Retinamides: hydrolytic conversion of retinoylglycine to retinoic acid in pregnant mice contributes to teratogenicity.

Retinamides are prominent among synthetic vitamin A derivatives (retinoids) which can prevent or reduce the incidence of certain carcinogen-induced neoplasms in animals. They also possess lower toxicity toward adult and developmental systems than natural retinoids, presumably because of the presence of an amide endgroup which resists ready hydrolysis. In this investigation, we compared the developmental toxicities in mice of N-(4-hydroxyphenyl)retinamide(4-HPR), N-ethylretinamide (ER) and two retinoylamino acids, N-(all-trans-retinoyl)glycine (RG) and N-(all-trans-retinoyl)-DL-leucine (RL), which are formed from retinoic acid and the alpha-amino acids; RG and RL were shown in a previous study to differ from each other and from retinoic acid in certain toxicity bioassays. We found that while 4-HPR, ER, and RL were only minimally embryotoxic, RG was uniquely active as a teratogen with potency equivalent to that of retinol, the precursor of retinoic acid. Since binding to cytoplasmic proteins and nuclear receptors is a function of the presence of an acidic endgroup in the retinoid molecule, we investigated if RG given to pregnant mice was converted to retinoic acid (RA) and if teratologically significant amounts were detectable in the embryo. A single 100 mg/kg dose of RG in oil vehicle was given orally to ICR mice on day 11 of gestation (plug day = day 0). Extraction and quantification by HPLC of the retinoids in the maternal plasma and in whole embryos were performed at hourly intervals for the first 10 h after dosing and at 26 h. RG was absorbed rapidly reaching peak levels in the maternal plasma at 1 h after the dose and maintained a level of 15 micrograms/mL for up to 4 h, before starting a decline. RG also transferred to the embryo reaching peak levels greater than 0.75 micrograms/g wet weight between 2 and 4 h after the dose. All-trans RA was detected in the maternal plasma and the embryo at 1 h after the dose, reaching peak levels at 2 h in both compartments (0.43 micrograms/mL or g), before starting a decline. Small quantities of 13-cis RG (a contaminant in the original solution comprising 2-3% by weight) and 13-cis RA were also detected in both compartments, but their amounts in the embryo were considered insufficient to contribute to teratogenicity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of HL-60 cell differentiation by water-soluble and nitrogen-containing conjugates of retinoic acid and retinol

Retinoids induce the promyelocytic cell line, HL-60, to differentiate along the granulocytic pathway in vitro. A number of water-soluble and nitrogen-containing retinoids were synthesized in our laboratory [retinoyl-glucose (RAGL), retinyl-glucose (ROGL), retinoyl-adenosine (RADS), retinoyl-adenine (RAD), retinoyl-beta-glucuronide (beta RAG), and retinoyl-alpha-glucuronide (alpha RAG)]. These retinoids (10(-5) to 10(-8) M), as well as retinoic acid (RA) and retinol (ROL), were tested for their ability to induce the differentiation of HL-60 cells in vitro and to affect cell growth and viability during a 24- to 72-h incubation period. Differentiation was assessed by measuring the percentage of cells expressing the Mac-1 antigen on their cell surfaces. RA and the conjugates of RA were all quite active in inducing HL-60 cell differentiation, whereas ROL and ROGL had much less activity at equimolar concentrations. beta RAG, alpha RAG, RADS, and RAD were less toxic, whereas the glucose conjugates of retinol and retinoic acid (ROGL and RAGL) were both considerably more toxic than either RA or ROL at equimolar concentrations. All retinoids affected cell growth in a dose-dependent fashion. At 24 h, free RA or ROL was not detected in the cells after incubation with any of the retinoid conjugates.     

Chemical synthesis of all-trans-[11-3H]retinoyl beta-glucuronide and its metabolism in rats in vivo.

All-trans-[11-3H]retinoyl beta-glucuronide (RAG) was synthesized in a single step from all-trans-[11-3H]retinoyl fluoride, with a 24% yield. After its intraperitoneal injection into rats, RAG was detected in the blood, liver, intestine and kidney during the following 24 h period. Although the concentration of radiolabelled metabolites decreased with time, RAG predominated at nearly all times in nearly all tissues. Small amounts of retinoic acid (RA) were also universally present, together with unidentified polar metabolites and small amounts of non-polar esters of RA. The major excretion products of RAG in faeces and urine were RA and polar metabolites. Thus RAG, although converted in part to RA in vivo, persists as a major component in blood and tissues for at least 24 h. These observations support the concept that the retinoid beta-glucuronides might serve a physiologically significant role in the function of vitamin A.     

Changes in Soluble CD18 in Murine Autoimmune Arthritis and Rheumatoid Arthritis Reflect Disease Establishment and Treatment Response

Introduction

In rheumatoid arthritis (RA) immune activation and presence of autoantibodies may precede clinical onset of disease, and joint destruction can progress despite remission. However, the underlying temporal changes of such immune system abnormalities in the inflammatory response during treat-to-target strategies remain poorly understood. We have previously reported low levels of the soluble form of CD18 (sCD18) in plasma from patients with chronic RA and spondyloarthritis. Here, we study the changes of sCD18 before and during treatment of early RA and following arthritis induction in murine models of rheumatoid arthritis.

Methods

The level of sCD18 was analyzed with a time-resolved immunoflourometric assay in 1) plasma from early treatment naïve RA patients during a treat-to-target strategy (the OPERA cohort), 2) plasma from chronic RA patients, 3) serum from SKG and CIA mice following arthritis induction, and 4) supernatants from synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) from 6 RA patients cultured with TNFα or adalimumab.

Results

Plasma levels of sCD18 were decreased in chronic RA patients compared with early RA patients and in early RA patients compared with healthy controls. After 12 months of treatment the levels in early RA patients were similar to healthy controls. This normalization of plasma sCD18 levels was more pronounced in patients with very early disease who achieved an early ACR response. Plasma sCD18 levels were associated with radiographic progression. Correspondingly, the serum level of sCD18 was decreased in SKG mice 6 weeks after arthritis induction compared with healthy littermates. The sCD18 levels in both SKG and CIA mice exhibited a biphasic course after arthritis induction with an initial increase above baseline followed by a decline. Shedding of CD18 from RA SFMC and RA PBMC cultures was increased by TNFα and decreased by adalimumab.

Conclusions

The plasma sCD18 levels were altered in patients with RA, in mice with autoimmune arthritis and in cell cultures treated with TNFα and adalimumab. Decreased levels of plasma sCD18 could reflect autoimmunity in transition from early to chronic disease and normalization in response to treatment could reflect autoimmunity in remission.     

Endotoxin induced increases in rat plasma pituitary-adrenocortical hormones are better reflected by alterations in tumor necrosis factorα than interleukin-1β

In order to assess the relative cytokine contribution to endotoxin stimulation of pituitary-adrenocortical hormone secretion, we measured plasma levels of interleukin-1β (IL-Iβ), tumor necrosis factorα (TNFα), adrenocorticotropin (ACTH) and corticosterone following lipopolysaccharide (LPS) challenge in rats. LPS administration induced robust increases in both plasma ACTH and corticosterone levels at 3 h after i.p. injection; while ACTH decreased towards control levels, corticosterone remained at peak concentrations at 6 h after LPS injection. Basal levels of plasma IL-1β were below the sensitivity of the ELISA and basal levels of plasma TNFα were 0.25 ± 0.12 pM. Small but highly variable non-significant increases in plasma IL-1β levels were seen at 3 h and 6 h after injection of LPS. The lack of functional consequences of the small increases in IL-1β levels was demonstrated by unchanged levels of [¹²⁵I]IL-1α binding in liver at 3 h after LPS injection. In contrast, dramatic increases in plasma TNFα concentrations were observed at 3 h and decreased to non-injected control levels at 6 h after LPS injection. There was a significant positive correlation between ACTH and TNFα after LPS injection, while no correlation was seen between ACTH and IL-1β. These data demonstrate differential regulation of IL-1β and TNFα by endotoxin treatment and suggest that TNFα may be a more potent mediator of LPS-induced ACTH secretion in rat.     

Vaginal hydrolysis of retinyl acetate: increase in plasma retinol and retinol binding protein in women with cervical dysplasias

We previously reported the clinical feasibility of a Phase I trial involving the topical administration of a RA gel applied cervicovaginally in women with mild or moderate cervical dysplasia. Now, we report hydrolysis and systemic absorption of the RA gel from the vagina. HPLC analysis of samples of residual gel obtained from the cervical canal after topical bolus application indicate that the RA undergoes prompt in vivo hydrolysis yielding retinol as a major metabolite. Venous blood samples of 41 subjects, who self-administered a RA gel, were analyzed for plasma retinol and RBP concentrations prior to and upon completion of a 7-day treatment course and upon return for follow-up examinations. An increase in both the concentrations of plasma retinol and RBP were detected after topical application of the RA gel. These elevated values receded after the gel administration was discontinued. No significant changes were observed in plasma retinol or RBP concentrations in placebo-treated subjects. The efficacy of RA as a chemopreventive agent in treating cervical dysplasias remains to be determined.     

Effect of α-Methylphenylalanine and Phenylalanine on Brain Polyribosomes and Protein Synthesis

Abstract: A chronic hyperphenylalanemia was effectively produced in developing mice by daily administrations of phenylalanine (2 mg/g body wt) and a phenylalanine hydroxylase inhibitor α-methyl-D, L-phenylalanine (0.43 mg/g body wt). The presence of α-methylphenylalanine in newborn mice inhibited 65–70% of hepatic phenylalanine hydroxylase activity within 12 h. Since this maximum inhibition persisted for 24 h or longer, decreased enzyme activity was maintained by daily administrations. Whereas concentrations of phenylalanine increased approximately 40-fold in both plasma and brain following injection of α-methylphenylalanine and phenylalanine, plasma levels of tyrosine were not altered significantly. Concomitant with changes in phenylalanine concentrations we observed the brain polyribosomes' disaggregation, which reached a maximum 3 h after injection and persisted as long as 18 h. Polyribosomes did not become refractory to as many as 10 daily injections of α-methylphenylalanine and phenylalanine. In addition to polyribosome disaggregation, chronic hyperphenylalanemia reduced the rates of polypeptide chain elongation on polyribosomes isolated from brain homogenates.     

Dynamic changes in the gonadotrope cell subpopulations during an estradiol-induced surge in the ewe

Whether estradiol targets a subpopulation of gonadotrope cells was investigated in this study. Ovariectomized ewes (OVX) or OVX ewes immunized against GnRH and treated with hourly pulses of GnRH analogue (OVX-IMG) were killed at 6, 12, 16, and 24 h after administration of 50 microg of 17beta-estradiol (E(2)). Control ewes received no E(2) treatment. In OVX or OVX-IMG ewes killed 6 h after E(2) injection, a decrease in gonadotropin plasma levels was observed compared with non-E(2)-treated ewes. In contrast, a surge in gonadotropin plasma concentrations occurred in ewes killed 16 h after injection. The percentage of total immunoreactive gonadotrope cells among the pituitary cells was lower in E(2)-treated ewes compared with nontreated animals. The proportion of monohormonal LH cells was constant throughout the experiment, except at the surge peak, where it was enhanced. In the OVX ewes, the proportion of bihormonal LH/FSH cells was lower in the E(2)-treated ewes compared to the nontreated ewes (P: < 0.001), with a more pronounced decrease 16 h after E(2) injection. A slight increase occurred 12 h after E(2) injection compared with 6 h after injection (P: < 0.05). A similar pattern was observed in the OVX-IMG ewes, except at 12 h after E(2) injection, when no increase occurred. In both OVX and OVX-IMG ewes, injection of E(2) decreased FSHbeta mRNA expression but did not alter the relative levels of LHbeta mRNA. These data suggest that the negative feedback of E(2) on LH and FSH secretion mainly targets the bihormonal cells and occurs, at least in part, directly at the pituitary level. During the gonadotropin surge, the sustained FSH release from the bihormonal cells would induce a switch from bihormonal cells to monohormonal LH cells by depleting these cells of FSH.     

Efficient clearance of poly(ethylene glycol)-modified immunoenzyme with anti-PEG monoclonal antibody for prodrug cancer therapy

The F(ab')(2) fragment of the anti-TAG-72 antibody, B72.3, was covalently linked to Escherichia coli-derived beta-glucuronidase that was modified with methoxypoly(ethylene glycol). The conjugate (B72.3-betaG-PEG) localized to a peak concentration in LS174T xenografts within 48 h after injection, but enzyme activity persisted in plasma such that prodrug administration had to be delayed for at least 4 days to avoid systemic prodrug activation and associated toxicity. Conjugate levels in tumors decreased to 36% of peak levels at this time. Intravenous administration of AGP3, an IgM mAb against methoxypoly(ethylene glycol), accelerated clearance of conjugate from serum and increased the tumor/blood ratio of B72. 3-betaG-PEG from 3.9 to 29.6 without significantly decreasing the accumulation of conjugate in tumors. Treatment of nude mice bearing established human colon adenocarcinoma xenografts with B72. 3-betaG-PEG followed 48 h later with AGP3 and a glucuronide prodrug of p-hydroxyaniline mustard significantly (p< or =0.0005) delayed tumor growth with minimal toxicity compared to therapy with a control conjugate or conventional chemotherapy.     

Kinetics of phospholipase A2, arachidonic acid, and eicosanoid appearance in mouse zymosan peritonitis

Intraperitoneal injection of zymosan into mice induces a peritonitis characterized by cellular influx, plasma leakage and the appearance of arachidonic acid (AA) metabolites. We report that zymosan injection also stimulates the accumulation of AA, docosahexaenoic acid, linoleic acid, and phospholipase A2 (PLA2) activity. The amount of the unsaturated fatty acids (UnFA) varies both with the zymosan dose and time. Significantly increased levels of UnFA were first detected 15 min after zymosan injection. Maximal levels of the UnFA were reached 1 to 2 h post zymosan injection (AA: 725 +/- 29 ng/mouse, docosahexaenoic acid: 296 +/- 23 ng/mouse, linoleic acid: 4489 +/- 179 ng/mouse) and declined to saline control levels by 8 h. PLA2 activity was significantly increased 5 to 15 min after zymosan injection. Maximal levels of PLA2 activity occurred 15 to 30 min after zymosan injection (31.8 +/- 9.1 nmol phospholipid/mg protein/h) and then decreased by 30% through 24 h. Neither the appearance of UnFA nor PLA2 activity correlated with cellular influx, but both were coincident with plasma exudation at 5 to 15 min after zymosan. However, maximal exudation occurred 1 to 2 h post zymosan injection similar to that seen with the UnFA but not PLA2. These latter results suggest that a significant portion of the UnFA found in the peritoneal cavity of zymosan-injected mice originates from the plasma. PLA2 activity at the early time points (5 to 15 min) may also contribute to the levels of UnFA via hydrolysis of tissue and/or cellular phospholipids.     

Effect of post-mating GnRH analogue (buserelin) treatment on PGF2alpha release in ewes and ewe lambs

The objectives of this study were to determine the effects of buserelin or saline treatment on ovarian function (Experiment 1), plasma PGFM concentrations and oxytocin stimulated prostaglandin F(2alpha) (PGF(2alpha)) release (Experiment 2) in ewe lambs and ewes. Welsh Halfbred ewes (n=26) and ewe lambs (n=24) were mated to vasectomised rams at synchronised oestrus and on Day 12 post-mating each animal was injected intramuscularly either normal saline or 4 microg buserelin. In Experiment 1, plasma progesterone and oestradiol concentrations were determined in samples collected by jugular venepuncture 1h before and at 0, 2, 4, 6, 8, 24, 48 and 72 h after treatment (n=7 per treatment group). Progesterone concentrations increased (P<0.05) from 2 to 8h after buserelin treatment and returned to basal levels after 72 h, whereas oestradiol concentrations were maximal at 2h post-treatment and returned to basal levels after 24h (P<0.05). Oestradiol concentrations were lower (P<0.05) in buserelin-treated animals than controls at 72 h post-treatment. Basal and post-treatment progesterone concentrations were greater (P<0.05) in ewes than in ewe lambs but oestradiol levels were similar for both age groups. Ovulation rate, determined by laparoscopy on Day 14, was similar for both age groups (ewes 1.1; ewe lambs 1.0). Buserelin treatment induced accessory corpora lutea in ewes (4/7; 57%) but not in ewe lambs (0/7; 0%). In the Experiment 2, plasma PGFM concentrations were determined in samples collected at 20-min intervals for 6h on Day 14 and at 20-min intervals for 1h before and at 10-min intervals for 1h and then at 20-min intervals for a further 3h period after an intravenous injection of oxytocin (1IU/kg body weight) on Day 15 post-oestrus. In this experiment there were five ewe lambs and six ewes per treatment group. There was no effect of buserelin treatment or age on basal PGFM concentrations on either Day 14 or 15. Although peak PGFM concentrations tended to be lower in buserelin-treated animals, the difference was not significant (P>0.05). However, peak duration following oxytocin challenge on Day 15 post-mating was shorter (P<0.05) in control ewes compared with control ewe lambs. In conclusion, buserelin treatment given on Day 12 post-oestrus enhances luteal function more in ewes than ewe lambs and after a transitory increase, reduces oestradiol concentrations in both ewes and ewe lambs. However, buserelin treatment does not significantly attenuate the luteolytic signal.     

The effect of dose and route of oestradiol benzoate administration on plasma concentrations of oestradiol and FSH in long-term ovariectomised heifers

Oestradiol (E(2)) suppresses FSH and affects follicle wave dynamics in cattle. However, neither the optimum dose of ODB required to suppress FSH nor the effect of route of ODB administration on blood concentrations of E(2) are known; hence, the aim of this experiment was to answer these questions. Ovariectomised heifers received Progesterone Releasing Intravaginal Device (PRID) for 7 days, and 4 days later heifers received one of eight ODB treatments at second PRID insertion as follows; (1) 0.0 mg (Control; n=3), (2) 0.5 mg (n=4), (3) 1.0 mg (n=4), (4) 2.5 mg (n=6), (5) 5.0 mg (n=4), (6) 10. 0 mg (n=4), (7) 5.0 mg (n=4), and (8) 10.0 mg (n=5). For treatments 2-6 inclusive, ODB was administered intramuscularly in oil, while for treatments 7 and 8, the ODB in powder form was administered topically in the vagina by gelatine capsule attached to the PRID. Blood samples were collected every 6 h for the first 48 h, every 12 h for the next 48 h, and twice daily for a further 6 days. The interval from ODB administration to peak E(2) concentration was similar (P0.05) for treatments 2-6 where ODB was administered intramuscularly (mean 13.4+/-1.24 h), and was longer (P<0.05) for the intravaginal capsule treatments (mean 25.5+/-2.84 h). Plasma concentrations of E(2) increased with increasing intramuscular dose of ODB injected, (plasma E(2)=-0.237+16.109 (dose)-0.74 (dose)(2), R(2)=0.75; P<0.05). Peak plasma concentrations of E(2) following the 5- and 10-mg capsules were similar to each other and to those following the 0.5-mg injection (P0.05), but were lower than concentrations obtained following injection of 1.0-5.0 mg (P<0.05). Across all treatments, both the maximum percentage decline in FSH and the interval to FSH nadir were related to the peak plasma concentrations of E(2) (maximum % decline in FSH=11.17+1.564 (peak E(2))-0.009 (peak E(2))(2), R(2)=0.75; P<0.01), (hours to FSH nadir=10.628+1.486(hours to peak E(2))-0.0282(hours to peak E(2))(2), R(2)=0.22; P<0.05). Concentrations of FSH increased as E(2) declined from its peak value, irrespective of maximum value achieved. It was concluded that the intramuscular administration of ODB in oil to ovariectomised heifers given a PRID results in higher plasma concentrations of E(2) and causes a greater reduction in FSH than administration topically by intravaginal gelatine capsule. E(2) transiently suppresses FSH in ovariectomised heifers, and the magnitude of the suppression is dose-dependent; however FSH concentrations begin to increase 1-2 days after ODB administration while concentrations of E(2) were declining but still high.     

Absence of effect of estrogen on dopamine, norepinephrine and beta-endorphin-like immunoreactivity in human plasma

The plasma concentrations of immunoreactive norepinephrine (NE), dopamine (DA), beta-endorphin (beta-E), luteinizing hormone (LH) and follicle stimulating hormone (FSH) were determined by RIA and HPLC every 6 h until 72 h after iv administration of conjugated estrogens during the midfollicular phase. The LH level showed a biphasic pattern after the injection of conjugated estrogens, i.e. significant suppression (-50%) for 6-42 h after the injection, followed by a rebound increase with a peak (+85%) at 72 h. The plasma levels of immunoreactive beta-E, NE and DA did not change significantly for 72 h after the injection.     

Kinetics, distribution, and biotransformation of the chemical HI-6 in the rat, dog, and rhesus monkey

Disposition of the bis-pyridinium mono-oxime, HI-6, following intramuscular injection in rats (200 mg/kg bw), beagle dogs (10 and 50 mg/kg bw), and rhesus monkeys (50 mg/kg bw) revealed that the oxime was absorbed rapidly and completely from the site of injection, was distributed rapidly in the tissues, and that tissue concentrations decreased below the limits of detection by 4 h after treatment. No overt signs of toxicity were observed in any species at the concentrations given. Tissue analysis for HI-6 and degradation products was conducted by extraction followed by ion-pair, reverse phase, HPLC chromatography. The estimated plasma half-life values were 20, 40-55, and 25-30 min for rats, dogs, and monkeys, respectively. HI-6 and the degradation products were excreted via the urine. A marked species difference in disposition was observed in that HI-6 selectively accumulated in the diaphragmatic muscle of the rat to a level 10- to 20-fold higher than the level in blood plasma, whereas in the dog and monkey, diaphragmatic concentrations were comparable with those in the plasma. Three degradation products of HI-6 were detected in the plasma of the three species. One excreted product formed spontaneously since it was also detected in buffered solutions used for abiotic stability studies. The second product, the picolinic acid analog of HI-6, appeared to be metabolically formed in vivo. A third product remains unidentified.     

Tumour necrosis factor-alpha,interleukin-2 soluble receptor and different inflammatory parameters in patients with rheumatoid arthritis

BACKGROUND AND AIMS: Although the participation of cytokines in the pathogenesis of rheumatoid arthritis (RA) seems to be unequivocal, their relationship with current serum markers of this disease is not clear. The present study analyses whether there is any correlation between the levels of tumour necrosis factor-alpha (TNF-alpha), interleukin-2 soluble receptor (sIL-2R) and the concentrations of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and beta(2)-microglobulin in a group of 21 patients with RA, all rheumatoid factor positive. METHODS: The levels of TNF-alpha and sIL-2R were analysed in association with other parameters of inflammation (ESR, CRP and beta(2)-microglobulin). RESULTS: In comparison with the control group, RA patients presented high median levels of both cytokines, TNF-alpha (6.4 pg/ml) and sIL-2R (56 pmol/L), as well as of ESR (34 mm/h), CRP (0.9 mg/dl) and beta(2)-microglobulin (1.6 mg/dl) (p < 0.01). However, only ESR levels in the RA group significantly differ from the control group (p < 0.01). No correlation was found between the inflammatory parameters. CONCLUSIONS: These results suggested that TNF-alpha and slL-2R levels are up-regulated in RA patients but did not significantly differ from the control group. Due to the chronic course of this disease, other inflammatory markers must be identified in order to provide early therapeutic strategies to these patients.     

Changes in plasma zinc,copper, iron,and hepatic metallothionein in adjuvant-induced arthritis treated with cyclosporin

The early changes in hepatic metallothionein (MT) and plasma zinc (Zn), copper (Cu), and iron (Fe) were investigated during the induction of adjuvant (AJ) arthritis in rats in conjunction with cyclosporin (CSA) treatment. Plasma Zn decreased after AJ injection (60% of control values at 8 h), and this was associated with a 4.5-fold increase in hepatic MT at 8 h. Plasma Zn was lowest at 16 h (40% of control), whereas hepatic MT concentrations increased to a maximum of 20-fold at 16 h. Changes in plasma Fe paralleled those of Zn, whereas plasma Cu levels were increased. Plasma metal and hepatic MT concentrations returned toward normal from d 1–7. At d 14, when marked paw swelling was apparent, hepatic MT and plasma Cu were again increased and plasma Zn decreased. Administration of CsA decreased MT induction in rats injected with AJ and also caused a marked recovery in plasma Zn and Fe levels. These changes were small but significant even in the early stages (up to 24 h) after AJ injection and were followed by a sustained improvement in all parameters, corresponding to the nonappearance of clinical arthropathy in CsA-treated rats. TNF-α and IL-6 production by peritoneal macrophages isolated from AJ-injected rats was significantly decreased by CsA treatment at d 7 and 14. The inhibition of hepatic MT induction during acute and chronic inflammation by cyclosporin emphasizes the role of the immune system in altered metal homeostasis in inflammation.     

Oestradiol-17beta, progesterone, FSH and LH in prepubertal calves induced to superovulate

Fluorogestone acetate (vaginal sponge for 4 days) and PMSG (i.m. injection at the time of sponge insertion) treatment was administered to seven 3-month-old calves to induce superovulation. Samples of peripheral plasma were taken every 4 h during treatment (4 days) and then every 2 h for 7 days. FSH, LH, oestradiol and progesterone were measured by radioimmunoassays. In all calves oestradiol concentrations increased 24 h after PMSG injection and reached the highest levels (41-502 pg/ml) during the preovulatory surge of both gonadotropins. The surge of LH and FSH occurred from 12 to 22 h after cessation of treatment. The maximum levels of LH and FSH were 11-72 ng/ml and 23-40 ng/ml respectively and occurred within 4 h of each other. Between 40 and 68 h after the LH peak the concentrations of progesterone began to increase from basal values, reaching 24.0-101.7 ng/ml when the animals were killed. A quantitative relationship was found between plasma oestradiol concentration and the numbers of ovulating follicles. Progesterone levels seemed to be related to the numbers of corpora lutea and also to the numbers of unovulated follicles. Gonadotrophin output was not quantitatively related to ovarian activity or to steroid secretion.     

NRF2 as a determinant of cellular resistance in retinoic acid cytotoxicity

Clinical use of retinoic acids (RA) is hindered by toxicity possibly related to oxidative stress. Recently, RA at relatively low concentrations was shown to inhibit NRF2 and the expression of its target antioxidative genes. This raises the possibility that RA toxicity may result from cellular inability to cope with resultant oxidative stress. Using in vitro cell and in vivo mouse models, we report that RA, specifically all-trans-RA (atRA) at concentrations implicated in toxicity, can activate NRF2 and induce NRF2 target genes, particularly the subunits of the rate-limiting enzyme of glutathione biosynthesis, glutamate cysteine ligase (GCLM/GCLC). RNA interference-mediated silencing of NRF2, but not of retinoid X receptor-alpha and -beta, reduced basal and atRA-induced GCLM/GCLC gene expression. Moreover, RA increased nuclear accumulation of NRF2, antioxidant response element (ARE) reporter activity, and NRF2 occupancy at AREs. 4-Hydroxynonenal, a lipid peroxidation product, was increased by RA. Inhibition of MEK1/ERK mitogen-activated protein kinases significantly suppressed atRA-induced NRF2 activation and ARE-regulated gene expression, reducing cell resistance against toxic concentrations of RA. NRF2-silenced cells were vulnerable to atRA-induced mitochondrial toxicity and apoptosis. In conclusion, toxic RA activates NRF2, thereby triggering an adaptive response against the resultant oxidative stress. NRF2 enhancement as a therapeutic target of retinoid toxicity awaits further investigation.     

Carboxypeptidase N (kininase I) activity in blood and synovial fluid from patients with arthritis

Carboxypeptidase N (CPN, kininase I) and kininase II (angiotensin converting enzyme) activities were measured simultaneously in blood plasma and synovial fluid in patients suffering from rheumatoid arthritis (RA), psoriatic arthritis (PA) and osteoarthritis (OA) and in the plasma of normal volunteers. CPN levels (defined as the rate of hydrolysis of furylacryloyl-Ala-Lys) in blood were modestly increased and correlated with erythrocyte sedimentation rate in RA and PA. Based on the hydrolysis of synthetic substrates, CPN activity was much higher than kininase II activity in synovial fluid (SF). SF kininase activities were always inferior to the blood levels in all patients and were correlated with the logarithm of SF leukocyte counts, an indicator of the intensity of inflammation. In addition, CPN and albumin levels in SF were highly correlated when expressed as a percent of the plasma concentrations. Biochemical properties of CPN in crude SF confirmed its similarity to blood CPN. Polymorphonuclear leukocytes derived from inflammatory SF did not release CPN. It is concluded that kininases diffuse from the blood into SF through increased vascular permeability and that CPN could be a major metabolic pathway for kinins in this form of exudate. CPN leads to the formation of des-Arg kinins, selective agonists of the B1 receptors for kinins.     

NRF2 as a determinant of cellular resistance in retinoic acid cytotoxicity

Clinical use of retinoic acids (RA) is hindered by toxicity possibly related to oxidative stress. Recently, RA at relatively low concentrations was shown to inhibit NRF2 and the expression of its target antioxidative genes. This raises the possibility that RA toxicity may result from cellular inability to cope with resultant oxidative stress. Using in vitro cell and in vivo mouse models, we report that RA, specifically all-trans-RA (atRA) at concentrations implicated in toxicity, can activate NRF2 and induce NRF2 target genes, particularly the subunits of the rate-limiting enzyme of glutathione biosynthesis, glutamate cysteine ligase (GCLM/GCLC). RNA interference-mediated silencing of NRF2, but not of retinoid X receptor-α and -β, reduced basal and atRA-induced GCLM/GCLC gene expression. Moreover, RA increased nuclear accumulation of NRF2, antioxidant response element (ARE) reporter activity, and NRF2 occupancy at AREs. 4-Hydroxynonenal, a lipid peroxidation product, was increased by RA. Inhibition of MEK1/ERK mitogen-activated protein kinases significantly suppressed atRA-induced NRF2 activation and ARE-regulated gene expression, reducing cell resistance against toxic concentrations of RA. NRF2-silenced cells were vulnerable to atRA-induced mitochondrial toxicity and apoptosis. In conclusion, toxic RA activates NRF2, thereby triggering an adaptive response against the resultant oxidative stress. NRF2 enhancement as a therapeutic target of retinoid toxicity awaits further investigation.