The effect of quinine and ascorbic acid on rat testes.

BACKGROUND: We have previously demonstrated that quinine is a testicular toxicant in Sprague-Dawley rat. OBJECTIVE: To describe the changes in the testicular levels of testosterone and lipid peroxidation secondary to quinine and ascorbic acid administration in rats. METHODS: Twenty male Sprague-Dawley rats per group were assigned to one of three treatment groups: 0 mg quinine and 0 mg ascorbic acid/kg body weight (control); 10 mg quinine/ kg BW; and 10 mg quinine plus 0.1 mg ascorbic acid/kg BW. Rats were intramuscularly administered their respective doses of quinine five days in a week and ascorbic acid three days in a week for eight weeks. All the animals were sacrificed at the end by decapitation. Seminal analysis was performed on tubular fluid from caudal epididymides. Evaluations were made for testicular levels of testosterone and lipid peroxidation through malondialdehyde (MDA). Testicular specimens were also processed for histology under light microscopy. RESULTS: Quinine significantly (p < 0.01) increased free radicals (from elevation of MDA) and decreased testosterone in the testis compared with those of the control group and those treated with a combination of quinine and ascorbic acid. The semen of rats treated with only quinine demonstrated a significantly (p < 0.001) lower sperm concentration and motility compared to the controls and those treated with quinine plus ascorbic acid. Microscopic examination of cross-sections of seminiferous tubules also showed that ascorbic acid partially protected against quinine -induced testicular effects. CONCLUSION: Ascorbic acid has beneficial effect and protects against quinine-induced testicular reduction of testosterone.

Hypothalamic-pituitary interactions during the periovulatory secretion of follicle-stimulating hormone in the rat.

We investigated the importance of anterior afferents to the medial basal hypothalamus (MBH) on the increases in plasma FSH during the periovulatory period in the 4-day cyclic rat. We served the anterior connections to the MBH either at 1200 h on proestrus (before the time of onset of the normal spontaneous LH surge in plasma and the associated first phase of FSH release) or near the end of the LH surge and first phase of FSH release at 2000 h on proestrus (before the onset of the second or selective phase of FSH release). Analyses of FSH and LH in blood collected through indwelling atrial catheters or from the trunk after decapitation showed that anterior deafferentation of the MBH at 1200 h on proestrus blocked the proestrous LH surge, the elevations in plasma FSH during proestrus and estrus, and ovulation. In contrast, when brain surgery was delayed until 2000 h on proestrus, the second phase of FSH release and ovulation occurred. In rats with retrochiasmatic transections made at 1200 h, a constant rate iv infusion of LHRH from 1500-1800 h on proestrus restored the LH surge, both phases of increased plasma FSH, and ovulation. The results suggest that 1) the prevolutory LH surge and the first phase of FSH release are dependent on rostral afferents to the MBH which result in hypothalamic LHRH release and 2) the role of rostral afferents to the MBH in the second phase of FSH release is solely to result in hypothalamic LHRH release during proestrus.

Further studies on the effectiveness with which exogenous luteinizing hormone and follicle-stimulating hormone stimulate the release of endogenous follicle-stimulating hormone during the rat oestrous cycle.

The effects of exogenous rat LH or FSH on the release of endogenous FSH in the cyclic rat have been investigated. Rats were administered phenobarbitone to block the spontaneous increases in gonadotrophins in plasma during pro-oestrus and oestrus and then cannulated through the jugular vein or cannulated and hypophysectomized during the late morning or early afternoon of pro-oestrus. Comparison of patterns of plasma FSH in hypophysectomized and intact rats after i.v. injection of 0.5 micrograms FSH at 17.00 h suggested that exogenous FSH stimulated the release of endogenous FSH in less than 5 h. Intravenous LH (2 micrograms at 16.00 and at 18.00 h) raised the level of FSH in plasma between 2 and 6 h after the first injection of LH. Both gonadotrophins stimulated FSH release by the pituitary gland during the morning of oestrus. Comparison of patterns of plasma FSH in hypophysectomized and intact rats after i.v. injection of 0.25 or 0.05 micrograms FSH at 14.00 h suggested that the latency between FSH injection and stimulation of some FSH release by the pituitary gland is as short as 2 h. Intravenous LH (3, 4 or 9 micrograms) at 14.00 h did not increase the level of FSH in plasma within 2 h and was only minimally effective in raising the level within 4 h. Intravenous LH (2 micrograms at 16.00 and at 18.00 h) on the afternoon of dioestrus day 2 was nearly as effective in increasing the levels of FSH in plasma as it was when administered to pro-oestrous rats. This procedure did not raise the plasma levels of FSH in rats used on dioestrus day 1. The results suggest that in the phenobarbitone-blocked, pro-oestrous rat (1) a small increase (less than that observed spontaneously) in plasma rat FSH during pro-oestrus is effective in stimulating FSH release by the pituitary gland, (2) an increase in plasma rat FSH can exert positive feedback on its own secretion within 2 h and (3) a large increase in plasma rat LH is not very effective in increasing the plasma level of FSH over a period of 4 h. The results also suggest that the spontaneous increase in plasma levels of FSH and, to a lesser extent, of LH is involved in causing the selective phase of FSH release which occurs during late pro-oestrus and the morning of oestrus, and that LH and FSH act differently, but not necessarily by way of a different mechanism, to stimulate release of FSH by the pituitary gland.

Morphometric analysis of the effect of chloroquine on rat foetal lung maturation.

Pregnant Sprague-Dawley rats were injected with chloroquine phosphate (40mg/kg b. wt 1.p) in the late canalicular (day 20) and early terminal-sac (day 21) and sacrificed in the late terminal sac (day 22 = term) stage of foetal lung development. The control animal received normal saline. Morphometric evaluation of the lungs by light microscopy revealed three important exposure-related lesions. They were: (a) reduction in the volume density of parenchyma and saccular space, (b) reduction in the volume of an average saccule and, (c) an increase in the number of saccules per unit volume (Numerical Density). The observations suggest that chloroquine retards foetal lung maturation by reducing the saccular expansion which takes place immediately preterm. However, the possible practical implications are unclear.

Effect of oral contraceptive on early DMBA carcinogenesis of the rat palatal mucosa.

Experiments were undertaken to find out whether administration of oral contraceptive (OC) per nasogastric tube could accelerate or inhibit early carcinogenesis of the rat palatal mucosa induced by painting 0.5% solution of 7,12-Dimethyl benz (a) anthracene (DMBA) in liquid paraffin. Forty female Sprague-Dawley rats were divided into 8 groups consisting of 5 animals per group. Group I--DMBA; Group II--DMBA + Oral Contraceptive I (13.0gm% Norethindrone + 8.7mg% ethyl estradiol); Group III--DMBA + Oral Contraceptive II (6.5mg% Norethindrone + 4.3% ethyl estradiol). Group IV--Oral Contraceptive I, Group V--Oral Contraceptive II, Group VI--liquid paraffin, Group VII--Corn oil and Group VIII--untreated control. Each animal was sacrificed at 18 weeks, the palatal mucosa degloved, fixed in 10% formal saline, sectioned in paraffin at 5u, stained with Hematoxylin and Eosin and evaluated histologically for features of epithelial dysplasia. Morphometric analysis was performed on epithelial and keratin thickness. Results indicate higher mean grade of epithelial dysplasia for group II when compared to group I (P less than 0.01) thereby suggesting a cocarcinogenic action for the Oral Contraceptive I. Lower dose Oral Contraceptive II was, however, unable to produce any significant mean grade of dysplasia in group III, suggesting further that the cocarcinogenic action of the Oral Contraceptive used was dose dependent. Morphometric analysis shows a decrease (P less than 0.01) in Keratin and palatal epithelium in DMBA treated animals and further decrease (P less than .001) in those two structures when Oral Contraceptive was used in combination with DMBA, suggesting that the cocarcinogenic action of Oral Contraceptive could result from the induced atrophy of keratin/epithelium and subsequent improved access of the DMBA to target cells in the basal layer of the epithelium. It is suggested that Oral Contraceptive accelerated the induction of early DMBA carcinogenesis of the rat palatal mucosa in this study.

An investigation into the effects of chloroquine on fertility of male rats.

We investigated the effect of chloroquine on the fertility of adult male rats. Rats were administered chloroquine at a daily intra-peritoneal dose of 10mg/kg body weight and 40mg/kg body weight per rat, five days a week for 16 weeks. Females mated with treated males showed a dose dependent decrease in the number of litter per female rat. In the in-vitro studies, more than 80% os spermatozoa were immotile in all concentrations of chloroquine tested. These results suggest that chloroquine brings about its antifertility effect by decreasing sperm motility.

The effects of chloroquine on pulmonary saccular growth near term.

The Antimalaria drug, chloroquine is an amphiphilic cationic compound and might therefore be suspected to interfere with foetal lung maturity as previously observed with other amphilphilic cationic drugs. Single doses of chloroquine phosphate (40mg/kg b. wt) was administered to pregnant rats on days 20 and 21 of gestation (term = 22 days). Morphometric analysis revealed a decrease in volume density (Vv) of parenchyma, saccular space and average saccular volume. Concurrent administration of single doses hydrocortisone on day 21 increased the Vv of the Parenchyma saccules and average saccular volume. This result suggests that the retardation of foetal lung maturity induced by chloroquine could be reversed by a concurrent administration of hydrocortisone. Questions concerning the mechanism by which these effects are produced must remain open.

Sterological estimation of seminiferous tubular dysfunction in chloroquine treated rats.

We have used the simple-point sampling of linear intercept lengths and the optical dissector method to describe the effects of chloroquine on rat testicular morphology. The rats were injected intraperitoneally with chloroquine phosphate (CQ), an antimalaria and amphiphilic drug known to induce generalized lipidosis. The result showed that CQ (10 mg/kg/day) treatment for 7 weeks significantly reduced (i) the weight of the testis, (ii) the daily sperm production count, (iii) the total spermatoid count per testis and (iv) the star volume of the seminiferous tubules. There was however an increase in the total spermatocyte count per testis. Together, these findings suggest that disruption of spermatogenesis accompanied a decrease in tubular size in CQ treated rats.

Chloroquine-induced retardation of foetal lung maturation in rats.

Chloroquine (CQ) is a widely used drug and its administration has been reported to increase surfactant- associated phospholipids in lungs. We have used histomorphometric techniques to study the effects of CQ on foetal lung maturation in rats. CQ (40mg/kg BW) or saline was administered to pregnant rats on days 20 and 21 of gestation. A third group received the same dose of CQ on days 20 and 21, and in addition, hydrocortisone (HD; 25 mg/kg BW) on day 21 of pregnancy. Foetuses were delivered by hysterotomy on day 22. The lungs were weighed, fixed in Bouin's fixative and embedded in paraffin. Morphometric studies were performed on 5 microns-thick sections. The lung weight/100g BW, the volume density of lung saccular spaces, and the cross-sectional area and volume of the saccular spaces were reduced but the numerical density of the saccules was not decreased in foetuses exposed to CQ. With the exception of lung weights, which were lower in the foetuses exposed to CQ and HD, there were no other differences between this group and that exposed to CQ only. The results suggest that CQ attenuates the expansion of saccular spaces which occurs in preparation for post-natal gaseous exchange, and thus CQ retards foetal lung maturation. Although HD further reduced lung weights as expected from its reported action on foetal lungs, it did not reverse the CQ-induced retardation in lung maturation.

A Comparative Study of the Wound Healing Properties of Moist Exposed Burn Ointment (MEBO) and Silver Sulphadiazine.

Burns expose the deeper tissues of the skin or body to invasive microbes. Topical preparations for treating burn wounds, to be useful, should ideally have antibiotic power and promote healing. Silver compounds have been the mainstay of topical burn treatment for decades. However, most chemical substances retard wound healing. Several natural agents such as honey and moist exposed burn ointment (MEBO) are believed to protect wounds from infection and promote healing without causing any of the adverse effects of purified chemicals. In this study, we compared the wound healing properties of MEBO, a herbal preparation of Chinese origin, with silver sulphadiazine (SSD), a long-standing conventional burn dressing. Ten adult Sprague Dawley rats were divided into two groups. They were housed in separate cages and received partial-thickness burn wounds on their dorsal skin. They were then treated with MEBO and SSD. The wounds were inspected daily until day 8, when all the animals were sacrificed, perfused with normal saline, and had their wounds excised and prepared for histology. It was found that animals in both groups were well preserved. No clinical infections occurred. Wound healing was at an advanced stage by day 8 in all the animals. Clinical and histological examination showed that the two agents gave the animals comparable protection and healing possibilities. It is concluded that MEBO is a suitable and efficacious alternative to conventional silver-based topical therapies for treating partial-thickness burn wounds.

Disruption of rat estrous cyclicity by the environmental estrogen 4-tert-octylphenol.

4-tert-Octylphenol (OP) is a prevalent environmental pollutant which binds to estrogen receptors and exerts estrogenic actions in vitro. The effects of OP in vivo on mammalian female reproduction are not known. We investigated whether (i) exposure of neonatal rats to OP interfered with the onset of vaginal opening or their ability to have regular estrous cycles as adults and (ii) exposure of adult rats to OP interfered with estrous cyclicity and ovulation. Injection of 1 mg OP in corn oil sc on the day after birth did not affect the day of vaginal opening. However, 9 of 11 OP-treated rats were in persistent vaginal estrus when examined at three months after birth compared with 0 of 9 corn oil-injected controls, which cycled regularly. Ten of eleven neonatal rats injected with 1.7 mg of the estrogenic pesticide methoxychlor also were in persistent estrus at 3 months after birth, and all 10 neonatal rats injected with 1 mg of 2,4,5-trichlorophenol, which is apparently nonestrogenic, cycled regularly. Injection of 20 or 40 mg OP in corn oil vehicle sc three times weekly into previously untreated adult cyclic rats caused persistent estrus in 2 of 6 and 16 of 21 rats, respectively. Injections were continued for three more weeks in 5 of the 16 rats rendered persistent estrus by the 40 mg OP treatment. These rats remained in persistent estrus for the additional 3-week period. The other 11 persistent estrous rats in the 40 mg treatment group started to cycle regularly within 5-7 days after the last injection. Unlike pentobarbital, injection of OP into cyclic rats during the afternoon of proestrus did not block ovulation. These results provide strong evidence that OP acts like estrogen in vivo in both neonatal and adult female rats to exert effects that block reproductive cyclicity.

Effect of chloroquine on oestrus cycle and ovulation in cyclic rats.

We conducted experiments to determine the effects of chloroquine (CQ) on the oestrus cycle and ovulation in 4-day cyclic rats. There were two treatment groups. Group I animals had chloroquine phosphate (40 mg kg-1 body wt.) administered intraperitoneally (i.p.) starting from dioestrus day 1, once a day, 5 days a week for 4 weeks. Oestrus smear was monitored by daily saline vaginal lavage. Control rats received an equal volume of physiological saline. All animals were sacrificed at the end of the 4th week of treatment. Group II animals also received chloroquine phosphate (40 mg kg-1 body wt.) administered in a single dose at either 0900 h or 1800 h pro-oestrus, and on the morning of oestrus the rats were killed. Trunk blood was collected in each group at the time of sacrifice, centrifuged and the serum stored for subsequent radioimmunoassay of luteinizing hormone (LH), follicle stimulating hormone (FSH) and oestrogen. The fallopian tube was dissected out and a search made for the ova. Results showed that administration of chloroquine altered the oestrus cycle (i.e. the rats showed a persistent dioestrus smear), lowered serum oestrogen and luteinizing hormone levels while serum FSH was unaltered, prevented the expected ovulation when injected at 0900 h pro-oestrus and did not affect ovulation in rats injected at 1800 h pro-oestrus. This study supports the notion that chloroquine has an adverse effect on hypothalamo-pituitary ovarian systems.

Effects of complete hypothalamic deafferentation on the estrous phase of follicle-stimulating hormone release in the cyclic rat.

We investigated whether neural afferents to the medial basal hypothalamus play an acute role in the estrous phase of FSH release in the 4-day cyclic rat. A cannula was inserted into the right atrium of the heart under brief ether anesthesia during the early afternoon of proestrus for subsequent blood collections and injection of LHRH. In some of the rats, the medial basal hypothalamus was surgically isolated from the rest of the brain with a small knife under brief ether anesthesia between 2000 h and 2130 h of proestrus. Control groups consisted of naive rats which were not treated during the night of proestrus and sham-operated animals in which the knife was lowered to the corpus callosum between 2000 h and 2130 h or proestrus. Rats were bled at 2200 h of proestrus and at 0200 h, 0600 h and 1000 h of estrus for radioimmunoassay of plasma FSH and LH. The plasma FSH levels in all 3 groups between 2200 h of proestrus and 1000 h of estrus were elevated above levels observed in other cannulated rats bled to the onset of the proestrous phase of FSH release at 1400 h of proestrus. There were no statistically significant differences in plasma FSH or LH concentrations at any of the time periods between the 3 groups of serially bled rats. The deafferentation procedure did not appear to impair the pituitary gland's ability to secret gonadotrophins as injection of 50 ng of LHRH after the bleeding at 1000 h of estrus caused substantial elevations in plasma FSH and LH concentrations which were not different between the 3 groups. The results suggest that neural afferents to the medial basal hypothalamus play no acute role in the estrous phase of FSH release in the cyclic rat.

Plasma LH patterns after LHRH infusion in long-term, unanesthetized ovariectomized rats. Evidence for neural control of the pulsatile LH phenomenon.

Experiments were conducted in vivo to investigate further if the control of the pulsatile plasma LH phenomenon in ovariectomized (OVX) rats is located in the brain or in the adenohypophysis. Luteinizing hormone releasing hormone (LHRH) was infused at a constant rate (2--100 ng/h) through an indwelling venous cannula in unanesthetized, unrestrained OVX rats. Blood samples were collected at 5-min intervals through a second venous cannula prior to and during LHRH infusion for subsequent radioimmunoassay of plasma LH. LHRH infusion at 12.5, 50 and 100 ng/h did not interfere with the magnitude or the periodicity of LH pulses in plasma but the range within which plasma LH fluctuated was elevated. Phenobarbital (75 mg/kg BW; i.p.) blocked the pulsatile plasma LH and maintained the plasma LH nearly constant at reduced levels. Pulse i.v. injections of LHRH but not constant rate i.v. infusions restored pulsatile LH patterns in phenobarbital-treated OVX rats. The results are consistent with the view that pulsatile LHRH release is responsible for the pulsatile nature of plasma LH in OVX rats. The results do not support the concepts of a short-loop feedback of LH or an ultra-short-loop feedback of LHRH on LH secretion at least on an acute basis.

Blockade of the selective increase in serum follicle-stimulating hormone concentration in immature female rats and its effects on ovarian follicular development.

We investigated whether administration of monosodium L-glutamate (MSG) to neonatal female rats would block the selective increase in serum follicle-stimulating hormone (FSH) concentration in immature rats in an attempt to provide a model in which to study the importance of the selective FSH rise on ovarian follicular development. In two separate experiments, s.c. injections of MSG (4 mg/g BW) on Days 1, 3, 5, 7 and 9 after birth blocked the selective increase in serum FSH concentration observed on Days 7 and 15 without blocking basal FSH secretion. Serum luteinizing hormone (LH) levels were unaffected in the first experiment and changed little in the second. MSG-treated rats had smaller ovaries on Days 15 and 23. The ovaries of MSG-treated rats on Day 15 showed decreased follicular growth as evidenced by a decrease in the number and percentage of follicles with diameters greater than 50 microns, in the number of follicles with greater than 1 layer of granulosa cells, and in the number of follicles beyond the primary stage of follicular development. These differences between MSG-treated rats and controls all but disappeared by Day 23. The results demonstrate that neonatal administration of MSG blocks the selective increase in serum FSH concentration in immature female rats and suggest that this selective increase in serum FSH levels plays a role in the normal acceleration of ovarian follicular development but is not needed for the development of preovulatory follicles by the sixth week after birth.