Inhibition of gastrin-stimulated cell proliferation by the CCK-B/gastrin receptor ligand CI-988.

The gastrointestinal hormone gastrin functions as a trophic factor for oxyntic mucosa as well as a secretagogue for gastric acid. In preclinical toxicology studies CI-988, a peptoid cholecystokinin (CCK) ligand with nanomolar affinity for the CCK-B/gastrin receptor, caused gastric gland degeneration and mucosal atrophy in cynomolgus monkeys, perhaps consistent with an expected pharmacological outcome of inhibition of the trophic effect of gastrin on stomach mucosa. Because of the expense and difficulty associated with experimental use of non-human primates, we investigated the effects of CI-988 on signal transduction pathways associated with gastrin-stimulated cell proliferation using the AR42J rat pancreatic tumour cell line as a model. The AR42J cell line was selected because it is known to express the CCK-B/gastrin receptor and because it is responsive to the growth promoting effects of gastrin in vitro. Gastrin-17 at 1 nM stimulated proliferation of AR42J cells 26% and 104% above control after 24 and 96 hours, respectively. CI-988 at 1 nM had no apparent effect on basal cell proliferation rates, but decreased gastrin-17 stimulated cell proliferation 13% and 47%, respectively, after 24 and 96 hours of treatment, consistent with competitive antagonism at the gastrin receptor. Because the trophic effect of gastrin towards AR42J cells has been linked to intracellular calcium ([Ca2+]i) mobilization and/or cyclic AMP, the effect of CI-988 on these second messengers were also investigated. Gastrin-17 at 10 nM stimulated both ([Ca2+]i) and cAMP, while CI-988 alone at 100 nM had no effect, but blocked the gastrin-stimulated increases in both mediators. Therefore, using the AR42J pancreatic tumour cell line as a model, the dipeptoid CCK-B/gastrin receptor ligand CI-988 behaves as an antagonist towards gastrin receptor-stimulated signal transduction pathways and cell proliferation in vitro.

In vitro 2,3,7,8-tetrachlorodibenzo-p-dioxin interference with the anterior pituitary hormone adrenocorticortropin.

Treatment of male Sprague-Dawley rats with a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to increase serum adrenocorticotropin (ACTH) and decrease serum corticosterone. The present in vitro study was designed to assess whether TCDD has a direct effect on the anterior pituitary under basal and stimulated conditions. Primary anterior pituitary cell cultures were prepared from normal 180- to 220-g male Sprague-Dawley rats and the cultures treated with 10(-9)-10(-19) M TCDD. Maximal secretion of ACTH occurred between 10(-11) and 10(-15) M TCDD for both medium (2-fold) and intracellular (1.5-fold) concentrations after 24 h TCDD exposure. TCDD treatment also caused an early (6 h) and persistent (10 days) increase in basal medium (1.4- to 2.8-fold) and intracellular (1.1- to 1.7-fold) ACTH concentrations. However, while stimulation with corticotropin-releasing hormone (CRH) increased intracellular ACTH 1.5- to 1.7-fold in pituitary cells treated for 24 h with 10(-9)-10(-13) M TCDD, ACTH secreted into the media was decreased by 30-50% compared with controls. Lastly, the secretagogue arginine-8-vaso-pressin (AVP), did not increase the amount of ACTH secreted above levels observed with basal TCDD exposure. From this study, it appears that TCDD stimulates in vitro synthesis and secretion of ACTH by the anterior pituitary under basal conditions, but decreases the pituitary's responsiveness to CRH and AVP stimulation.

Gastric effects of the CCK-B/gastrin receptor ligand CI-988 in cynomolgus monkeys.

We have previously demonstrated that the CCK-B/gastrin receptor ligand CI-988 induces gastric gland degeneration and atrophy in cynomolgus monkeys, an effect consistent with gastrin receptor antagonism and inhibition of gastrin's trophic effects on oxyntic mucosa. However, gastrin receptor ligands of the dipeptoid chemical series to which CI-988 belongs have been reported to act as agonists or antagonists towards gastrin-related events, depending on the animal model and the functional endpoint examined. To investigate further these apparently conflicting data, basal gastric acid secretion was monitored acutely in conscious monkeys given CI-988 orally at 10 mg/kg or intravenously at 0.01 mumol/kg/hr and histological changes in gastric mucosa were evaluated in monkeys given CI-988 orally at 5, 25 or 75 mg/kg/day for 4 weeks. Degeneration and atrophy of gastric glands occurred at 25 and 75 mg/kg with statistically significant decrements in gastric mucosal height at 75 mg/kg. In addition, CI-988 stimulated gastric acid secretion when given either orally or intravenously. Co-administration of the structurally unrelated CCK-B/gastrin antagonist L-365,260 completely blocked CI-988-stimulated acid secretion, confirming that CI-988's agonist effect on acid secretion is mediated by the gastrin receptor. Assuming that gastric mucosal degeneration is the result of inhibition of gastrin's trophic activity, CI-988 appears to induce paradoxical agonist and antagonist gastrin-receptor mediated effects.

Evidence for Ah receptor mediation of increased ACTH concentrations in primary cultures of rat anterior pituitary cells exposed to TCDD.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to increase plasma ACTH concentrations in male Sprague-Dawley rats and in male rat primary anterior pituitary cell cultures. The present study examined whether the anterior pituitary effects observed after TCDD exposure are mediated via the Ah receptor (AhR). Primary anterior pituitary cell cultures were prepared from normal 180- to 220-g male rats and the cultures treated with alpha-naphthoflavone (ANF), an antagonist; beta-naphthoflavone (BNF), an agonist; BNF + TCDD; 3,3',4,4',5-pentachlorobiphenyl (PCB), which is known to bind to the AhR; and 2,2',4,4',5,5'-hexachlorobiphenyl (HCB), which does not bind the AhR. Support for the TCDD-AhR-mediated increases in ACTH concentrations is provided by the following observations: (1) ANF inhibited both the 1.3- to 2-fold TCDD-induced increase in basal medium and intracellular ACTH concentrations and the 30% TCDD-induced decrease in medium ACTH levels and the 1.2-fold increase in intracellular ACTH levels in corticotropin-releasing hormone (CRH)-stimulated cells, (2) BNF increased basal medium (1.7-fold) and intracellular (1.3-fold) ACTH concentrations, (3) BNF + TCDD demonstrated additivity by increasing basal medium (2.4-fold) and intracellular (1.7-fold) ACTH concentrations, (4) PCB increased basal medium (1.8- to 2.1-fold) and intracellular (1.3- to 1.8-fold) ACTH concentrations and inhibited medium ACTH secretion in CRH stimulated cells by 24-43%, and (5) HCB did not effect basal or CRH stimulated medium and intracellular ACTH concentrations. From this study it appears that TCDD-induced changes in ACTH secretion and synthesis by cultured anterior pituitary cells is mediated through the Ah receptor.

Gastric gland degeneration induced in monkeys by the CCK-B/gastrin receptor antagonist CI-988.

Gastric effects of subchronic treatment with the cholecystokinin-B (CCK-B)/gastrin receptor antagonist CI-988 were investigated in cynomolgus monkeys. In preliminary range-finding studies, CI-988 was given orally to 1 monkey per sex for 14 days at doses of 50, 100, 200, and 500 mg/kg/day. Subchronic studies of CI-988 were subsequently conducted using 5 monkeys per sex at doses of 0, 5, 25, and 75 mg/kg for 4 or 13 wk. High-dose monkeys were dosed initially at 100 mg/kg, but the dose was not well tolerated and was decreased to 75 mg/kg after 8 days of treatment. One male monkey at 75 mg/kg was euthanatized in extremis on day 23. In the range-finding study, minimal to moderate, multifocal to diffuse degeneration of gastric glands, primarily in the fundic region, was observed at 100 mg/kg and above, with frank gastric mucosal atrophy occurring at 200 and 500 mg/kg. Minimal to mild gastric gland degeneration was also observed in the subchronic study after 4 wk at 25 and 75 mg/kg, but histopathologic gastric changes were remarkably absent after 13 wk. Mucosal height in the stomach fundus was decreased 19.8% in 75-mg/kg males at week 4, and although gastric mucosa appeared histologically normal after 13 wk, mucosal height remained 28.6% less than that of controls. In females at 75 mg/kg, fundic mucosal height was decreased 7% and 5% at weeks 4 and 13, respectively, but decreases were not statistically significant. Mean serum gastrin concentrations were increased 10-fold in males only after 4 wk at 75 mg/kg, but were comparable to controls during week 13. CI-988-induced gastric gland degeneration is consistent with antagonism of gastrin's trophic activity toward gastric mucosa. Notwithstanding decrements in gastric mucosal height, disappearance of mild histopathologic findings despite continued treatment with the ligand suggests some degree of adaptation to subchronic CCK-B/gastrin inhibition, although the mechanism of accommodation has yet to be delineated.

Cytochrome P450 2: peroxidative reactions of diversozymes.

Cytochrome P450, the most versatile biological catalyst known, was originally named as a pigment having a carbon monoxide difference spectrum at about 450 nm and no known function. Recent progress in many laboratories has revealed that the P450 superfamily has immense diversity in its functions, with hundreds of isoforms in many species catalyzing many types of chemical reactions. We believe it is safe to predict that each mammalian species may be found to have up to a hundred P450 isoforms that respond in toto to a thousand or more inducers and that, along with P450s from other sources, metabolize a million or more potential substrates. Accordingly, the name DIVERSOZYMES is proposed for this remarkable family of hemoproteins. This paper reviews the peroxidative reactions of Diversozymes, including peroxides as oxygen donors in hydroxylation reactions, as substrates for reductive beta-scission, and as peroxyhemiacetal intermediates in the cleavage of aldehydes to formate and alkenes. Lipid hydroperoxides undergo reductive beta-cleavage to give hydrocarbons and aldehydic acids. One of these products, trans-4-hydroxynonenal, inactivates P450, particularly alcohol-inducible 2E1, in what may be a negative regulatory process. Although a P450 iron-oxene species is believed to be the oxygen donor in most hydroxylation reactions, an iron-peroxy species is apparently involved in the deformylation of many aldehydes with desaturation of the remaining structure, as in aromatization reactions.

Inactivation of ethanol-inducible cytochrome P450 and other microsomal P450 isozymes by trans-4-hydroxy-2-nonenal, a major product of membrane lipid peroxidation.

Of the microsomal P450 cytochromes, the ethanol-inducible isoform, P450 2E1, is believed to be predominant in leading to oxidative damage, including the generation of radical species that contribute to lipid peroxidation, and in the reductive beta-scission of lipid hydroperoxides to give hydrocarbons and aldehydes. In the present study, the sensitivity of a series of P450s to trans-4-hydroxy-2-nonenal (HNE), a known toxic product of membrane lipid peroxidation, was determined. After incubation of a purified cytochrome with HNE, the other components of the reconstituted system (NADPH-cytochrome P450 reductase, phosphatidylcholine, and NADPH) were added, and the rate of oxygenation of 1-phenylethanol to yield acetophenone was assayed. Inactivation occurs in a time-dependent and HNE concentration-dependent manner, with P450s 2E1 and 1A1 being the most sensitive, followed by isoforms 1A2, 3A6, and 2B4. At an HNE concentration of 0.24 microM, which was close to the micromolar concentration of the enzyme, four of the isoforms were significantly inhibited, but not P450 2B4. In other experiments, the reductase was shown to be only relatively weakly inactivated by HNE. P450s 2E1 and 2B4 in microsomal membranes from animals induced with acetone or phenobarbital, respectively, are as readily inhibited as the purified forms. Evidence was obtained that the P450 heme is apparently not altered and the sulfur ligand is not displaced, that substrate protects against HNE, and that the inactivation is reversed upon dialysis. Higher levels of reductase or substrate do not restore the activity of inhibited P450 in the catalytic assay. Our results suggest that the observed inhibition of the various P450s is of sufficient magnitude to cause significant changes in the metabolism of foreign compounds such as drugs and chemical carcinogens by the P450 oxygenase system at HNE concentrations that occur in biological membranes. In view of the known activities of P450 2E1 in generating lipid hydroperoxides and in their beta-scission, its inhibition by this product of membrane peroxidation may provide a negative regulatory function.

Subcellular localization, aggregation state, and catalytic activity of microsomal P450 cytochromes modified in the NH2-terminal region and expressed in Escherichia coli.

This laboratory previously expressed cDNAs encoding rabbit liver cytochrome P450 2E1 (the ethanol-inducible isoform) and the corresponding protein lacking amino acids 3-29, a proposed membrane anchor, in Escherichia coli. Unexpectedly, the shortened protein, like the full-length form, was found to be predominantly located in the bacterial inner membrane rather than the cytosol and to have full catalytic activity. Additional proteins with alterations in the NH2-terminal region of P450 2E1 or P450 2B4 (the phenobarbital-inducible isoform) were similarly expressed, and it was concluded that such modifications can change the cytochrome to an increased cytosolic localization and that the first two hydrophobic segments are not uniquely involved in attachment to the bacterial membrane (Pernecky et al., 1993, Proc. Natl. Acad. Sci. USA 90, 2651-2655). In the present study, three chimeric cytochromes were produced to determine the effect on subcellular localization: 2E1:2B4, in which the first 17 residues of 2E1 (delta 3-29) replaced the corresponding 17 residues in 2B4 (delta 2-27), and BM-3:2B4 and BM-3:2E1, in which the first 19 residues of P450BM-3 replaced the first 17 in 2B4 (delta 2-27) and 2E1 (delta 3-29), respectively. Of the total cytochrome expressed, the localization in the E. coli cytosol was about 60, 70, and 80% for the respective chimeras, with 80% being the highest for any P450 we have examined. A plot of the extent of membrane binding versus hydropathy of the NH2-terminal region showed that the terminal sequence strongly influences the subcellular distribution and that a group of 2E1 proteins and a group of 2B4 proteins each have other regions that characteristically determine the extent of membrane attachment. The role of the NH2-terminal region in the high level of aggregation of purified full-length P450 is indicated by the finding that the multimeric state of 2E1 or 2B4 is unaffected by sodium cholate at concentrations that convert 2E1 (delta 3-29) or 2B4 (delta 2-27) to the monomeric state. In contrast to our earlier experience with P450 2E1, purified P450 2B4 (delta 2-27) has on the average only about half the activity of full-length 2B4 with substrates that undergo oxidative dealkylation or oxygenation at a hydroxyl group.

Lipid peroxidation in the adrenal glands of male rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

This study was performed to determine whether TCDD (50 micrograms/kg; single oral dose) could induce adrenal microsomal lipid peroxidation, which might be correlated to decreased levels of cytochrome P-450 and 21-hydroxylase activity. The amount of malondialdehyde (MDA) formed was significantly higher than controls at days 1 through 5 following TCDD treatment. Microsomal cytochrome P-450 levels were depressed after lipid peroxidation at days 1, 3, and 5, and 21-hydroxylase activity decreased at day 5 after TCDD treatment. This study shows that TCDD stimulates adrenal microsomal lipid peroxidation which is associated with decreased cytochrome P-450 levels and 21-hydroxylase activity.

TCDD alters pituitary-adrenal function. I: Adrenal responsiveness to exogenous ACTH.

Plasma ACTH concentrations in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rats (50 micrograms/kg; single, oral dose) were 2.1-, 2.1-, 2.9-, 1.7-, 1.5-, 2.0- and 3.0-fold greater than control values, respectively, at days 1, 3, 5, 7, 10, and 14. At days 1 and 5 plasma corticosterone concentrations were increased 5.1- and 8.0-fold, respectively; whereas, at days 10 and 14 they were depressed to values of 50% and 39% of controls, respectively. Adrenal glands were excised from rats treated with TCDD and corticosterone production was assessed. Basal corticosterone concentrations produced by treated adrenals were depressed to 81%, 72%, and 71% of control values at days 5, 7, and 14, respectively. Corticosterone secretion by ACTH stimulated adrenals was equivalent to controls. These findings suggest that TCDD exposure decreases the bioactivity of the ACTH secreted by the anterior pituitary.

TCDD alters pituitary-adrenal function. II: Evidence for decreased bioactivity of ACTH.

The present study assessed the ability of primary cultures of rat anterior pituitary cells to secrete bioactive ACTH in the presence of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The bioactivity of the secreted pituitary cell ACTH was determined by its ability to stimulate secretion of corticosterone from primary cultures of rat adrenal cells. ACTH from basal or CRH stimulated pituitary cells treated with TCDD was found to be less capable of stimulating corticosterone secretion from primary rat adrenal cell cultures than equimolar concentrations of ACTH purchased from a commercial supplier. Corticosterone secretion from adrenal cell cultures treated with ACTH from basal or CRH stimulated pituitary cell cultures exposed to TCDD was decreased by 60 and 70%, respectively. The decreased ability to stimulate corticosterone secretion can be overcome when extracts of ACTH from pituitary cell cultures treated with TCDD are supplemented with commercial ACTH. These findings indicate that TCDD may alter the bioactivity of secreted ACTH from the anterior pituitary gland.

Stimulation by voluntary exercise of adrenal glucocorticoid secretion in mature female hamsters.

The possibility that habitual voluntary running induces a chronic change in adrenal glucocorticoid synthesis and secretion was examined in freely running mature female hamsters, in whom this behavior accelerates growth, reduces body fat levels, and elevates core temperature. Hamsters were free to run on horizontal discs or in vertical wheels between 32 and 80 days, in 14L:10D or in 10L:14D photoperiods, and at the end of this period, corticosterone and cortisol steroidogenesis and serial plasma corticosterone concentrations during day and night were used as measures of the chronic stimulation of adrenal cortical activity. Habitual voluntary running significantly increased steroidogenesis of both glucocorticoids and plasma corticosterone concentrations and alone accounted for all the variance in enhanced synthesis and secretion of corticosterone. Acute exercise and/or the nocturnal phase of circadian period enhanced the chronic stimulatory effects of exercise on cortisol. Despite its voluntary and apparently stress-free nature, running induces chronic increases in basal glucocorticoid secretion in mature female hamsters. Putative oversecretion of corticotropin releasing factor in freely running hamsters could account for increased steroidogenesis, acceleration of growth, reduced body fat levels, and core temperature elevation.

Evidence for pteridine regulation of lead-mediated inhibition of uroporphyrinogen and heme formation in rat bone marrow.

Uroporphyrin I (URO I) accumulation has been reported in the bone marrow of rats exposed to lead, suggesting a sensitivity of uroporphyrinogen III cosynthase (COSYN) to this heavy metal. Furthermore, it has been reported that a polyglutamated folate derivative may serve as a coenzyme for the catalytic action of hepatic uroporphyrinogen III cosynthase. These findings raised the question of whether depletion of polyglutamated folate could enhance the susceptibility of bone marrow COSYN to lead and potentially interfere with the formation of heme. Nitrous oxide, an anesthetic agent capable of causing bone marrow tetrahydrofolate deficiency, depressed total bone marrow polyglutamated folate content by 42% with significant reductions in all three chain lengths (5-7) identified in the bone marrow during an exposure period of 7 days at 4 hr/day. Lead acetate (15 mg/kg) administered by ip injection at Days 0 and 2 during a 7-day exposure to nitrous oxide resulted in an 84% increase of bone marrow URO I content, which was markedly higher than the increases of 22 and 38% seen with sole administration of lead or nitrous oxide, respectively. The combination of agents also produced a 48% rise in COPRO I, a 39 and 43% decrease in COPRO III and protoporphyrin, respectively, and a 42% decline in the activity of microsomal 7-ethoxycoumarin O-deethylase, which is hemoprotein, cytochrome P-450 mediated. Heme oxygenase activity was not altered by nitrous oxide, lead, or their combination. These results suggest that bone marrow folate deficiency may render COSYN more sensitive to lead as characterized by increased uroporphyrin I and coproporphyrin I isomer content, decreased coproporphyrin III and protoporphyrin content, and depressed microsomal hemoprotein, cytochrome P-450-mediated drug-metabolizing capability.

Tetrachlorodibenzo-p-dioxin alters rat hypothalamic endorphin and mu opioid receptors.

The present study was undertaken to assess if hypothalamic beta-endorphin (beta E) and/or brain mu opioid receptors are associated with 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) (50 micrograms/kg)-induced hypophagia and body weight decline in rats. Hypothalamic beta E concentrations were initially increased to 166% of controls on day 1, and then were depressed to 39% and 49% of control values on days 2 and 3, respectively. Brain mu opioid receptor number was increased 60% in TCDD-treated rats at day 3 without a change in the binding affinity. Food-restricted rats did not exhibit changes in hypothalamic beta E concentrations or brain mu opioid receptor number. These results indicate that TCDD causes early perturbations in hypothalamic beta E concentrations and brain mu receptor number, which may contribute to the mechanisms by which TCDD leads to decreased food intake and progressive weight loss.

Human chorionic gonadotropin treatment prevents depressed 17 alpha-hydroxylase/C17-20 lyase activities and serum testosterone concentrations in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats.

It is known that administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes decreased serum testosterone concentrations in the rat. Previous studies in this laboratory have shown that in rats TCDD exposure results in decreased 17 alpha-hydroxylase and C17-20 lyase activities. The decreases in these activities paralleled decreases in testicular microsomal heme and cytochrome P450 contents. As reported herein, neither testicular mitochondrial cytochrome P450 content nor the activity of cholesterol side-chain cleavage was altered in rats exposed to TCDD. Since the production of testosterone in the testis is dependent on LH, it is important to determine the early effects of TCDD on serum LH concentrations in the rat. Male Sprague-Dawley rats were given a single, oral dose of TCDD (50 micrograms/kg). Serum LH concentrations were determined by RIA on Days 1, 2, 3, 5, and 7 following TCDD treatment. Rat serum LH concentrations were decreased to 60% of controls as early as Day 1 and continued to be depressed on Days 2 and 3 at 53% and 59% of control values, respectively. Rat serum LH returned to control values by Day 5 in spite of continued depression of serum testosterone concentrations. The early depression in serum LH levels caused by TCDD may be related to the subsequent androgenic deficiency in the rat. Treatment of rats with hCG was found to be able to prevent the depression of the activities of testicular microsomal 17 alpha-hydroxylase and C17-20 lyase and serum testosterone concentrations caused by TCDD. These data indicate that TCDD decreases serum testosterone by decreasing P450(17 alpha) and C17-20 but not P450sec activities and that hCG treatment prevents the TCDD-induced decrease.

Chronic ethanol consumption depresses hypothalamic-pituitary-adrenal function in aged rats.

In separate experiments, nine (n = 20) and fifteen (n = 12) month old rats were treated with either 6% ethanol or 12% sucrose (to balance caloric intake) in the drinking water to examine the effect of chronic ethanol consumption on the hypothalamic-pituitary-adrenal axis of aged rats. Rats were maintained on these treatment regimens for thirty days and were killed by decapitation. Blood was collected and plasma concentrations of adrenocorticotropin (ACTH) and corticosterone were determined by radioimmunoassay. Adrenal glands were cleaned, quartered and used to test in vitro responsiveness to ACTH. Anterior pituitary glands from all 15 month old rats and one half of the nine month old rats were collected, frozen and extracted for measurement of tissue ACTH concentration. The remaining anterior pituitary glands from the nine month old rats were challenged with corticotropin releasing hormone (CRH) to test in vitro responsiveness. In nine month old rats, chronic ethanol consumption decreased plasma ACTH and corticosterone (P less than 0.05). Pituitary ACTH concentrations were unchanged in treated nine month old rats, but the amount of pituitary ACTH released in response to CRH was decreased (P less than 0.05) in rats consuming ethanol. In vitro responsiveness of the adrenal gland to ACTH in nine month old rats consuming ethanol was unchanged (P greater than 0.05). Plasma ACTH and corticosterone concentrations were also decreased in 15 month old rats chronically consuming ethanol (P less than 0.05). No differences were noted in responsiveness of the adrenal gland or in the amount of pituitary ACTH due to ethanol consumption in 15 month old rats (P greater than 0.05). The results of these experiments indicate that chronic ethanol consumption decreases hypothalamic-pituitary-adrenal function in aged rats.

Effects of lead on haem biosynthesis during erythroid differentiation in vitro.

Murine erythroleukaemia cells (MELC) are erythroid precursor cells that undergo erythroid differentiation in the presence of the inducer hexamethylene bisacetamide (HMBA). The effects of lead on haem biosynthesis in MELC following HMBA-induced differentiation were studied. MELC were induced with HMBA in the presence of 20, 40 and 80 mum-lead acetate and cell density, haem content, incorporation of (14)C-labelled delta-aminolaevulinic acid (ALA) into haem, and the activities of the enzymes delta-aminolaevulinic acid dehydratase (ALA-D), uroporphyrinogen I synthetase (URO-S) and ferrochelatase (FERRO) were determined. MELC exposed to 80 mum-lead showed significant erythroid hypoplasia (40-50%) and a significant decrease (30-50%) in haem content at 2, 4 and 6 days after induction in comparison with the controls. Significant inhibition of ALA-D, the most sensitive index, was noted at 20 mum-lead, and at 80 mum-lead ALA-D activity was decreased by 60-80% in comparison with the controls. URO-S and FERRO showed significant decreases of 34% and 50%, respectively, at 80 mum-lead. A decrease of 50% in the incorporation of [(14)C]ALA into haem at 80 mum-lead indicated an impairment in haem synthesis. The results suggest that the impairment of haem formation by lead is coincident with the production of severe erythroid hypoplasia.

Time course of electrophysiologic effects induced by di-n-butyl-2,2-dichlorovinyl phosphate (DBCV) in the adult hen.

Previous work in our laboratory indicated that di-n-butyl-2,2-dichlorovinyl phosphate (DBCV) produced electrophysiologic changes in hen peripheral nerve that coincided with the development of histopathologic changes and neurologic signs of peripheral neuropathy. The purpose of the present study was to follow the time course for the development of the electrophysiologic changes and to determine whether pretreatment with the phosphinate analog of DBCV (DBCV-P), a nonageable organophosphorus compound, prevented these effects. Although significant electrophysiologic deficits occurred in the tibial and sciatic nerve 24 h after DBCV treatment, the most marked changes coincided with the onset of clinical signs of organophosphorus-induced delayed neuropathy (14-21 d). The sciatic and tibial nerves were equally susceptible to DBCV in producing deficits characterized by changes in the relative refractory period and an increased strength-duration threshold. Pretreatment with DBCV-P prevented the clinical signs and also attenuated the electrophysiologic deficits induced by DBCV treatment. These data suggest that electrophysiologic deficits occur before clinical signs of organophosphorus-induced delayed neuropathy (OPIDN) and may be indicative of a link between neurotoxic esterase (NTE) inhibition and onset of overt clinical toxicity.

Human placental lipid peroxidation--II. NADPH and iron dependent stimulation of microsomal lipid peroxidation by paraquat.

Paraquat, a widely used herbicide, was found to cause a marked stimulation of lipid peroxidation in the human placental microsomes in vitro. Both NADPH and chelated iron were necessary to observe paraquat-stimulated lipid peroxidation. The malondialdehyde accumulation in the incubation medium increased with increase in time, protein and paraquat concentration. The reaction did not exhibit the initial lag phase suggesting that endogenous membrane-bound antioxidants in human placental microsomes are either absent or present in extremely small quantities.