Hepatic differentiation of human adipose tissue-derived mesenchymal stem cells and adverse effects of arsanilic acid and acetaminophen during in vitro hepatic developmental stage.

In the present study, we differentiated hepatocyte-like cells (HLCs) from human adipose tissue-derived mesenchymal stem cells (AT-MSCs). The hepatic differentiation was confirmed by increases in hepatic proteins or genes, the cytochrome P450 (CYP) activities, albumin secretion, and glycogen storage. To determine the developmental toxic effect of arsanilic acid (Ars) and acetaminophen (AAP) on the hepatic development, the differentiating cells were treated with the test chemicals (below IC12.5) from day 4 to day 13. The enzymatic activities of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) did not significantly differ in response to Ars treatment. AAP treatment increased the activities of all enzymes in a dose-dependent manner, significantly at concentrations of 2.5 and 5 mM of AAP. On the expressions of hepatic genes for Ars, the expressions were significantly inhibited by more than 0.5 mM for Albumin (ALB), but only 2.5 mM for α-feto protein (AFP). In the AAP-treated group, the expressions of ALB and AFP were significantly decreased at the concentrations exceeding 0.625 mM. The activities of CYP3A4 were not changed by both treatments. The activities of CYP1A2 were increased by AAP, whereas it was decreased by Ars treatment. In conclusion, AAP could cause serious adverse effects during the hepatic development as compared to Ars.

Synthesis of a fluorescently labeled compound for the detection of arsenic-induced apoptotic HL60 cells.

Arsenic compounds have shown medical usefulness since they proved to be effective in causing complete remission of acute promyelocytic leukemia. In this work we obtained a fluorescently labeled arsenic compound that can be used with current fluorescence techniques for basic and applied research, focused on arsenic-induced apoptosis studies. This compound is an arsanilic acid bearing a covalently linked FITC that was chemically synthesized and characterized by fluorescence, UV-Vis, mass and FTIR spectrometry. In addition, we assessed its apoptotic activity as well as its fluorescent labeling properties in HL60 cell line as a leukemia cell model through flow cytometry. We obtained a compound with a 1:1 FITC:arsenic ratio and a 595 m/z, confirming its structure by FTIR. This compound proved to be useful at inducing apoptosis in the leukemia cell model and labeling this apoptotic cell population, in such a way that the highest FITC fluorescence correlated with the highest arsenic amount.

Signaling pathway of glutamate in the vestibular nuclei following acute hypotension in rats.

Acute hypotension induces excitation of electrical activity and expression of c-Fos protein and phosphorylated extracellular signal-regulated kinase (pERK) in the vestibular nuclei. Expression of c-Fos protein and pERK is mediated by the excitatory neurotransmitter, glutamate. In this study, in order to investigate the signaling pathway of glutamate in the vestibular nuclei following acute hypotension, expression of the NR2B subunit of glutamate N-methyl-D-aspartate (NMDA) receptors and the GluR1 subunit of glutamate alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors was measured by Western blotting in the medial vestibular nucleus (MVN) following acute hypotension in bilateral labyrinthectomized (BL) rats. In intact labyrinthine animals, acute hypotension increased expression of pGluR1 and pNR2B in the MVN. Expression of pGluR1 Ser831 and Ser845 peaked at 5 and 30 min after acute hypotension and expression of pNR2B peaked at 60 min after acute hypotension, respectively. In BL animals, expression of pGluR1 Ser831, pGluR1 Ser845, and pNR2B was decreased significantly compared to intact labyrinthine animals following acute hypotension. These results suggest that excitatory afferent signals from the peripheral vestibular receptors, resulting from acute hypotension, release glutamate into postsynaptic neurons in the vestibular nuclei and the excitatory signals are transmitted through the GluR1 subunit of the AMPA receptors and the NR2B subunits of the NMDA receptors in the vestibular system.

Effect of arsanilic acid on performance and residual of arsenic in tissue of Japanese laying quail.

Three hundred seventy-five 63-d-old laying Japanese quail were randomly distributed into 3 experimental groups (125 birds per group) and fed the following diets for 40 d, with 30 d on the experimental diets, followed by a 10-d withdrawal period: 1) control; 2) 50 mg of 4-arsanilic acid/kg of feed; and 3) 100 mg of 4-arsanilic acid/kg of feed. Each treatment consisted of 5 replicates of 25 birds. During the first 30 d of the experiment, all eggs were recorded, collected, individually weighed daily, and feed consumption was determined every 10 d. Five quail from each replicate in the experiment were euthanized by cervical dislocation at 0, 30, 35, and 40 d. Tissue samples from the liver, kidney, heart, gizzard, and the muscle on the breast and leg were collected for determination of As residue. The feces and eggs at 0, 30, 35, and 40 d of the experiment were selected for determination of As. Results showed that dietary inclusion of 50 and 100 mg/kg of 4-arsanilic acid significantly improved feed utilization and egg production, but the concentration of As in the tissues and feces in groups fed 4-arsanilic acid was higher than in control group. The results of the present study demonstrate that the use of organic As compounds as feed additives in diet is a matter for argument.

Human sperm immobilizing activity of aminophenyl arsenic acid and its N-substituted quinazoline, pyrimidine, and purine derivatives: protective effect of glutathione.

We examined the potential toxicity of pentavalent organic arsenicals for human sperm. We used computer-assisted sperm analysis to examine the effects of three aminophenyl arsenicals and their nine N-substituted quinazoline, pyrimidine, and purine derivatives on human sperm motility and kinematics in human semen and medium. Among the arsenicals examined, (aminophenylazo)-phenyl arsonic acid and its N-substituted pyrimidine derivative PHI-370 (2-methylthio-4-[(4'-aminophenylazo)-phenylarsonic acid] pyrimidine) exhibited rapid sperm immobilizing activity in medium with EC(50) values of 77 and 82 microM, respectively, and t(1/2) of < 3 min. Molecular modeling analysis indicated that sperm-immobilizing organic arsenicals exhibit high dipole moments (>7 Debyes). Sperm immobilizing activity of these arsenicals was completely abrogated in the presence of seminal plasma. Furthermore, coincubation of motile sperm with PHI-370 in the presence of reduced glutathione (GSH) resulted in dose-dependent protection of sperm motility and sperm motion parameters. Coincubation of the arsenical with GSH at a molar ratio of 1:20 resulted in 95% retention of sperm progressive motility. The mean values of the other sperm movement characteristics also showed > 90% protection. These observations suggest that the rapid sperm immobilizing activity of these pentavalent arsenicals may be as a result of direct binding of the arsenical with the sperm thiol components essential for sperm motility as well as induction of oxidative damage by disruption of sperm cell's antioxidant system. Sodium arsanilate and its N-substituted pyrimidine derivative, PHI-370, are useful probes to further evaluate the mechanism of pentavalent arsanilate-induced human sperm dysfunction.

Prevention of vestibular deafferentation-induced spontaneous nystagmus with pretreatment of Ca2+ channel/N-methyl-D-aspartic acid receptor antagonists in guinea pigs.

Involvement of Ca2+ channel and N-methyl-D-aspartic acid (NMDA) receptors with induction of the spontaneous nystagmus (SN) after unilateral labyrinthectomy (UL) was evaluated by examining the effect of verapamil and MK-801 in guinea pigs. An injection of verapamil or MK-801 before the intratympanic application of arsanilate significantly (p < 0.05) suppressed the frequency of the SN towards the arsanilate-applied side. The frequency of the SN towards the intact side in these drug-treated animals was much lower than in the control animals until 36 h after the application of arsanilate. In addition, 60 days subsequent to induction of the UL, application of the drug before we injected the arsanilate into the opposite middle ear suppressed the SN towards the second arsanilate-injected side, but not towards the first injection side. We suggest that Ca2+ channel and NMDA receptor may be involved in the induction of the SN, and that the pretreatment of their antagonists could be applied in preventing the vestibular deafferentation-induced SN.

Evidence for the involvement of NMDA receptors in vestibular compensation.

The involvement of NMDA (N-methyl-D-aspartic acid) receptors in the initial stage of the vestibular compensation was evaluated by examining the effect of MK-801 on this compensation in guinea pigs. MK-801, injected 30 min before induction of unilateral labyrinthectomy by an arsanilate, significantly (P < 0.05) reduced the maximum frequency of the spontaneous nystagmus (SN) towards both the arsanilate-applied and the intact sides. In addition, injection of arsanilate into the opposite middle ear, 60 days subsequent to induction of the unilateral labyrinthectomy, suppressed the SN towards the second injected side, but had no effect on the SN towards the first injected side. These results suggest that NMDA receptors may be linked to the initiation of the vestibular compensation.

Characterization of the limited specificity of antigen recognition in earthworms.

Earthworms respond to parenteral stimulation with a protein antigen by the formation of an antigen-binding protein. Earthworms were parenterally stimulated with different proteins and the protein binding was estimated in vitro on both humoral and cellular levels. The binding was significantly higher when the same protein was used for in vivo stimulation. The degree of specificity of the antigen-binding protein after the secondary in vivo challenge increased, but even so it was considerably lower than that of vertebrate immunoglobulins.

Repeated antigenic challenge induces earthworm coelomocyte proliferation.

Annelids are able to protect themselves against foreign materials by natural and acquired cellular immunity. The aim of this study was to characterize the kinetic of antigen induced proliferation of earthworm coelomocytes. The proliferative activity of free coelomocytes of Eisenia foetida decreased after a second contact with the same antigen. Precursor cells in the mesenchymal lining of the coelomic cavity responded to stimulation immediately. These results indicate that for a successful triggering of the proliferative response of the free coelomocytes repeated contact with stimulating agents is needed.

Sodium arsanilate-induced vestibular dysfunction in meadow voles (Microtus pennsylvanicus): effects on posture, spontaneous locomotor activity and swimming behavior.

Vestibular dysfunction was chemically induced in male meadow voles (Microtus pennsylvanicus) by intratympanic injections (30 mg per side) of sodium arsanilate (atoxyl). The control group received intratympanic injections of isotonic saline. After a one-week recovery period the voles were behaviorally assayed for integrity of their labyrinthine systems. All subjects were tested for the presence of the air-righting reflex and body rotation-induced nystagmus. Three weeks later a multivariate assessment of spontaneous motor activity of the voles was carried out in the automated Digiscan Activity Monitor. In addition, the swimming behavior of the voles was examined. Voles with vestibular dysfunction exhibited pronounced postural abnormalities (head dorsiflexion), were not able to swim with their nose above the water for a 1 min test period, and displayed disorientation and thrashing movements. In the Digiscan activity test the atoxyl-treated voles displayed significantly more activity in the horizontal measures (Ps less than 0.01), including greater distance travelled per movement and greater speed of movements, relative to the control animals. The labyrinthectomized group also spent significantly (P less than 0.05) less time in vertical movements and exhibited significantly more time in stereotypic behavior (P less than 0.01), relative to controls. Atoxyl-treated voles also showed significantly less thigmotaxis (wall-hugging) than the control animals (P less than 0.01). In general, changes in spontaneous behavior observed in the sodium arsanilate-treated voles were consistent with the presence of postural and balance abnormalities and a redirecting of exploratory vertical movements toward horizontal locomotion to the extent that these animals were clearly hyperactive in this dimension. The multivariate behavioral assessment available in the Digiscan Activity Monitoring system, thus seems to be especially useful in the examination of behavioral components affected by vestibular dysfunction.

Influence of virginiamycin on growth and efficiency of large white turkeys.

Efficacy of virginiamycin (22 mg/kg) in combination with no drug, amprolium, carbarsone, halofuginone, or monensin, was studied. Male and female turkeys were raised to market age in five experiments conducted from 1983 to 1987. Body weights and feed:gain responses to virginiamycin for males and females were positive and significant (P less than .05). Virginiamycin resulted in mean 5.2 and 6.3% body weight responses and 3.3 and 2.2% feed:gain responses for males at 19 or 20 wk of age and for females at 16 or 17 wk of age, respectively. Mortality rates were low in all studies, and were not influenced by virginiamycin. In a processing study, virginiamycin in combination with halofuginone did not affect shrinkage, yield, or market grade. Feed was utilized by males and females 3.9 and 3.0%, respectively, more efficiently than expected with dietary virginiamycin, compared with results predicted by a simulation modeling technique. Profitability was considerably greater with dietary virginiamycin using actual data than with simulated feed consumption data.

Sodium arsanilate-induced vestibular dysfunction in rats: effects on open-field behavior and spontaneous activity in the automated digiscan monitoring system.

Vestibular dysfunction was chemically induced in Long-Evans rats by intratympanic injections (30 mg per side) of sodium arsanilate (atoxyl). Following a one-week recovery period the rats were behaviorally assayed for integrity of the labyrinthine systems. All subjects were tested for presence of the air-righting reflex, the contact-righting reflex (by lightly holding a sheet of Plexiglas against the soles of the rat's feet), and body rotation-induced nystagmus. All animals were then tested for their ability to remain on a small (15 x 15 cm) platform. Next, the subjects were given two 10-min open-field tests during which ambulation, rearing, grooming, and defecation responses were recorded. Four to five weeks later all rats were tested twice (60 min per session) in the automated Digiscan Activity Monitor which provides a multivariate assessment of spontaneous motor activity. The rats with vestibular dysfunction (Group VNX) took significantly less time to fall off the platform (p less than 0.01). They also exhibited significantly more open-field ambulation but fewer rearing responses (ps less than 0.01). An examination of group correlation coefficients for open-field variables and the platform test scores revealed some interesting group differences (ps less than 0.05). In the Digiscan tests the atoxyl-treated rats exhibited fewer number of horizontal movements, but increased speed for these movements (ps less than 0.05). Vertical movements did not differ significantly in incidence, but these movements were greatly reduced in duration (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Reactions of lipoamide dehydrogenase and glutathione reductase with arsonic acids and arsonous acids.

Lipoamide dehydrogenase reacts irreversibly with arsonous acids, RAs(OH)2, and arsonic acids, RAs(O)(OH)2, to form enzyme-inhibitor complexes. The formation of inactive enzyme requires NADH and is kinetically first order in the presence of excess arsonous acid. The second-order rate constant for formation of the enzyme-inhibitor complex was 545 min-1 M-1 for phenylarsonous acid, C6H5As(OH)2, and 5640 min-1 M-1 for methanearsonous acid, CH3As(OH)2. The kinetics of formation of inactive enzyme in the presence of arsonic acids was found to obey a rate law predicted by a two-step mechanism in which a rate-limiting reduction of an arsonic acid to the corresponding arsonous acid by reduced enzyme, E(SH)2, preceded formation of an inactive binary complex of reduced enzyme and arsonous acid: ES2 + NADH + H+ = E(SH)2 + NAD+; E(SH)2 + RAs(O)(OH)2 = ES2 + RAs(OH)2 + H2O; and E(SH)2 + RAs(OH)2 = ES2AsR + 2H2O. GSSG reductase reacts reversibly with C6H5As(OH)2 to form an inactive binary addition compound in the presence of NADPH. The value of the association constant for formation of enzyme inhibitor complex at pH 7.0 was 119 M-1. The initial rate of the GSSG reductase-catalyzed oxidation of NADPH by GSSG was insensitive to MeAs(OH)2. The kinetics of inhibition of GSSG reductase by arsenite and C6H5As(O)(OH)2 were found to obey the rate law described for lipoamide dehydrogenase and arsonic acids. GSSG reductase catalyzed the oxidation of NADPH by p-arsanilic acid. The initial rate of oxidation of NADPH was linearly dependent on enzyme concentration. The turnover number for GSSG reductase with p-arsanilic acid as an oxidant was 0.13 mol NADPH mol FAD-1 min-1.

[Effect of arsanilic acid on various growth parameters and sugar transport in the rat]. / Influencia del ácido arsanílico sobre algunos factores de crecimiento y el transporte de azúcares en rata.

The effects of arsanilic acid as promoter of faster growth on the nutrition index and intestinal absorption of galactose and arabinose has been studied in growing rats. The rats that received arsanilic acid 0.7 mg/day added to water showed gain in body weight and feed intake, I.T. (feed conversion ratio) decrease and P.E.R. (protein efficiency ratio) increase. A dose of 1.4 mg/day produced harmful effects. The in vitro intestinal transport of galactose and arabinose decreased and increased respectively in animals that received additive in comparison with controls.

Development of a method for the incorporation of substitution-inert metal ions into proteins. Site-specific modification of arsanilazotyrosine-248 carboxypeptidase A with cobalt(III).

This investigation demonstrates the use of substitution-inert metal ions as site-specific amino acid modifying reagents. The approach involves the production of a chelating agent at the site of interest with the subsequent in situ oxidation of substitution-labile cobalt(II) to exchange-inert cobalt(III) with H2O2. We have produced the chelate complex ethylenediamine-N,N'-diacetato(arsanilazotyrosinato-248 carboxypeptidase A)cobalt(III) [CoIII(EDDA)(AA-CPA-Zn)]. Model CoIII(EDDA)(azophenolate) complexes have helped to define the reaction conditions necessary to produce the enzyme derivative and have proved invaluable in the spectral analysis of the cobalt(III)-enzyme complex. The modified enzyme contains one active-site zinc and one externally bound cobalt per enzyme monometer. Circular dichroism and visible spectra of the derivative and apoenzyme substantiate the site-specific nature of the incorporation. Concimitant with CoIIIEDDA incorporation, the enzyme loses its peptidase activity yet maintains with FeIIEDTA returns the original properties of the arsanilazotyrosine-248 enzyme.

Phosphonic and arsonic acids as inhibitors of human red cell acid phosphatase and their use in affinity chromatography.

1. In order to obtain an effective ligand for affinity chromatography of the low molecular weight acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) from human red cells nine phosphonic and two arsonic acid substrate analogues were investigated as potential inhibitors. The two forms of acid phosphatase type B (b1 and b2) were isolated and partially purified using conventional methods and the inhibitory action of the substrate analogs investigated. 2. Four of the phosphonic acids were relatively effective competitive inhibitors. It appears that certain structural and electronic requirements have to be fulfilled by the phosphonic acids in order to exhibit significant affinity for the enzyme. A high affinity appears to require the presence of a bulky, hydrophobic moiety which has to be separated from the phosphorus atom by the distance of one atom. 3. p-Aminobenzylphosphonic acid exerted the highest affinity for acid phosphatase with a pH optimum at 6.5. Ki values of 4 . 10(-4) and 6 . 10(-4) M were found for the b1 and b2 forms, respectively. 4. Coupling of p-aminobenzylphosphonic acid to Agarose yielded an effective and specific affinity medium. By means of affinity chromatography using this medium, acid phosphatase was purified 500-fold in a single step.

Studies on delayed hypersensitivity to protein antigen. Induction of delayed hypersensitivity by chemically modified antigen.

it was shown in our previous paper that mice primed with chemically modified bacterial alpha-amylase (BaA), which was neither cross-reactive with anti-BaA antibody nor able to induce a humoral anti-BaA response, developed enhanced responses to a subsequent challenge with native BaA and that the magnitude of the immunological memory was closely related to the priming dose of modified BaA. This paper describes the experimental conditions for induction of delayed hypersensitivity (DH) by modified BaA in relation to the development of immunological memory for antibody response to native BaA. Mice primed with either an intraperitoneal (i.p.) or subcutaneous (s.c.) injection of modified BaA in complete Freunds adjuvant (CFA) developed enhanced anti-BaA as the immunogen and modified BaA as the eliciting antigen, the relationship of anti-BaA responses to a subsequent challenge with BaA. In contrast, when mice were immunized with an s.c. injection of the modified BaA only, a significant level of DH to native BaA could be induced, as measured by the footpad reaction (FPR). The highest degree of DH was observed in mice given 50 micrograms of modified BaA. DH was detectable within 5 days and persisted for 25 days after immunization. In the reciprocal combination of native BaA as the immunogen and modified BaA as the eliciting antigen, the relationship of anti-BaA responses to DH was examined. The primary anti-BaA responses induced by an i.p. injection of large doses of BaA was markedly higher than those induced by an s.c. injection, while DH was exhibited only in mice given s.c. injection of BaA in CFA. With respect to DH to native BaA induced by the modified BaA, it was shown that C3H/He mice were high and C57BL/6 mice were low responders.