Acute toxicity of organic chemicals to Gammarus pulex correlates with sensitivity of Daphnia magna across most modes of action.

We investigated the sensitivity of the freshwater crustacean amphipod Gammarus pulex towards organic xenobiotic compounds in comparison to the sensitivity of the crustacean cladoceran Daphnia magna. In addition we studied the influence of the chemical's mode of action on the relationship between the sensitivity of G. pulex and that of D. magna. We tested the acute toxicity of twelve compounds (Malathion, Aldicarb, Carbofuran, 2,4-dichloroaniline, 2,4-dichlorophenol, 1,2,3-trichlorobenzene, 4,6-dinitro-o-cresol, 2,4,5-trichlorophenol, Ethylacrylate, 4-nitrobenzyl-chloride, Sea-nine, Imidacloprid) with different modes of action and physicochemical properties towards the freshwater amphipod G. pulex in laboratory experiments. Additional toxicity data was collected from the peer-reviewed literature and databases (data pairs for 44 chemicals in total). The chemicals were assigned to seven mode of action groups. The relationship between the sensitivity of G. pulex (48h-LC50s and 96h-LC50s) and that of D. magna (48h-EC50s) was investigated using regression analysis and correlation plots. G. pulex is two to three orders of magnitude more sensitive towards neonicotinoids than D. magna (P=0.0046, n=3). For organophosphates we found that D. magna is more sensitive than G. pulex by approximately a factor of six (P=0.0256, n=6). There was no significant difference between the sensitivity of D. magna and that of G. pulex in any of the other mode of action groups; however chemicals with the same mode of action grouped together in the same area of the correlation plot. Without the neonicotinoids 75% of all G. pulex toxicity data were within one order of magnitude of the D. magna data and 100% within two orders of magnitude. The regressions with all data and with all data minus neonicotinoids were both significant linear relationships with slopes around one and intercept around zero. Thus, G. pulex is generally equally sensitive towards organic xenobiotics as D. magna.

Adaptations in microbiological populations exposed to dinitrophenol and other chemical stressors.

Microbiological populations in natural and engineered systems may experience multiple exposures to chemical stressors, which may affect system functions. The impact of such exposures on the metabolism of a population of Pseudomonas aeruginosa was studied using respirometry. Two serial exposures to low concentrations of 2,4-dinitrophenol (DNP), pentachlorophenol (PCP), or N-ethyl maleimide (NEM) did not affect metabolism beyond that expected for a single exposure. However, at higher concentrations, three exposures to DNP led to a combination of metabolic stress and resilience in the population. At a low DNP concentration of 400 mg/L, multiple exposures led to increased stress but indicated no development of resilience. At a high DNP concentration of 1,200 mg/L, no biological activity was observed, indicating that the population did not survive the exposure. At intermediate concentrations of 800 and 900 mg/L DNP, stress was observed, but it was found to decrease after multiple exposures. This, combined with the observation that the size of the population decreased, indicated that resilience in the population had developed because of elimination of the weaker organisms in the population. In contrast, the lack of resilience at the lower DNP concentration was attributed to the survival of the strong as well as weak members, lowering the resilience of the population as a whole. The development of resilience within a window of stressor concentrations is an important finding with implications for predicting the performance of biotreatment processes and biosensor technologies and for interpreting ecotoxicity risk assessments.

Discriminating between different acute chemical toxicities via changes in the daphnid metabolome.

Currently, there is widespread interest in exploiting "omics" approaches to screen the toxicity of chemicals, potentially enabling their rapid categorization into classes of defined mode of action (MOA). Direct infusion Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) metabolomics provides a sensitive and nontargeted analysis of potentially a thousand endogenous metabolites. Our previous work has shown that mass spectra can be recorded from whole-organism homogenate or hemolymph of single adult Daphnia magna. Here we develop multivariate models and discover perturbations to specific metabolic pathways that can discriminate between the acute toxicities of four chemicals to D. magna using FT-ICR MS metabolomics. We focus on model toxicants (cadmium, fenvalerate, dinitrophenol, and propranolol) with different MOAs. First, we confirmed that a toxicant-induced metabolic effect could be determined for each chemical in both the hemolymph and the whole-organism metabolome, with between 9 and 660 mass spectral peaks changing intensities significantly, dependent upon toxicant and sample type. Subsequently, supervised multivariate models were built that discriminated significantly all four acute metabolic toxicities, yielding mean classification error rates (across all classes) of 3.9 and 6.9% for whole-organism homogenates and hemolymph, respectively. Following extensive peak annotation, we discovered toxicant-specific perturbations to putatively identified metabolic pathways, including propranolol-induced disruption of fatty acid metabolism and eicosanoid biosynthesis and fenvalerate-induced disruption of amino sugar metabolism. We conclude that the metabolic profiles of whole-daphnid homogenates are more discriminatory for toxicant action than hemolymph. Furthermore, our findings highlight the capability of metabolomics to discover early-event metabolic responses that can discriminate between the acute toxicities of chemicals.

Effect of chlorogenic acid on mast cell-dependent anaphylactic reaction.

Chlorogenic acid (CGA), a naturally occurring polyphenol compound, has a number of biological activities. However, roles of CGA in the mast cell-dependent anaphylactic reaction have not been fully examined. In the present study, the effect and mechanism of CGA on mast cell-dependent anaphylactic reaction were investigated using in vivo and in vitro models. CGA inhibited compound 48/80-induced systemic anaphylactic shock in mice and skin vascular permeability in rats. CGA also inhibited anti-dinitrophenyl (DNP) IgE-mediated passive cutaneous anaphylaxis (PCA). Moreover, CGA dose-dependently reduced histamine and TNF-alpha release from RBL-2H3 cells activated by anti-DNP IgE. Pretreatment with CGA suppressed IgE-antigen complex induced calcium uptake into RBL-2H3 cells. When CGA was added, the level of intracellular cyclic adenosine monophosphate (cAMP) in RBL-2H3 cells was significantly elevated compared with the untreated cells. Decreased calcium uptake and increased cAMP level might be involved in the inhibitory effect of CGA on mast cell activation. These results suggest a possible therapeutic application of CGA in allergic diseases.

Review of testicular toxicity of dinitrophenolic compounds, 2-sec-butyl-4,6-dinitrophenol, 4,6-dinitro-o-cresol and 2,4-dinitrophenol.

The present review paper summarizes the data available in the literature concerning dinitrophenolic compounds and evaluates male reproductive toxicity in experimental animals. Gavage and feeding doses of 2-sec-butyl-4,6-dinitrophenol (dinoseb; CAS No. 88-85-7) manifested testicular toxicity, and 4,6-dinitro-o-cresol (DNOC; CAS No. 534-52-1) showed similar but weaker testicular toxicity in laboratory animals. Consecutive doses of dinoseb and DNOC by gavage seemed to induce spermatotoxicity by disturbing spermiogenesis or the maturation process of sperm in the epididymis, and the most probable target cells of spermatotoxicity were thought to be testicular spermatids in rats. Prolonged exposure to dinoseb and DNOC in the diet also induced testicular toxicity in rats. However, the feeding dose of dinoseb irreversibly affected the early stage of spermatogenesis and produced infertility in rats. On the other hand, 2,4-dinitrophenol (DNP; CAS No. 51-28-5) did not show testicular toxicity in laboratory animals according to available literature. Further studies in laboratory animals with nitrophenolic compounds are required for clarification of their testicular toxicity and for risk assessment in humans.

Decreased epithelial barrier function evoked by exposure to metabolic stress and nonpathogenic E. coli is enhanced by TNF-alpha.

A defect in mitochondrial activity contributes to many diseases. We have shown that monolayers of the human colonic T84 epithelial cell line exposed to dinitrophenol (DNP, uncouples oxidative phosphorylation) and nonpathogenic Escherichia coli (E. coli) (strain HB101) display decreased barrier function. Here the impact of DNP on macrophage activity and the effect of TNF-alpha, DNP, and E. coli on epithelial permeability were assessed. DNP treatment of the human THP-1 macrophage cell line resulted in reduced ATP synthesis, and, although hyporesponsive to LPS, the metabolically stressed macrophages produced IL-1beta, IL-6, and TNF-alpha. Given the role of TNF-alpha in inflammatory bowel disease (IBD) and the association between increased permeability and IBD, recombinant TNF-alpha (10 ng/ml) was added to the DNP (0.1 mM) + E. coli (10(6) colony-forming units), and this resulted in a significantly greater loss of T84 epithelial barrier function than that elicited by DNP + E. coli. This increased epithelial permeability was not due to epithelial death, and the enhanced E. coli translocation was reduced by pharmacological inhibitors of NF-kappabeta signaling (pyrrolidine dithiocarbamate, NF-kappabeta essential modifier-binding peptide, BAY 11-7082, and the proteosome inhibitor, MG132). In contrast, the drop in transepithelial electrical resistance was unaffected by the inhibitors of NF-kappabeta. Thus, as an integrative model system, our findings support the induction of a positive feedback loop that can severely impair epithelial barrier function and, as such, could contribute to existing inflammation or trigger relapses in IBD. Thus metabolically stressed epithelia display increased permeability in the presence of viable nonpathogenic E. coli that is exaggerated by TNF-alpha released by activated immune cells, such as macrophages, that retain this ability even if they themselves are experiencing a degree of metabolic stress.

Enhancement of allergic responses in vivo and in vitro by butylated hydroxytoluene.

The effect of butylated hydroxytoluene (BHT), which is used widely as an antioxidant, on IgE-dependent allergic responses in vivo and in vitro was investigated. For in vivo study, passive cutaneous anaphylaxis (PCA) was elicited in rats by i.d. injection of anti-DNP IgE and 48 h later by i.v. injection of DNP-HSA. BHT was i.p. given immediately after anti-DNP IgE injection. For in vitro studies, the rat mast cell line RBL2H3 sensitized with monoclonal anti-dinitrophenol (DNP) IgE was challenged with the multivalent antigen DNP-human serum albumin (DNP-HSA) in the presence or absence of BHT. beta-Hexosaminidase and histamine released from RBL2H3 cells, as indicators of degranulation of the cells, the concentration of intracellular Ca2+, the level of phosphorylated-Akt, and global tyrosine phosphorylation as indicators of mast cell activation, were measured. The results showed that BHT given to anti-DNP IgE-sensitized rats augmented DNP-specific PCA in a dose-dependent manner. In the presence of BHT, IgE-induced releases of beta-hexosaminidase and histamine from RBL2H3 cells were increased. BHT also further elevated IgE-mediated increased concentrations of intracellular Ca2+ and the levels of phosphorylated-Akt, but did not affect global tyrosine phosphorylation, in RBL2H3 cells. Moreover, the PI3K inhibitor LY294002 inhibited IgE-dependent degranulation and its enhancement by BHT. These findings indicate that BHT may upregulate PCA by enhancing mast cell degranulation associated with enhancements of intracellular Ca2+ concentration and PI3K activation, suggesting that BHT might affect allergic diseases such as allergic rhinitis and asthma.

Perivascular mast cells promote atherogenesis and induce plaque destabilization in apolipoprotein E-deficient mice.

BACKGROUND: Mast cells are major effector cells in allergy and host defense responses. Their increased number and state of activation in perivascular tissue during atherosclerosis may point to a role in cardiovascular disorders. In the present study, we investigated the contribution of perivascular mast cells to atherogenesis and plaque stability in apolipoprotein E-deficient mice. METHODS AND RESULTS: We show here that episodes of systemic mast cell activation during plaque progression in mice leads to robust plaque expansion. Targeted activation of perivascular mast cells in advanced plaques sharply increases the incidence of intraplaque hemorrhage, macrophage apoptosis, vascular leakage, and CXCR2/VLA-4-mediated recruitment of leukocytes to the plaque. Importantly, treatment with the mast cell stabilizer cromolyn does prevent all the adverse phenomena elicited by mast cell activation. CONCLUSIONS: This is the first study to demonstrate that mast cells play a crucial role in plaque progression and destabilization in vivo. We propose that mast cell stabilization could be a new therapeutic approach to the prevention of acute coronary syndromes.

Pathogenetic transition in the morphology of abnormal sperm in the testes and the caput, corpus, and cauda epididymides of male rats after treatment with 4,6-dinitro-o-cresol.

In order to elucidate the pathogenesis of tailless sperm, 4,6-dinitro-o-cresol (DNOC) was administered to Jcl:SD male rats at daily oral doses of 0, 10 or 15mg/kg for 5 days. Sperm were collected from the caput, corpus, and cauda epididymides on days 1, 7 and 14 after the last dosing (D1, D7 and D14, respectively), counted and examined morphologically by phase-contrast and scanning electron microscopy. The incidence of abnormal sperm was significantly increased in the DNOC 15mg/kg group. On D1, peeled sperm (loss of mitochondrial sheath at the proximal end of the middle piece) was frequently observed in the caput epididymides, whereas sperm in the corpus and cauda epididymides had normal morphology. Distribution of the peeled sperm changed as time passed and the corpus epididymides showed a peak incidence on D7. On D14, the highest incidence of abnormal sperm was observed in the cauda epididymides, where the major abnormality was tailless. Similar effects were also found in the 10mg/kg group but were less potent. Transmission electron microscopy of testicular sperm on D1 revealed the presence of elongated spermatids that lacked the mitochondrial sheath at the proximal end of the middle piece, although the round and elongating spermatids looked normal. These results suggest that DNOC exposure of male rats primarily causes partial loss of the mitochondrial sheath in the testicular elongated spermatids, and that the affected sperm become tailless by D14 after reaching the cauda epididymides.

Anti-allergic effects of Lycopus lucidus on mast cell-mediated allergy model.

The current study characterizes the mechanism by which the aqueous extract of Lycopus lucidus Turcz. (Labiatae) (LAE) decreases mast cell-mediated immediate-type allergic reaction. The immediate-type allergic reaction is involved in many allergic diseases such as asthma and allergic rhinitis. LAE has been used as a traditional medicine in Korea and is known to have an anti-inflammatory effect. However, its specific mechanism of action is still unknown. LAE was anally administered to mice for high and fast absorption. LAE inhibited compound 48/80-induced systemic reactions in mice. LAE decreased the local allergic reaction, passive cutaneous anaphylaxis, activated by anti-dinitrophenyl (DNP) IgE antibody. LAE dose-dependently reduced histamine release from rat peritoneal mast cells activated by compound 48/80 or anti-DNP IgE. Furthermore, LAE decreased the secretion of TNF-alpha and IL-6 in phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187-stimulated human mast cells. The inhibitory effect of LAE on the pro-inflammatory cytokine was p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) dependent. LAE attenuated PMA plus A23187-induced degradation of IkappaBalpha and nuclear translocation of NF-kappaB, and specifically blocked activation of p38 MAPK, but not that of c-jun N-terminal kinase and extracellular signal-regulated kinase. Our findings provide evidence that LAE inhibits mast cell-derived immediate-type allergic reactions and involvement of pro-inflammatory cytokines, p38 MAPK, and NF-kappaB in these effects.

Comparative studies on the spermatotoxic effects of dinoseb and its structurally related chemicals.

Three dinitrophenolic compounds, dinoseb (DNBP; 7.5 mg/kg b.w.), 4,6-dinitro-o-cresol (DNOC; 4, 7.5, 15 mg/kg b.w.), and 2,4-dinitrophenol (DNP; 7.5, 15, 30 mg/kg b.w.) were administered orally to sexually matured Jcl:SD male rats for 5 consecutive days. Half of the males in each group were necropsied at 3 (D3) and 14 (D14) days after the last dosing, respectively, and examined for the effects of dinitrophenols on spermato-/spermiogenesis. DNBP (7.5 mg/kg), DNOC (15 mg/kg), and DNP (30 mg/kg) caused 1, 5, and 0 deaths, respectively, as well as a decreased body weights during the treatment. Although examinations on D3 revealed no treatment-related alterations, DNBP and DNOC resulted in reduced sperm motility and increased incidence of tailless sperm in the cauda epididymis on D14. DNP also caused slightly increased incidence of tailless sperm on D14. These results demonstrate that DNBP, DNOC, and DNP manifest similar spermatotoxic effects at or around a lethal dose in rats.

Effects of dinoseb, 4,6-dinitro-o-cresol, and 2,4-dinitrophenol on rat Sertoli-germ cell co-cultures.

The effects of dinoseb (DNBP), a known testicular toxicant in the rat, on germ cells were investigated in Sertoli-germ cell co-cultures. Two DNBP-related dinitrophenolic compounds, 4,6-dinitro-o-cresol (DNOC) and 2,4-dinitrophenol (DNP), were also examined, as testicular toxicity of these compounds had not been elucidated. Cultures were exposed to each compound (10(-7)-10(-4)M) for 24h and examined for the number and viability of detached cells and morphologic alterations under a light microscope. DNBP significantly increased the number of detached cells (10(-5) and 10(-4)M) and suppressed their viability (10(-6)-10(-4)M). Morphologic observations revealed degenerative alterations in the germ cells and Sertoli cells. Similar effects as observed after DNBP exposure were evident at 10(-4)M DNOC and 10(-4)M DNP. These results demonstrate that DNBP, DNOC, and DNP have in vitro toxicity to these cell populations at high concentration, and suggest the possibility that DNOC and DNP also cause testicular damage in experimental animals and humans.

Ixeris dentata green sap inhibits both compound 48/80-induced aanaphylaxis-like response and IgE-mediated anaphylactic response in murine model.

We studied the inhibitory effect of the green sap of Ixeris dentata (IXD) on compound 48/80-induced anaphylaxis-like response in a murine model. IXD dose-dependently inhibited the anaphylaxis-like response induced by compound 48/80 in mice. IXD inhibited the anaphylaxis-like fatal response at the dose of 0.1 g/kg by 75%. IXD had a significant inhibitory effect on compound 48/80-induced ear swelling response at the doses of 0.05 and 0.1 g/kg. IXD (0.1 g/kg) also inhibited passive cutaneous anaphylaxis mediated by anti-dinitrophenyl IgE by 81.45%. When IXD was given as a pretreatment at concentrations ranging from 0.005 to 0.1 g/l, the histamine release from rat peritoneal mast cells induced by compound 48/80 was reduced in a dose-dependent manner. These results indicate that IXD may possess antianaphylactic activity.

Toxicity of 2,6-Di-tert-butyl-4-Nitrophenol (DBNP).

U.S. Navy submarines reported a yellowing of metal surfaces on their internal surfaces. The yellowing was initially identified on the painted steel bulkheads but further examination indicated that it was not limited to steel surfaces and included bedding, thread tape, Formica, plastisol covered hand-wheels, and aluminum lockers. Crew members also reported to the medical department that their skin turned yellow when they came in contact with these contaminated surfaces and requested information on the effects of exposure. Studies conducted by General Dynamics' Electric Boat Division (EBD) determined that the agent was 2,6-Di-tertbutyl-4-Nitrophenol (DBNP). 2,6-Di-butylphenol (DBP) is an antioxidant additive used in lubricating oils and hydraulic fluids. In the enclosed atmosphere of a submarine, the oil mist could be spread throughout the boat by venting the lube oil to the atmosphere. Submarines use electrostatic precipitators (ESP) to clean the air of particulate materials. During passage through the ESP, oil mist containing DBP is nitrated to DBNP, which is then moved throughout the boat in the ventilation system. Analysis of the EBD data indicated 24-hour exposure concentrations to be in the range of <3.0 to 122 ppb in the laboratory and submarine settings. Submarine crews may be exposed to these concentrations for as many as 24 hours/ day for 90 days during underway periods. Toxicity studies regarding the oral and dermal uptake of DBNP were conducted. From the literature the lethal dose to 50 percent of the population (LD50) of DBNP (rat) was reported by Vesselinovitch et al. in 1961 to be 500 mg/kg. Our studies indicated that the LD50 is in the range of 80 mg/kg in the rat. Our work also includes dermal absorption studies, which indicated that DBNP is not well absorbed through intact skin. Within this study, no no-observable adverse effect level (NOAEL) or lowest observable adverse effect level (LOAEL) was identified. Calculation of a reference dose was completed using standard methods based on the LD50 as a numerator with several uncertainty and modifying factors. EBD's determination of airborne concentrations aboard submarines fall in the range of these anticipated allowable concentrations and could indicate significant chronic exposures. No adverse effects from DBNP exposures have been reported to date.

Monitoring impact of a pesticide treatment on bacterial soil communities by metabolic and genetic fingerprinting in addition to conventional testing procedures.

Herbogil (dinoterb), a reference herbicide, the mineral oil Oleo (paraffin oil used as an additive to herbicides), and Goltix (metamitron) were taken as model compounds for the study of impacts on microbial soil communities. After the treatment of soil samples, effects on metabolic sum parameters were determined by monitoring substrate-induced respiration (SIR) and dehydrogenase activity, as well as carbon and nitrogen mineralization. These conventional ecotoxicological testing procedures are used in pesticide registration. Inhibition of biomass-related activities and stimulation of nitrogen mineralization were the most significant effects caused by the application of Herbogil. Even though Goltix and Oleo were used at a higher dosage (10 times higher), the application of Goltix resulted in smaller effects and the additive Oleo was the least-active compound, with minor stimulation of test parameters at later observation times. The results served as a background for investigation of the power of "fingerprinting" methods in microbial ecology. Changes in catabolic activities induced by treatments were analyzed by using the 95 carbon sources provided by the BIOLOG system. Variations in the complex metabolic fingerprints demonstrated inhibition of many catabolic pathways after the application of Herbogil. Again, the effects of the other compounds were expressed at much lower levels and comprised stimulations as well as inhibitions. Testing for significance by a multivariate t test indicated that the sensitivity of this method was similar to the sensitivities of the conventional testing procedures. The variation of sensitive carbon sources, as determined by factor weights at different observation times, indicated the dynamics of the community shift induced by the Herbogil treatment in more detail. DNA extractions from soil resulted in a collection of molecules representing the genetic composition of total bacterial communities. Distinct and highly reproducible community patterns, or genetic fingerprints, resulting from application of the different herbicides were obtained by the sequence-specific separation of partial 16S rDNA amplification products in temperature gradient gel electrophoresis. Significant pattern variations were quantified. For detailed analysis, application-responsive bands from the Herbogil and Oleo treatments were sequenced and their tentative phylogenetic positions were identified. Data interpretation and the potentials and biases of the additional observation windows on microbial communities are discussed.

Chromosome aberrations in vitro related to cytotoxicity of nonmutagenic chemicals and metabolic poisons.

Chromosome aberrations can occur by secondary mechanism(s) associated with cytotoxicity, induced by chemicals that do not attack DNA. Aberrations are formed from DNA double-strand breaks, and DSBs are known to be induced by nonmutagenic (Ames test negative) noncarcinogens at toxic levels [Storer et al. (1996): Mutat Res 368:59-101]. Here, 8 of 12 of these chemicals caused aberrations in CHO cells at cytotoxic doses, and often only when cell counts (survival) at 20 hr approached < or =50% of controls. Five of eight noncarcinogens (2,4,-dichlorophenol, dithiocarb, menthol, phthalic anhydride, and ethionamide) and one of two equivocal carcinogens (bisphenol A) caused aberrations, usually over a narrow dose range with steeply increasing cytotoxicity. Phthalic anhydride and ethionamide were positive only at doses with precipitate. Phenformin was negative even at toxic doses and ephedrine and phenylephrine were negative and gave little toxicity. Aberrations were also induced by metabolic poisons, 2,4-dinitrophenol, (uncouples oxidative phosphorylation), and sodium iodoacetate, (Nal; blocks ATP production). Five of the chemicals that induced aberrations in CHO cells were tested in human TK6 cells and four were positive, the fifth being equivocal. Stable aberrations (translocations) were induced in human cells by Nal. Clearly, chemicals can give "false-positive" results in the chromosome aberration assay at cytotoxic levels, though cytotoxicity does not always produce aberrations, so that further information (e.g., DNA reactivity) is needed to determine whether a result is a "false-positive." Primary DNA-damaging chemicals such as alkylators are also cytotoxic, but give strong increases in aberrations without marked initial toxicity by the measures used here, although the aberrations they induce do reduce long-term survival in colony-forming assays.

Adenosine A3 receptors promote degranulation of rat mast cells both in vitro and in vivo.

OBJECTIVE: To investigate the effects of adenosine receptor agonists and antagonists on 5-HT release from rat isolated pleural mast cells and on plasma protein extravasation in the skin of conscious rats. In vitro METHODS: Rat isolated pleural mast cells were loaded with [14C] 5-HT, sensitised with mouse monoclonal anti-DNP and then challenged with human serum albumin-DNP. DNP-stimulated 5-HT release from mast cells was determined. In vivo METHODS: Rats, loaded intravenously with [125I] human serum albumin, were injected intradermally with adenosine agonists at sites on the back. 30 min later plasma protein extravasation at each injection site was determined. RESULTS: In isolated mast cells, each adenosine agonist enhanced DNP-induced 5-HT release, N6-(3-iodobenzyl)-5-(N-methyl-carboxamidoadenosine), (IB-MECA), being the most potent agonist. The adenosine A1/A2 antagonist, 8-phenyltheophylline (8-PT), had no effect on the response to IB-MECA. In contrast, 3-(4-amino-iodobenzyl)-8-[4-[[[carboxy]methyl]oxy]phenyl]-1-propylxanthi ne, (I-ABOPX), inhibited (pA2 6.2) the IB-MECA responses. In the skin of conscious rats, intradermal IB-MECA produced a marked plasma protein extravasation (PPE) which was mimicked by N6-2-(4-aminophenyl)-ethyladenosine (APNEA). The PPE produced by IB-MECA was not affected by either 8-PT or CGS15943A, but was virtually abolished by cyproheptadine and in rats pre-treated with Compound 48/80. CONCLUSIONS: These results indicate that stimulation of adenosine A3 receptors both enhances degranulation in vitro and directly produces degranulation of rat mast cells in vivo.

Effects of Poncirus trifoliata on type I hypersensitivity reaction.

A study was carried out to examine the effect of an aqueous extract from immature fruit of Poncirus trifoliata L. (Rutaceae) (PTIFE) on the type I hypersensitivity reaction. Forty-eight hour PCA (passive cutaneous anaphylaxis) in rats was significantly inhibited by the oral administration of PTIFE (200 mg/kg). It also inhibited histamine release from rat peritoneal mast cells (RPMC) induced by mouse anti-dinitrophenyl (DNP)-IgE and dinitrophenyl-human serum albumin (DNP-HSA). These results suggest that PTIFE has anti-allergic action against the type I hypersensitivity reaction.

Comparative effects of the metabolic inhibitors 2,4-dinitrophenol and iodoacetate on mouse neuroblastoma cells in vitro.

The toxic effects of two metabolic inhibitors, dinitrophenol and iodoacetic acid, were compared. Mouse neuroblastoma cell cultures (Neuro-2a) were exposed to different concentrations of the toxic compounds for 24, 48 and 72 h to study basal toxicity effects (cell proliferation by quantification of total protein content (PR) and relative neutral red uptake (RNRU) by lysosomes). The following biochemical indicators assessed in the in vitro test system were: cytosolic phosphofructokinase (PFK) and enolase (ENL) activities in glycolysis; mitochondrial succinate dehydrogenase (SDH) activity in the citric acid cycle; lysosomal beta-galactosidase (GAL) activity; and neuronal acetylcholinesterase (AChE) activity. The effects of the two metabolic inhibitors on the various indicators differed. Iodoacetic acid was found to be far more toxic than dinitrophenol to neuroblastoma cell proliferation at 24 h exposure. Though 2,4-dinitrophenol and iodoacetic acid both inhibited cell proliferation of the neuroblastoma cells, their effects on the other endpoints were opposite. Dinitrophenol was a general activator of the metabolism, particularly affecting lysosomal function. Iodoacetic acid did not significantly alter general metabolism, but considerably modified lysosomal function and AChE activity. The modification of lysosomal function of Neuro-2a cells by the two compounds was quite different: dinitrophenol increased RNRU and GAL activity, and iodoacetic acid decreased both parameters.

Thiols metabolism is altered by the herbicides paraquat, dinoseb and 2,4-D: a study in isolated hepatocytes.

This report is an extension and complement of a previous study reporting the effect of three herbicides (paraquat, dinoseb and 2,4-D) on cell viability, GSH oxidation, NADH and ATP depletion (Arch. Toxicol. 68:24-31, 1994). Here we report additional data and findings aimed at a better understanding of the toxicity mechanisms induced by these herbicides. Biochemical mechanisms of cytotoxicity induced by the herbicides paraquat (1,1'-dimethyl-4,4'-bipyridylium dichloride), dinoseb (2-sec-butyl-4,6-dinitrophenol) and 2,4-D (2,4-dichlorophenoxyacetic acid) were investigated in freshly isolated rat hepatocytes. Herbicide metabolism, especially paraquat and 2,4-D, rapidly depletes GSH and protein thiols. Paraquat and 2,4-D (1-10 mM) decrease the GSH/GSSG ratio, promote loss of protein thiol contents and induce lipid peroxidation. Dinoseb, the most effective cytotoxic compound under study (used in concentrations 1000-fold lower than paraquat and 2,4-D), had moderate effects upon the GSH/GSSG ratio and lipid peroxidation, causing a depletion of protein thiols of about 20%. The results indicate that the herbicides paraquat and 2,4-D are hepatotoxic and may induce cell death by decreasing cellular GSH/GSSG ratio and protein thiols, and by inducing lipid peroxidation. The cytotoxic action of dinoseb is likely to be related with the uncoupling of oxidative phosphorylation in mitochondria. Therefore, it is likely that liver damage observed during the first phase of herbicide-intoxication is related to these metabolic processes.