Non-immunological toxicological mechanisms of metamizole-associated neutropenia in HL60 cells.

Metamizole is an analgesic and antipyretic, but can cause neutropenia and agranulocytosis. We investigated the toxicity of the metabolites N-methyl-4-aminoantipyrine (MAA), 4-aminoantipyrine (AA), N-formyl-4-aminoantipyrine (FAA) and N-acetyl-4-aminoantipyrine (AAA) on neutrophil granulocytes and on HL60 cells (granulocyte precursor cell line). MAA, FAA, AA, and AAA (up to 100 µM) alone were not toxic for HL60 cells or granulocytes. In the presence of the myeloperoxidase substrate H2O2, MAA reduced cytotoxicity for HL60 cells at low concentrations (<50 µM), but increased cytotoxicity at 100 µM H2O2. Neutrophil granulocytes were resistant to H2O2 and MAA. Fe2+ and Fe3+ were not toxic to HL60 cells, irrespective of the presence of H2O2 and MAA. Similarly, MAA did not increase the toxicity of lactoferrin, hemoglobin or methemoglobin for HL60 cells. Hemin (hemoglobin degradation product containing a porphyrin ring and Fe3+) was toxic on HL60 cells and cytotoxicity was increased by MAA. EDTA, N-acetylcystein and glutathione prevented the toxicity of hemin and hemin/MAA. The absorption spectrum of hemin changed concentration-dependently after addition of MAA, suggesting an interaction between Fe3+ and MAA. NMR revealed the formation of a stable MAA reaction product with a reaction pathway involving the formation of an electrophilic intermediate. In conclusion, MAA, the principle metabolite of metamizole, increased cytotoxicity of hemin by a reaction involving the formation of an electrophilic metabolite. Accordingly, cytotoxicity of MAA/hemin could be prevented by the iron chelator EDTA and by the electron donors NAC and glutathione. Situations with increased production of hemin may represent a risk factor for metamizole-associated granulocytopenia.

GSSG/GSH ratios in cryopreserved rat and human hepatocytes as a biomarker for drug induced oxidative stress.

The formation of reactive oxygen species (ROS) could cause cellular damage and eventually lead to apoptosis and necrosis. The ratio between oxidized glutathione and reduced glutathione (GSSG-to-GSH ratio) has been used as an important in vitro and in vivo biomarker of the redox balance in the cell and consequently of cellular oxidative stress. This paper optimizes a LC-MS/MS method for the simultaneous determination of GSH and GSSG. The proposed method is based on the derivatization of reduced GSH using iodoacetic acid (IAA) in order to prevent its rapid oxidation to GSSG during sample preparation. The optimized analytical method was applied to evaluate the effect of different pharmaceutical agents on GSSG-to-GSH ratio in cryopreserved rat and human hepatocytes in culture. Hepatocyte viabilities were also determined at the same time by using the WST-1 assay as a direct measurement of cell mitochondrial respiration. The results obtained demonstrate that cryopreserved rat and human hepatocytes in culture are reliable in vitro models for the evaluation of cellular oxidative stress. In addition, the GSSG-to-GSH ratio measurements could be a biomarker of hepatotoxicity providing similar results to those of cytotoxicity assay.

Photodegradation study of three dipyrone metabolites in various water systems: identification and toxicity of their photodegradation products.

The photochemical behaviour of three relevant metabolites of the analgesic and antipyretic drug dipyrone, 4-methylaminoantipyrine (4-MAA), 4-formylaminoantipyrine (4-FAA) and 4-acetylaminoantipyrine (4-AAA), was evaluated under simulated solar irradiation (Suntest system). For 4-MAA, different aqueous solutions (synthetic seawater, freshwater and Milli-Q water) as well as different operational conditions were compared. According to the experimental results, 4-MAA resulted as being an easily degraded molecule by direct photolysis, with half-life times (t1/2) ranging from 0.12 to 0.58 h, depending on the irradiation conditions. Faster degradation was observed in synthetic waters, suggesting that the photolysis was influenced by the salt composition of the waters. However, no effect on the degradation rate was observed by the presence of natural photosensitizers (dissolved organic matter, nitrate ions). 4-FAA and 4-AAA showed slower photodegradation kinetics, with t1/2 of 24 and 28 h, respectively. A study of photoproduct identification was carried out by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS) (ESI positive mode), which allowed us to propose a tentative photodegradation pathway for 4-MAA and the identification of persistent by-products in all the cases. Finally, the application of an acute toxicity test (Daphnia magna) showed an increase in toxicity during the photolytic process, a consequence of the formation of toxic photoproducts.

Effects of antioxidant 1-O-hexyl-2,3,5-trimethylhydroquinone or ascorbic acid on carcinogenesis induced by administration of aminopyrine and sodium nitrite in a rat multi-organ carcinogenesis model.

The effect of antioxidant, 0.25% 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ) or 0.25% ascorbic acid (AsA), on carcinogenesis induced by administration of 0.05% aminopyrine (AP) and 0.05% sodium nitrite (NaNO2), was examined using a rat multi-organ carcinogenesis model. Groups of twenty F344 male rats were treated sequentially with an initiation regimen of N-diethylnitrosamine, N-methyl-N-nitrosourea, N-butyl-N-(4-hydroxybutyl)nitrosamine, N,N'-dimethylhydrazine and 2,2'-dihydroxy-di-n-propylnitrosamine during the first 4 weeks, followed by AP+NaNO2, AP+NaNO2+HTHQ, AP+NaNO2+AsA, NaNO2+HTHQ, NaNO2+AsA, each of the individual chemicals alone or basal diet and tap water as a control. All surviving animals were killed at week 28, and major organs were examined histopathologically for development of preneoplastic and neoplastic lesions. In the AP+NaNO2 group, the incidences of hepatocellular adenomas and hemangiosarcomas were 95% and 35%, respectively. When HTHQ or AsA was simultaneously administered, the incidences decreased to 58% and 11%, or to 80% and 15%, respectively. On the other hand, in the AP+NaNO2 group and the NaNO2-alone group, when HTHQ, but not AsA, was simultaneously administered, the incidence of carcinomas in the forestomach significantly increased. The results suggest that HTHQ can prevent tumor production induced by AP and NaNO2 more effectively than AsA. On the other hand, an enhancing or possible carcinogenic effect of simultaneous administration of HTHQ and NaNO2 only on the forestomach is suggested, while simultaneous treatment with the same dose of AsA and NaNO2 may not be carcinogenic to the forestomach or other organs.

[Comparative study of the effect of paracetamol, phenacetin and amidopyrine on mitochondrial function in isolated rat hepatocytes]. / Sravnitel'noe izuchenie deistviia paratsetamola, fenatsetina i amidopurina na funktsiiu mitokhondrii v izolirovannykh gepatotsitakh krys.

Effects of amidopyrine, phenacetin and paracetamol on the viability and energetic state of isolated rat hepatocytes were compared. During incubation in minimal salt solution all drugs in concentrations above 1 mM in dose-dependent manner decreased the viability of hepatocyte suspension assessed by trypan blue dye inclusion and inhibited the ATP synthesis and the respiratory activity. Cytotoxic effect of chemicals decrease in the order: amidopyrine-->phenacetin-->paracetamol. The inhibition of the rate of endogenous respiration were accompanied by stimulation of oxygen consumption in nonmitochondrial systems. An uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, and disruption of plasma membrane by digitonin followed by substrate succinate addition did not restore the respiratory activity of hepatocytes up to the level of control cells. These data show that cytotoxicity of the chemicals is determined by their interaction with enzymes of mitochondrial respiratory chain.

Oxidation of aminopyrine by hypochlorite to a reactive dication: possible implications for aminopyrine-induced agranulocytosis.

Aminopyrine is associated with a high incidence of agranulocytosis. It is known to be oxidized by peroxidases and hypochlorous acid to a blue cation radical. It has been proposed that the mechanism by which hypochlorous acid oxidizes aminopyrine to a cation radical involves N-chlorination followed by loss of a chlorine radical. Another possible mechanism is loss of HCl to form an iminium ion and subsequent reaction with another molecule of aminopyrine and a hydrogen ion to form two radical cations. This mechanism would lead to incorporation of a hydrogen from water; however, using a deuterated analog, we found no hydrogen incorporation, thus providing strong evidence against this mechanism. Using a stopped-flow diode array spectrophotometer to study the reaction between aminopyrine and hypochlorous acid, an intermediate with a lambda max at approximately 420 nm was observed in the formation of the cation radical. We propose that this represents a dication formed by the loss of chloride ion from N-chloroaminopyrine. This intermediate is very reactive, with a half-life of approximately 15 ms, and in addition to being the precursor of the cation radical, it also appears to react with two molecules of water to form several other products that were observed and are consistent with the proposed dication intermediate. Similar stable products were formed when amino-pyrine was oxidized by the combination of myeloperoxidase, hydrogen peroxide, and chloride or activated neutrophils. The reactive dication formed by neutrophil-derived hypochlorous acid could be responsible for aminopyrine-induced agranulocytosis.

[Cytochrome P-450 activity and lipid peroxidation in rat liver and lung microsomes during hypoxia and followed by pressure oxygenation]. / Aktivnost' tsitokhroma P-450 i perekisnogo okisleniia lipidov v mikrosomakh pecheni i legkikh krys v usloviiakh gipoksii i posleduiushchei barooksigenatsii.

Influence of hypoxia (0.029 MPa, I h) followed by hyperoxia (0.2 MPa, I h) on microsomal oxidation and lipoperoxidation was studied in rat liver and lungs. Distinct increase of cytochrome P-450 catalytic activity with amidopyrine and benzo-a-pyrene as substrates of the I type was found after hypoxia, subsequent hyperoxia resulted in significant increase of amidopyrine and benzo-a-pyrene metabolism in liver and lung tissues and of aniline metabolism in liver tissue. Both hypoxia and hyperoxia led to increase in content of diene conjugates and Schiff bases in liver and lungs, while the increase of diene conjugates in liver and both diene conjugates and Schiff bases in lungs were observed under hyperoxic conditions.

[Induction of cytochrome P-450 by triterpensaponins in Vietnamese ginseng]. / Induktsiia tsitokhroma P-450 triterpensaponinami V'etnamskogo zhen'shenia.

It was found that rat liver cytochrome P-450 is induced by the Vietnamese ginseng triterpensaponines mixture (TSM) as well as by K5VN Panaxozides-11 (VP-11) purified from this mixture. Addition of TSM and VP-11 accelerates benz(alpha)pyrene and aminopyrine hydroxylation and increases the content of cytochrome P-450 isoforms with Mr of 57 kDa and 54 kDa in rat liver microsomes. Since VP-11 accounts for about 50% of TSM, the results obtained suggest that the microsomal monooxygenase system induction is caused by this triterpensaponine. Induction by TSM and VP-11 was compared to that by phenobarbital (PB) and 3-methylcholanthrene (MC). It was shown that according to their inductive action TSM and VP-11 belong neither to the PB- nor to the MC-type. Cytochrome P-450 induction may play an important role in the triterpensaponine action on the organism, because this enzyme participates in the metabolism of such endogenous compounds as prostaglandins, steroid hormones, cholesterol, etc.

The carcinogenic danger of nitrie pollution of the environment.

Presented are the literature data as well as the results of our own investigations on the genotoxic and carcinogenic effects of sodium nitrite (SN). The carcinogenicity of SN detected in animal experiments appears to be related to the formation of nitroso compounds from endogenous nitrosable precursors. Sodium nitrite possesses transforming and promoting effects in cell cultures, as well as mutagenic effects in the bacterial systems, where the predominant effect of SN was compared to that of N-nitrosodimethylamine (NDMA). Prolonged pretreatment with SN amplifies the liver DNA damage in rats in case of NDMA endogenous synthesis.

Lack of tumorigenicity of aminopyrine orally administered to B6C3F1 mice.

To test the tumorigenic potential of aminopyrine, an antipyretic analgesic, it was administered in drinking water at levels of 0 (control), 0.04 and 0.08% to 50 male and 50 female B6C3F1 mice for 100 weeks, and the mice were subsequently maintained without aminopyrine for a further 4 weeks. The most frequent types of tumor, in both treated and control groups, were hepatocellular tumor in male mice and malignant lymphoma/lymphoid leukemia in female mice. No statistically significant differences were observed in the incidences of these tumors between treated and control groups. The incidences of several other tumors in male and female mice also showed no statistically significant differences between treated and control groups. Therefore, no tumorigenic effect of orally administered aminopyrine in B6C3F1 mice was apparent in the present study.

[Inhibiting effect of the polyphenolic complex from Plantago major (plantastine) on the carcinogenic effect of endogenously synthesized nitrosodimethylamine]. / Ingibiruiushchee vliianie polifenol'nogo kompleksa iz podorozhnika bol'shogo--plantastina--na kantserogennyi éffekt éndogenno sintezirovannogo nitrozodimetilamina.

Amidopyrine administered in combination with sodium nitrite in the long-term experiment produces the toxic damage of the liver and tumors in rats in connection with endogenic synthesis of carcinogenic nitrosodimethylamine. The inclusion into the animal diet of the polyphenolic complex from Plantago major--plantastine as an inhibitor of the carcinogen synthesis reduced the toxic damage of the liver that was indicated by normalization of biochemical parameters and also decreased the tumor yield from 87.5% to 33.3%. The data obtained may be the basis for the combined use of plantastine with nitrosated drugs that would contribute to carcinogenesis prevention.

Mutagenicity of 4-aminoantipyrine and 4-nitrosoantipyrine, the C-nitroso derivative of antipyrine.

The drug antipyrine and its 4-substituted analogs, 4-aminoantipyrine, 4-dimethylaminoantipyrine (aminopyrine) and 4-nitrosoantipyrine were tested for mutagenicity against the screening array of Salmonella typhimurium tester strains TA100, TA98, TA97, TA102 and TA104. Antipyrine and aminopyrine were nonmutagenic to all 5 tester strains even in the presence of S9. 4-Aminoantipyrine was directly mutagenic to TA97 only and the presence of S9 slightly increased its activity. 4-Nitrosoantipyrine was directly mutagenic to all tester strains used and S9 decreased its activity except with strain TA102. The possible long-term hazards of C-nitroso compounds derived from drugs and dietary constituents are discussed in view of their pluripotent direct genotoxicity.

Generation of high yields of Syrian hamster cholangiocellular carcinomas and hepatocellular nodules by combined nitrite and aminopyrine administration and Opisthorchis viverrini infection.

Combined administration of 0.1% nitrite and 0.1% aminopyrine in the drinking water for eight to ten weeks resulted in subsequent development of both hepatocellular nodules and cholangiofibrotic lesions/cholangiocellular carcinomas in Syrian golden hamsters. Additional prior dosing with Opisthorchis viverrini metacercariae (100/animal) induced inflammatory and proliferative changes in the livers of infected hamsters and was associated with a significant increase in yields of hepatocellular and cholangiocellular preneoplastic and neoplastic lesions. Thus, environmental factors thought to be casually related to the high levels of human liver cancer observed in the Northeastern provinces of Thailand were sufficient to bring about development of equivalent tumors in experimental animals. The results indicate that parasite associated liver injury and non-specific compensatory regeneration may play an important role in generation of both hepatocellular and cholangiocellular carcinomas in man.

Strain differences in susceptibility to the embryotoxic effects of aminopyrine in mice.

Embryotoxic effects induced by aminopyrine were studied in two inbred strains of mice, C57BL/6N and DBA/2N. Aminopyrine was given by subcutaneous injection at a dose of 200 mg/kg on day 7, 8 and 9 of gestation. In both strains, aminopyrine-induced malformations such as omphalocele, club foot and kinky tail were observed, but the incidence of malformations was significantly higher in C57BL/6N than in DBA/2N. Maternal plasma levels of aminopyrine and its metabolites were significantly higher in C57BL/6N, compared to DBA/2N. Further, reciprocal crosses between these two strains were used to clarify whether the maternal or fetal genotype is more important in aminopyrine-induced embryotoxicity. F1 hybrid embryos developing in C57BL/6N or DBA/2N mothers were as resistant as inbred DBA/2N. These results suggest that strain differences in susceptibility to aminopyrine may depend on fetal genotype rather than maternal factors.

Does pyrazolone-induced renal injury exist?

On the basis of animal experiments and clinical findings, pyrazolones are found to have adverse renal effects. However, the latter are minor, very rare, and of practically no clinical relevance. In animals, pyrazolones induce proteinuria, oliguria, retention of substances excreted via the urine, and probably, in rare cases, papillary necrosis. Oliguria is rare in humans. A contribution of pyrazolone drugs in a specific case of papillary necrosis and in rare cases of acute interstitial nephritis is not proven, yet possible. Pyrazolone drugs induce renal injury less frequently than do the other classical analgesics.

Histological studies into rat liver following long-term application of aminophenazone, phenazone, and propyphenazone.

Morphological studies by means of light microscopy are important for toxicological drug testing. More complete information on potential damage can be obtained only by combination of pharmacological, biochemical, hepatofunctional, and morphological tests and studies. No systematic tests had been applied to rats, in the past. Therefore, aminophenazone, phenazone, and propyphenazone were tested for periods up to 16 weeks. Long-term application of the three pyrazolone derivatives resulted in the following dose-dependent and time-dependent alterations: hepatocyte enlargement, fatty degeneration and reactive-inflammatory changes along with single-cell necrosis, round cellular infiltration of periportal fields, and Kupffer cell activation. The alterations differed in intensity by the following order: aminophenazone greater than phenazone greater than propyphenazone.

Protective action of ascorbic acid against mutagenicity of aminopyrine plus nitrite.

Mutagenicity of aminopyrine and of aminopyrine plus nitrite was tested by the micronucleus test in bone marrow of mice and by host mediated mutagenicity assay with mice as host animals and S. typhimurium strain G 46. In parallel the possibility of the protective action of ascorbic acid was studied. Aminopyrine at the dose of 90 mg/kg po when administered to mice together with potassium nitrite induced a significant increase in the frequency of micronuclei in polychromatic erythrocytes and proved to be mutagenic for a Salmonella strain. In both systems mutagenicity of the combination of aminopyrine at this dose plus nitrite was abolished completely by ascorbic acid (373 or 622 mg/kg po). Ascorbic acid neither induced a significant increase in the frequency of micronuclei nor was mutagenic for the strain G 46. A formulation of aminopyrine with ascorbic acid is proposed.

Acute and genetic toxicity of 1-nitropyrene and its fate after single oral doses to rats.

The mammalian acute and genetic toxicity of 1-nitropyrene (NP) was studied because this and other nitroarenes are highly mutagenic toward bacteria and have been identified in emissions from combustion processes. A suspension of NP did not cause observable signs of acute toxicity and was not lethal when administered to male and female rats at single oral doses as high as 5.0 g/kg. Histological examination of stomach, intestine, lung, heart, spleen, pancreas, adrenal, and kidney from rats euthanized at 4 and 14 d after treatment revealed no detectable differences from control rats. Urine and feces were collected for 4 d after treatment with 5.0 g/kg. About 70% of the dose was present in the feces as NP, and about 2% was present as the reduced metabolite 1-aminopyrene (AP). Sulfate and glucuronide conjugates of AP were present in small amounts (less than 1%) in the urine, showing that at least some of the dose was absorbed. Bone marrow cells from female rats given NP orally at 0.5, 1.5, and 5.0 g/kg showed a slight dose-related increase in the frequency of sister chromatid exchanges. Both NP and AP showed low mutagenicity in Chinese hamster ovary cells in vitro. Evidence of reductive metabolism of NP in rats raises concern about the potential exposure of humans to this compound. However, the weak in vivo and in vitro genetic toxicity of NP at high dose levels in mammalian systems suggests that the potential hazard may not be as high as predicted from bacterial mutagenicity data.