Abnormalities of sexual development in male rats with in utero and lactational exposure to the antiandrogenic plasticizer Di(2-ethylhexyl) phthalate.

Several members of the phthalate ester family have antiandrogenic properties, yet little is known about how exposure to these ubiquitous environmental contaminants early in development may affect sexual development. We conducted experiments to determine effects of in utero and lactational exposure to the most prevalent phthalate ester, di(2-ethylhexyl) phthalate (DEHP), on male reproductive system development and sexual behavior. Sprague-Dawley rats were dosed with corn oil or DEHP (0, 375, 750, or 1,500 mg/kg/day, per os) from gestation day 3 through postnatal day (PND) 21. Dose-related effects on male offspring included reduced anogenital distance, areola and nipple retention, undescended testes, and permanently incomplete preputial separation. Testis, epididymis, glans penis, ventral prostate, dorsolateral prostate, anterior prostate, and seminal vesicle weights were reduced at PND 21, 63, and/or 105-112. Additional dose-related effects included a high incidence of anterior prostate agenesis, a lower incidence of partial or complete ventral prostate agenesis, occasional dorsolateral prostate and seminal vesicle agenesis, reduced sperm counts, and testicular, epididymal, and penile malformations. Many DEHP-exposed males were sexually inactive in the presence of receptive control females, but sexual inactivity did not correlate with abnormal male reproductive organs. These results suggest that in utero and lactational DEHP exposure also inhibited sexually dimorphic central nervous system development. No major abnormalities were found in any of eight control litters, but DEHP caused severe male reproductive system toxicity in five of eight litters at 375 mg/kg/day, seven of eight litters at 750 mg/kg/day, and five of five litters at 1,500 mg/kg/day. These results demonstrate that the male reproductive system is far more sensitive to DEHP early in development than when animals are exposed as juveniles or adults. The effects of DEHP on male reproductive organs and sexual behaviors and the lack of significant effects on time to vaginal opening and first estrus in their littermates demonstrate that DEHP (and/or its metabolites) affects development of the male reproductive system primarily by acting as an antiandrogen. The pattern of effects of in utero and lactational DEHP exposure differed from patterns caused by other phthalate esters, and the preponderance of anterior prostate agenesis appears to be unique among all chemicals. These results suggest that DEHP acts partly by mechanisms distinct from those of other antiandrogens.

Maximizing profitability in a hospital outpatient pharmacy.

This paper describes the strategies employed to increase the profitability of an existing ambulatory pharmacy operated by the hospital. Methods to generate new revenue including implementation of a home parenteral therapy program, a home enteral therapy program, a durable medical equipment service, and home care disposable sales are described. Programs to maximize existing revenue sources such as increasing the capture rate on discharge prescriptions, increasing "walk-in" prescription traffic and increasing HMO prescription volumes are discussed. A method utilized to reduce drug expenditures is also presented. By minimizing expenses and increasing the revenues for the ambulatory pharmacy operation, net profit increased from +26,000 to over +140,000 in one year.

Comparative pyrogenic potency of endogenous prostanoids and of prostanoid-mimetics injected into the anterior hypothalamic/preoptic region of the cat.

In both pyrogen-induced fever and fever subsequent to acute hypothalamic trauma, pyrexia is believed to be mediated by cyclooxygenase products acting within the anterior hypothalamic/preoptic (AH/PO) region of the brain. The goal of the present study was to assess, through a potency analysis, the likely contributions of various prostanoids to pyrexia production. Prostanoids and prostanoid-mimetics were injected bilaterally into the AH/PO region of conscious, indomethacin pretreated cats, and partial dose-response curves for pyrexic activity were obtained. ED1 degrees doses (doses producing a 1 degree C fever) for PGE2, PGE1 and 6-keto-PGE1 (a metabolite of PGI2 and/or of the PGI2 hydrolysis product, 6-keto-PGF1 alpha) ranged between 2 and 15 pmol. PGF2 alpha and the stable PGI2-mimetics, iloprost and 6-beta-PGI1, required doses of 900-1100 pmol. PGD2 and 6-keto-PGF1 alpha had ED1 degrees doses of 2200-2400 pmol. PGI2, thromboxane (TX) B2 and the TXA2/PGH2-mimetics, SQ26655, 9,11-azo-PGH2 and U46619, were incapable of producing a 1 degrees C rise at the maximum dose of 30,000 pmol. The results offer no support for an involvement in fever of PGF2 alpha, PGD2, TXA2, TXB2, PGH2, PGI2 or 6-keto-PGF1 alpha. Only the 3 E-series prostaglandins were sufficiently potent to merit serious consideration as mediators of pyrexia. Of these, only PGE2 is known to be produced in abundance by cat brain; no information is available regarding PGE1 production, and our results with PGI2 and 6-keto-PGF1 alpha indicate that cat brain may not synthesize 6-keto-PGE1. The results thus suggest an important role for PGE2 in fever production in the cat and are compatible with an involvement of PGE1.

Examination of the subdiencephalic rat brain for sites mediating PGE1-induced pyrexia.

The subdiencephalic rat brain was mapped for sites capable of mediating prostaglandin-induced pyrexia. In conscious rats, PGE1, 200 ng in a volume of 1 microliter, was injected unilaterally into 412 sites between the midmesencephalon and the caudal medulla. Injections into only 12 sites caused a reproducible, short-latency core temperature increase of at least 0.5 degrees C. None of these was located in the paramedian brainstem, which was considered a likely site of PGE1 action because of the presence there of thermosensitive and pyrogen-sensitive neurons. Rather, the reactive loci were found in the hippocampus (5 sites) and in the vicinity of the cochlear nuclei (7 sites). Injections into only 2 sites in the latter region failed to produce pyrexia. In the hippocampus, however, injections at 31 sites in the same frontal planes as the reactive loci produced no effect. The possibility that the active hippocampal sites were associated with a distribution of injectate to PGE1-sensitive neurons located within hippocampal cleavage planes rather than in a circumscribed region is discussed.

Studies on the pyrexic effect of PGE1 injected into the region of the cochlear nuclei.

In 30 of 33 unanesthetized rats, unilateral injection of PGE1 (100 ng in 1 microliter) into or near the cochlear nuclei (CN) produced a body temperature increase of at least 0.5 degrees C. Usually, the rise started within the first minute after injection. Bilateral destruction of the highly PGE1-sensitive anterior hypothalamic/preoptic region (AH/PO) eliminated the response. In approximately two-thirds of rats in which concentrated dye or 3H-PGE1 was injected into CN, there was evidence that a small portion of the injectate had rapid access to the AH/PO. In the remaining rats, the tracer material did not reach AH/PO tissue. In the rats involved in the tracer studies, there was no correlation between the magnitude of the response to PGE1 injected into CN and the extent of transport of tracer from CN to AH/PO. The results suggest that PGE1 injected into CN produced pyrexia through an action on tissue at or near the injection site and that neuronal pathways in AH/PO are necessary for the expression of the response.

Assessment of the pyrogenic potency of the thromboxane A2-mimetics, SQ26655 and U46619, and thromboxane B2 by intrapreoptic injection in the cat.

Using bilateral injection into the anterior hypothalamic/preoptic region of the conscious, indomethacin pretreated cat as an assay system, the pyrogenic activity of two thromboxane A2-mimetics (SQ26655 and U46619) and thromboxane B2 was assessed. PGE2 was used as a reference pyrogenic agent. Thromboxane B2 and both of the thromboxane A2-mimetics were at least 5263 times less potent than PGE2. The results do not support the putative role of thromboxanes as mediators of fever.

Intracerebroventricular and preoptic injections of leukotrienes C4, D4, and E4 in the rat: lack of febrile effect.

Experiments were performed to ascertain the effect on core temperature in the rat of central administration of 3 products of the lipoxygenase pathway of arachidonate metabolism. The agents tested were leukotrienes C4, D4, and E4 (LTC4, LTD4, LTE4). In one series of rats, the leukotrienes were injected into the ventral aspect of the third cerebral ventricle (5 microliter injection volume). Each rat received, in separate experimental sessions, an injection of a control solution, of 1 microgram of prostaglandin E1 (PGE1) and of 1 microgram of LTC4, LTD4, or LTE4. In another series of rats, bilateral 1 microliter injections into the tissue of the preoptic region were made. Each animal received a control solution, 40 ng PGE1 (20 ng/side) and 400 ng LTC4, LTD4, or LTE4 (200 ng/side). Neither the intraventricular nor the preoptic injections of any of the leukotrienes produced a significant increase in colonic temperature. However, PGE1 injected intraventricularly or into the preoptic region produced a large, rapidly developing core temperature rise. The strong febrile response to PGE1 and the results of dye distribution studies indicate that the lack of effect of the leukotrienes was not due to incorrect injection cannula placement. The ineffectiveness of the leukotrienes also cannot be attributed to loss of biological activity of these agents during storage. Near the end of the study, samples of each leukotriene were assayed using the guinea pig tracheal strip method and were found to be highly active. The results suggest that, at least in the rat, these 3 arachidonate metabolites are not likely to be important mediators of fever.

Sites and mechanism of action for the effects of intrathecal noradrenaline on thermoregulation in the rat.

1. In unanaesthetized rats, intrathecal injection of 0.30 mumole noradrenaline (NA) at the level of the lumbar enlargement produced a transient rise in core temperature followed by prolonged hypothermia and tail skin vasodilation.2. Studies of the distribution of [(3)H]NA injected at the lumbar enlargement revealed that at least 97% of the activity recovered from the central nervous system was located in the spinal cord, primarily within the thoracic and upper lumbar segments. Thus, the thermoregulatory effects of intrathecal NA are not likely to be due to an action at supraspinal sites.3. Studies with [(3)H]NA also indicated that significant levels of unmetabolized labelled NA were present in plasma as early as 3 min after intrathecal injection. However, by 15 min after injection, 75% of this radioactivity had disappeared. There was a good temporal correlation between the transient appearance of high levels of NA in plasma and the initial hyperthermic effect of NA. Moreover, the hyperthermia was not inhibited by mecamylamine-induced ganglionic blockade. These results indicate that the initial hyperthermic effect of intrathecal NA is due to a direct action of this monoamine at peripheral sites subsequent to leakage from the spinal subarachnoid space.4. In anaesthetized rats, NA (0.30 mumole) and clonidine (0.035 mumole) injected intrathecally at the lumbar enlargement produced a sustained decrease in neural activity recorded from the lumbar sympathetic chain, a finding which suggests that the vasodilation and hypothermia produced by intrathecal NA are due to an inhibition of sympathetic outflow.5. To investigate the spinal site of action of NA on thermoregulation, rats were prepared with spinal catheters which extended either to the upper cervical region or the lower sacral area. Studies with [(3)H]NA showed that these modifications of the catheter lengths altered the accessability of NA to the intermediolateral nucleus (i.m.l.) of the spinal cord. Injections of NA through the cervical and sacral catheters elicited thermoregulatory effects which differed from those elicited by injections near the lumbar enlargement. The differences were consistent with the hypothesis that the hypothermia and tail skin vasodilation elicited by NA injected at the lumbar enlargement are mediated, at least in part, via a direct inhibitory effect of this monoamine on sympathetic preganglionic neurones located in the i.m.l.6. The effect on mean arterial blood pressure of intrathecal injection of NA (0.30 mumole) at the lumbar enlargement was examined in unanaesthetized rats fitted with chronic arterial catheters. These injections produced an immediate increase in blood pressure. However, this effect was transient and, during most of the time when NA-induced vasodilation and hypothermia were present, blood pressure was normal or only slightly elevated. Thus, it is not likely that a baroreceptor-mediated reflex inhibition of sympathetic outflow contributed significantly to the vasodilatory or hypothermic action of NA.

The thermoregulatory effects of noradrenaline, serotonin and carbachol injected into the rat spinal subarachnoid space.

1. We have examined the effects on thermoregulation in the rat of noradrenaline bitartrate (NA), 5-hydroxytryptamine hydrochloride (5-HT) and carbamylcholine chloride (CCh) injected into the lumbar spinal subarachnoid space via a chronic indwelling catheter.2. Intrathecal injections of the monoamines and CCh reproducibly affected thermoregulation, whereas injections of control solutions had no effect.3. Intrathecal injections of NA (0.01-0.30 mumol) produced a dose-dependent hypothermia associated with a decrease in tail skin vasomotor tone. Shivering activity was not depressed during the hypothermia and sometimes increased. Intrathecal administration of the alpha-adrenergic agonist clonidine (0.0175-0.070 mumol) elicited changes in T(c) and T(sk) similar to those induced by intrathecal NA.4. Intrathecal 5-HT (0.030-0.90 mumol) elicited a dose-dependent hyperthermia accompanied by increased tail skin vasomotor tone and increased shivering.5. CCh injected intrathecally (0.001-0.06 mumol) evoked a dose-dependent hyperthermia. During the period when core temperature was rising, tail skin vasomotor tone increased and shivering-like activity was present. Once the maximum core temperature had been reached, tail skin vasodilatation occurred. Vasodilatation persisted until core temperature had returned to normal.6. Intravenous injections of 5-HT (0.30 and 0.90 mumol) or CCh (0.006 and 0.03 mumol) caused no thermoregulatory effect. The effects of these agents injected intrathecally were therefore not due to an action in the periphery.7. Intravenous infusions of NA (0.06 and 0.10 mumol) produced hypothermia and transient tail skin vasodilatation. We suggest that an action at peripheral sites may have contributed to the effects produced by intrathecal injection of this monamine.8. These findings suggest that spinal noradrenergic, serotonergic and cholinergic synapses may be importantly involved in the control of body temperature in the rat. The possible functional roles of these synapses and the putative spinal sites of action of the injected substances are discussed.

The effects of intrathecal sympathomimetic agents on neural activity in the lumbar sympathetic chain of rats.

Intrathecal injections of norepinephrine (0.30 mumol) and clonidine (0.035 mumol) produced decreases in neural activity recorded from the lumbar sympathetic chain and caused changes in rat thermoregulation consistent with a reduction in efferent sympathetic activity. These results support the conclusion that bulbospinal noradrenergic pathways inhibit sympathetic outflow. The data also indicate that the spinal cord is a potential site of action for clonidine.

Multiplicative interaction between narcotic agonisms expressed at spinal and supraspinal sites of antinociceptive action as revealed by concurrent intrathecal and intracerebroventricular injections of morphine.

In rats in which the fourth ventricular exits had been acutely occluded, morphine sulfate was injected concomitantly into the spinal subarachnoid space (0-10 micrograms/4 microliter) and into the third cerebral ventricle (0-50 micrograms/5 microliter). The combinations of intrathecal (i.t.) and intracerebroventricular (i.v.t.) dosages used were selected to yield particular ratios of supraspinal to spinal (SS:S) agonisms. Dose-response lines for both the tail-flick and hot plate responses were constructed for each SS:S ratio, with the abscissa representing i.v.t. morphine dosage. It was observed that the analgetic potency of morphine injected i.v.t. was profoundly potentiated by the concurrent administration of morphine i.t. Dose-response lines for i.v.t. morphine were shifted progressively to the left as the spinal dose of morphine was increased. At the optimal balance of spinal and supraspinal dosage (SS:S = 1:1), the ED50 values for i.v.t. morphine for the hot plate and tail-flick tests were reduced by factors of 45 and 29, respectively. A similar, but less profound, potentiation of the analgetic potency of morphine injected i.t. by concurrent administration of morphine i.v.t. was observed. Isobolographic analysis of the data revealed that the isobols were hyperbolas having extreme negative curvature of all effect levels. Inspection of the isobols indicated that, at all ratios of spinal to supraspinal agonism which could conceivably be obtained when morphine is given systemically, the spinal-supraspinal interaction is multiplicative. The results suggest that narcotic agonism at both spinal and supraspinal narcotic-sensitive sites is essential to the production of analgesia by systemically administered morphine and that neither site can logically be deemed the "primary" site of narcotic action.

Sites of antinociceptive action of systemically injected morphine: involvement of supraspinal loci as revealed by intracerebroventricular injection of naloxone.

Dose-response lines for the tail-flick and hot plate tests were obtained in rats which had received systemic injections of morphine (10-150 mg/kg i.p.) and intracerebroventricular (i.v.t.) injections of naloxone (3-20 micrograms). Diffusion studies indicated that the antagonist remained localized within supraspinal structures. Between the doses of 3 and 10 micrograms i.v.t. naloxone produced a dose-dependent rightward shifting of the morphine dose-response lines, the shift produced by the 10-micrograms dose being sufficient to abolish the analgetic action of morphine doses as large as 75 mg/kg. Higher doses of naloxone (e.g., 20 micrograms) produced no additional rightward shift, indicating an inability of i.v.t. naloxone to antagonize the analgesia produced by high systemic doses of morphine. These findings seem to suggest that analgesia produced by low to moderate systemic doses of morphine is mediated entirely by an action of the narcotic upon supraspinal structures. However, the results of an analogous study in which naloxone was injected into the spinal subarachnoid space indicated that analgesia produced by morphine given systemically in low to moderate doses is mediated exclusively by an action on the spinal cord. This apparent paradox can be resolved if one assumes that the total analgesia observed after a low to moderate systemic dose of morphine is a result of a multiplicative, rather than an additive, interaction of narcotic agonisms expressed at the spinal and supraspinal sites of action, respectively.

Thermoregulatory characteristics of neurogenic hyperthermia in the rat.

1. The thermoregulatory characteristics of the neurogenic hyperthermia produced in rats by unilateral mechanical destruction of the rostral hypothalamic/preoptic region were studied. 2. The investigational methods employed included (a) observing the thermoregulatory effector activities which were responsible for generation of hyperthermia, (b) observing the thermoregulatory reactions elicited by forcefully elevating or lowering core temperature during neurogenic hyperthermia and (c) observing the effect of ambient temperature on hyperthermia magnitude. 3. At 26 degrees C, hyperthermia was effected by a transient increase in shivering thermogenesis and a concomitant minimization of heat loss through the tail. 4. At 26 degrees C, perturbations of core temperature during the plateau phase of hyperthermia were induced by internal or external heating and cooling. The disturbances elicited compensatory changes in shivering activity and in tail vasomotor tonus, and core temperature was rapidly and precisely returned to its pre-perturbation level. 5. The magnitudes of hyperthermias experienced by rats lesioned at 10, 15, 26 and 32 degrees C, as measured by the change in colonic temperature and by the area under the fever curve, were not significantly different. At 36 degrees C, rats were hyperthermic prior to lesioning, and the magnitude of the lesion-induced hyperthermia was significantly attenuated. 6. The results indicate that the neurogenic hyperthermia produced by unilateral hypothalamic puncture in the rat is generated by a coordinated modulation of thermogenic and heat retentive effectors and that the plateau level of hyperthermia is well regulated. These characteristics are compatible with the hypothesis that neurogenic hyperthermia is mediated by prostaglandins released from injured tissue and acting on surviving rostral hypothalamic tissue.

A dose ratio comparison of the interaction between morphine and cyclazocine with naloxone in rhesus monkeys on the shock titration task.

Dose-response curves for morphine (0.5-20 mg/kg) were obtained in 4 rhesus monkeys performing the discrete trials shock titration task. Naloxone (5-500 microgram/kg) uniformly produced a dose dependent, parallel shift of the morphine dose-response curves to right. Plots for the 4 animals of the Log (dose ratio--1) versus --Log (naloxone) revealed that the line had a slope of -0.96. The value of the pA2 was 7.2. Similar experiments were carried out in two rhesus monkeys using cyclazocine. Naloxone (50-1000 microgram) similarly produced a rightward dose dependent, parallel shift of the cyclazocine dose response curves. Unlike the results obtained with morphine, the pA2 for naloxone obtained with cyclazocine was 6.3 and the slope of the dose ratio line was -1.6.

Antagonism by indomethacin of neurogenic hyperthermia produced by unilateral puncture of the anterior hypothalamic/preoptic region.

1. In unanaesthetized rats, restrained at an ambient temperature of 24 degrees C, the anterior hypothalamic/preoptic (AH/PO) region was lesioned unilaterally by acute mechanical puncture.2. In control (no pre-treatment) rats, unilateral AH/PO puncture produced a neurogenic hyperthermia which began immediately, reached its peak magnitude (mean peak magnitude = +2.3 degrees C) within 60-90 min and persisted usually for 8-16 hr. At defervescence, core temperature fell to a level near that of the pre-lesioning base line.3. The prostaglandin synthesis inhibitor, indomethacin, administered I.P. at doses of 5 and 15 mg/kg 1 hr before puncture of the AH/PO region, attenuated the lesion-induced hyperthermia in a dose dependent fashion. The higher dose reduced peak magnitude by 80% and the 6 hr Fever Index by 88%. The vehicle used to dissolve the indomethacin (60% DMSO/40% saline) did not significantly attenuate the hyperthermia.4. In rats that were hyperthermic after AH/PO damage, indomethacin (10-15 mg/kg I.P.) caused core temperature to fall promptly to near the prelesion base line. Reversal occurred whether the indomethacin was injected while core temperature was still rising or late in the plateau phase of the hyperthermia.5. It is suggested that the neurogenic hyperthermia elicited by unilateral lesioning of the AH/PO region was mediated by prostaglandins released from injured tissue and possibly from extravasated blood. Evidence is cited indicating that the most likely sites of action of the released prostaglandins are the surviving portion of the AH/PO region on the punctured side and the intact contralateral AH/PO region.