Estradiol induces galanin gene expression in the pituitary of the mouse in an estrogen receptor alpha-dependent manner.

Estradiol imprinting plays an important role in the regulation of galanin (GAL) gene expression in the rat. In the anterior pituitary gland, GAL gene expression is greatly induced by estrogen. The relative involvement that the two estrogen receptor subtypes, alpha and beta, have in regulating this induction is not known. We have utilized ER alpha-knock-out (ER alphaKO) mice to discriminate the roles of ER alpha and ER beta in the regulation of GAL gene expression in the anterior pituitary gland. Our goals were to measure the effects of estradiol on GAL gene expression by solution hybridization ribonuclease protection assay in wild-type mice and to determine the roles of ER alpha and, indirectly, ER beta by measuring the same response in the ER alphaKO mice. Estradiol treatment for one week elevated GAL gene expression 30-40 fold in the wild-type mouse pituitary. Evaluation of estrogen effects on GAL gene expression in the anterior pituitary of ER alphaKO animals revealed that ER alpha is essential, because no response to estrogen was observed in these animals. Since ER beta mRNA was identified in the anterior pituitary by RT-PCR, but estrogen had no effects on GAL gene expression in the ER alphaKO mice, the beta subtype of ER does not appear to participate in estrogen-evoked GAL gene expression in the mouse anterior pituitary.

A global model to define the behavior of partial agonists (bell-shaped dose-response inducers) in pharmacological evaluation of activity in the presence of the full agonist.

The dose-response models for full agonists and for a particular type of partial agonist can be described by sigmoidal curves and bell-shaped curves, respectively. The methods currently used to evaluate the interaction of a full agonist and a partial agonist require a large number of experimental units and base their analysis on nonlinear regression analysis, which may not be statistically appropriate. We propose an appropriate design and a global nonlinear model to evaluate such interactions. The new model allows us to estimate the interaction parameters and the parameters that characterize the individual partial agonist curve and the full agonist curve.

Induction of promiscuous G protein coupling of the follicle-stimulating hormone (FSH) receptor: a novel mechanism for transducing pleiotropic actions of FSH isoforms.

Under physiological conditions, FSH is secreted into the circulation as a complex mixture of several isoforms that vary in the degree of glycosylation. Although it is well established that the glycosylation of FSH is important for the serum half-life of the hormone and coupling of the receptor to adenylate cyclase, little is known concerning how physiologically occurring glycosylation patterns of this hormone affect receptor signaling. In this study, we have examined the biological activity of deglycosylated human FSH (DeGly-phFSH), recombinant mammalian-expressed hFSH (CHO-hFSH), and insect cell-expressed hFSH (BV-hFSH, alternatively glycosylated) as compared with that of purified human pituitary FSH (phFSH) using a Chinese hamster ovarian cell line stably expressing the hFSH receptor (3D2 cells). Differentially glycosylated forms of FSH did not bind to the FSH receptor in the same manner as phFSH. Although all hormones showed similar potency in competing for [125I]phFSH binding to the hFSH receptor, competition curves for deglycosylated and insect cell-produced FSH were steeper. Similarly, glycosylation of FSH had a profound effect on bioactivity of the hormone. Purified hFSH produced a sigmoidal dose-dependent stimulation in cAMP production, whereas DeGly-phFSH and BV-hFSH induced biphasic (bell-shaped) dose-response curves. BV-hFSH also elicited biphasic effects on steroidogenesis in primary cultures of rat granulosa cells. The cellular response to BV-hFSH was dependent on the degree of receptor-transducer activation. BV-hFSH bioactivity was strictly inhibitory when combined with the ED80 of phFSH. Lower concentrations of phFSH resulted in a gradual shift from inhibition to a biphasic activity in the presence of the ED20 of phFSH. Biphasic responses to BV-hFSH were attributed to activation of different G protein subtypes, since treatment of 3D2 cells with cholera toxin or pertussis toxin differentially blocked the two phases of BV-hFSH bioactivity. These data suggest that alternative glycosylation of FSH leads to a functionally altered form of the hormone. Functionally different hormones appear to convey distinct signals that are transduced by the receptor-transduction system as either stimulatory or inhibitory intracellular events via promiscuous, glycosylation-dependent G protein coupling. Promiscuity in signaling of the FSH receptor, in turn, may represent a potentially novel mechanism for FSH action, whereby the gonad may respond in diverse ways to complex hormonal signals such as those presented by circulating FSH isoforms.

Prolactin receptor messenger ribonucleic acid expression in the ovary during the rat estrous cycle.

We have investigated the relative amounts and sites of synthesis during the rat estrous cycle of the two ovarian mRNAs encoding the long and short PRL receptors (PRL-R). Quantitative analysis has revealed that the mRNA encoding the short PRL-R is consistently present throughout the cycle in lower quantities than the long receptor mRNA. Both receptor mRNAs are at maximal levels during proestrus, decline to their lowest level of expression during estrus, then gradually rise in metestrus and diestrus. By in situ hybridization, both receptor mRNAs are present during early proestrus in corpora lutea, in the granulosa cell layers of large Graafian follicles, and in the interstitial cells closely associated with these follicles. The short PRL-R mRNA was detected at significant levels in the granulosa-derived cumulus oophorus and in the thecal cell region at this time, whereas the long PRL-R mRNA was only weakly expressed in these cell types. In contrast, the long PRL-R mRNA was present at higher levels, compared to the short receptor mRNA, in the granulosa cells of preantral follicles in the interior of the ovary. On late proestrus, the long PRL-R mRNA was found predominantly in the mural granulosa cells of large Graafian follicles and in corpora lutea, but by estrous morning this mRNA appeared to be mostly restricted to the corpora lutea. This distribution was maintained through estrous evening and metestrous morning. On diestrus, both mRNAs were present in some corpora lutea and in the granulosa cell layer in a subset of the larger Graafian follicles, but were detected at even higher levels in the interstitial cells surrounding these follicles; again, the long receptor mRNA appeared to be only weakly expressed in the thecal cell region of these follicles. These results indicate that the levels and locations of PRL-R mRNA expression in the ovary, and therefore, the potential responsiveness of the ovary to PRL, change throughout the reproductive cycle. Furthermore, the presence of both receptor mRNAs in several different ovarian cell types suggests that both of these receptor forms play important roles in PRL physiology in the ovary.

Infusion of dopamine at low concentrations stimulates the release of prolactin from alpha-methyl-p-tyrosine-treated rats.

Prolactin (PRL) secretion from the anterior pituitary gland is inhibited by dopamine (DA) released into the hypophysial portal vasculature from neurons in the hypothalamus. We have shown previously that DA also stimulates PRL secretion in vitro. Here we report that DA has a dual effect on PRL release in vivo. Injection of rats with alpha-methyl-p-tyrosine (200 mg/kg, ip) induced an immediate 35-fold enhancement of PRL secretion which reached a plateau by 90 min after injection on diestrus 1. When DA was infused intravenously at varying doses beginning at 90 min after alpha-methyl-p-tyrosine, differing effects on PRL secretion were observed. These effects were dose dependent: higher doses of DA (1000 ng/kg/min) inhibited and lower doses (10 ng/kg/min) stimulated PRL secretion. These data suggest that DA may be an important stimulator of PRL secretion in vivo.

Activity of vasoactive intestinal peptide and serotonin in the paraventricular nucleus reflects the periodicity of the endogenous stimulatory rhythm regulating prolactin secretion.

PRL secretion in the female rat is regulated by an endogenous stimulatory rhythm (ESR) of prolactin-releasing factors of hypothalamic origin which has a bimodal periodicity with distinct nocturnal (N) and diurnal (D) phases. The N phase reaches peak magnitude by 0300 h and the D phase reaches peak magnitude by 1700 h. This rhythm was first unmasked in ovariectomized rats by correctly timed injection of a dopamine antagonist. OT, vasoactive intestinal peptide (VIP), and serotonin (5-HT) are differentially involved in generating the ESR. Pharmacological studies suggest that OT is the neurohormone and VIP and 5-HT are neuromodulators which act to stimulate OT release. Recently, we reported that activity of OTergic neurons in the paraventricular nucleus (PVN) and OT concentrations in the anterior pituitary mirror the periodicity of the ESR. The present experiments were conducted to determine if VIP and 5-HT activity in the hypothalamus also mirrors the periodicity of the ESR. Push-pull cannulae were surgically implanted in the PVN of ovariectomized female rats. Following recovery, push-pull perfusion was conducted from either 0600-1400 h, 1400-2200 h, or 2200-0600 h. VIP was measured in perfusates by RIA. There was no difference in VIP pulse frequency between rats perfused during the three periods studied. However, animals perfused from 2200-0600 h had significantly greater pulse amplitude as compared to rats at either 0600-1400 h or 1400-2200 h. Activity of 5-HTergic neurons in the hypothalamus was studied by estimating the turnover of 5-HT 10 min following the injection of pargyline. Hypothalamic nuclei were dissected using Palkovits' punch technique and 5-HT concentration assayed by HPLC in conjunction with electrochemical detection. Turnover of 5-HT was estimated by calculating the slope of the accumulation of 5-HT over 10 min at differing times of day using least squares regression analysis. There was a distinct diurnal rhythm of 5-HT accumulation in the PVN. Rats killed at 1700 h had significantly greater slopes of 5-HT accumulation in the PVN than rats killed at either 0300 or 1200 h. Similarly, there was a diurnal rhythm of 5-HT turnover in the suprachiasmatic nucleus. Rats sampled at either 1200 or 1700 h had significantly greater slopes of 5-HT accumulation in the suprachiasmatic nucleus than rats sampled at 0300 h. There was no diurnal rhythm of 5-HT turnover evident in either the median eminence or the supraoptic nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)

Activity of oxytocinergic neurons in the paraventricular nucleus mirrors the periodicity of the endogenous stimulatory rhythm regulating prolactin secretion.

PRL secretion is regulated by an endogenous stimulatory rhythm of PRL-releasing factors within the hypothalamus. The endogenous rhythm has a bimodal periodicity with a nocturnal component which peaks at approximately 0300 h and a diurnal component that peaks at approximately 1700 h. Several PRL-releasing factors are known to be involved in this rhythm. Among these are oxytocin (OT), vasoactive intestinal peptide, and serotonin. We have proposed that OT is the neurohormone that stimulates PRL release from the lactotroph. In this study, we examined the activity of OTergic neurons in the paraventricular nucleus using the expression of the protooncogene c-fos (Fos) as a marker of neuronal activity. Ovariectomized rats were killed at either 0300, 1200, or 1700 h and brains quickly fixed by perfusion with 2.5% acrolein in 4% paraformaldehyde. Brains were blocked and processed for OT/Fos immunohistochemistry. Rats killed at 0300 and 1700 h had significantly greater proportion of Fos expressing OTergic neurons than control rats (1200 h). Percent of Fos-positive OTergic neurons were 2- and 1.5-fold greater at 0300 and 1700 h than 1200 h, respectively. The majority of these neurons were located in the medial parvocellular paraventricular nucleus and periventricular area. In another experiment, groups of OVX rats were killed every 2 h over a 24-h period and OT extracted from their anterior and posterior pituitaries. OT was present in the anterior pituitary in a bimodal rhythm. OT concentrations were greatest at approximately 0400 h and slowly declined to baseline by 1000 h. Another peak of OT was present in the anterior pituitary at approximately 2000 h and quickly declined to baseline by 2400 h. This rhythm of OT was not reflected in either the posterior pituitary or trunk blood. These data suggest that activity of a specific population of OTergic neurons of the paraventricular nucleus is rhythmic. The periodicity of these neurons mirrors that of the endogenous stimulatory rhythm. Furthermore, the anatomical location of these neurons suggests that they may project to the median eminence. Indeed, this heightened activity is reflected in a bimodal rhythm of OT in the anterior pituitary. Taken together, the data presented here provide compelling support for the role of OT as the neurohormone in the mechanism of the endogenous stimulatory rhythm.

Ontogeny of the endogenous stimulatory rhythm regulating prolactin secretion in immature female rats.

PRL secretion in the female rat is regulated by an endogenous stimulatory rhythm (ESR). This rhythm consists of two components: a nocturnal (N) component whose activity is greatest by 0300 h and a diurnal (D) component that peaks at approximately 1700 h. This periodicity coincides with the periods of the N and D surges of PRL in responses to the mating stimulus. Furthermore, we have shown that the ESR is involved in the regulation of mating-induced PRL surges. Mating causes a lowering of dopaminergic tone which reveals the ESR for PRL. The ability of immature female rats to express PRL surges induced by copulomimetic stimuli begins at 25 days of age. In this study we investigated the ontogeny of the ESR in immature female rats in order to observe the relationship between the onset of PRL secretion induced by copulomimetic stimuli and the development of the ESR. Immature female rats were raised in our colony and kept with their dams until used in an experiment or weaned at 23 days of age where appropriate. At 15, 20, 23, 24, 25, or 30 days of age female rats received a single ip injection of domperidone (DOM; 5 mg/kg) or saline vehicle at 0300, 1200, or 1700 h. Thirty minutes after the injection the rats were decapitated, and trunk blood was collected. PRL was measured by RIA. DOM had no effect on PRL secretion as compared to that in saline-treated controls at 15 days of age. However, in all other age groups DOM induced a significant increase in PRL levels compared to those in saline-treated animals regardless of the time of injection. In addition, there was no time of day difference in the PRL secretory response to DOM in rats 15-23 days of age. However, rats treated with DOM at 0300 h at 24 days of age secreted approximately 2-fold greater PRL than rats treated similarly at 1200 or 1700 h. Moreover, at 25 and 30 days of age, rats treated with DOM at either 0300 or 1700 h secreted significantly greater PRL than rats treated similarly at 1200 h. These results suggest that the ESR for PRL secretion begins by 24 days of age. In addition, they indicate that the hypothalamic developmental event preceding and required for expression of mating-induced surges of PRL is the establishment of the ESR.

The endogenous stimulatory rhythm regulating prolactin secretion is present in the lactating rat.

Prolactin (PRL) secretion in the female rat is regulated by an endogenous stimulatory rhythm (ESR), which is normally under the tonic inhibition of dopamine (DA). The ESR consists of a nocturnal (N) component which peaks at approximately 03.00 h, and a diurnal (D) component which peaks at approximately 17.00 h. This ESR has been shown to be present in ovariectomized and cervically stimulated rats. We have proposed that the ESR is continuously present in the female rat and that any suppression of the tonic inhibitory influence on PRL secretion can reveal its existence. In this study, the effects of the DA-lowering stimulus of suckling was investigated at different times of day in lactating rats. In addition, the pattern of PRL secretion in freely lactating rats throughout a 24-hour period was studied. Female rats were separated from their pups for 6 h prior to reunion at either 03.00 (coincident with the N component), 12.00 (control) or 17.00 h (coincident with the D component) at various stages of lactation. Blood samples were collected from intra-atrial cannulae immediately before separation of pups and dams, immediately before reunion of pups and dams (0 time), and 15, 30, 60 and 120 min following reunion of pups and dams. Four days following parturition, dams suckled at either 03.00 or 17.00 h secreted significantly greater PRL than rats suckled at 12.00 h. Peak levels of PRL were 60-, 90- and 25-fold greater than 0 time levels, at 03.00, 17.00 and 12.00 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Hysterectomy-induced alterations in prolactin secretion of lactating rats.

The contribution of the uterus to the regulation of PRL secretion in lactating dams and cycling female rats was investigated. Lactating animals were hysterectomized or sham operated 2 days after parturition, and the number of pups was adjusted to eight. Blood samples for PRL RIA were obtained through intra-atrial cannulae implanted 2 days before experimentation. In order to study the PRL secretory profile in undisturbed freely lactating rats, blood samples were taken every 2 h for 24 h starting at 1400 h. During early lactation (days 7-8), hysterectomy did not alter the PRL secretory profile compared to that of sham-operated controls. On days 14-15 post partum, PRL secretion followed a characteristic bimodal pattern showing two PRL surges at 1800 h and 0600 h. After hysterectomy, the early morning PRL surge disappeared and PRL secretion showed an unimodal daily rhythm reaching its peak at 1800 h. The possible effect of the absence of the uterus on suckling-induced PRL release at various stages of lactation was studied. On days 7-8, suckling stimuli after 4 h of pup deprivation induced robust PRL release. Hysterectomy did not significantly alter PRL release at this earlier stage of lactation. In control groups, the suckling-induced PRL secretory response markedly declined as the postpartum period advanced. On the other hand, the hysterectomized animals retained significantly greater responsiveness to suckling during the second half of lactation. These data indicate an inhibitory influence of the uterus on PRL secretion. The onset of this uterine effect is considerably delayed, and its influence became prominent only at a later phase of lactation. The effect of length of pup deprivation preceding the suckling stimulus, in combination with hysterectomy, was also investigated. Hysterectomy significantly increased suckling-induced PRL release after 4 and 24 h separation compared to the sham-hysterectomized animals. When the separation was longer than 48 h, the inducibility of PRL release by suckling declined and was not influenced by hysterectomy. In order to study the possible influence of the uterus on PRL secretion during the estrous cycle, regularly cycling female rats were hysterectomized at diestrus 1. Twelve days later the animals were cannulated, and serial blood samples were taken during the subsequent proestrus. Hysterectomy did not alter the PRL surge which occurred on the afternoon of proestrus indicating that the uterus does not have a major function in regulating PRL secretion on proestrus. In conclusion, hysterectomy significantly delayed the extinction of suckling-induced PRL release revealing the active role of the uterus in the regulation of this neuroendocrine reflex.

Oxytocin, vasoactive-intestinal peptide, and serotonin regulate the mating-induced surges of prolactin secretion in the rat.

In female rats the mating stimulus induces a bimodal pattern of PRL secretion. A surge of PRL occurs at approximately 0300 h, called the nocturnal surge (N). Another surge occurs at 1700 h on the same day, called the diurnal surge (D). By lowering dopaminergic tone pharmacologically, we have recently demonstrated the existence of an endogenous rhythm stimulatory to PRL secretion in female rats. The periods of this stimulatory influence coincide with the periods of the N and D surges of PRL that occur in mated rats. In addition, we have shown that the 0300 h component of this endogenous rhythm is regulated by oxytocin (OT) and vasoactive intestinal peptide (VIP), and the 1700 h component is regulated by OT and serotonin (5-HT). In this study, we investigated the roles of OT, VIP, and 5-HT in controlling the N and D surges of PRL in ovariectomized (OVX) rats receiving a physiological dopamine-lowering stimulus, copulomimetic stimulation. Blood samples were obtained the day before the experiments between 1700 and 1900 h to verify that the rats used were having surges of PRL in response to cervical stimulation (CS). The role of OT was studied by infusing the OT antagonist 1-deamino-2-D-Trp4-Val8-Orn-OT (OT-A; 0.5 micrograms/kg.min) beginning at either 0100 or 1500 h and continuing for 5 h on day 2 after the last CS. Serial blood samples were obtained immediately before infusion and 60, 90, 120, 150, 180, 240, and 300 min after the start of infusion. The samples overlapped either the N or D surge of PRL. All rats used in these studies demonstrated D surges of PRL the day before the experiment. Saline infusion had no effect on either the N or D surge of PRL in OVX-CS rats. However, infusion of OT-A completely blocked both the N and D surges of PRL. The role of VIP was studied by infusing the VIP antagonist [4-D-Cl-Phe6-Leu17]VIP (VIP-A; 01 micrograms/kg.min) beginning at either 0100 or 1500 h and continuing for 5 h. VIP-A completely blocked both the N and D surges of PRL. To study the role of 5-HT, rats received an acute treatment with p-chlorophenylalanine (PCPA; 250 mg/kg, sc) at either 0100 or 1500 h, and blood samples were taken as before. PCPA had no effect on the N surge of PRL.(ABSTRACT TRUNCATED AT 400 WORDS)

Hypothalamic factors involved in the endogenous stimulatory rhythm regulating prolactin secretion.

We recently reported that acute pharmacologic depression of dopaminergic tone at different times of day unmasks a sex-specific endogenous stimulatory rhythm regulating PRL secretion. The PRL secretory responses of ovariectomized rats to the dopamine antagonist domperidone (DOM) were higher at 0300 and 1700 h than at 1200 h. These are the times during which surges of PRL appear in mated rats. This experimental paradigm was used to investigate the roles of the putative PRL-releasing factors (PRFs) oxytocin (OT), vasoactive intestinal peptide (VIP), and serotonin (5-HT) in this rhythm. The role of OT was studied by infusion of the OT antagonist 1-deamino-2-D-Trp-4-Val-8-Orn-Oxytocin (OT-A, 0.5 microgram/kg min) for 6 h. Two hours after beginning the OT-A infusion DOM was administered, as a single injection of 200 micrograms/kg iv at either 0300, 1200, or 1700 h. Serial blood samples were collected immediately before and 5, 10, 20, 30, 60, 120, 180, and 240 min after DOM administration. Infusion of OT-A attenuated the heightened PRL secretory responses to DOM given at both 0300 and 1700 h but did not affect the response at 1200 h. The role of VIP was studied by infusing the VIP antagonist [D, 4-Cl-Phe6,Leu17] VIP (VIP-A, 0.1 microgram/kg.min) as described above. VIP-A infusion had no effect on the PRL secretory responses to DOM given at 1200 or 1700 h but attenuated the heightened response at 0300 h. In order to study the role of 5-HT in the rhythm, rats were pretreated with p-chlorophenylalanine (250 mg/kg sc) 48 and again 24 h before the experiment. Pretreatment with p-chlorophenylalanine had no effect on the PRL secretory responses to DOM given at 0300 or 1200 h, but it attenuated the augmented PRL secretory response at 1700 h. These data suggest that both VIP and OT act as endogenous PRFs at 0300 h and 5-HT and OT act as PRFs at 1700 h. We propose that VIP and 5-HT are continuously active oscillatory neurotransmitters regulating OT release into pituitary portal blood and that these daily events only eventuate in PRL release when the mating stimulus has release the lactotroph from the inhibitory effects of dopamine.

A sex-specific endogenous stimulatory rhythm regulating prolactin secretion.

In the rat, PRL secretion is under inhibitory control by tuberoinfundibular dopaminergic neurons. The levels of dopamine (DA) in hypophysial portal blood decline during surges of PRL secretion (e.g. suckling and cervical stimulation). However, this decline alone is not sufficient to account for the amount of PRL released. In this study we investigated the possible existence of an endogenous stimulatory rhythm for PRL secretion that may be masked by the tonic inhibitory tone of DA and unmasked by the DA-lowering effects of cervical stimulation. The PRL secretory response to pharmacological depression of DA-ergic tone was studied in ovariectomized (OVX) female, adult castrated (AC) male, neonatally androgen-sterilized (TP) female, and neonatally castrated (NC) male rats. Since mated rats have serum PRL surges at 0300 and 1700 h, these groups were treated with 200 micrograms/kg domperidone (DOM), iv, at 0300 h, 1700 h, or the intersurge interval, 1200 h. Serial blood samples were collected immediately before and at frequent intervals after DOM injection. OVX female rats had significantly greater serum PRL responses to DOM at 0300 and 1700 h than at 1200 h. AC male rats secreted significantly less PRL in response to DOM compared to OVX rats, and their PRL responses to DOM were similar at all three times. TP female rats had PRL secretory responses similar to those of the OVX rats at 1200 h, and the responses at 0300 and 1700 h were similar. NC male rats had PRL secretory responses similar to those of AC male rats. There was no difference between the PRL secretory profiles at any time after DOM injection in NC rats. These data provide evidence for an endogenous stimulatory rhythm for PRL secretion that is specific to female rats. They further suggest that the neonatal steroid environment is critical for differentiation of some sexually specific characteristics.

Iron chelation as a possible mechanism for aspirin-induced malondialdehyde production by mouse liver microsomes and mitochondria.

To investigate the possibility that lipid peroxidation is the mechanism responsible for aspirin-induced liver damage, pure neutralized acetylsalicylic acid (ASA), 0.6-90.9 mM, was added to calcium-aggregated mouse liver microsomes followed by incubation in NADPH buffer at 37 degrees C for 60 min and subsequent measurement of malondialdehyde (MDA). MDA production at ASA concentrations from 1.2 to 4.6 mM was greater than control (P less than 0.004). Peak MDA values were observed with 4.6 mM ASA, 39.58 +/- 6.73 nmol MDA/mg protein vs. 16.16 +/- 2.85 (P less than 0.004). Higher concentrations of ASA were inhibitory compared with the value at 4.6 mM (P less than 0.001). Aspirin had similar effects on MDA production by mouse liver mitochondria. MDA production with either ASA or buffer was completely suppressed by the potent iron-chelating agents desferrioxamine and alpha,alpha' dipyridyl when these were added to the microsomal preparations. Since MDA production in this system is known to be affected by iron-chelating agents (enhanced at low concentration, inhibited at higher concentration), the iron-chelating properties of ASA were investigated. Conductivity titration curves of Fe(OH)3 added to water or ASA suggested that the ASA was complexing with iron. The presence of an iron-ASA complex was established by high pressure liquid chromatographic analysis of the solution from this study. We conclude that aspirin enhances MDA production by hepatic microsomes and mitochondria via an aspirin-iron chelate and that this represents at least one mechanism by which aspirin may produce liver damage.