intromissions. In rats, a minimum number of intromissions of sufficient strength is required for a female to become pregnant^[31]. These two effects on intromission may also contribute to the inhibition in fertility evident in this study.  

Paracetamol caused a profound elevation of pre-implantation losses suggesting an impairment in the fertilizing potential of spermatozoa. Defects in sperm functions via a direct and/or an indirect action could be the main mechanism of the antifertility effect of paracetamol. Motility of both epididymal and ejaculated sperm were severely depressed which would undoubtedly impair sperm transport. It is possible that paracetamol may induce its antimotility action either acting directly on spermatozoa through its nitric oxide inhibiting activity^[4] or indirectly by altering the microenvironment of the epididymis through its inhibitory action on cyclooxygenase enzyme^[6,32]. Furthermore, an inhibition in hyperactivated sperm motility was evident with paracetamol. There is evidence that nitric oxide is important in sperm hyperactivation^[33] and therefore paracetamol could inhibit this event. Onset of hyperactivated motility is accepted to be a concomitant feature of sperm capacitation. Capacitation is mandatory for acrosome reaction, sperm ovum binding and subsequent fertilization^[34]. Paracetamol induced inhibition of hyperactivated sperm motility would be likely to interrupt these events leading to successful fertilization. Further, high doses of paracetamol can lead to lipid peroxidation^[35] and lipid peroxidation is known to impair the fertilizing potential of spermatozoa^[36].

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